Transcript
Bio
Surface innovation Optimising cell adhesion
Evaporation & edge effect In cell based assays
Take a deep breath Culturing cells under low oxygen
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Welcome This publication marks our 6th edition of the Bio-Innovation series, a magazine dedicated to showcasing new and emerging trends relevant to the field of life science for our Australian and New Zealand customers.
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Our latest issue is filled with new developments in the field of Cell Biology – with a focus on Cell Culture. We spotlight several new developments including optimisation of cell adhesion, for example an innovative new monomer that is “folded” at 37 Celcius, and “unfolds” at 25 Celcius, offering a non-enzymatic mechanism to release cells from the growth surface whilst maintaining the cell protein matrix intact, newly designed microplates that reduce edge effects, maximising cell culture performance and assessing cell viability in high density cryogenic storage.
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As cell culturing advances and newer technologies are introduced, cells themselves or cell products will be used more and more for human therapies, therefore, the products used become even more important. With this in mind, we endeavour to provide the most innovative options currently available.
Editor Mika Mitropoulos: mika.mitropoulos@thermofisher.com Art & Design Andrew Dennis: andrew.dennis@thermofisher.com Reno Leuc: reno.leuc@thermofisher.com Cover Image: Fibroblast cells. Fluorescent light micrograph of two fibroblast cells, showing their nuclei (green) and cytoskeleton. The cytoskeleton is made up of microtubules of the protein tubulin (yellow) and filaments of the protein actin (blue). The cytoskeleton supports the cell's structure, allows the cell to move and assists in the transport of organelles and vesicles within the cell. Fibroblasts are cells forming connective tissue, and are responsible for secreting connective tissue proteins such as collagen.
Lindsay Allan Category Manager Life Science & Lab Equipment Thermo Fisher Scientific
Contents Surface innovation – optimising cell adhesion .......................................................04 Optimising neuron adhesion & growth ..................................................................08 Maximise cell culture production & performance ...................................................10 Take a deep breath – consider culturing cells under low oxygen ..............................12 Growth of & recombinant protein production in Sf-9 insect cells ..............................15 DNA from microbial cultures – magnetic bead based isolation.................................16 Viability of mammalian cells in high density cryogenic storage.................................18 Primary cell analysis ...........................................................................................20 Odour from irradiated polystyrene has no effect on cell growth & performance ..........23 Breakthrough ClipTip technology .........................................................................30 Cell biology workflow ..........................................................................................24 Turbofect™ in vitro transfection reagent...............................................................31 Rapid-flow membrane ........................................................................................32 Copper CO2 incubators: why your next incubator should be 100% pure copper .........34 Expansion & differentiation of human mesenchymal stromal cells ...........................36 Upcell surface versus trypsinisation......................................................................39 The unique design of Thermo Scientific Carrier Plates ............................................40 Raindance thunderstorm.....................................................................................42 Evaporation & edge effect in cell based assays ......................................................44 Low-frequency tumour allele detection .................................................................46 Sample preparation for high-throughput live cell imaging .......................................48 Q&A ..................................................................................................................51
by D by Drr Jo Joseph ose seph ph G Granchelli ra ranc anc nche nch helll i
Surface innovation optimising cell adhesion The value and contribution of surface chemistries to the success of in vitro cell culture are emphasised in this article, and the critical issues associated with the development of such technologies, as well as the relevant developments up to date, are discussed and reviewed. As an important tool for the study of cell biology, the production of proteins and the discovery and characterisation of new drugs, in vitro cell biology has uses within many academic, clinical and life science laboratories, as well as applications in pharmaceutical R&D. Cell-based assays are widely recognised as relevant in vitro models, and techniques such as high content analysis (HCA) now form a key approach to drug discovery research. As such, the quality and consistency of in vitro cell culture is of paramount importance in order to obtain the most accurate and reproducible results possible. Experimental success or failure will depend on numerous variables that require careful control, including the techniques and protocols used, cell lineage, growth conditions, media formulation and culture surfaces. In adherent cell culture, the surface used can have a significant impact on cell viability, the consistency and purity of a culture, and consequently on the reproducibility and accuracy of the obtained results. When culturing cells, the aim is to maintain their physiology to be representative of the in vivo model, and engineering mechanical attachment to artificial cell surfaces via normal cellular processes can help to achieve this. Here we discuss the principles of cell adhesion, along with the basic technologies used to optimise surfaces for improved
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adhesion and expansion in addition to the effects of various surface compositions on cell growth. A review of more recent technological developments of surface chemistries will also look at the preferences of more fastidious cell types and the differential use of culture surfaces for different applications.
Cell interaction and communication Cells respond to external stimuli such as nutrients, growth factors, cytokines, O2 [1], pH and mechanical/shear stress, through cell surface interactions and the activation of internal signalling pathways (classified as outside-in interactions). In contrast, inside-out intracellular signal transductions allow the cell to remodel its external microenvironment and to up-regulate or change the structure of cell surface receptors. This remodelling is often required for growth, survival, intracellular adhesion or migration. Integrins are one group of cell surface receptors that are critical for such processes and consequently normal cell function. Their transmembrane heterodimeric structure allows them to act as mediators between the cells and molecules within the external microenvironment and the internal signaling pathways – they are essentially communicators. Influential extracellular matrix (ECM) components In vivo, cells adhere to biological substrates via the basal lamina, a tissue-specific, self-assembling, acellular glycoprotein matrix, which is secreted predominantly by the cells themselves. The most proximal part of the basal lamina, the extracellular matrix (ECM) contains a combination of different glycoproteins (approximately 45), including laminin, collagen, perlecan and nidogen. Modifications to
by Dr Joseph Granchelli Thermo Fisher Scientific
the cell surface, (i.e. the up- or down-regulation of integrins), are commonly required for cellular events such as growth, migration, or apoptosis, which through complex signalling pathways induce a rapid reconfiguration of the ECM. Furthermore, the binding affinities of glycoproteins allow homologous and heterologous interactions resulting in the formation of a complex network of molecules, with numerous integrin binding sites for cell attachment. One unit of the cell surface integrin molecule confers binding specificity, while the other confers binding affinity to the corresponding ECM component. This binding initiates conformational changes of the integrin, activating complex signaling pathways for communication to the intra-cellular components. As a result, significant effects on cell physiology are observed [2]. Consequently, cellular interaction and adhesion are essential contributors to cell survival. When deprived of the correct attachment signal via their integrin receptors, catastrophic effects on the cell culture population as a whole may result. Even if they adapt to survive in suspension, their physiology and morphology may have greatly altered. Cells are likely to divide into sub-populations with distorted physiology, or be stimulated to undergo significant cellular events such as differentiation or apoptosis. The result is a heterogeneous population which has moved away from its true phenotype and may exhibit a varied response to controlled experimental stimuli. In order to maintain cell physiology and phenotype in vitro, the provision of culture conditions specific for that cell type can reduce or eliminate these potential changes. Normal cell attachment and growth can be facilitated through the application of an appropriate artificial culturing surface.
In vitro substrates Novel culture surfaces must be capable of facilitating rapid reattachment to quickly re-establish normal cell function and recovery from any integrin disruption that may have occurred as a result of the enzymatic treatment used for cell dissociation and passaging [3]. However, the materials used need to be structurally similar to the glycoprotein molecules of the ECM. Many scientists have used coatings for their culture vessels, for example in-house manufactured rat tail collagen isolates [4]. Maintaining a consistent level of purity and coating uniformity across all culture flasks is not easy and the use of in-house preparations containing animal derivatives can provide a potential source of contamination. Thus, manufacturers have worked to develop surfaces using both natural glycoproteins and man-made alternatives. Many of these alternatives such as polystyrene, PETG, thermanox and permanox are thermoplastic polymers, which are easy to mould and manufacture, and are very uniform. However, they are hydrophobic in nature, so will exhibit a very different surface topology from the ECM. With no functional oxygen or nitrogen groups, even more hydrophilic surfaces such as polycarbonate and glass require some form of surface modification to further facilitate cell attachment and growth.
Common surface modifications Energetic oxidation This method enables the creation of small hydrophilic regions on the polymer surface, which can stabilise the protein’s conformational changes that are essential for binding. There are a variety of treatments that can be used to do this, such as corona discharge, plasma treatments, or UV [5] and gamma irradiation, all of which effectively bombard the surface with
References: 1. Brevig T et al. Artificial Organs 2006; 30: 915-921 2. Drug Discovery & Development magazine: November 2008; Vol. 11, No. 11: 44-47 3. Gräbner R. Cytometry 2000; 40: 238-244 4. Brevig T et al. Biomaterials 2005; 26: 3039-53 5. Bacáková L. Virchows Arch 2002; 440: 50-62 6. Faucheux N et al. Biomaterials 2004; 25: 2721 -2730 7. Faucheux N et al. Biomaterials 2006; 27: 234-245 8. Broedel SE Jr & Papiak SM. The Case for Serum-Free Media. BioProcess International. February 2003 9. Hata H et al. J Thorac Cardiovas Surgery 2006; 132: 918-924 10. Miyahara Y et al. Nature Medicine 2006; 12: 459-465 11. Sumide T et al. The FASEB Journal 2006; 20: 392-394 12. Nishida K et al. Transplantation 2004; 77: 379-385
Atom % C=O Plasma
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C=O or O-C-O 11
Corona UV
8 12
5 1
Gamma
3
0
Functional Group Key O=C=O
COOO
C-O
Alcohol/Hydroxyl
7
5
C=O
Aldehyde or Ketone
2 1
0 0
O-C=O
Carboxylic Acid or Ester
COOO
Alkylperoxide
0
0
Figure 1.
Figure 2.
ions, electrons or photons. The result is a subtle difference between the chemical groups formed. This bombardment breaks some of the bonds in the polymer chains as well as the gaseous material surrounding them, providing readily available free-radicals on the culture surface. With the ability to quench nearby molecules, the distribution of chemical groups will become slightly hydrophilic to facilitate the protein unfolding required to expose the hydrophobic domains for binding [Figure 1]. However, without the coating of animalderived substrates, the cells must use proteins for integrin binding from the surrounding media. Ready-made cell culture media commonly contains animal serum, which includes fibronectin and vitronectin. These will self-assemble in critical concentrations and bind to the artificial substrate, providing a ligand-rich surface for cell binding [6, 7]. Each surface will exhibit different affinities for each of the soluble proteins and the conformation and orientation will vary, demonstrating a unique binding pattern that is more suited to some cell lines than others [8]. Consequently, other surface modifications have been developed in order to further improve on the surfaces available.
important recognition domain; or incorrect refolding can cause denaturation, blocking any future cell adhesion events [2].
Passive adsorption Passive adsorption is one surface modification that involves coating the polymer surface with naturally occurring, selfassembling protein molecules from the ECM (i.e. collagen) [3] . During passive adsorption, binding occurs when the ligand collides with the surface by random thermal motion in the correct orientation with enough energy to interact non-covalently. The binding mechanism uses hydrophobic interactions between the polymer chains and the protein hydrophobic domains, and the proteins will bind to the polymers in a random orientation. The proteins used are typically coated in an aqueous solution and the hydrophobic regions are internally positioned for polymer binding. As such, protein chains must partially unfold in order to melt into the polymer surface during binding. This conformational change may have significant negative implications, such as: the activation or deactivation of critical functionality; masking an
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The main driver for using passive adsorption is to promote normal cell adhesion with molecules that are naturally occurring in the ECM. Although some more sensitive cell lines may require a biological coating, many may just demonstrate better function and proliferation [Figure 2]. However, the degree of experimental success can be dependent upon cell type. There is no standard rule that applies and each set-up may require further optimisation. Surfaces modified using this method can be more costly to produce than alternatives and while these products can be certified aseptic, they are not terminally sterile (to FDA standards), since the methods required for this can alter the biological activity of the protein-coated surface. Passive adsorption can also demand a binding process that is not energetically favourable. Proteins in aqueous solutions do not always readily unfold their tertiary structure to expose the hydrophobic domains which are required for binding [2]. Fine-tuning Further optimisation is often essential if the self assembling matrix is not suitable for a specific, and maybe more sensitive, cell type to adhere. For example, a cell line will naturally adapt, over a number of passages, to a less sophisticated surface and reduce its requirement for serum. Where there are no serumderived proteins to aid cell attachment, it is thought that the cells themselves will temporarily produce attachment factors until they are able to produce their own ECM. This is commonly known as serum reduction [Figure 3], but again the degree to which this is possible and successfully implemented is often dependent on cell and surface type [Figure 4]. Serum reduction can be of particular value within bioproduction, where the use of serum necessitates downstream protein filtration steps. Adventitious agents can be introduced which can accrue significantly over time [8]. Thermo Scientific Nunclon Delta is an example of a surface that can be used for such purposes.
Figure 3.
Figure 4.
A novel surface modification The covalent grafting of polymers is one rather new and exciting surface modification, which is useful for attaching molecules that do not efficiently adhere through passive adsorption, such as proteins which do not exhibit highly hydrophobic regions. The Thermo Scientific Nunc Immobiliser cell line is one example of this, as it uses a group that reacts with polystyrene, an anthroquinone group which is coupled to a spacer arm. Various chemical functionalities are attached to the other end, allowing the covalent coupling of biomolecules. For example, the reactive group of Immobiliser amino will covalently couple primary amines, while Immobiliser streptavidin will couple biotinylated molecules.
graft area can be overcome; and bioengineered human corneal endothelial cell sheets can be produced in vitro to retain native morphology and functional characteristics. Conversely, for the cultivation of single cells and cell clusters in suspension, there are surfaces available that have been designed not only to prevent cell attachment, but to also allow the growth of cells that are sensitive to unwanted activation and differentiation signals arising from cell adhesion. The Nunc HydroCell Surface is a thin layer of a covalently-immobilised, super hydrophilic polymer, which enables a more homogenous suspension culture. Higher cell yields and cell-secreted protein production can be achieved across many cell culture applications, including those involving monocytes/macrophages and stem cells. Furthermore, the adsorption of culture medium-derived proteins and cell-secreted proteins to the surface is minimal.
Stimulus-sensitive surfaces can modify or change their chemistry in response to external stimuli, and can provide significant benefits for cell growth, dissociation and passaging. One such surface, the Thermo Scientific Nunc UpCell, utilises the covalent modification of polystyrene to poly-n-isopropylacrylamide (NIPAM), to provide a material which can modify its chemical conformation upon temperature change. This enables the simple control of cell adhesion: at raised temperatures NIPAM becomes more hydrophobic and interacts more readily with proteins; whereas at lower temperatures numerous hydrophilic groups are exposed to prevent protein interaction. Furthermore, enzymatic methods such as trypsinisation, which can cleave important surface molecules, can be avoided during cell dissociation and passaging. The structure of the basal lamina and cell surface receptors are conserved, allowing whole sheets of cells to be removed. This method has proved especially important for tissue engineering research applications. For example the Nunc UpCell has been used to provide a feasible and effective method for treating heart failure [9,10] as well as for a range of corneal regenerative therapies [11,12]. Problems related to the intramyocardial injection of cells, including cell loss and limited
The future Today, cell culture is widely used and extremely valued across numerous clinical and discovery applications.Cell-based research is constantly providing the industry with new and exciting discoveries and novel in vitro models for further study. As such, it is a constantly evolving discipline which poses new questions on an ongoing basis. The requirements for culture optimisation are also evolving as the types of cells used are expanding and experimental procedures developing in complexity. To meet these demands, scientists and manufacturers alike understand that variables such as the surface, media and the cells themselves all require optimisation to produce strong adhesion, independent of the chemistry used. While it is possible to design surfaces which are good for the majority of cells, more difficult cell types require more advanced modifications. Partnering with companies such as Thermo Fisher Scientific can provide extensive application expertise and practical advice to jointly aid surface selection and development. Furthermore, phenotypic stability and experimental consistency can be significantly improved.
Figure 1. Energetic oxidation of polystyrene – the original surface is hydrophobic but the oxidised surface has both hydroxyl and carboxyl functional groups making it more hydrophilic. The table demonstrates the different surface configuration using the selection of oxidative treatments. Figure 2. Preferential growth of PC-12 and MDCK cells on different culture surfaces. Figure 3. Cell line Hel-299 demonstrates differential adaptation response to serum reduction with Corona and N2O plasma oxidised surfaces. Figure 4: Vero cells adaptation to serum free media with Corona and N2O plasma oxidised surfaces.
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optimising neuron adhesion & growth Choosing the right surface chamber slide References: Bartlett W.P., & Banker G.A. (1984) An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture: 1. Cells which develop without intercellular contacts. J. Neurosci. 4: 1944-1953. Bottenstein, J.E., & Sato G.H. (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. USA 76: 514-519. Corey J.M., Wheeler B.C., & Brewer G. J. (1991) Compliance of Hippocampal Neurons to Patterned Substrated Networks. Journal of Neuroscience Research 30: 300-307. Healy K.E., Thomas C.H., Rezania A., Kim J.E., McKeown P.J., Lom, B., & Hockberger P.E. (1996) Kinetics of bone cell organisation and mineralisation on materials with patterned surface chemistry. Biomaterials 17: 195-208. Kleinfeld D., Kahler K.H., & Hockberger P.E. (1988) Controlled Outgrowth of Dissociated Neurons on Patterned Substrates. Journal of Neuroscience 8 (11): 4098-4120.
In general, chamber slides are used for cellular imaging studies of adherent cells. Their ability to attach to various culture surfaces is cell type specific. For example, epithelial and fibroblast cell lines attach and proliferate readily on cell culture-treated plastic surfaces. Biological coatings and chemical modifications of the culture surfaces may alter the proliferation status of these cells. On the other hand, neurons do not adhere easily on standard cell culture-treated plastic surfaces. Their survival and growth depend heavily on certain biological coatings or chemical modifications of the culture surface. The Thermo Scientific Nunc Lab-Tek Chamber Slides are designed for simplifying imaging analysis involving microscopic examination of cultured cells. The multi-chambered design allows for parallel studies of multiple conditions and stimuli required by many applications. Downstream cytostaining process is greatly facilitated by the removal of the upper structures (wells). The standard footprint of a slide is compatible with all imaging equipment and makes the microscopic examination very convenient.
Lom B., Healy K.E., & Hockberger P.E., (1993) A versatile technique for patterning biomolecules onto glass coverslips. Journal of Neuroscience Methods 50: 385-397. Matsuzawa M., Potember R.S., Stenger D.A., & Krauthamer V. (1993) Containment and growth of neuroblastoma cells on chemically patterned substrates. Journal of Neuroscience Methods, 50: 253-260. Schaffner A.E., Barker J.L., Stenger D.A., & Hickman J.J. (1995) Investigation of the factors necessary for growth of hippocampal neurons in a defined system. Journal of Neuroscience Methods 62:111-119. Spargo B.J., Testoff M.A., Nielsen T.G., Stenger D.A., Hickman J.J., & Rudolph A. S. (1994) Spatialy controlled adhesion, spreading and differentiation of endothelial cells on self assembled molecular monolayers. Proc. Natl. Acad. Sci. USA Vol. 91: 11070-11074.
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In this study, we examined the correlation between neuronal cell adhesion and the chamber slide surface modifications.
Methods Cell Culture – PC-12 (ATCC CRL 1721) cells were maintained in Thermo Scientific HyClone medium and supplements including MEM containing L-glutamine, antibiotic antimycotic, non-essential amino acids, sodium pyruvate, 10% horse serum and 5% bovine calf serum. Three days after plating on test surfaces, the medium was replaced with a N2.1 defined medium (modification of Bottenstein and Sato, 1979; Bartlett and Banker, 1984) containing progesterone (20 nM), putrescine (100), selenium dioxide (30 nM), 100 g/ml transferrin (bovine), 5 g/ml insulin (solubilised in 0.01N HCI), and 0.5 mg/ml ovalbumin in DMEM with 15 mM
(PDL; 1 mg/ml) in borate buffer (boric acid 3.1 g/l,
Glass and plastic surfaces must be adequately
borax 4.8 g/l in water). They were washed with
modified to support adhesion and differentiation
sterile water and dried before use.
of primary neurons. Culturing primary neurons is particularly challenging since they do not continue
Quantification of Primary Amines Primary amines were quantified using an o-phthalaldehyde
proliferating after dissociation. Primary neuron survival in culture depends on cell adhesion and
based assay (Thermo Scientific Pierce). All
differentiation, which can be facilitated by altering
measurements were done in soda lime glass chamber slide products or in chambered-
the culture surface with biological coating or chemical modification (Figure 2). Neuron adhesion
coverglass assemblies using a fluorescence plate
and growth requires more than just the hydrophilic
reader. A fluorescence/concentration curve was generated using polylysine as the standard. The
surface provided by glass and standard cell culturetreated plastic surface. Therefore, very few neurons
concentrations of amines on the modified surfaces are expressed in relative terms of g polylysine/slide.
survived on bare glass or cell culture treated Permanox (Figure 2A, 2B, 2C). The application of fresh polylysine to the culture surface provides
HEPES (pH 7.36). Nerve growth factor (NGF; 100 ng/ml) was added after 4 days in culture. Cells were fixed 2 days after the addition of NGF. All cells were passaged on a weekly basis. Primary chick brain cultures were prepared from 11 day chick embryos. Cortices were dissected, minced, incubated with trypsin for 20 minutes, carefully washed, and dissociated by drawing through a pipette. Cells were counted and plated at 105 cells/ cm2 in 10% horse serum/DMEM (Thermo Scientific HyClone). The next day the medium was changed to N2.1 defined medium and on day 4, cells were treated with a mitotic inhibitor. After 10 days in culture the neurons were fixed in 4% formaldehyde and mounted in 90% glycerol or a permanent mounting buffer. Modification of glass – Slides were dipped in 1% N-(2-aminoethyl)-3-aminopropyl trimethoxy silane in 95% ethanol/water, washed two times with ethanol and baked for 10 minutes at 100°C. This generated a surface with attached diaminopropyl silane (DAPS) groups. The procedure for the preparation of the CC2 surface is proprietary information. All slides were assembled into chamber slide products and sterilised before use in cell culture. Chamber slide products requiring polylysine treatment were coated by incubating for 4 to 24 hours with filter sterilised poly-D-lysine
Culture Surface
Polylysine Equivalent (g/slide)
Polylysine-coated glass 78
adequate surface chemistry for the primary neuron growth both on PDL-coated glass and PDL-coated Permanox chamber slides (Figure 2E, 2F, 2G), comparable to that of the PDL-coated polystyrene culture dish (Figure 2H). On the other hand, even
CC2 glass
30
DAPS glass
5
without the PDLcoating, CC2 chemically modified
Non-modified glass
<1
glass provides a superior surface for primary neuron adhesion and differentiation (Figure 2D).
Table 1: Quantification of Primary Amines on Glass Surfaces
Results The rat pheochromocytoma cell line PC12 shows differential behaviour on modified and non-modified glass. PC-12 cells cultured on non-modified glass slide form cell clumps with fibroblast-like morphology. Cell death occurred in a significant portion of the culture indicating sub-optimal growth conditions for these cells on the non-modified glass (Figure 1A). PC-12 cells appear less aggregated when grown on DAPS modified glass, although a large number of the cells still developed fibroblastlike morphology (Figure 1B). PC-12 cells cultured on CC2 glass demonstrate good survival. They are less fibroblast-like with rounder bodies. The neuronal process outgrowth is more pronounced on CC2 glass than that on the non-modified glass and the DAPS glass (Figure 1C).
Figure 1 : PC12 cells displayed superior morphology and growth on the CC2 modified glass chamber slide to those on the non-modified glass and the DAPS glass.
1A. Non-modified
1B. DAPS glass
1C. CC2 glass
Figure 2: The growth and survival of primary neuron requires biological coating (e.g. polylysine) or chemical modification (e.g. CC2
2A. Permanox
2E. Permanox/Polylysine
2B. Glass Slide
2F. Glass Slide/Polylysine
2C. Coverglass
2G. Coverglass/Polylysine
2D. CC2 Glass Slide
glass) of the chamber slide surface. Standard cell culture-treated plastics and bare glass fail to support primary neuron culture.
2H. Polystyrene/Polylysine
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Maximise Cell Culture Production & Performance
Many biologically active products, including monoclonal antibodies, recombinant proteins and viruses for vaccines, are produced using animal cell culture. The quantity and performance of these biologicals directly correspond to the quality of cell culture reagents used in the manufacturing process.
Metabolic Pathway DesignTM High performance with Thermo Scientific HyClone Serum-Free Media for specific cell culture platforms is achieved through the Metabolic Pathway DesignTM technology. This approach to media formulation development ensures cell productivity during growth and production phases of cell life. This extensive work in media development has resulted in products that provide a consistent nutrient supply in a form acceptable to cultured cells.
The Thermo Scientific Research and Product Development team leads the industry in characterising sera, identifying critical cell culture components and developing a premier line of serum-free media. Using the latest culture and analytical equipment to determine the needs of cells, this research team has developed media for a variety of applications. By applying its core competence in the nutrition of eukaryotic cells, this research team has developed media that enables scientists to meet cell culture objectives.
The Metabolic Pathway DesignTM Approach: t Balances nutrient supply against metabolic waste accumulation t Determines the effective dose of nutrients critical to the production of recombinant proteins t Provides complex lipids and phospholipids that facilitate delivery through the cell membrane
By applying an understanding of the metabolism and characteristics of major cell platforms employed in modern manufacturing processes, Thermo Scientific is able to provide media optimisation services. In addition, we are able to offer a wide variety of options for development collaboration meeting specific needs for most cell types, culture configurations and selection/amplification systems.
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The objective for developing serum-free media through Metabolic Pathway Design is to provide the intended cell line with the nutrients needed to promote cell growth and stimulate productivity. Serum-Free Media Applications for HyClone Serum-Free Media (SFM) can be found in many biotechnology fields, including the production of human and animal biopharmaceuticals, diagnostic reagents and bio-agricultural products. Due to increased attention on producing biologicals representative of their native form, our Serum-Free Media focuses on
Developed through Metabolic Pathway Design™ to Support Maximum Cell Growth and Productivity
supporting mammalian and invertebrate cell culture platforms. Some of the critical cell culture applications of these platforms include the expression of recombinant proteins, monoclonal antibodies, viral vectors for gene therapy, and viral vaccines. These applications constitute the majority of biopharmaceutical research, development and manufacturing employing eukaryotic cell culture. Developed through the Metabolic Pathway DesignTM approach, the Serum-Free Media (SFM) support superior performance in multiple cell culture platforms. This development process produces SFM targeted to increase process yields for each respective cell platform. Current HyClone SFM applications include hybridoma, NS0, insect, Chinese Hamster Ovary (CHO), PER.C6®, HEK 293, MDCK, MDBK, COS-7 and Vero cell cultures. The use of serum-free media provides many time- and cost-saving advantages, including: t Eliminating the need to pre-screen serum lots t Simplified regulatory documentation t Consistent media performance t Reduced downstream purification challenges Media Optimisation HyClone Serum-Free Media is developed to support the growth of major cell platforms used in eukaryotic cell-based manufacturing. A key to improved performance in biopharmaceutical development and manufacturing is identifying the optimal
medium. Our optimisation program ensures that the medium selection process is a success. Thermo Scientific offers standard serum-free media formulations suitable for key cell culture platforms. While standard serum-free media will offer superior performance, many cases have unique cell line or process specific criteria requiring further optimisation to achieve the highest level of success. By combining our knowledge of cell culture requirements with state-of-the-art analytical and development equipment, we have the ability to assess and meet the specific needs of customer cell culture processes. Our optimisation program has the ability to thoroughly evaluate the nutrient demands of customer cell culture systems, potential key component toxicities, necessary process specific supplementation and many other critical cell culture elements. Throughout the optimisation program, recommended changes in medium composition are carefully evaluated for manufacturing suitability. This assessment ensures that the finished, optimised medium can be manufactured according to end user requirements. Rapid Response Production Customised and optimised prototypes for industrial applications must be evaluated quickly to facilitate final product selection and implementation. In research settings, minor modifications of existing media and reagent formulations are necessary to accommodate specific goals and objectives.
The Rapid Response Production™ facility is a short-turnaround media manufacturing service designed for the production of clientspecific media and reagents. Requests are typically manufactured within seven days. The Rapid Response Production facility is a non-cGMP media and reagents manufacturing suite capable of preparing up to 200 L liquid lot sizes and up to 20 kg dry powdered lot sizes. Liquid lots are packaged in 100, 500 and 1000 mL bottles, or in Thermo Scientific HyClone BioProcess Container™ (200 L maximum) systems. Dry powdered lots are packaged in volumespecific bottles or in bulk containers. Process Supplements A successful process supplements platform has been developed to support demand in the area of fed-batch cell culture. Each supplement is developed through the Metabolic Pathway Design™ approach to provide specific nutrient combinations for a variety of applications, and successfully tested in CHO, hybridoma, NS0, HEK 293, PER.C6 and other cell lines. For convenient evaluation of these supplements, the HyClone Cell Boost Process Supplement Kit is available (HYCSH30890). This kit contains all six Cell Boost supplements and has demonstrated an ability to improve culture productivity by more than 10 times what is currently achieved.
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Take a deep breath consider culturing cells under low oxygen
By Mary Kay Bates, Thermo Fisher Scientific
Abstract: While for many decades, animal cells have been cultured in air supplemented with carbon dioxide, new applications for cells or cell products as human therapies mean that, as much as possible, we must try to mimic conditions existing in tissues where the cells originated. For most cells, this means a much lower oxygen concentration of 1-12%, instead of the 21% oxygen in the air that we breathe. Oxygen affects nearly all cellular processes and evidence shows that cells cultured in low, physiological oxygen, or hypoxia, grow faster, live longer, and have much lower stress. Cell culture incubators (“tri-gas”) incubators that provide nitrogen gas in addition to carbon dioxide gas are the best way to achieve hypoxic conditions similar to those in the tissues.
Introduction: The air that you breathe Take a deep breath. Feel your diaphragm and abdomen expand. Let it out. You just fed oxygen to all your tissues (and carried away carbon dioxide waste), and you do it constantly without thinking about it. Obviously, all your cells need oxygen to function, to survive. And it turns out that oxygen itself regulates nearly every cellular process, from cell metabolism, to differentiation, to cell division. In the laboratory, we commonly grow cells in air supplemented with 5 to 10% carbon dioxide gas to complement the sodium bicarbonate buffer in the growth medium, which balances the pH. And since we breathe air, taking it into our bodies through our lungs and bloodstream, it makes sense to culture cells in vitro in the same air that we breathe. Or does it?
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Within human tissues, oxygen is very low It turns out that inside our bodies, within the tissues, oxygen concentrations are much lower than the 20-21% oxygen that is in the atmosphere. In fact, oxygen in arterial blood is only about 12%. Deeper in the tissues, oxygen concentrations vary, depending on distance from arterial blood vessels. For example, in the brain, oxygen ranges from 0.5-7%, in the eyes from 1-5%, liver, heart and kidneys 4-12%, and in the uterus, 3-5%.[1][2] Clearly, oxygen concentrations in the body are much lower than in the atmospheric air.
of replacement organs. Personalised medicines produced from cultured cells are also advancing rapidly; some, such as monoclonal antibodies for cancer and autoimmune treatments, are already used in the clinic. For pharmaceutical and cosmetic manufacturers, using cultured cells can reduce use of animals for testing, and it is vital to use culture conditions that will best predict responses when these products are used in humans. These revolutionary techniques, using cultured cells for human therapy, mean that more attention is being paid to better mimicking conditions in the body, including providing oxygen at physiological levels.
A historical perspective Cell lines have grown quite happily for decades in atmospheric conditions supplemented with carbon dioxide gas. When cell culture was in its infancy, cells were grown in glass dishes on the benchtop. Most cell lines, beginning with HeLa, were derived from aggressive cancers that quickly adapted to new, less than ideal conditions. But as culture skills developed, as understanding grew, as tissue culture expanded to more laboratories, conditions for cells also improved. Time and testing brought better growth media and supplements, humidified incubators heated to body temperature, CO2 gas to carefully maintain the proper pH, and so on. Along with developing and fine-tuning culture media, researchers sometimes considered the contributions from oxygen too. Alan Richter and colleagues in 1972[3] first noted that culturing cells in low oxygen increased the plating efficiency, or the number of cells that successfully adapted to growing in a plastic dish. In 1977, Packer and Fuehr[4] demonstrated that culturing in 10% oxygen dramatically increased the lifespan of human fibroblasts, extending their lifespan by 25% compared to cells grown in “normal” 20% oxygen. Over the succeeding years, researchers continued to learn more about the biology of physiological oxygen concentration and how it affects cells, including on the molecular level. But in general, low oxygen, or hypoxic, culturing has not received much attention, and many cell culturists are not even aware of this approach. In the twenty-first century, cell culture has come of age and many advanced applications are now routine; including culturing human embryos for in vitro fertilisation (IVF), nurturing a few simple human tissues such as replacement skin for burn victims, and growth of new blood vessels and bladders. Research is continually pushing frontiers for cell-based therapies such as stem cell treatments and growth
Culturing cells at lower oxygen gives better results In the last ten to fifteen years, many researchers have demonstrated that culturing different cell types in low oxygen provides myriad beneficial results. For example, when primary Mouse Embryonic Fibroblasts (MEFs) were cultured in 3% oxygen instead of 20% oxygen, they avoided the senescence that commonly occurs after about 28 days of growth[5] (see Figure 1). In addition, the cells in lower oxygen grew faster, showed less DNA damage, and had fewer stress responses. A different group showed that immune cells cultured at low oxygen behaved as though they were in a healthy body, but the same cells cultured at atmospheric oxygen sent signals as though they were fighting off an infection.[6] So culturing cells under oxygen conditions that are “normal” for cells in the body makes the cells healthier, grow better, less damaged, and less stressed. Stem cells are routinely cultured in low oxygen.[7] Many scientists find that culturing them at low oxygen is critical to maintain their normal stem cell characteristics and to keep them from differentiating. Recently, Lengner et al. [8] showed that human embryonic stem cells (hESC) must be cultured in 5% oxygen to retain their pluripotency. Culturing these cells at atmospheric (20%) oxygen caused chromosomal aberrations. In concert with these findings, IVF clinics commonly culture human embryos at 3-5% oxygen for procedures, to ensure proper development.
Established cell lines and tumours also affected While most hypoxia research has focused on the most physiologically relevant cells, i.e. primary cells and stem cells, Richter also found that neoplastic cells grew better in low oxygen[3]. As early as 1958, suspension cells were found to
References: [1] D. M. Panchision, “The role of oxygen in regulating neural stem cells in development and disease.,” Journal of cellular physiology, vol. 220, no. 3, pp. 562–8, Sep. 2009. [2] Z. Ivanovic, “Hypoxia or in situ normoxia: The stem cell paradigm.,” Journal of cellular physiology, vol. 219, no. 2, pp. 271–5, May 2009. [3] A. Richter, K. Sanford, and V. Evans, “Influence of oxygen and culture media on plating efficiency of some mammalian tissue cells,” J Natil Cancer Inst, vol. 49, pp. 1705–1712, 1972. [4] L. Packer and K. Fuehr, “Low oxygen concentration extends the lifespan of cultured human diploid cells,” Nature, vol. 267, pp. 423–425, 1977. [5] S. Parrinello, E. Samper, A. Krtolica, J. Goldstein, S. Melov, and J. Campisi, “Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.,” Nature cell biology, vol. 5, no. 8, pp. 741–7, Aug. 2003. [6] K. R. Atkuri, L. A. Herzenberg, A.-K. Niemi, T. Cowan, and L. A. Herzenberg. Importance of culturing primary lymphocytes at physiological oxygen levels. Proc Nalt Acad Sci 104(11): 4547–52, 2007. [7] D. Wernerspach, J. Morris, and M. Wight. Oxygen: Too Much of a Good Thing. Laboratory Equipment, 2009. [8] C. J. Lengner, A. A. Gimelbrant, J. A. Erwin, A. W. Cheng, M. G. Guenther, G. G. Welstead, R. Alagappan, G. M. Frampton, P. Xu, J. Muffat, S. Santagata, D. Powers, C. B. Barrett, R. A. Young, J. T. Lee, R. Jaenisch, and M. Mitalipova. Derivation of pre-X inactivation human embryonic stem cells under physiological oxygen concentrations. Cell 141(5): 872–83, 2010.
13
[9] P. Cooper, A. Burt, and J. Wilson. Critical effect of oxygen
grow better in low oxygen [9]. Long-established cell lines including K562 [10], MCF7, A549 and more [11] are affected by hypoxic growth. In humans, solid tumours are well known to have hypoxic centers, not perfused by the blood stream. These areas contain necrotic cells which are resistant to chemotherapy and radiation [12]. Thus, it makes sense to use hypoxic culture for cells used in cancer models, in search of new therapies or testing drug effectiveness and toxicities.
Options exist for hypoxic culturing There are currently a few ways to generate hypoxic conditions for cultured cells. One involves using modular gas chambers inside a standard CO2 incubator. For investigators who want to test hypoxia effects for their own cells and projects, these small chambers could be a good introduction. They hold up to twelve 10cm plates and require additional equipment for gassing the chamber, including regulators, tubing and pumps. They must be recharged after each entry, and currently cannot be monitored for internal conditions. Humidity is maintained by including an extra dish with sterile water. Most hypoxic culturing is performed in a so-called “tri-gas” incubator, though this is a misnomer. Only two gases are supplied; carbon dioxide (as usual) and nitrogen, to reduce the oxygen levels. Generally oxygen can be lowered to 0.5-1%. While some manufacturers may claim oxygen levels as low as 0.1%, this is not truly possible due to sensor detection limits, plus residual oxygen remaining in culture media and plastic culture vessels[3]. Some small incubators purport to offer hypoxic conditions, but these are not ideal since they use a gas mixture from a single tank, and they generally do not have separate sensors, so it is impossible to ensure that cells are receiving proper amounts of carbon dioxide, oxygen and nitrogen gases.
conserve gases as well as reduce chances of contamination entering the incubator. For human applications, it is important to ensure that the chosen incubator is certified for use with human patient samples. Consider contamination control technologies, since any contamination in cells for therapies or products for use in humans could be disastrous. It is important to have an automated decontamination system that eliminates handling of internal parts, and it should be proven effective by independent testing. In addition, a contamination prevention feature which is constantly working is also vitally important for these sensitive applications.
Conclusions As cell culturing advances and new technologies mean that cells themselves or cell products will be used for human therapies, culturing conditions become even more important. One critical aspect is to best mimic conditions in the body where the cells originated, including providing hypoxic conditions which simulate physiological oxygen concentrations. “Tri-gas” incubators efficiently provide these conditions, but similar technologies do not necessarily produce the same results, so it is important to carefully evaluate manufacturer data when choosing an incubator that provides variable oxygen culturing.
tension on rate of growth of animal cells in continuous suspended culture. Nature 182: 1508–1509, 1958. [10] G. H. Danet, Y. Pan, J. L. Luongo, D. A. Bonnet, and M. C. Simon. Expansion of human SCID-repopulating cells under hypoxic conditions. J Clin Invest 112: 126–135, 2003. [11] N. Simiantonaki, C. Jayasinghe, R. Michel-Schmidt, K. Peters, M. I. Hermanns, and C. J. Kirkpatrick. Hypoxia-induced epithelial VEGF-C/VEGFR-3 upregulation in carcinoma cell lines. Int J Oncol 32: 585–592, 2008. [12] M. C. Brahimi-Horn, J. Chiche, and J. Pouysségur. Hypoxia and cancer. J Mol Med 85(12): 1301–7, 2007.
Figure 1 : Newly isolated mouse embryonic fibroblast (MEF) cells avoid senescence and grow faster at physiological (3%) oxygen compared to atmospheric (20%) oxygen.
Thermo Scientific offered the first commercially available tri-gas incubator in 1979, only two years after the first paper was published [4] proving that cells in hypoxia grew better, healthier and with longer lifespans. Designing a tri-gas incubator is complicated, since adding in a large amount of nitrogen gas (75-95%) affects all the other conditions, including CO2, temperature and, of course, humidity. A divided or segmented inner glass door can help
14
Adapted from Parrinello et al. Nature Cell Biology 2003.
Growth of & recombinant protein production in Sf-9 insect cells Insect cells are important host systems for recombinant protein production. Sf-9 cells are typically grown in Spinner or Erlenmeyer flasks. TPP have introduced disposable orbitally shaken vessels, the TubeSpin® bioreactor 50 (“TubeSpins” or TS) and TubeSpin®
Xiao Shen, Patrik O. Michel, Lucia Baldi, David L. Hacker, and Florian M. Wurm Laboratory of Cellular Biotechnology, EPFL, CH-1015 Lausanne, Switzerland
bioreactor 600 (“Maxi-TubeSpins” or MTS, TPP, Trasadingen, Switzerland) for the cultivation of Sf-9 cells at working volumes of 10 mL and 300 mL, respectively.
Biomass 3.5
10
3
Cell growth Sf-9 cells were cultivated as described (Xie et al., 2011) 011) /mL and reached a maximum density of 1.1 x 107 cells/mL as with a packed cell volume (PCV) of 3%. Viability was essels studied. maintained above 90% for over 7days in the two vessels
2.5 8
PCV (%)
Viable cell density (10e+6/mL)
Cell growth 12
6
2 1.5
4 1 2 0
0.5 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
TS MTS 0
1
2
3
4
5
6
7
8
9 10 11 12 13
Baculovirus infection and GFP expression
EGFP expression 70
EGFP positive cells (%)
60
Infection with a recombinant baculovirus coding for green fluorescent protein (GFP) was performed with Sf-9 cells grown in TS and MTS. Infections were performed as described (Xie et al. 2011).
50 40 30 20 10 0
0
1
2
3
4
Sf-9 cells 24 h post transfection with GFP coding plasmid
days
Average intracellular plasmid
Sf-9 cells were co-transfected with the enhanced green fluorescent protein (EGFP) gene (5%) and the tumour necrosis factor receptor-Fc fusion (TNFR-Fc) protein gene (95%) using a baculovirus immediate early promoter (IE-1) as described in (Shen et al., 2011). By 2 days post-transfection, the EGFP-positive cell population was greater than 50 % in both TS and MTS vessels. qPCR data revealed that the plasmid delivery was efficient with negligible loss after 3 days. The volumetric yield of recombinant TNFR-Fc was greater than 60 mg/L as determined by ELISA in TS and MTS.
Plasmid delivery efficiency
Average intracellular plasmid (% of supplied)
100%
6e+4
4e+4
2e+4
0
0
1
2
3
4
5
TS MTS 80%
60%
40%
20%
0%
0.25
days
1
2
3
days
TNFR-Fc production 100 TS MTS
Reduced Sf-9 Sf-9 CHO
Non-reduced Sf-9 CHO Sf-9 CHO
80
TNFR-Fc titer (mg/L)
Plasmid copy number per cell
8e+4
PEI-mediated transient gene expression
150 KDa
Our results confirm the efficacy of the disposable, orbitally shaken systems TubeSpin® bioreactor 50 and TubeSpin® bioreactor 600 for the cultivation of SF-9 cells and for recombinant protein production via both viral and non-viral gene delivery methods.
60
References: 75 KDa
40
50 KDa
0
t 4IFO 9 .JDIFM 10 9JF 2 )BDLFS %-BOE8VSN '. 5SBOTJFOUUSBOTGFDUJPOPGJOTFDU4GDFMMTJO5VCF4QJO® bioreactor 50 tubes. BMC Proc 5 (Suppl 8): P37.
20
0
1
2
3
4 days
5
6
7
t 9JF 2 .JDIFM 10 #BMEJ - )BDLFS %- ;IBOH 9BOE8VSN '. 5VCF4QJOCJPSFBDUPSGPSUIFIJHIEFOTJUZDVMUJWBUJPO of Sf-9 insect cells in suspension. Biotechnol Lett 33(5): 897-902. t "DLOPXMFEHNFOUT8FUIBOL%S-FPOB(JMCFSUPGUIF6OJWFSTJUZPG+ZWÊTLZMÊ'JOMBOEGPSSFDPNCJOBOU('1CBDVMPWJSVT5IJTXPSLIBT been supported by the KTI-Program of the Swiss Economic Ministry and by the Swiss National Science Foundation (SNSF) and the Academy of Finland (decision no. 135820). TPP (Trasadingen, Switzerland) is acknowledged for providing TubeSpin® bioreactors.
15
DNA from microbial cultures Magnetic bead based isolation There is a growing need for rapid, high-throughput methods that enable isolation of high quality, pure DNA from microbial cultures as well as samples containing PCR inhibitors, such as swabs and cultured foods. While highthroughput purification of DNA from microbial cultures can be achieved using 96 well silica plates, this method is not hands-free or walk-away.
Methods currently Moreover, magnetic bead based technologies are c urrently the preferred MO method for hands-free, automated DNA isolation. M O BIO Laboratories has method developed a magnetic bead based purification met thod that enables walkKingFisher away purification on the Thermo Scientific KingFish her Flex and KingFisher® Duo magnetic particle processors. Using mechanic mechanical cal force in the presence of an enhanced microbial lysis buffer, cells are efficiently ntly broken using a 96 well plate shaker and then centrifuged to remove debris. PCR inhibitors, including lipids and polysaccharides are also removed. The removal of inhibitors ensures isolation of pure DNA even from difficult bacterial samples such as skin, feacal or vaginal swabs, and food cultures of red pepper, chocolate, or coffee. KingFisher Flex and the KingFisher Duo enable hands-free hands free purification of nucleic acids using magnetic bead based technology. With the KingFisher Flex it is possible to purify 96 samples per run and with the KingFisher Duo 12 samples per run. The PowerMag Microbial DNA Isolation Kit (27200-4) has been optimised specifically for use on KingFisher Flex and KingFisher Duo magnetic g particle processors. Here we describe the e successful purification and analysis of DNA from cultures microbial and food cultu tures purified using the PowerMag Microbial DNA Isolation Kit on the KingFisher Duo. The combination of bead beating and salt-free magnetic bead ad purification enables high DNA purity and yields from microbes using a 96 6 well w purification system.
16
Pure Microbial Cultures E. faecalis, B. subtilis and E. coli cultures were grown over night and 1.8 ml aliquots were dispensed into a 2 ml Collection Plate. Following centrifugation and removal of supernatant, cells were resuspended d d in i 350μl 350 l off MicroBead Mi B d Lysis L i Solution/RNase A and were transferred into the PowerMag™ Bead Plate. Cell lysis was performed in a 96-well plate shaker. Following clearance of the inhibitors by centrifugation, the cleared lysates were transferred to a new block. The remaining purification steps were performed on the robotic deck of the KingFisher Duo. Sample quality and yield were evaluated using a Thermo Scientific NanoDrop 2000 Spectrophotometer ometer and by running 5 μl of each sample mple on a 1.2% TAE agarose ge gel. Food od Cultures Lis isteria monocytogenes was grown Listeria overnight in a 10% ground beef (22% o fat) or in dark chocolate (86% cacao) culture. 1.8 ml of culture was used for DNA isolation. Samples were purified using either the PowerFood™ Microbial DNA Isolation Kit (silica spin filter method) or the PowerMag Microbial DNA Isolation Kit with the Inhibitor Removal Technology step included. RNase A was used in all samples. 16S rDNA universal primers were used with the Kapa2G Fast HotStart ReadyMix for endpoint PCR. 1μl of R eac ach sam mpl ple e was used along with a each sample 1:10 1: 10 0 dilution dililut utio ut ion io on to check che c eck c for amplification inhi in hibi hi b tit on bi o . inhibition.
Vince Moroney, Heather Martinez, Heather Callahan, Suzanne Kennedy, MO BIO Laboratories, Carlsbad, CA, USA Marika Suomalainen, Thermo Fisher Scientific, Vantaa, Finland
Results Pure Microbial Cultures
Food Cultures
When performing high-throughput purification, it is essential to achieve consistent, reproducible results with respect to DNA quality and yield. Here, we examined DNA isolated from replicate samples of overnight E. faecalis, B. subtilis and E. coli cultures using the PowerMag Microbial DNA Isolation Kit on the KingFisher Duo. Gel electrophoresis analysis demonstrated high quality, high molecular weight DNA in all samples (Figure 1). DNA yield and purity were examined using a NanoDrop 2000 spectrophotometer, revealing consistent yields ranging from 25.5 – 69.5μg, with A260/A280 ratios ranging from 1.93 – 2.10 (Table 1).
Inhibition of downstream reactions due to co-purification of contaminating substances, such as lipids and polysaccharides, is a major obstacle when isolating DNA from cultured food and swabs. To overcome this problem, we included patented Inhibitor Removal Technology step in the PowerMag Microbial DNA Isolation Kit. Here, we cultured Listeria monocytogenes in 10% ground beef or dark chocolate (86% cacao). Starting with 1.8 ml of each culture, DNA isolation was performed using ether the PowerFood Microbial DNA Isolation Kit (silica spin filter technology) or the PowerMag Microbial DNA Isolation Kit (SwiftMag technology) with the optional Inhibitor Removal Technology step. Samples were analysed via gel electrophoresis, and no difference in DNA quality or molecular weight was observed between DNA isolated using the silica spin filter versus SwiftMag technology (Figures 2 and 3, Table 2).
E. faecalis
B. subtilis
DNA yield and purity were next examined using a NanoDrop 2000 spectrophotometer. Interestingly, we observed increased average yields in samples isolated using SwiftMag technology versus samples isolated using spin filters (Table 2). Beef Spin Filter
E. coli
Beef SwiftMag
Figure 1.
Sample
A260/A280 Yield (μg)
E. faecalis
1.98
43.3
E. faecalis
1.93
45.7
B. subtilis
2.05
25.5
B. subtilis
2.06
26.0
E. coli
2.07
69.5
E. coli
2.10
64.0
Chocolate Spin Filter
Chocolate SwiftMag
Beef Spin Filter
Beef SwiftMag
Chocolate Spin Filter
Chocolate SwiftMag
Figure 2.
Figure 3.
Figure 1: DNA isolated from a panel of six samples High quality, high molecular weight DNA was observed in each sample with yields varying based on microbial load. No differences in yield or quality were observed between the replicate samples. Figure 2. Listeria monocytogenes was grown overnight in a 10% ground beef (22% fat) or in dark chocolate (86% cacao) culture. 1.8 ml of culture was used for DNA isolation with the PowerFood Microbial DNA Isolation Kit (silica spin filter method) or the PowerMag Microbial DNA Isolation Kit. Samples were examined on a 1% agarose gel and no difference was observed between DNA isolated using the silica spin filter versus SwiftMag
#
Sample
Isolation Method
Average A260/A280
Average Yield (μg)
1
Beef
Silica spin filter
1.83
8.1
Figure 3. 16S rDNA universal primers were used with the Kapa2G Fast HotStart ReadyMix for endpoint PCR.
3
Beef
SwiftMag magnetic beads
2.02
12.9
5
Chocolate
Silica spin filter
1.86
7.0
7
Chocolate
SwiftMag magnetic beads
1.85
17.8
technology.
1μl of each sample described in Figure 2 was used along with a 1:10 dilution to check for amplification inhibition. All samples amplified successfully. Table 1: Consistent yields of
Conclusion
pure DNA isolated from E. faecalis, B. subtilis and E. coli cultures using the PowerMag
The PowerMag Microbial DNA Isolation Kit is the first successful method of magnetic bead based automated DNA purification for microbial cultures, food cultures and swabs. A combination of Inhibitor Removal Technology and the SwiftMag magnetic bead technology enables DNA from pure microbial cultures, swabs and food cultures known to be high in PCR inhibitors to be purified on the KingFisher Duo magnetic particle processors. Here, we have shown that consistent yields of high quality DNA can be obtained from pure cultures of E. faecalis. Additionally, we cultured Listeria monocytogenes overnight in a 10% ground beef or in dark chocolate and demonstrated that high quality DNA can be isolated, followed by successful PCR amplification. In conclusion, the PowerMag Soil DNA Isolation Kit, part of MO BIO’s growing line of magnetic bead based nucleic acid isolation kits, together with the KingFisher Duo enables rapid, hands-free isolation of high quality, inhibitor-free DNA from microbial cultures, food cultures and swabs.
Microbial DNA Isolation Kit on the KingFisher Duo. Table 2. Increased average yields of pure DNA isolated Listeria monocytogenes using the PowerMag Microbial DNA Isolation Kit (SwiftMag technology) compared with the PowerFood Microbial DNA Isolation Kit (silica spin filter technology).
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Viability of mammalian cells in high density cryogenic storage In most laboratories, space is a premium – particularly in the freezer. With the need to store more samples for longer periods of time, dense storage formats for optimal freezer utilisation, such as the Nunc CryoBank System have been developed. While this optimisation of freezer space is important, the bigger factor is the long term preservation of the samples and viability. In this article we discuss the viability of mammalian cells in the high density storage Nunc CryoBank System. We examine samples stored in a dense storage format and the viability of retrieved cells. To do this, we cryo-preserved two primary cell types and two cell lines in the CryoBank and compared cell viability in thawed vials placed in the peripheral positions of the rack and in vials placed in the central positions of the rack. Additionally, centrifugation of thawed vials may be used for quick removal of the cryogenic freezing agent in order to get a high viability of retrieved cell populations. We tested whether the CryoBank system could withstand repeated and extensive centrifugation.
Materials and Methods CHO (Chinese Hamster Ovary) cell line, MDCK (Madin-Darby Canine Kidney) cell line, HUVEC (Human umbelica vein endothelia cells) primary cell, HDFa (Human derived fibroblast from adult skin) primary cells were cultured according to standard protocols>
Cell Viability: Cells were grown to 75-80% of confluence, harvested with Trypsin-EDTA (Cambrex BE17-161E), and dispensed in CryoBank vials. Six vials placed in the peripheral positions of the rack and 4 vials positioned in the central positions of the rack (Figure 1.a) were filled with 1.0 mL of cell suspension (containing 1.0 x 106 cells and 7.5% v/v DMSO). The remaining 86 vials in the rack were filled with 1.0 mL culture medium supplemented with 7.5 % DMSO. The rack was placed in a box of expanded polystyrene, and incubated over night at –80°C (an approximate cooling rate of 1°C/min was attempted by using this method). The rack was then transferred to the vapour phase of a liquid-nitrogen freezer and incubated over night. For determining the cell viability, the rack was transferred to –80°C, and three vials were assayed at a time. The vials were thawed at 37°C, and cell viability was immediately determined. For determination of the total cell number the thawed cell suspension was diluted 1:1 with medium and 300μL cell suspension was mixed with equal volume of lysis buffer and vortexed. 300 μL stabilisation buffer was added to the cell lysate and vortexed. This cell lysate was then analysed with a fluorescent cell counter. This method used fluorescent propidium iodide, which intercalates with DNA in the cell nuclei. During analysis the fluorescent signal is counted and correlated to total cell count. For determination of non-viable cells, 300 μL diluted cell suspension was analysed. The cell viability was calculated by: % cell viability = (total cell number – number of non-viable cells)/total cell number x 100. Comparisons of cell viability for the 4 cell types were performed using an independent, unpaired t-test and analysis of variance (ANOVA), and a significance level of 0.05.
Centrifugation Test: Twenty CryoBank vials were filled with 1.0 mL culture medium (E-MEM with 10% FBS) supplemented with 7.5% DMSO, and then frozen and thawed as described above. The thawed vials were centrifuged in the rack: three times for 10 min at a stress of 300 – 2000g. The vials were visually inspected for deformities and appearance of stress lines after each 10-min centrifugation step. In order to expose the vials to as much stress as possible, vials centrifuged at 300g were also centrifuged at 500g, 1200g and 2000g.
18
Marwood Kristensen T, Sehested A, Weitzmann L. Thermo Fisher Scientific, Roskilde, Denmark
Figure 1.A Cell viability measured on vials placed in
Results: Cell Viability Determination of cell viability for HUVEC, CHO and MDCK (Figure 1.B, C and D) showed that viability of retrieved cells was not compromised, irrespective of the vials being placed in peripheral or central positions of the storage rack. For the primary cell type, HDFa (Figure 1.E), a significant variability in viability of retrieved cells in vials in peripheral and central positions was observed. In order to investigate if the variability was due to a decreased retrieval of viable cells in vials placed in central positions or due to an improved retrieval of viable cells in vials placed in peripheral positions, we compared cell viability of the HDFa cells in CryoBank vials with cell viability in standard Cryovials. The results show that the viability of retrieved cells in Cryovials is identical to viability of retrieved cells in vials placed in central positions (Cryovials 90.6% ± 1.8, CryoBank 90.6% ± 0.8, data presented as means ± SEM). It was concluded that the variability observed using HDFa in the CryoBank system is due to improved retrieval of viable cells in vials placed in peripheral positions rather than lower retrieval of viable cells from vials in the central positions. a
b
c
peripheral positions of the rack is shown by blue colour and vials placed in central positions are shown in red. The remaining positions in the rack were occupied with vials filled with culture medium only. This set-up was repeated 3 times for each cell type. Figure 1.B Determination of cell viability for HUVEC revealed no significant difference after cryopreservation in peripheral or central positions of the rack. Bars represent means ± SD (n=3). Figure 1.C Determination of cell viability for CHO revealed
d
e
no significant difference after cryopreservation in peripheral or central positions of the rack. Bars represent means ± SD (n=3)
f
Figure 1.D Determination of cell viability for MDCK revealed no significant difference after cryopreservation in peripheral or central positions of the rack. Bars represent means ± SD (n=3). Figure 1.E Determination of cell viability for HDFa cells revealed a higher variability of cells frozen in and retrieved from
Centrifugation of the CryoBank system
Figure 2 Centrifugation period
G-value Stress
Lines/deformities
3 x 10 minutes
300
None
3 x 10 minutes
500
None
3 x 10 minutes
1200
None
3 x 10 minutes
2000
None
The CryoBank vials and rack was investigated for appearance of stress-lines or other deformities after centrifugation. Twenty vials first centrifuged at 300g were exposed to centrifugation at 500g, 1200g and finally 2000g. No observations of either stress lines or deformities of the vials were observed.
peripheral positions of the rack (P<0.0002). Bars represent means ± SD (n=3). Figure 1.F....... Figure 2. The CryoBank vials and were exposed to repeated and extensive centrifugation. No observations of either stress lines or deformities of the vials were observed.
Conclusion Determination of cell viability for two primary cell types and two cell lines cryo-preserved in the Nunc CryoBank storage system shows that viability of retrieved cells was not compromised due to the dense format of the NuncCryoBank storage system. An improved retrieval of viable HDFa from CryoBank vials placed in peripheral positions was observed and might be because the CryoBank vials are slimmer than standard Cryovials, thus allowing a quicker freezing of the cell suspension.The SBS-footprint of the CryoBank rack allows it to be subjected to centrifugation. Figure 2 shows that CryoBank vials and rack can tolerate extensive centrifugation, without being damaged. This feature can ensure quick removal of the cryogenic freezing agent and support protocols in achieving a high level of viable cells.
19
As clinical researchers investigate new therapeutic methods, they are turning more & more to the body’s own cells to repair cellular damage and fight disease.
primary
cell analysis
for Immunotherapy, Regenerative Medicine & Transplantation Oncology immunotherapy involves isolating and engineering T cells to fight tumours. Researchers in the field of regenerative medicine are exploring the use of stem cells and progenitor cells from various tissues to treat damaged organs. Transplantation to treat haematologic disorders such as leukaemia, lymphoma, and severe aplastic anaemia (SAA) involves stem cells from bone marrow, cord blood, and mobilised peripheral blood. With the increase in approved therapeutic methods and clinical research involving immunotherapy, regenerative medicine, and transplantation, a universal method for accurate analysis of a wide variety of cell and sample types is essential.
20
Determination of Cell Concentration and Viability
Trypan Blue Viability Staining / Analysis Procedure
The Cellometer Auto 2000 Cell Viability Counter features a pre-optimised dual-fluorescence viability assay for a wide variety of primary cell types, including PBMCs, stem cells, total nucleated cells, and splenocytes. For this experiment, we tested viability using a trypan blue method as well as a dual-fluorescence AO/PI method. The AO/PI method is highly recommended for samples containing debris or red blood cells.
1. Mix sample well by pipetting up and down at least ten times 2. Combine 20μl of sample and 20μl of trypan blue dye solution and mix well by pipetting up and down 3. Load 20μl of sample into the disposable counting chamber 4. Allow cells to settle in chamber for 1 minute 5. Insert Counting Chamber into the Cellometer 6. Select the Trypan Blue Assay 7. Enter your Sample ID and a Dilution Factor of 2 8. Optimise the Focus and Click Count
Primary Reagents: t Bone marrow, cord blood, peripheral blood, and mobilised peripheral blood samples (All Cells) t Trypan Blue Stain t Cellometer ViaStain™ AO/PI (acridine orange / propidium iodide) Staining Solution (Nexcelom Bioscience, Part# CS2-0106-5ML)
AO/PI Viability Method Acridine orange (AO) dye stains DNA in the cell nucleus of all cells to obtain a total nucleated cell count. Propidium iodide (PI) DNA-binding dye is used to stain dead cells and determine cell viability. Healthy cells with intact membranes are impermeable to the PI dye. Dead cells stained with both AO and PI fluoresce red due to fluorescence resonance energy transfer (FRET). Live nucleated cells fluoresce green and dead nucleated cells fluoresce red. Because Cellometer AO/ PI live and dead cell counts are conducted in the fluorescent channels and debris / non-nucleated cells do not fluoresce, there is no interference from debris and non-nucleated cells.
AO/PI Viability Results In order to validate the Cellometer Auto 2000 Cell Viability Counter for primary cell analysis, samples at various stages of processing were evaluated. For some samples, Trypan Blue and AO/PI staining methods were compared. In some cases, Trypan Blue staining was used to compare manual and automated counting. Representative images for several of the sample types tested are shown below. The bright field images show the differences in sample complexity at various stages of processing. Nucleated cells, red blood cells, platelets, and debris are visible in bright field images. Only nucleated cells are visible in the dual-fluorescence images. Live nucleated cells are circled in green. Dead nucleated cells are circled in red.
Bone Marrow Aspirate (fresh) Bone marrow aspirate contains red blood cells (erythrocytes) white blood cells (leukocytes), and platelets (thrombocytes). As seen in the fluorescent image below in which only nucleated cells are visible, nucleated white blood cells make up a very small percentage of total cells in the aspirate sample.
AO/PI Viability Staining / Analysis Procedure 1. Mix sample well by pipetting up and down at least ten times. 2. Combine 20μl of sample and 20μl of AO/PI dye solution and mix well by pipetting up and down 3. Load 20μl of sample into the disposable Cellometer Counting Chamber 4. Allow cells to settle in chamber for 1 minute 5. Insert Counting Chamber into the Cellometer 6. Select the AO/PI Viability Assay 7. Enter your Sample ID and a Dilution Factor of 2 8. Optimise the Focus and Click Count
Trypan Blue Viability Method The trypan blue dye enters cells with compromised membranes, making them appear dark upon bright field imaging. This dark colour enables the Cellometer software to identify and count dead cells independently of live cells. In the bright field / trypan blue method, size differentiation is used to exclude platelets and debris from cell counts, but red blood cells may be counted.
Image 1 and 2. Bright field and dual-fluorescence counted image for a bone marrow aspirate.
Bone Marrow MNC Sample following Density Gradient Separation (cryopreserved) The majority of platelets and red blood cells are removed upon density gradient separation. The amount of remaining red blood cell contamination will vary from sample to sample. A cell concentration / viability analysis method that is not affected by red blood cell contamination is recommended at this stage of sample processing. As shown by the circles in the images below, non-nucleated cells do not appear in fluorescent images used for Cellometer AO/PI cell counting. Image 3 and 4. Bright field and dual-fluorescence counted image for bone marrow mononuclear cells following density gradient separation.
21
Bone Marrow CD34+ Sample following Density Gradient Separation and Immunomagnetic Separation (fresh)
Trypan Blue vs. AO/PI Viability Results
Following immunomagnetic separation, samples contain little to no red blood cell contamination. A simple trypan blue viability method or dual-fluorescence method can be used at this stage.
Thirty processed samples (via density gradient separation and / or immuno-magnetic separation) were tested using both manual trypan blue and automated AO/PI viability methods. There was a very high degree of correlation between the two methods. (R2 value of 0.96%).
Image 5 and 6. Bright field and dual-fluorescence counted image for CD34+ cells from bone marrow following density gradient separation and immunomagnetic separation
The number and concentration and percent viability of nucleated cells measured for representative bone marrow samples is listed in the table below. For each sample, 20μl of sample was loaded into the Cellometer Counting Chamber and four different quadrants were imaged and counted. The total number of nucleated cells counted and the concentration of nucleated cells in the original sample and the percent viability are automatically generated by the Cellometer system.
Sample*
Bone Marrow Aspirate (fresh)
Bone Marrow MNC (cryopreserved)
Bone Marrow CD34+ (fresh)
Cell Count Concentration % Viability Population (# of cells) (cells / mL) Total
1,456
5.06 x 106
Live
1,460
5.05 x 106
Dead
4
1.4 x 104
Total
3,436
1.19 x 107
Live
1,980
6.84 x 106
Dead
1,456
5.04 x 106
Total
500
1.74 x 106 6
Live
460
1.61 x 10
Dead
40
1.39 x 105
Live
2,475
8.64 x 106
Dead
199
6.90 x 105
99.7%
57.6%
92%
The six samples displaying the largest discrepancy in percent viability were the bone marrow mononuclear cell samples and CD34-depleted bone marrow samples containing varying amounts of red blood cell contamination. Manual counts included red blood cells in total cell counts, resulting in higher percent viability calculations for these samples. CD14+, CD19+, and CD34+ cell samples (no RBC contamination) showed excellent correlation between trypan blue and AO/PI. Due to the density of cells and degree of non-nucleated cell contamination in the fresh cord blood, bone marrow aspirate and peripheral blood samples, they could not be tested using the trypan blue viability method. A dual-fluorescence method is highly recommended for analysis of fresh patient samples.
*All samples tested were from different donors.
Conclusion With the Cellometer Auto 2000, researchers can use a single method to accurately evaluate the viability of cells at various stages of processing. Switching to the AO/PI enables accurate total nucleated cell (TNC) counts and viability characterisation of fresh patient samples and eliminates potential over-estimation of cell viability caused by red blood cell contamination and counting following density gradient separation. Researchers have the option to perform automated trypan blue or AO/PI analysis for purified samples. In addition to improving accuracy, Cellometer automated analysis decreases test time by as much as 80%, eliminates the inter-operator variability associated with manual counting, and significantly cuts training time for new technicians. Archived images and automated data reports provide a permanent QC record for each sample tested and enable remote troubleshooting and data-sharing. In addition to primary nucleated cells, proprietary Cellometer software has been used to count more than 1,600 cell lines, including clumpy tumour cell lines and irregular-shaped cells.
22
tech note
All tests were carried out by an independent testing consultancy (Embryotech Laboratories, Mass, USA).
Odour from irradiated polystyrene has no effect on cell growth and performance Over the years, many users have expressed concern that the unpleasant odour from radiation sterilised polystyrene products may influence cell growth and performance. This has been of particular concern in the IVF field, where safety is at a premium. In order to examine the effect of irradiated polystyrene on embryos and sperm, a Mouse Embryo Assay (MEA) and a Human Sperm Survival Assay (HSSA) were performed on irradiated polystyrene products.
Methods MEA: Embryos used in this test were derived from a cross between B6C3F1 female mice mated to B6D2F1 male mice (B6C3F1x B6D2F1). The resulting embryos were cultured directly in ‘embryo tested’ HTF medium overlaid with light culture oil in the Thermo Scientific Nunc 4 Well Dishes with 21 embryos per dish in triplicate. Three of the four wells received seven 1-cell stage embryos in 0.5 mL of medium. All incubations were done at 37°C with 5% CO2. After 96 hours, the dishes were removed from the incubator and the embryos were examined microscopically. Those embryos that were determined to have reached the blastocyst stage were scored as viable (Fig. 1). HSSA: Human semen samples were thawed for 30 minutes at room temperature before mixing with pre-tested Ham’s F10 medium supplemented with 2% BSA. After spinning and re-suspending in a fresh aliquot of the same
medium, the swim up fraction was collected. The motile fraction was determined, and a suitable volume of the sperm suspension was added to the Thermo Scientific 4 Well Dish. Motility was recorded after 24 hours. Since it is known that the odour is most intense immediately after irradiation and dissipates with time, dishes were tested at both 2 and 10 weeks* after irradiation. It is also common practice to open packages and let them air overnight before use. Therefore, one pack of dishes from each set was opened 24 hours prior to MEA and HSSA testing, and one pack was opened immediately before testing. Three dishes from each of these sets were tested in each assay and monitored for embryo survival at the 2-cell and blastocyst stage in MEA, and for sperm cell motility in HSSA.
These criteria were met for both assays under the described conditions (Figs. 2 and 3).
Conclusion No significant differences in embryo or sperm survival were observed between groups, whether dishes were tested at 2 or 10 weeks, or the packs were opened to allow the odour to dissipate overnight. Furthermore, in both assays the product performance exceeded the acceptance criteria, confirming that odour has no detrimental effect on embryo survival and sperm motility.
* Due to the nature of the sterilisation logistics, two weeks is the earliest possible time after irradiation that a user could receive products.
Results MEA acceptance criterion ≥ 80% (Embryotech ≥ 70%) embryos have developed to blastocysts within 96 hours after fertilisation.
Fig. 2: Mouse Embryo Assay (MEA) in IVF dishes unpacked 0 or 1 day before use at 2 and 10 weeks after irradiation. No significant difference between these set-ups was seen in blastocyst formation 4 days after fertilisation.
HSSA acceptance criterion ≥ 70% (Embryotech ≥ 60%) sperm cells remain motile 24 hours after sample preparation.
Early Embryonic Development Fig. 1.Diagrammatic representation of the developmental stages immediately after fertilisation.
Fig. 3: Human Sperm Survival Assay (HSSA) in IVF dishes unpacked 0 or 1 day before use at 2 and 10 weeks after irradiation. No significant difference between Day 0 Fertilisation
Day 1 Two Cell Stage
Day 3 Morula
Day 4 Early Blastocyst
these set-ups was seen in sperm motility 24 hours after sample preparation.
23
cell biology ce b o ogy workflow
The Cell Culture Café is a science-based community where techniques, tips, and questions concerning cell culture are presented and discussed. Monthly webinars explore topics ideally suited to your biggest concerns in the lab.
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Cell Isolation & Preparation
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30
Product Focus: TurboFect™ in vitro transfection reagent Highly efficient, reliable & effective TurboFect™ – superior transfection reagent
TurboFect™ in vitro Transfection Reagent is a highly efficient, easy-to-use, non-immunogenic transfection
100
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1500
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reagent. It is a sterile solution of a proprietary cationic polymer in water. The polymer forms compact, stable, positively charged complexes with DNA. These complexes protect DNA from degradation and
0
0 TurboFect™
ExGen 500
Vendor ndor A
Vendor B
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facilitate gene delivery into eukaryotic cells. GFP positive cells (%)
Mean fluorescence intensity
Reliable Co-Transfection Triple transfection of HeLa cells. HeLa cells were co-transfected with a 1:1:1 mixture of plasmids encoding eGFP GFP localised in the cytoplasm, eCFP with a nuclear localisation signal and monomeric DsRed Red protein with membrane localisation signal. The cells were transfected with TurboFect™ t™ in vitro Transfection Reagent.
Effective even in hard-to-transfect cell lines EGFP expression in primary mouse bone marrow derived dendritic cells transfected sfected with TurboFect™ in vitro Transfection Reagent.
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31
Rapid-Flow Membrane: A comparison of flow rate and throughput of cell culture media and serum Marci Moore, Stephanie Carter, Robert Scott, Joseph Granchelli, Thermo Fisher Scientific, Rochester, NY, USA
Sterile filtration of liquids used in cell culture is a critical step in maintaining a contamination-free culture. Thermo Scientific Nalgene filter units and bottle top filters have the unique Rapid-Flow membrane support plate designed to increase the rate at which liquids can be filtered. The Rapid-Flow system provides more uniform membrane support and stability than does the radial spoke design used by other filter manufacturers. Here we test whether this design increases the rate at which liquids can be filtered and the maximum volume of filtered liquid, compared to competitors’ filters. We find that the Rapid-Flow system offers significant performance advantages over similar size filters from other manufacturers.
Introduction The first Nalgene vacuum filter was introduced in 1965, and since then filter units and bottle-top filters have become crucial tools in the sterile filtration of media in the laboratory. Such filtration is essential in maintaining contaminationfree media, serum, and buffers for cells
in culture. Since filtration takes time, advancements that increase the flow rate of liquid would be welcomed by cell culture researchers. Thermo Scientific Nalgene filter units and bottle top filters have the unique Rapid-Flow membrane support system. The Rapid-Flow system consists of a multi-column array that provides more uniform membrane support and stability than does the radial spoke design used by other filter manufacturers. Rapid-Flow is designed to allow increased flow rates and greater fluid throughput. Flow rate and throughput advantages of Rapid-flow have already been demonstrated in Nalgene 0.1 micron PES filters1. The purpose of this study is to compare the performance of Nalgene 0.2 micron PES Rapid-Flow filters with 0.2 micron PES filters from Corning and Millipore.
Test Fluids: Cell Growth Media: DMEM + 10% FBS (feotal bovine serum), RPMI 1640 + 10% BGS (bovine growth serum). Both
are typical cell growth media. Serum: A mixture of 100% BGS (bovine growth serum) and 100% BCS (bovine calf serum) was used. 100% serum is a common component of cell growth media and also a severe filtration challenge.
Test Method: Flow Rate and Maximum Filtration Volume The vacuum system was started and adjusted to full vacuum (27.4” Hg) in the 80L vacuum buffer and 25” Hg at the test stand. Testing did not begin until the vacuum buffer registered ≥ 25” Hg. The filter unit being tested was attached to a calibrated 2L glass receiver and to vacuum. Nalgene Rapid-Flow sterile filter units, Millipore Steritop- GP filter units, and Corning filter units were tested in random order. The filter units were filled to the top with test solution and the vacuum stopcock at the test stand was opened. Time points were taken every 200mL for DMEM + 10% FBS or RPMI 1640 + 10% BGS, and every 50mL for 100% serum until 2 Litres (L) of fluid had
Table 1. Filters Investigated Description
Catalog #
Membrane Pore Size and Material
Membrane Diameter (mm)
Nalgene 1000mL Rapid-Flow Filter
567-0020
0.2μm PES
90
Millipore 1000mL Steritop-GP Filter
SCGPT10RE
0.22μm PES
70
Nalgene 500mL Filter
595-4520
0.2μm PES
75
Corning 500mL Filter
431097
0.22μm PES
63x63mm (square)
Table 2. Flow rate results
32
Description
Flow Rate with RPMI (mL/ttmin)
Flow Rate with DMEM (mL/min)
Flow Rate with BCS/ BGS (mL/min)
Maximum Volume of 100% Serum filtered
Nalgene 1000mL Rapid-Flow Filter
698.4
827.4
224.8
567
Millipore 1000mL Steritop-GP Filter
471.6*
626.8*
173.1
450
Nalgene 500mL RapidFlow Bottle Top Filter
508.7
618.1
180.6
367
Corning 500mL Filter
397.6*
508.7*
123.4*
267
been filtered or clogging was reached (defined as less than 1 drop of fluid per second, or a flow rate such that the time to filter 100ml exceeded 2 min). Fluid was replenished as needed such that at least 200mL of test solution was in the filter unit at all times to prevent the filter unit from running dry. Six filters of each type were tested with RPMI and DMEM; three filters of each type were tested with 100% serum.
Data Analysis: Two-sample t-tests assuming unequal variances were used to determine the significance (p<0.05) of filter performance (flow rate and clogging volume) for DMEM + 10% FBS, RPMI 1640 + 10% BGS and 100% serum between a) 1000mL Nalgene Rapid-Flow filters and 1000mL Millipore Steritop-GP filters, and b) 500mL Nalgene Rapid-Flow filters and 500mL Corning filters.
Discussion The improved performance of the new Rapid-Flow support plate was apparent when comparing filter performance under these media and serum conditions. The 1000mL Rapid-Flow filters showed significantly faster flow rates over those from Millipore, with a 48% improvement for RPMI + serum and a 32% improvement for DMEM + serum. 1000mL Rapid-Flow filters
Figure 1. Flow Rate using RPMI + 10% BCS
800
showed a 30% higher average flow rate than 1000mL Millipore filters using 100% serum but were not significantly faster per t-test. For 500mL filters, Nalgene Rapid-Flow filters showed statistically faster flow rates over Corning filters of 28% for RPMI + serum, 22% for DMEM + serum and 46% for 100% serum. While the filters used for this study are designed to filter 1000mL or 500mL of liquid, all were capable of filtering more than 2000mL of media plus serum without clogging. 100% serum, however, was a more challenging fluid for all filters tested, with throughputs of 600mL or less. Although the Rapid-Flow filters filtered more serum on average than did the other filters, there were no statistically significant differences seen in volume of serum filtered between the 500mL filters or the 1000mL filters tested. Contamination is the enemy of anyone maintaining cell cultures. While close attention to sterile technique can reduce the chance of introducing contamination, it is critical to ensure that the materials used to maintain the culture are sterile at the outset. Final filtration in the laboratory is an easy and effective way to remove microbial and particulate contamination from any liquids that will come in contact with the cells in culture. Conventional filter units are designed with membrane
support plates that contain ribs that radiate from the centre. When vacuum is applied to filters with this design, the spacing of the ribs allows distortion of the membrane resulting in a reduction of fluid flow.
filter units with cell culture media. Application note #ANLSPFILT01PES, 2012.
Conclusion Rapid-Flow 1000mL filters flowed significantly faster than Millipore filters with media + serum and were equivalent with 100% serum. Rapid-Flow 500mL filters flowed significantly faster than Corning filters with both media + serum and 100% serum. Neither 500mL nor 1000mL filters showed significant differences in throughput for media + serum or 100% serum.
Figure 3. Flow Rate using 100% BCS/BGS
250
1000
Figure 4. Maximum Volume filtered using 100% BCS/BGS
600
Nalgene Rapid-Flow Millipore Corning
*
700
200
600
*
500
800
*
500
*
150
600
100
400
400
400 300
300
200
200
200
50
100
100 0
of Thermo Scientific Nalgene 0.1 micron Rapid-Flow PES
To overcome this effect Nalgene Rapid-Flow filters contain a multi-column array that provides more uniform and less restrictive support to the membrane; this unique design permits higher flow rates and throughput. Rapid-Flow offers increased rates of flow in 1000mL units when filtering media + serum compared to filter units offered by Millipore. In 500mL units, the Nalgene Rapid-Flow filters offer increased flow rates compared to Corning filters for media + serum and for pure serum. The new design may also offer improvements in the maximum volume filtered when filtering 100% serum.
Figure 2. Flow Rate using DMEM + 10% FBS
*
References: 1. Flow rate and throughput
1000mL Filter Units
500mL Filter Units
0
1000mL Filter Units
500mL Filter Units
0
1000mL Filter Units
500mL Filter Units
0
1000mL Filter Units
500mL Filter Units
No significant differences were found.
33
Copper CO2 Incubators: Why your next incubator should be 100% pure copper
CO2 incubators inc cub uba a ators at provide an excellentt growth en nvi viro ronmen nt for cell cultures. However, the environment m and humid conditions also susta an ain ai same warm sustain the growth of potentially contaminating microorgan nisms(1). Records from early microorganisms s demonstrate that copper can in nhibit civilisations inhibit ms. the growth of many different microorganism microorganisms. Reviews off modern literature (2) indicate thatt ws or stops growth of many orga nisms, copper slow slows organisms, b fungi, algae and yeast. including bacteria,
34
Copper ions bond to contaminants and then disrupt key proteins and processes that are critical to microbial life. The suppression of bacterial colonisation by solid copper was demonstrated in Porton Down, England by the Centre for Applied Microbiology and Research (CAMR)(3). In that study, CAMR showed that copper piping reduces the growth of Legionella pneumophila, causative agent of Legionaire’s Disease. It has also been demonstrated that nothing matches the contamination fighting efficiency of pure 100% copper. Research measuring the viability of methicillin resistant Staphylococcus aureus on various copper alloys and stainless steel (Figure 1) has shown that low copper content alloys and copper plated stainless steel are less effective than 100% copper. Similar results have been documented against typical contaminants For this reason, reason the Thermo Scientific incubator contaminants. Heraeus CO2 incubators are available with interiors made from 100% pure copper.
Methicillin resistant S. aureuss (CFU/ml)
Reduced Copper Content Results In Reduced Antimicrobial Effectiveness
1.00E+06 1.00E+04 1.00E+02 1.00E+00
0
90
180
270
Time (minutes)
99% Copper
80% Copper
55% Copper
360
Stainless Steel
Figure 1: Reduced Copper Content Results In Reduced Antimicrobial Effectiveness (Adapted from Michels HT, Wilks SA, Nocye JO and Keevil CW. Copper alloys for human infection disease control. Materials Science and Teaching Conference, 2005.)
Efficiency of 100% Copper Incubators Improves with time The antimicrobial efficiency of 100% pure copper interiors improves as the surface oxidises over time, visible as tarnishing. Figure 2 shows a study that demonstrates that as copper ages, the tarnishing effect provides an increased amount of cupric ions to attack contaminating microorganisms. Tarnished and untarnished (bright) 1cm2 copper and copper alloy samples were tested for their antimicrobial activity. E.coli bacteria were applied to each
coupon and air dried. At several time points, the bacteria were collected and the number of viable organisms determined. Only tarnished, high copper content carriers exhibited increased antimicrobial performance with age. Untarnished alloys with limited copper had almost no effect. Antimicrobial Efficiency Improves With Tarnish E. coli 0157:H7 Viability (CFU/ml)
How does it work?
References: (1) Incubators with Thermal Disinfection Cycles (2000). Genetic Engineering News. 20:37. (2) Copper Development Association, 260 Madison Avenue, New York, NY 10016, 212-251-7200 Ph,
1.00E+08
212-251-7234 Fax, Staff@cda. copper.org, www.copper.org
1.00E+07
(3) The Influence of Plumbing Material, Water Chemistry and Temperature on Biofouling of Plumbing Circuits with Particular Reference to the Colonization of Legionella Pneumophilaa (1993). ICA Project 437B
1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 0
15
30
45
60
Time (minutes) 55% Copper, Copper Untarnished
90% Copper, Copper Untarnished
99% Copper, Copper Untarnished
55% Copper, Tarnished
90% Copper, Tarnished
99% Copper, Tarnished
I addition In dditi to t the th antimicrobial ti i bi l nature t off copper, there th are other th significant advantages to the 100% copper incubator: t No special handling is required for copper, and maintenance is minimal minimal. There is no need to risk exposure of cultures or personnel to toxic chemical disinfectants or UV light, which becomes less effective as a decontamination source over time. t 5IFBOUJNJDSPCJBMQSPQFSUZPGQVSFDPQQFSJTalways working. It provides continuous protection against contamination on contact, all day every day. t $PQQFSJTsafe for cells. Because copper ions do not become airborne, they pose no threat to precious cells incubated in culture vessels on copper shelves. 100% pure copper surfaces protect the entire incubator chamber, including walls, shelves and humidity water reservoir, to provide you peace of mind that your cells are safe from contamination introduced by routine door openings and sample access. When looking into your next incubator, be sure to consider copper interiors. Additionally, the percentage of copper should be confirmed to ensure optimal antimicrobial performance. The Thermo Scientific Heraeus CO2 incubators have 100% pure copper interiors, in addition to the traditional decontamination protocols for your peace of mind.
35
Expansion & differentiation of human mesenchymal stromal cells Louise Gjelstrup and Thomas Stelzer, Thermo Fisher Scientific
Human mesenchymal stromal cells (hMSC) are candidates for clinical use because they are readily expanded in culture, have immunomodulatory potential and can differentiate into the osteogenic, chondrogenic and adipogenic lineages. Their therapeutic potential is currently studied as part of clinical trials to treat diseases such as graft-versus-host disease1 and osteoarthritis2, as well as in the regeneration of cardiac muscle following myocardial infarcts3.
Whether the requirements are for clinical or research use, obtaining a substantial number of cells can constitute a bottleneck for the investigator. hMSC display some plasticity in their culture conditions, but several investigators report a higher growth index and increased differentiation potential at lower seeding densities4,5. We here present a protocol enabling the clinician or researcher to rapidly expand a population of hMSCs on Thermo Scientific Nunclon Delta cell culture treated surface utilising the potential of Thermo Scientific HyClone AdvanceSTEM Mesenchymal Stem Cell Basal Medium, developed specifically for the optimal expansion and maintenance of undifferentiated hMSCs. A definitive test of multi-potency is a functional test. In consequence, we subjected the expanded hMSC to differentiation. The cells were differentiated into osteoblast or adipocytes in Thermo Scientific Nunc 48 well multidishes.
Methods Cultivation of hMSC. Human mesenchymal stromal cells (Lonza, USA) were maintained in ˞-MEM medium containing 10% FBS, 1% Penicillin/Streptomycin and 2mM UltraGlutamine, or AdvanceSTEM™ Mesenchymal Stem Cell Basal Medium supplemented with 10% AdvanceSTEM
36
Growth Supplement. In order to test the effect of cell disassociation on the growth and differentiation of the hMSCs. Cell culture: hMSC was incubated at 37°C in a humidified atmosphere of 5% CO2 in air using a Thermo Scientific Revco Ultima II Series CO2 Incubator. Cell counting: Cells were counted using an integrated automated fluorescence microscope (Nucleocounter, Chemometec, Denmark).
Growth curves: To develop the protocol, growth curves of hMSC cultivated in AdvanceSTEM and -MEM growth media were established. hMSCs in passage 2 were seeded at 100, 350, 1000 and 4000 cells/cm2 in Nunclon Delta treated T25 flasks. The cultures were placed in an IncuCyte Plus and incubated at 37°C in a humidified atmosphere of 5% CO2. The IncuCyte Plus is an automated imaging platform, configured to fit inside a CO2 incubator, and designed to provide kinetic, non-invasive live cell imaging by acquiring phase-contrast images of the cells at user-defined times and locations within the cultures. The primary metric of the instrument is culture confluence, that is, the fraction of the surface that is covered by cells. The cells were cultivated for 12 days with media change every 4 days. Differentiation protocol: After the expansion protocol cells were harvested with trypsin and re-seeded in AdvanceSTEM or -MEM in 48 well multidishes at a density of 5000 cells/cm2 for differentiation into osteoblasts and adipocytes.The cells were incubated for 48 hours at which time the media in the 48 well multidishes were changed to either AdvanceSTEM Adipogenic or osteogenic differentiation media with AdvanceSTEM Growth
The differentiation cultures were assayed on Days 3, 7 and 18. The growth of hMSCs seeded at four different densities in two different media (-MEM medium and AdvanceSTEM Mesenchymal Stem Cell Basal Medium) was investigated. hMSCs were cultured for 12 days and the growth was followed in the incubator using the IncuCyte Imager. For the first 50 hours, at 1000 and 4000 cells/cm2, the growth of hMSCs in -MEM medium and AdvanceSTEM Mesenchymal Stem Cell Basal Medium displayed a similar growth pattern (Fig. 1A), but then the cultures diverged. The growth rate of hMSC in -MEM medium declined and reached a plateau at approximately 80% culture confluence after approximately 100 hours for cultures seeded at 4000 cells/cm2 and 150 hours for cultures seeded at 1000 cells/cm2.
120
100
AdvanceSTEM - 100 cells/cm 2
AdvanceSTEM - 350 cells/cm 2
80 AdvanceSTEM - 1000 cells/cm 2
% Confluence
Supplement. The cells were incubated for 18 days with the media being changed every 4-5 days. The cells were assayed for differentiation using the following commercial kits: For Osteogenic differentiation, the OsteoImage PA-1501 kit was used. The kit measures specific staining of the hydroxyapatite portion of the bone-like nodules deposited by cells. For adipogenic differentation, the AdipoRed PT-7009 was used. The kit utilises Nile Red to dye the intracellular lipid droplets formed inside the differentiating adipocytes.
AdvanceSTEM - 4000 cells/cm 2
60 α-MEM - 100 cells/cm 2 α-MEM - 350 cells/cm 2
40
α-MEM - 1000 cells/cm 2
20
α-MEM - 4000 cells/cm 2
0 0
50
100
Fig. 1.
150
200
250
300
350
Incubation time (Hours)
Fig 1A: Growth of hMSC in T25 flasks with Nunclon Delta surface in -MEM medium or AdvanceSTEM Mesenchymal Stem Cell Basal Medium. Cells were seeded at four different densities: 100-350-1000-4000 cells/cm2. Cultures were incubated for 12 days under standard culture conditions (5% CO2 with media change every fourth day. Culture confluence was measured using automated microscopy in the incubator (IncuCyte Imager) every three hours. Each data point represents the mean of 50 measurements in one flask.
The growth of cultures of hMSCs seeded at 1000 and 4000 cells/cm2 in AdvanceSTEM Mesenchymal Stem Cell Basal Medium continued to grow past 80% culture confluence and were able to reach 96-99% confluence. At 350 cells/cm2, the cultures seeded in -MEM medium plateaued at around 75% confluence after 180 hours and cells seeded in AdvanceSTEM Mesenchymal Stem Cell Basal Medium reached the same level after approximately 250 hours. At the lowest seeding density of 100 cells/cm2, hMSC cultured in -MEM medium reached a plateau of 67% at approximately 235 hours. The cells seeded in AdvanceSTEM Mesenchymal Stem Cell Basal Medium were still in a slow exponential growth phase at the experiments end at 300 hours with a confluence of approximately 40%. The purpose of our protocol is to expand a relatively low number of cells; we thus chose the relatively low seeding density, which displayed good exponential growth, of 350 cells/cm2. The morphology of hMSCs cultured in the two media was similar (Figs.1B & 1C).
Fig 1B: hMSC morphology in AdvanceSTEM Mesenchymal Stem Cell Basal Medium after 7 days of incubation. Seeding density: 350 cells/cm2.
Fig 1C: hMSC morpholgy in -MEM medium after 7 days of incubation. Seeding density: 350 cells/cm2.
Nunclon Delta treated TripleFlasks an effective format for the cultivation of hMSC The growth of hMSC in -MEM medium and AdvanceSTEM Mesenchymal Stem Cell Basal Medium TripleFlasks using our in-house developed protocol was effective in generating a large population of hMSCs for either differentiation or cryogenic storage.
hMSC seeded in Nunclon Delta treated T175 EasyFlask at 350 cells/cm 2 (220500 cells/CF1). hMSC cultured u r for 8 dayss in T175
TripleFlasks harvested (2.6-3.0x10 7 cells/CF4). 9.0x10 5 cells seeded in TripleFlask (350 cells/cm 2).
T175 EasyFlas a k harvested (1.2-1.5x100 7 cells/CF1). 5 9.0x10 cells l seeded in Nunclon Del D ta treated TripleFlask (350 3 cells/cm 2). hMSC C cultured r for 8 days in TripleFl r ask
2.64x10 5 cells Nunc 48 well multidish seeded for osteogenic and adipogenic differentiation (5000 cells/ cm 2).
Nunc Cryotube with hMSC
Differentiation assay
37
Impact of AdvanceSTEM Mesenchymal Stem Cell Basal Medium on differentiation Marked differences between hMSCs expanded in AdvanceSTEM Mesenchymal Stem Cell Basal Medium and hMSCs expanded in -MEM medium was observed during differentiation. hMSCs expanded in AdvanceSTEM Mesenchymal Stem Cell Basal Medium displayed a 59% higher signal using the OsteoImage assay compared to cells expanded in -MEM medium (Fig. 2). Regarding adipogenic differentiation, hMSCs expanded in -MEM medium were unable to differentiate into adipocytes displaying baseline signals in the AdipoRed assay. In contrast, hMSCs expanded in AdvanceSTEM Mesenchymal Stem Cell Basal Medium differentiated successfully into adipocytes (Fig. 3).
18000 16000 14000
AdvanceSTEM, Trypsin α-MEM, Trypsin
10000 8000
4000 2000 0 Day 3 Day7 Day 18 Fig. 2. Osteoimage data from hMSCs osteogenically differentiated in 48 well multidish and assayed at day 3, 7, and 18 post-change to differentiation media. Data displayed as relative fluorescence units.
4000
The lowest seeding concentration displaying good exponential growth was 350 cells/cm2. In consequence a seeding density of 350 cells/cm2) was chosen for the expansion protocol. In our experiment we expanded the original hMSC population. Differentiation into adipocytes and osteoblasts were possible with cells expanded in HyClone AdvanceSTEM Mesenchymal Stem Cell Basal Medium, but only osteogenic differentiation, at a lower yield, was possible with cells expanded in -MEM medium.
3500
AdvanceSTEM, HyQtase
t/VODMPO%FMUBUSFBUFETVSGBDFTJODMVEJOH5SJQMF'MBTLTBSFBOFGGFDUJWFGPSNBU for the easy and rapid expansion of hMSC t&YQBOTJPOPGI.4$PO/VODMPO%FMUBVTJOH"EWBODF45&..FTFODIZNBM Stem Cell Basal Medium does not compromise the osteogenic and adipogenic potential of the hMSCs " TVCTUBOUJBMMZ IJHIFS ZJFME PG PTUFPCMBTU JT BDIJFWFE XIFO DFMMT BSF FYQBOEFE t"TVCTUBOUJBMMZIJHIFSZJFMEPGPTUFPCMBTUJTBDIJFWFEXIFODFMMTBSFFYQBOEFE in AdvanceSTEM Mesenchymal Stem Cell Basal Medium compared to -MEM medium.
AdvanceSTEM, Trypsin
3000 Fluorescence units
Conclusions
AdvanceSTEM, un-differentiatedcontrol
6000
Results
AdvanceSTEM, HyQtase
12000 Fluorescence units
hMSCs maintain their multi-potency after large scale expansion in Nunclon D lt ttreated t dT i l Fl k Delta TripleFlasks In order to verify that the cells had maintained their ability to differentiate, the cells expanded in TripleFlasks, in both types of growth media, using our in-house protocol, was differentiated into osteoblasts and adipocytes in 48 well multidish at a density of 5000 cells/cm2. The differentiation was induced using either osteogenic or adipogenic differentiation media. The differentiation was monitored at Days 3, 7, and 18 using commercial kits.
2500
α-MEM, Trypsin AdvanceSTEM, un-differentiatedcontrol
2000 1500 1000 500 0
Fig. 3.
Day 3
Day7
Day 18
ApidoRed data from hMSCs differentiated into adipocytes in 48 wells multidish. The cells were assyed at Days 3, 7, and 18 post-change to differentiation media. Data displayed as relative fluorescence units.
References Referenc Refe rences es 1. Koelling S, Miosge N. Stem cell therapy for cartilage regeneration in osteoarthritis. Expert Opin Biol Ther. 2009 Nov;9(11):1399-405. 2. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005 Feb 15;105(4):1815-22. Epub 2004 Oct 19. 3. Baldazzi F, Ripa RS, Jørgensen E et al. Release of bio-markers of myocardial damage after direct intramyocardial injection of gene and stem cells via the percutaneous transluminal route. Eur Heart J 2008;29:1819-26. 4. Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis CN, Papamichail M. Characterization of the optimal culture conditions for
Composite OsteoImage/Hoechst stained hMSCs differentiating into osteoblas ts. The cells are photographed at day 18 post induction. The hMSC were expanded in Mesenchyy mal Stem Cell AdvanceSTEM Mesenchymal Basaa l Medium Bas Medium and and passaged pas assa sage gedd using usinn g trypsin. usi tryp tr ypsin sin. Basal
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Composite AdipoRed /Hoechst stained hMSCs differentiating into adipocy tes. The cells are photographed at day 18 post induction. The hMSC were expanded in Advan Ad vancc eS eSTE TEM M Mesenchymal Mesenc Mes enchy hyma mall Stem St em Cell C Cel elll AdvanceSTEM Basa l Medium Basa M dium Medi Med ium and andd passaged pas passa sage gedd using usin usi in g trypsin. tryp tr yp sin sin. i Basal
Composite AdipoRed /Hoechst stained hMSCs differentiatin g into adipocy tes. The cells are photographed at day 18 post induction. The hMSC were expanded in -ME -MEM MEM Mm medi medium ediuu m an aand nd pa pass passaged ssage agedd usin uusing singg tryp tr yp sin sin. i trypsin.
clinical scale production of human mesenchymal stem cells. Stem Cells. 2006 Feb;24(2):462-71. 5. Reger RL, Wolfe MR. Freezing harvested hMSCs and recovery of hMSCs from frozen vials for subsequent expansion, analysis, and experimentation. expe p rimeentat a ion. o Methods Meethod odss Mol Mool Biol. o 2008;449:109-16. 2008 008;449 ; 9:109 09-16. 6
application
note UpCell Surface versus Trypsinisation in Preservation of Surface Proteins During Cell Harvesting
Harvest of cells from the UpCell Surface using temperature reduction t The dish was incubated at 20°C for 30 min. (no change of culture medium) t Detached cells were transferred to a 15 mL conical-bottom tube
Grey: anti-CD140a White: isotype control for non-specific binding (background)
Human bone marrow cells Cells harvested from traditional cultureware by trypsinisation Relative Cell Number
Cells harvested from UpCell Surface by temperature reduction Relative Cell Number
Cells harvested by temperature reduction or trypsinisation were washed twice by centrifugation (300 x g, 5 min.). Supernatants were discarded, and aliquots of 5.0 x 105 cells were incubated with 200 μL of PE-conjugated mouse monoclonal antibody against human CD140a (5 μg/mL) or PE-conjugated mouse IgG2a isotype-control antibody (5 μg/mL). After incubation at 4°C for 60 min. cells were washed with PBS and analysed by flow cytometry.
UpCell Surface Preserves the Integrity of CD140a
Log Fluorescence Intensity
Log Fluorescence Intensity
Human preadipocytes Results Human bone marrow cells and preadipocytes harvested from the UpCell Surface by temperature reduction had preserved cell surface CD140a, whereas CD140a on cells harvested from traditional cultureware by a short (3 min) trypsinisation could barely be detected. This demonstrates that using UpCell Surface and temperature reduction preserves the integrity of cell surface proteins to a higher degree than using traditional cultureware and enzymatic cell harvesting.
Cells harvested from UpCell Surface by temperature reduction
Cells harvested from traditional cultureware by trypsinisation Relative Cell Number
Methods: Human bone marrow cells or human preadipocytes in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) were seeded at 6.8 x 10³ cells/cm2 in a 6 cm dish with UpCell Surface and also in a traditional 6 cm dish. Cells were incubated for 24 hours at 37°C in a humidified atmosphere of 5% CO2 in air, then harvested using one of the following procedures:
Harvest of cells from traditional cultureware using trypsinisation t Cells were gently washed once with Ca2+– and Mg2+–free phosphate-buffered saline (PBS) t 2.0 mL of 0.25% trypsin/EDTA was added, and the dish was incubated at 37°C for 3 min. t 10 mL culture medium was then added to the dish, and the cells were transferred to a 15 mL conical-bottom tube
Relative Cell Number
Enzymatic cell harvesting often compromises the integrity of cell surface proteins. By contrast, the UpCell Surface allows cell harvesting simply by reducing the temperature of the cell culture, resulting in cell populations with preserved cell surface proteins. This application note compares the integrity of CD140a (a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family) on human bone marrow cells and preadipocytes harvested from the UpCell Surface by temperature reduction and from traditional cultureware (tissue culture-treated polystyrene) by trypsinisation. The cells were stained using a phycoerythrin (PE)-conjugated antibody against human CD140a and subsequently analysed by flow cytometry.
Log Fluorescence Intensity
Log Fluorescence Intensity
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Robert Scott, Jim Schantz, Joe Granchelli & Cindy Neeley Thermo Fisher Scientific
application
note The unique design of Thermo Scientific Carrier Plate enables precise adjustment of height for Cell Culture Inserts in multiwell dishes Abstract The Thermo Scientific Cell Culture Insert can be seated in a multiwell dish via either the 1 mm feet at the bottom of the insert or the 3 hanging tabs on the side of the insert. The Thermo Scientific Carrier Plate with the hanging slots allows for adjustment of insert height by engaging different hanging tabs on the insert. Here we determine, through physical measurement of the complete product system, the height of the growth surface of cell culture inserts when hanging in carrier plates. Measurements were taken for all three hanging positions, and recommended growth media volumes were calculated. Inserts were determined to hang with approximately 0.9 mm, 3.3 mm, and 6.3 mm between the well-bottom and insert growth surface for both 12- and 24-well plate formats. Introduction To facilitate versatility in the usage of cell culture inserts, carrier plates are designed to hold cell culture inserts in multiple positions above the growth surface of the multi-welled dish containing the insert. An important consideration for cell culture researchers is the height of the growth surface of the insert within the well, and the resulting volume of media required to grow cells at each height. Media height and volume are variables which may need to be controlled to optimise such things as gas exchange, oxygen tension, pH, availability of growth factors, or pH. Adjustment of the insert allows more than one physical position relative to the bottom of the carrier plate. Each position has associated to it minimum requirements of media volume to cover the cells. Physical measurements were made of inserts and carriers and those measurements, as well as recommended media volumes are reported right.
A. Distance from tab bottom to insert bottom B. Polycarbonate film thickness Hanging Slot C. Distance from well bottom interior to hanging position in hanging slot
Height of the cell culture insert growth surface
H=C-(A-B) Figure 1 Depiction of measurements taken from cell culture inserts and carrier plate/multiwell dishes.
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Reference: Thermo Scientific Nunc Solutions for Cell Culture and Growth brochure.
Materials Examined t NUN141002 – 24-well carrier plate/multiwell plate, 0.4 μm pore cell culture insert t NUN141082 – 12-well carrier plate/multiwell plate, t 8.0 μm pore cell culture insert Methods To first determine the hanging height in the carrier plate, the distance from bottom surface of the insert to the bottom of each heightadjusting tab on the insert was measured using a height gauge and stationary plate (See Figure 1, A). All 3 tabs on each insert were measured, and for each carrier plate format (12- and 24-well) a sample of n=3 inserts was included. Since the top of the polycarbonate film is the growth surface, the thickness of the film must be accounted for in measuring height (Figure 1, B). This was measured using a thickness gauge, and the film thickness was subtracted from measurement A to determine the distance from the bottom of the height-adjusting tab to the insert growth surface. When placed in the carrier plate, the bottom of the height-adjusting tab rests on the hanging slot of the carrier plate. Therefore, the hanging height of the growth surface (measurement A minus measurement B) is identical to the distance from the bottom of the hanging slot to the insert growth surface. To determine the well height of the insert growth surface from the bottom of the well plate, the height of the hanging slot from the well bottom was measured (Figure 1, C).
Results and discussion Table 1 contains overall results averaged from all plates and inserts for each format. Surface areas used for volume calculations are 3.5 cm2 for 12-well plates, and 1.8 cm2 for 24-well plates. Working volumes are calculated to give 5 mm growth media over the growth surface. Media volumes shown are suggestions, different volumes may be necessary depending on the cell type and experiment being performed. Hanging Position This was performed using a height gauge and stationary plate. Three different carrier plate/ multiwell dish samples were measured for each plate format, and measurements were taken from three locations in six different wells. Measured wells were chosen to sample as much of the plate as possible, at all four corners and two wells near the plate center (well B2 and B3 in 12-well, B3 and C4 in 24-well). Individual well measurements were averaged, and the calculation H = C - ( A - B ) was performed using these means for each height-adjusting tab size. The resulting dimensions (height H, see Figure 2 for clarification) represented the height of the cell culture insert growth surface from the bottom interior of the well in each hanging position. Using height H calculated above, the volume of cell growth media needed to cover the insert growth surface was also calculated. To cover cultures with media 5 mm deep, 5 mm was added to height H for each hanging position. This height was then multiplied by the growth surface area in individual wells for each plate format. The resulting volume is given as the recommended media volume at each hanging position.
Low Medium High
Measurements
24-well Carrier Plate
12-well Carrier Plate
0.9 ±0.1
0.9 ±0.1
Height (mm) Working Volume (mL) Height (mm) Working Volume (mL) Height (mm) Working Volume (mL)
1.0
2.0
3.3 ±0.1
3.4 ±0.1
1.5
3.0
6.3 ±0.1
6.4 ±0.1
2.0
4.0
Table 1: The height of the cell culture insert growth surface for all 3 hanging positions with recommended working volumes that allow 5 mm medium coverage of the insert. High Position 6.3mm
Medium Position 3.3mm
Low Position 0.9mm
Figure 2: Depiction of cell culture inserts hanging in carrier plate, showing three different hanging positions. Dimensions given represent the height of the growth surface of the cell culture insert from the interior bottom of the well (Height H). Dimensions in figure are rounded to the tenth of a millimeter.
Conclusion Carrier Plates are convenient for implementing versatility in the experimental design of cell culture inserts. The three hanging positions in the Carrier Plate allow for distance of approximately 0.9 mm, 3.3 mm, or 6.3 mm between the well-bottom and insert growth surface for both 12- and 24-well plate formats. Working volume should be adjusted according to the hanging position to suit the application needs.
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RainDance ThunderStorm The RainDance ThunderStorm System is a fully automated high-throughput targeted sequencing solution that enables researchers to process more samples per day and generate higher-quality data faster than ever before. The system features true walkaway capabilities that will automatically process up to 96 samples and access up to eight different primer panels. The ThunderStorm System leverages RainDance’s proven single molecule PCR technology that generates millions of unique microdroplet PCR reactions, enabling scientists to target up to 20,000 genomic loc loci in a single sample. The RainDance ThunderStorm System combines premium performance and a proven technology platform to deliver unprecedented daily sample throughput with the most attractive running economics and minimal hands-on time.
Sample
Target Selection
Next-Gen Sequencing Targeted Sequencing Workflow
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Fully automated, high-throughput targeted sequencing system enables researchers to process more samples, and generate higherquality data faster and easier than ever before.
Key Applications The ThunderStorm System was designed for a wide range of sequencing applications including: candidate gene screening, Genome Wide Association Study (GWAS) follow up, deep sequencing of heterogeneous tumour samples, medical genetics and targeted methylation studies. The system supports RainDance’s full portfolio of turn-key targeted sequencing solutions, including the DeepSeq™ FFPE Solution and MethylSeq™ Solution, as well as the ASDSeq™, XSeq™, ADMESeq™ and the HLASeq™ Research Screening Panels.
System Highlights t Fully automated high-throughput targeted sequencing system t Process 1 to 96 samples per run t Compatible with all NGS platforms: – Illumina’s HiSeq™ and MiSeq™ – Ion Torrent’s PGM™ – Life’s 5500 SOLiD™ – PacBio’s RS – Roche’s GS FLX™ – GS Junior™ Systems t Flexible format — Access up to 8 different primer panels in the same run t Low cost per sample — Prepare 2 samples per chip and eliminate sequencing library preparation costs
System Features t High-throughput processing of 1 to 96 samples without any user intervention t Supports all RainDance Targeted Sequencing applications (custom and defined content targeted sequencing, medical genetics, DeepSeq FFPE and MethylSeq Solutions
System Advantages t Comprehensive genomic coverage: Determine all variation contained in any desired region of the genome t Unparalleled target selection: Increased accuracy and greater coverage uniformity; requires less sequencing per sample than alternative enrichment methods
t Production ready for clinical research lab: Enable high-throughput screening and validation for mutation analysis and interpretation for genetic disorders using next-generation sequencing t Reduced sample input requirements: Use as little as 250 ng of unamplified genomic DNA t Elimination of downstream NGS library processing: Integrate sequencing adaptors directly into sequenceready targets with RainDance’s Tailed Primer Assay t Fast turnaround and simple workflow: Generate sequence-ready samples in as little as 4 hours from sample to sequencer t Designed for the future: Enabled for amplicon sizes from 150 bp to 1.5 kb today and up to ~10 kb in the future
Smart Consumables The ThunderStorm System includes new HeatWave TS Chips developed in collaboration with Sony DADC, the leading commercial manufacturer of smart consumables. These single-use chips provide a consistent, scalable and simple workflow that can be easily adapted and customised for a broad range of targeted sequencing applications.
FFPE and MethylSeq Solutions t Process samples in a standard 96-well microtitre plate format t Flexible run design: process up to 8 different primer panels t Localised temperature control to maintain sample integrity throughout the run t Enhanced workflow capability to import and export Excel-ready files for laboratory organisation t Configurable email notification of run success or error detection t Software run design allows users to define and queue runs ahead of time for easy workflow organisation t Graphical workflow design to instruct user on how to set up t Integrated bar-coding to record all reagents and consumables
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Evaporation & edge effect
in cell based assays In cell culture, a volume loss as small as 10% concentrates media components and metabolites enough to alter cell physiology, in some cases, severely.
Typically in microplate cell based assays, the outer wells of a 96 -well plate are often not used as this ‘edge effect’ is more pronounced in the outer and corner wells. The edge effect phenomenon can impact results and commonly is attributed to a lack of constant humidity and temperature resulting in uneven evaporation. In addition the lack of inclusion of the 36 outer wells of a 96-well plate results in a reduction in sample throughput by 38% of total capacity. With this in mind, the latest advancement in plates for cell culture is the Nunc Edge 96-well plates. These plates incorporate large perimeter evaporative buffer zones that eliminate well-to-well variability, while dramatically reducing the overall plate evaporation rate to lower that 2% after seven days of incubation. Tests have shown this leads to more viable and healthy cell yields. In this application note we evaluate the use of either water or a jellying je y g agent age suc such as aga agarose ose in the e reservoir ese o a and d co compare pa e the evaporation from the wells in the Edge plate to a normal plate plate..
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Fig. 1: Total evaporation % from the wells in standard plates and in Edge plates filled with water or agarose reservoirs. The figures are the average from 13 plates divided on three experiments. The large standard deviations on Edge plate evaporations are mainly due to the fact that in several plates no evaporation at all could be detected.
Materials and methods Nunc™ Edge plates were filled with 1.75mL water or 2.5mL 0.5% agarose (dissolved in water by heating in a microwave oven for one minute) per reservoir compartment. After setting of the agarose for half an hour at RT, the Edge plates together with standard F96 plates were filled with 100μL 0.002% aqueous crystal violet solution per well. The plates were incubated on the middle incubator shelf at 37°C, 5% CO2 and 95% relative humidity for four days, during which the incubator door was opened 90° for 15 seconds every hour seven times a day in order to simulate realistic conditions. The total evaporation from the wells and the reservoirs were determined by appropriate plate weighing before and after incubation, and the distribution of the evaporation on the individual wells were determined colorimetrically by the OD values at 590nm in 50μL well aliquots according to the following approximation:
Results The protection against evaporation by the Edge plate agarose reservoir is substantial, but maybe not quite as effective as by liquid water reservoir (Fig. 1) – although the amounts of evaporation from the respective reservoirs are the same (Fig. 2). Like that of water, the effect of agarose is not only a reduction of the total evaporation from the wells, but also an elimination of the evaporation edge effect (Fig. 3). The incubator humidity, which determines the evaporation velocity, is dependent on incubator door openings, so the 95% relative humidity is decreased to about 90% by frequent door openings and is only restored by the tranquility observed overnight (Fig. 4).
% well evaporation = g total water loss from wells g total water added to wells x
ODwell ODaverage
Fig. 2: Average evaporation amounts from the Edge plate reservoirs.
x 100
Fig. 4: Average incubator humidity readings from the three experiments just before the incubator door openings. The spike values reaching 95% relative humidity (violet line) appeared as the first readings after night tranquility. The concomitant temperature and CO2 readings (not shown) were on the other hand practically constant at 37˚C and 5% CO2 respectively.
Conclusion The Nunc Edge plates significantly reduce volume loss, effectively maintaining sample concentrations over long periods of incubation. The unique buffer zones enable the use of all 96 wells on the plate and greatly reduce the edge effect commonly experienced in cell culture and maintaining data consistency throughout the plate.The use of a 0.5% agarose gel in the Edge Plate reservoir is almost as effective as liquid water for protection against evaporation from the plate wells. This may be applied in order to eliminate the risk of spill from the reservoir by plate movement particularly when used in association with robotics. Additionally, the use of agarose gel, could without risk, increase the reservoir volume, thus prolonging the protection period for long-term uses before the reservoir gets exhausted. Fig. 3: Average evaporation percent from the individual wells throughout the 96 well plate matrix for standard plates, and for Edge plates with water and agarose reservoirs respectively.
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Low-Frequency tumour allele detection
The RainDance RainDrop System transforms the performance of molecular assays by enabling digital answers across a number of important applications including low-frequency tumour allele detection, gene expression, copy number variation, and SNP measurement. A variety of genetic alterations exist in human cancer, including deletions, amplifications, rearrangements and point mutations. DNA from tumour cells is released into clinical samples such as blood, lymph, stools and urine. In order to use these mutations as biomarkers, they must be detected in a large excess of non-mutated DNA from normal cells. Sensitive methods for detecting these somatic mutations can serve an important tool for clinical decision making and outcome in the oncology patient population.
References: RRefe efe ef fere fe reenc ren ennnccees: esss:: 1. PPezkin 1. ezzk ezki eezk zki zkin D, D et. eett. al. aall.l. Quantitative Q Qua Quuant uuaanntttiititaati ativ at ttiv tiiivve sensitive and and nd ssen se sens ennssitiv en ittivve detection ititi dette de tect eecct ect ctiion ioon on of of rare rare aarre re mutations mut mu muta m uut uta tations ttio tion io ion ons using on usin us ing nngg droplet-based ddro rrop ro oopplet pletleet ba let bbase bas aase assseed microfluidics, micr m iic icr croflu oflui ofl flui uidics dics cs,, Lab cs Lab on La on a Chip, Chi hip hhip, ipp,, ip 2011 201 201 20 0011 1111 Weaver 2.W 2. .W eeave eav avveer S, S, Dube Dub Duub D ube S, S, Mir M Miiirr A, A, Qin Qi Q in J, in J, Sun Suunn G, G, Ramakrishnan Rama Ra makr mak m akkrriishn issshn ish shhn hnaann RR, Jo JJones onnes oone es RC, RC R Livak LLiv iv ivaakk KJ. KKJJJ.T J.. Taking Taakin aki ak kki kin iinng qPC qP qPCR PPCR C CR R to to a higher hhiigher ghe ggh hher heeerr level: lev le lev evel el:l: analysis el: anna an anal nal aallysis ysi sis is is CNV ooff C NVV rreveals N evea ev eeve vveea vea eals ls tthe hhee power power owe ow wer wer er of o high higgh tthroughput thr th thro hrrroough hhro uughp ug ghhppuutt qPCR ghp qPCR PC PC CR R too enhance enhan en nhan nha anccee quantitative resolution. qqu uuaaanntita ttiita tit itta tattitive ivee res iv ive re esolu esolut es olut olut uttion. iion oon. n. Methods n. Met Meeth M eetthods hhood hod ods ods ds 200011100;;5550 22010;50:271-276 50: 0:227177171 1-227 1276 76 76 Q.. ZZhong, 33.. Q Zhhong, ongg S. on S. Bhattacharya, Bhh ta Bhat tac tach ach chharya arrya aary yaa, a, SS.. KKotsopoulos, otso oot tts tso soopoul poul pou u os os, os, s, J. J Olson, Olson lson, V. V.T . Taly, Tal Ta Taally ly, y, AA.D. ..D D. Griffiths, Gri Grriifffith Gr ffit ffi fith fifitthhs, s, D.R. D..R. R. Link R. LLin Li ink ink and in aannd nd J.W. J.W J W. J. Larson, LLar Lars La aar ars rrssoon, onnn,, Lab LLaaabb on on a Chip, Chi Ch C hi hip, p 2011, 2011, 01111 01 011 2167-2174. 1111,, 2216 111, 21 11667-21 -217 --2 217 221 174. 17
46
Traditiona attempts to detect low frequency alleles have been Traditional met with ttwo fundamental limitations. The assay may yield a false nega negative because the amount of starting DNA is too low to detect the rare mutation (inadequate sensitivity) or a stochastic false posit positive result because rare random mutations are present (in (inadequate specificity). Quantitative PCR assays are typically re reported to provide 0.5 to 1% sensitivity levels. The RainD RainDrop System leverages the power of up to 10 million individual PCR reactions to overcome these challenges to provide confident detection and quantification of rare mutations in high background of wild-type DNA at levels better than tha 1 part in 200,000. Additionally, digital PCR (dPCR) methods do not rely on standard curves for quantitation, which improves the ability to make run-to-run or cross-laboratory comparis comparisons, thereby improving the “transportability” of low frequency allele results. Assay
Mutation Type
Total Runs
EGFR_c.2573TG EGFR_c.2
T>G
5
>1/1,000,000
Assay B
C>G
3
>1/1,000,000
platform that is able to provide the required level of precision to measure smaller CNV differences in more challenging samples like cfDNA in a cost effective manner. Histogram (heat map) of droplet fluorescence intensities, for the 5-plex assay against a synthetic model. Standard techniques were used to compensate for spectral overlap of FAM and VIC signals. The six droplet populations correspond to the five individual assays plus the empty droplets.
LLOD
Assay C
C>A
6
>1/500,000
Assay D
C>G
4
>1/500,000
Assay E
A>G
3
>1/200,000
Figure 1: Shows a dilution series with decreasing mutant to wild-type ratios against several no template control samples.
Table 1 shows the the Lower Limit of Detection (LLOD) for several sev point mutation assays.
RainDanc RainDance ce continues to develop a and report performance on important importtant cancer assays including includ variants in EGFR, BRAF, KRAS, KR RAS, PIK3CA, ALK and M MLH1 to demonstrate the power of 10 million droplets. The c combination of superior sensitivity y, unprecedented multipl sensitivity, multiplexing, and flexibility in experime nt design make the RainDrop Rain experiment System a powerful genomic a analysis platform for grou ground-breaking research in cancer inc cluding rare variant detec including detection, absolute quantitation of biomar rkers, B profiling, and the ability to monitor residual biomarkers, disease.
Results of the SMA pilot study on 20 different patient samples from the Coriell cell repositories: 4 afflicted with SMA, 1 SMA carrier, and 15 negative controls. The measured genotypes of the different patients were consistent with their disease conditions (unafflicted, carrier, or afflicted). The patients afflicted with SMA each had zero copies of SMN1 (numbers SMA 17–20 in the figure), the carrier had just one copy, and the negative controls all had two or three copies (patients 1–15).
Copy number variation Copy number variations (CNVs) ar are involved in a large number of complex human diseases including includ many cancers and genetic conditions. An importan important measurement challenge in translational research involv involves identification of small CNV changes with high confi confidence. Cell free DNA (cfDNA) in blood plasma, for example, promises a readily available source of genetic material for tumour or pre-natal diagnosis diagno with minimally invasive sampling techniqu techniques. Early eff efforts using dPCR have repor reported the ability to detect a 1.25 1.25-fold difference in copy num number. Additionally, the bina binary nature of dPCR means that the precision is more indepe independent of variation in assay amplification, making it easier to optimise and standardise between laboratories. The RainDrop System is the only dPCR
47
Sample preparation for high-throughput live cell imaging References: Bjorkblom B, Ostman N, Hongisto V, Komarovski V, Filen JJ, Nyman TA, Kallunki T, Courtney MJ, Coffey ET (2005). Constitutively active cytoplasmic c-Jun N-terminal kinase 1 is a dominant regulator of dendritic architecture: role of microtubule-associated protein 2 as an effector. J Neurosci 25:6350−61. Cao J, Semenova MM, Solovyan VT, Han J, Coffey ET, Courtney MJ (2004). Distinct requirements for p38alpha and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death. J Biol Chem 279:35903−13. Cao J, Viholainen JI, Dart C, Warwick HK, Leyland ML, Courtney MJ (2005). The PSD95-nNOS interface: a target for inhibition of excitotoxic p38 stress-activated protein kinase activation and cell death. J Cell Biol 168:117−26. Courtney MJ, Lambert JJ, Nicholls DG (1990). The interactions between plasma membrane depolarisation and glutamate receptor activation in the regulation of cytoplasmic free calcium in cultured
Cell-based multi-well assays using high-content analysis and high-throughput screening instruments are becoming increasingly common in biomedical research. However, analytical methods usually need multiple wash steps and incubation of reagents. These steps can be extremely laborious when performed manually, thus it is preferable to automate these steps with a microplate washer. While using a microplate washer on fixed cells is usually not difficult, it can be quite problematic when working with live cells, especially for some loosely adherent cells. In this application note, we evaluated the performance of the Wellwash Versa, a microplate washer specifically designed for working with cells, for washing rat cerebellar granule neurons, a standard primary culture model widely used for studying neuronal function that is relatively loosely adherent and delicate, possessing a substantial amount of fine neuronal processes (Bjorkblom et al., 2005). Some tips for optimising the use of the Microplate washer will also be covered. Introduction Cell-based assays in 96-, 384-well or higher density plates are not only restricted to simple colorimetric/fluorometric endpoint microplate reader assays, but also include high content analysis and live cell imaging assays. Reducing well size reduces the consumption of experimental reagents and materials, and the generation of waste without decreasing the sample size, thereby permitting substantial increases in the range of conditions evaluated. This is particularly important in both cell-based high content analysis (HCA) and high-throughput screening (HTS), which usually involve a large number, sometimes up to millions, of screening conditions.
cerebellar granule cells. J Neurosci 10:3873−9. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A (2004). Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA 101:10554−9.
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In HCA/HTS, cells are usually cultivated in Microplates and the assay also takes place within the well. However, the culture medium, in which the cells are maintained, is often not optimal for imaging assays. It may be necessary to transfer cells to an imaging buffer with low background fluorescence and defined chemical composition, or cells are loaded with a fluorescent probe, which may need removal before image acquisition.
Many assays perform best in a specific buffer, and may involve multiple wash steps with different buffers. The manual pipetting steps required to wash cells, define well volumes and for assay setup are laborious and inefficient. In such situations, a microplate washer can improve efficiency. The use of microplate washers for in vitro assays, such as ELISA is routine. However, the application to cell-based assays requires that they be sufficiently gentle. For example, primary cultured neurons have a complex and fragile structure. They may easily be detached by sudden movements, or be stressed by vigorous fluid flow inside the well. The Wellwash Versa provides adjustable aspiration and dispensing speeds and positions. The dispense head is angled and the solution can be dispensed to the well side walls and flow slowly to the well bottom. This offers gentle but efficient washing and maintains the cells in a good condition for experiments. In this application note, we describe the development and optimisation of protocols for Wellwash Versa to accomplish specific needs and add some technical tips for using the unit for cell washing.
Materials Ma ate t ri rial als and d Me Meth Methods thodss Cell culture and wash/imaging wash/ima aging buffer Rat cerebellar granule cells w were isolated from cerebella of P7 Wistar rats essentially as des scribed (Courtney et al., 1990) and described maintained in minimal essent tial medium (MEM) containing 10% essential FBS, 33 mM glucose, 2 mM Glutamine, G 20 mM KCl, Pen/Strep (5 U / 5 μg/ml) and 10 μM Ara aC (applied 24 h after plating) for AraC 6−10 days before using for a allll tests in this application note. The wash/imaging buffer used in this application note is Locke’s buffer, which is composed p off 154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 5.6 mM Glc, 5 mM HEPES and 1.3 mM CaCl2 at pH 7.4 (Cao et al., 2004). Basic cell wash Brightfield images were acquired when the cells were in culture medium before any wash step. Plates were then washed by the Wellwash Versa using Locke’s buffer and the cells were kept in Locke’s buffer during the second acquisition for the images after the wash. To monitor depolarisation-induced intracellular calcium increases in neurons, cells expressing the genetically encoded calcium reporter YC3.60 (Nagai et al., 2004, Cao et al., 2005) were washed with calcium-containing Locke’s buffer either manually or using the Wellwash Versa, and kept in a 100μl volume before performing live cell imaging. Images through YFP and CFP emission filters (542nm/27 and 483nm/32 bandpass filters, AHF) under CFP excitation (438nm/20 filter) were captured over a period of 1 min to obtain a baseline. Then the cells were depolarised by addition of 30 mM KCl, using the dispensing device of the automated imaging system, and imaged for a further 5 min. The increase in the YFP/ CFP emission ratio during the experiment indicated the increase in intracellular calcium level. Sipping protocol To quantify liquid volumes accurately and maintain surfacetension properties, 0.002% bromophenol blue was added into the medium for cerebellar granule cells and distributed to the wells of a 96-well plate. A single aspiration step with different aspiration heights was applied to different columns as indicated. The standard curve was prepared by manual pipetting. The absorbance of the wells was measured at 595 nm and the volume of the medium after aspiration was calculated according to the standard curve.
Results and Discussion The flexibility to optimise parameters is necessary for every microplate washer, especially when working with microplates containing live cells. The Wellwash Versa provides sufficient parameters for optimising performance for different cell types and plate types, including the positioning of the aspiration and dispensing head, aspiration and dispensing speed and height. The most important criterion is to be gentle enough to the cells without compromising the washing efficiency. Primary neurons can be quite loosely attached on tissue culture plates. The neurites of differentiated neurons form a mesh of processes interconnecting individual soma. This can catch liquid flow and result in the entire cell sheet peeling off during wash steps. First, we aimed to find a basic wash protocol that can wash
the well adequately but with minimal disturbance to the loosely attached cells. Table 1. Parameters for the optimised wash step: Wash volume (μl)
500
Wash cycle
1/2
Soak/Shake
Off
Aspirate height
5 / 6 mm (2 / 3 mm from bottom)
Wash head speed
10
Aspirate speed
Medium
Aspirate time (s)
1
Dispense height start
7
Dispense height end
9
Dispense offset
1.2
Dispense tip touch
1.3
Final aspirate
No
Table 2. A595
Standard Deviation
No dye
0.041
0.003
Unwashed
0.570
0.028
2 mm from bottom, 1 cycle
0.055
0.004
2 mm from bottom, 2 cycles
0.041
0.003
3 mm from bottom, 1 cycle
0.070
0.009
3 mm from bottom, 2 cycles
0.042
0.003
A two-cycle procedure, using settings shown in Table 1, included an aspiration step down to 2−3 mm from the bottom, followed by an aspiration-dispensing step, i.e., simultaneous aspiration during the dispensing of 500 μl wash buffer per well at a higher position. This was shown to be adequate to rinse the well, as evaluated by comparing the absorbance of a well containing dye labelled buffer to a dye-free well (blank) before and after washing with different parameters (Table 2). Primary cultured neurons and their neurite network were therefore washed thoroughly without showing physical signs of disturbance (Figure 1).
Franz Ho and Michael Courtney, Molecular Signalling Laboratory, Department of Neurobiology, A.I. Virtanen Institute, University of Eastern Finland, Finland
Table 2. Absorbance at 595 nm of a 96-well plate with 0.002% bromophenol blue after performing wash steps with different aspiration height and cycles. 150 μl of dye was distributed into all wells except those designated “no dye” (blank control), and the wells were washed by washing steps as indicated below. A final aspiration step, defining a remaining volume of 100 μl, was applied to the whole plate to normalise the pathlength of all the wells for subsequent quantitation of washing efficiency. The numbers show the mean absorbance of 16 wells.
One concern of using a microplate washer is that the processes of aspiration and mechanical dispensing may interfere with the functional behaviour of cells, ultimately resulting in incorrect conclusions from misleading experimental results. It is well known that when cerebellar granule neurons are depolarised by extracellular potassium, extracellular calcium will enter through calcium channels and result in a sudden increase in intracellular calcium (Courtney et al., 1990). In order to demonstrate that cerebellar granule neurons give the same response after the washing steps by Wellwash Versa as after manual washing, the optimised washing process was used to prepare cells for measurement of intracellular calcium upon depolarisation. Intracellular calcium levels were detected by a fluorescent protein based FRET probe. The response of
49
Figure 1. Images before (A) and after (B and C) wash using Wellwash Versa. No significant cell loss can be seen. A magnified image was also acquired after wash (C) to illustrate that the appearance of cells, including soma and neurites, is normal. Images were captured by 4X objective (A and B) and 20X objective (C) with the transmission white LED of the BD Pathway 855 automated imaging system and digital contrast enhancement. Scale bars indicate 1 mm (A and B) and 100 μm (C).
cerebellar granule cells upon depolarisation is similar between those washed manually with a multichannel pipette or by Wellwash Versa (Figure 2). These results indicate Wellwash Versa can replace manual pipetting even when working on loosely attached primary cultured neurons. This makes high-throughput work much easier and less laborious. It also saves considerable time, especially for some cell-based assays which involve multiple changes of assay reagents and buffers before data acquisition. Sipping protocol Cells are often cultured for a relatively long time, for example, for dividing cells to reach a particular level of confluence or differentiating cells to reach a defined level of maturity, compared with the duration of the cell-based assay. Therefore, it is necessary to culture cells in a larger volume than is necessary for the actual assay. However, during the treatment of cells with screening targets, or when cells are stained by synthetic fluorescent probes for image acquisition, a decrease in the medium volume is usually preferred for minimising reagent usage and conserving valuable large-scale libraries. It is laborious and time consuming to remove or set by manual pipetting a defined amount of medium in multi-well plates before assay. For assays or assay steps that can be performed directly in cell culture medium, we made use of the aspiration function of the microplate washer and developed a protocol which can sip medium from the wells to leave the desired volume in each well.
be made for different plate types and different vendors because the dimensions of the plates may vary. Table 3: Volume remaining in the well versus the aspiration height Aspiration Height (mm)
Calculated Vol (μl)
StDev (μl)
5.4
71.78
3.63
5.6
78.67
3.36
5.7
82.51
2.64
5.8
86.66
2.37
6.0
95.34
2.58
6.2
104.43
3.07
Conclusion The Wellwash Versa is easy to use and to set up protocols for washing plates seeded with cells. It can suit different cell types as numerous parameters defining the behaviour of the washer heads can be adjusted to optimise the required task. It can be integrated into an automated imaging pipeline via serial port commands as well as used as a standalone device without computer connection. The user interface is clear and easy to follow. In practice, the Wellwash Versa is gentle enough for loosely attached cells without compromising washing efficiency.
Figure 2. Intracellular calcium levels of cerebellar granule cells before and after depolarisation. Blue: Cell washed by manual pipetting. Green: Cell washed by Wellwash Versa (error bars show the S.E.M. of average values from 6 wells).
Table 3 shows the volume remaining in the well versus the aspiration height. Manual pipetting is used for the standard and the volume is estimated by absorbance of each well with medium supplemented with 0.002% bromophenol blue as explained above. Aspiration from centre of the well without stopover time (“Aspiration time = 0”) give the highest reproducibility (data not shown). Note that adjustments have to
50
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