Transcript
BUILDING EMERGENT BIOLOGICAL MACHINES (AND OTHER COOL THINGS AT SMALL SCALE!) Rashid Bashir University of Illinois at Urbana-Champaign Department of Bioengineering Micro and Nanotechnology Laboratory University of Illinois at Urbana-Champaign http://libna.micro.uiuc.edu/
Caroline Cvetkovic Ritu Raman Vincent Chan, Ph.D. Acknowledgments to: Prof. Taher Saif (UIUC) Prof. Hyunjoon Kong (UIUC) Prof. Roger Kamm (MIT)
1
On Size and Scale ! 10mm
System on a chip
Tissue
1mm
Ants
Plant and Animal Cells
10µm 1µm
3D printing at micron to cm scale
Most Bacteria
100nm
12nm Feat. of MOS -T (in 2015)
Virus
10nm
MEMS
Proteins Helical Turn of DNA
1nm
Nano pores -
CNT, QD, NS, NWs, AAO
0.1nm
Bottom-up Biological
Bottom-Up Chemical Self-Assembly
Gate Insulator
Nanoelectronics and Nanoscale Sensors
Feature Size
100µm
Microelectronic and MEMS
System on A board
Organs
100mm
Top-Down Silicon Fab
Fabrication Techniques
Diagnostics for Global Health and Food safety
Silicon Nanofabrication
CD4+ Counting for HIV
3-D Bio- Stereolithography
Silicon MEMS Fabrication
PDMS Fabrication 70µm
Point of Care PCR – Droplet Heating
3-4 Terminal Nanopores for DNA Methylation and Sequencing Emergent Behavior of Cells
2 mm
Cells
Functionalized Nanosensor Array
Movement
Vascular Patch for Angiogenesis
Biological Robots
3-D Biofab. and Cellular Systems
Physical properties of adherent live cells
Dr ain
So ur
ce
Nanowires for Electrical Detection of miRNA
Cancer and Individualized Medicine
Can we detect HIV from a drop of blood ?
Bill Rodriguez MGH, Daktari
Mehmet Toner, MGH
Global Prevalence of HIV Infection
Global Prevalence of HIV Infection
• • •
33 M people living with HIV world wide (1.4M deaths annually) Only 1 in 8 are able to be tested for HIV/AIDS HIV/AIDS testing not widely available in 70% of countries with epidemic
Lysing of RBC in Whole Blood
Can we perform a CBC from a drop of blood ?
Sub-Types • Gen 3: A point of care CBC from a drop of blood -
-
WBC, Neutrophils, Monocytes, Platelets, RBCs CD4+, CD8+ CD64+ Neutrophils
Sepsis • Leading cause of death in critical care • 1,150,000 cases in the US per year • 20-50% die! 215,000 deaths per year • Estimated $26 billion annual cost to the U.S. healthcare system • (mean cost: $20,000 per case)
Detection of DNA Methylation for Cancer Diagnostics Using Solid State Nanopores R. Bashir (UIUC), A. Nardulli (UIUC), G. Vasmatzis, Mayo Clinic) • Solid state nanopores for detection of DNA Methylation • MBD proteins bind to CpG dinucleotide on dsDNA • Nanopores discriminates between unmethylated and methylated DNA Solid State Nanopores
Iqbal, et al., Nature Nanotechnology, 2007; Venkatesan, et al. Advanced Materials, 2009. Venkatesan, et al. Advanced Functional Materials, 2010, Venkatesan, et al. ACS Nano, 2012; Banerjee, et al. ACS Nano, 2013;
Shim, et al. Sci Rep, 2013.
Can we build machines and systems with cells?
Biological Machines 13
Terminator-2
Prosthetics
Biological Machines Nature […] has been pleased to construct […] organized bodies with a very large number of machines, which are of necessity made up of extremely minute parts so shaped and situated, such as to form a marvelous organ […] -- Marcello Malpighi Piccolino, M. Biological Machines: From Mills to Molecules. Nat. Rev. Mol. Cell Biol. 1, 149–152 (2000).
Size and Length Scale of the Human Body 10-9 m Biomolecules
Cells 10-6 m
Tissues 10-3 m
10o m
Functional Units •
Sensing
•
Information processing
•
Actuation
•
Protein expression
•
Transport
Human Body Organs 10-1 m
McMahon, T., & Bonner, J. T. On Size and Life. Scientific American, 1983.
14
Pathways to a “Biological Machine”: ‘Developmental Biology’ versus ‘Engineered’
Stem cells: Ludovic Vallier, University of Cambridge. Movie: R. Karstrom & D Kane, Development, 1996 http://www.depauw.edu/news/index.asp?id=17734
Impact of Cellular Systems “… engineering of multicellular biological machines represents a major step beyond what is currently being done … [and] has the potential to be truly transformative …”
Health
R. D. Kamm, et al., Mech. Eng., Nov 2010
Exemplary Cellular Systems: Organ mimics for drug testing
Security
Biological robots Implantable systems for drug sensing, synthesis, and release Self-replicating organisms for toxic waste clean-up
Environment
Biological Machines Design Move in one direction
(i) A Walking BioBot ‘An Inchworm’
(ii) A Swimming BioBot ‘A Sperm’
BioBot Road Map Primary Cardiomyocyte Biobot moves in one direction Optogenetic muscle Light driven Biobot move/stop in one direction
YouTube
High level description: A BioBot that walks or swims directionally but is unresponsive
Muscle & Motor Neurons NMJ drives/controls the motion
More formal specification: Repeat Pulsing muscle contraction with directional movement generated by asymmetry
Neurons that are inhibitory or excitatory
Biological Microrobots – ‘BioBots’ Prescribed tasks include sensing, information processing, transport, protein expression, and movement. Minibody ReleaseMinibody
Neuron Circuits and Control
release
philanthotoxin Philanthotoxin
Neuromuscular Junctions
Supply channel
Cell-instructive microenvironment
Protein expression
Sensors Transport
Controlled channel
Muscle Net motion Actuators
Information processing
Vasculature & Cell-Based Factory
Memory and Control
Bio-Bot
3-D Bottom up ‘Stereo-Lithography’ 3D Systems SLA 250/50
A
OH
OH
B
OH COO -
O
COO -
O
O
OH
O
y
x
Irradiated alginate
1. Oxidization
NaIO4 COO -
O
OH COO -
O O
H H
O
OH
x
O
y
O
Oxidized alginate (OA)
Modified 2. Acrylation Mini-Platform O
O H
OH
NH
O
O O
O
O
O
COO -
H
1.8 mm PEG 1000
EDC / HOBt
OH
x
O
y
Oxidized methacrylic alginate (OMA) Viper si2 SLA
System Type
SLA 250/50 3. Functionalization EDC / HOBt C HeCd (Gas) Nd:YVO4 (Solid-State) RGD peptid Wavelength 325 nm 355 nm e Power 40 mW 100 mW x y XY Resolution 250 µm 75 µm RGD conjugated OMA (Beam ф) 4. Crosslinking + O Z Resolution 0.1 mm 0.1 mm I-2959 / UV (Layer) O O
O
H
H
OH
10 mm
O
O
NH
O
Time (Day)
NH
O
O
OH
O
O
O
nO
Poly(ethylene glycol) methacrylate
1 cm
3-D Cell Based ‘StereoLithography’ Top view optical image of printed heart 2mm
Chan, et al. Lab Chip, 2010
CAD Model
2mm
Side view image of printed heart Chan, et al. Lab Chip, 2010
NIH 3T3 cells
Fabrication of Walking Biobots
1mm
V. Chan, et al., Sci. Rep. 2, 857; DOI:10.1038/srep00857, 2012
Achieving Net Movement?
1mm 7mm
7mm
7mm
Speed of Biobots
From Popular Science, 2013
A Muscle-Tendon-Bone (MTB) Inspired Design 26
• •
Bone – cell E > 40kPa, Overall E > GPa Tendon – connective tissue between bone and muscles, Overall E > 200MPa bone
muscle
joint
tendon Basic MTB biomechanical unit Low Stiffness
High Stiffness
Google images
Skeletal Muscle Cell Driven BioBots
Cvetkovic, Raman, et al. PNAS, 2014
Characterizing the Skeletal Biobots
Cvetkovic, Raman, Chan, et al. PNAS, 2014
How Can Contraction be Controlled with Light? Light-gated ion channels ChR2 Cation channel Depolarizing Blue light, Excitatory
NpHR Anion channel Hyperpolarizing Yellow light, Inhibitory
Fenno et al, Ann Rev Neurosci, 2011
31
Control with Light Directional Locomotion of Muscle Ring-Powered Bio-Bot with Asymmetric Geometry Optical Stimulation (2Hz)
Zero Net Locomotion in Symmetric Muscle Ring-Powered Bio-Bot with Symmetric Geometry FEA Simulation
Directional Locomotion of Muscle Ring-Powered Bio-Bot with Symmetric Geometry Targeted Optical Stimulation (2Hz)
Rotational Locomotion of Muscle Ring-Powered Bio-Bot with Symmetric Geometry FEA Simulation of Targeted Optical Stimulation
Rotational Locomotion of Muscle Ring-Powered Bio-Bot with Symmetric Geometry Targeted Optical Stimulation (2Hz)
Integrate NMJ with BioBots • Next: control contraction of myotubes and locomotion via activation of motor neurons and neurotransmitter release Embryoid Bodies
Muscle Strip Pearson Education, 2011
Embryoid Bodies
GFP+ MN MF20 DAPI
Embryoid Body
Neurite extensions Hb9-GFP GFAP (Glia) DAPI
Neuronal Circuits Neuron type A Toggle
Neuron type B philanthotoxin
Minibody release
Net motion
Electrical stimulation of Neuron type A, modulate toggle and record electrical response in Neuron type B
From Steve Stice, Univ. of Georgia
Bruce Wheeler, Univ of Florida
Can we control blood vessel growth in tissue?
(b) 1cm
Chick chorioallantoic membrane (CAM) assay
With Prof. Hyunjoon Kong Jeong, et al. Adv. Func. Mat. 2012
ogical Patterning ng of blood ls
of Blood Vessels in-vivo
Lots of neovessels! 300um
Jeong, et al. Adv. Func. Mat. 2012
Written in Blood ‘Vessels’ !
A Cardiac Patch to Restore Heart Function with
Prof. Larry Prof. Schook, Hyunjoon UIUC Kong, UIUC
*
*
*
Melhem, et al. Submitted, 2015
Multi-cell “components”: Vascular networks Perfused with microbeads
From Roger Kamm
45
Where we are, and where we are headed? 46
• • • • • • •
3 generations of walking machines demonstrated Control with electric fields and light Complex 3D geometries Neuronal control Vasculature Self repair? Exo-skeleton?
Some Possibilities 47
• • • • • • •
“Hyper-Organs” for implants and drug delivery High throughput drug screening Smart plants Surgical micro robotics Surveillance Energy production/harvesting “Emergent manufacturing”
Important Ethical Considerations 48
• At what level of complexity does a biological machine become a living organism? • And what features distinguish one from the other? • What if the biological machines can selfreplicate?
Ethics Modules Creating Biological Machines--2013 http://ebics.net/ethics/module-1/creating-biologicalmachines-bio-bot Hyper-organs and Engineering Biological Functions--2014 http://ebics.net/ethics/module-2-hyper-organs-andengineered-biological-functions Ethical Issues in the Conduct of Multi-institutional, Collaborative, Interdisciplinary Research--2015 http://ebics.net/content/module-3-ethical-issuesconduct-multi-institutional-collaborativeinterdisciplinary-research
Outreach Soft robotics walker
•
•
Micron and millimeter soft robotics • platforms are critical platforms for biologically powered machines • This macroscale demo demonstrates the functionality and mobile potential of soft robotics structures
“Optogenetic” robot
Provides macroscale analogy of optogetic biobots Explains utility of selectively stimulating distinct actuators to obtain a directed output
Outreach • • • •
3D Printing Temporal Dynamics Soft Robotics Demo Belousov-Zhabotinsky Reaction • Optogenetic Robot
• • • •
Science at the Market Engineering Open House NanoSTRuCT/BTW STEM Night/Lab Visits
B I O B LO C K S Build
with
Week
M
Lecture
1
8/24
2
8/31
3 3 4 6 7
9/7 9/14 9/21 9/28 10/5
8
10/12
9
10/19
10
10/26
11
11/2
12
11/9
13
11/16
13 14 16
11/23 11/30 12/7
Course Overview: Biological Machines, Cell Culture Review Lab 1: Biocompatibility, cell death and cell proliferation No class Lab 1: Imaging/Microscopy, Viability Assays Lab 2: Gene Delivery Lab 2: Transdifferentiation Lab 3: 3D Cell Culture, Cell/Cell + Cell/Matrix interactions, Collagen Gels Lab 3: 3D Microenvironments - Mechanical and Biochemical Lab 4: BioBots Lecture on Stereolithographic 3D Printing Lab 4: Design and fabrication of BioBot Muscle Ring Actuators Lab 4: Optogenetics, Exercise Conditioning, Effect of external factors (IGF, ACA) Lab 4: Mechanics and Movement, Viscoelastic models Lab 5: Different types of Bioactuators, Group Brainstorm new designs! No class Lab 5: Group Ethics Discussion Dry runs
Biology Lab
W
Lab
Cell Culture Review + Aseptic Technique PRACTICAL
8/26
Labor Day Lab 1: MTS Assay Part 1 Lab 2: Prep cells and Plasmids Lab 2: Transduction Lab 3: Collagen gel start
9/9 9/16 9/23 9/30 10/7
Cell Culture Review + Aseptic Technique Lab 1: Start Cultures in 12-well plates and treatment Lab 1: Live/Dead Staining Lab 1: MTS Assay Part 2 Lab 2: Transfect cells Lab 2: Imaging Lab 3: LV transductions
Lab 3: Gel release
10/14
Lab 3: Collect data, stain
Lab 4: CAD Design and 3D Lightyear Preparation Lab 4: Seed Muscle Rings
10/21
Lab 4: Exercise Conditioning
11/4
Lab 4: Stereolithographic Fabrication of BioBot skeleton Lab 4: Transfer Muscle Rings to BioBots Lab 4: Exercise Conditioning
Lab 4: Optical Stimulation/Electrical Stimulation Lab 5: Fabrication of skeleton + Seeding Thanksgiving Break Lab 5: Exercise Training/Caretaking Lab 5: Collect data, prepare presentation
11/11
9/2
10/28
11/18
Lab 4: Quantification of performance Lab 5: Transfer to BioBot
11/25 12/2 12/9
Thanksgiving Break Lab 5: Test BioBots, Clean Up Student Presentations
LAB 1
Biocompatibility
LAB 2
Lentiviral Transduction
LAB 3
3D Cell Culture in Natural Hydrogel
Students will learn standard methods of culturing cells in 3D hydrogel matrices (good practice for and alternative to fibrin hydrogels which will be used in following labs). They will assess the effect of culture in a 3D environment that mimics native ECM on cell adhesion, proliferation, and morphology.
LAB 4
Build a Walker BioBot
Students will learn how to design and fabricate living biological machines powered by engineered skeletal muscle that are capable of controlled directional locomotion in response to optical and electrical signaling. They will test the effects of "exercise" training on BioBot functional response and learn how to quantify force production using viscoleastic models of BioBot mechanics.
Students will test the effects of two different types and concentrations of commonly used stereolithography photoinitiators (I2959 - biocompatible in low concentrations, I651 - cytotoxic) on cell viability. They will assess viability via live/dead staining and MTS assay. Students will learn standard methods for delivering genes with high efficiency to cell lines typically considered "hard to transfect." The students will transduce cells with the master transcription factor MyoD and ChR2 to achieve a myogenic phenotype that is light inducible.
Acknowledgements Current Researchers (2014): • Dr. Bobby Reddy • Dr. Eric Salm • Dr. Jiwook Shim • Dr. Sangjo Shim • Olaoluwa Adeniba • Shouvik Banerjee • Caroline Cvetkovic • Greg Damhorst • Gelson Josue Pagan Diaz • Carlos Duarte • Anurup Ganguli • Tanmay Ghonge • Umer Hassan • Ritu Raman • Oluwayemisi Sonoiki • Vikhram Swaminathan
Funding Agencies (2014) • Abbott • NSF IGERT CMMB • NSF STC EBICS • NIH NCI M-CNTC • NSF IUCRC CABPN • TSMC • NIH NCI • National Science Foundation • USDA ARS, Center for Food Safety Engineering at Purdue
Faculty Collaborators • Prof. A. Alam (ECE, Purdue) • Prof. H. Asada (MIT) • Prof. A. Bhunia (Food Science, Purdue) • Dr. Andy Fischer (Abbott) • Prof. R. Kamm (MIT) • Prof. W. P. King (MechSE, UIUC) • Prof. H. J. Kong (CheBE, UIUC) • Prof. M. Ladisch (Ag& Bio Engr, Purdue) • Prof. Ann Nardulli (UIUC) • Prof. Gabi Popescu • Prof. W. Rodriguez (Daktari) • Prof. John Rogers (UIUC) • Prof. T. Saif (MechSE, UIUC) • Prof. M. Toner (Harvard Med School) • Dr. G. Vasmatzis (Mayo Clinic)
Thank you !
