1. CELL-ENVIRONMENT INTERACTION (OUTSIDE-IN) Yuan Liu

2. 1 ST SOFT LITHOGRAPHY 2 ND THE EFFECT OF CELL SHAPE ON CELL SURVIVAL

3. SOFT LITHOGRAPHY IN BIOLOGY AND BIOCHEMSITRY George M. Whitesides et al. Annu Rev Biomed Eng 2001

4. Soft lithography is a set of techniques for microfabrication using soft polymer stamps. It offers fine structural control of surfaces and has wide applications in biological research. Abstract of abstract

5. Outline  Photolithography to soft lithography  Features of soft lithography  Applications

6. Phtolithography  Photolithography  Disadvantages:  Expensive  Poor surface control  Time consuming

7. Soft Lithography  Soft Lithography Soft polymer stamps for microfabrication  Advantages:  Inexpensive  Fine surface control  Convenient  Lot of applications KU Jayhawks stamp

8. Surface Engineering  Self-Assembled Monolayers (SAMs)  Alkanethiolates HS(CH2)nX on gold  Surface properties determined by X  Inert Surface  No absorption and adsorption

9. Surface Engineering  Attaching Ligand  Bind to specific protein  Promote adhesion  Homogeneous and Heterogeneous

10. PDMS Stamps  Poly-dimethylsiloxane  Elastomeric  Nontoxic  Commercially available  Replica molding

11. μ CP  Micro-contact printing  Use PDMS stamp  Small feature  Contact  Print  Sub-micron  SAM on gold  Other materials  AFM writing

12. Dry Lift-Off  Lift-off  Wet traditionally  Elastomeric membrane for dry lift-off Membrane, method, and patterned cells Image from Biotherm

13. Microfluid System  By soft lithography  Laminar flow  Not turbulent  Contact at the interface  Use for patterning

14. Microfluid System  Generate gradient  Mimic biological system Gradient produced by microfluid system

15. Applications in Biochemical Assays  Miniaturize biochemical assays  Increase # of assays in parallel  Microfluid structures  Easily done by μ CP  Lab on chip  μ TAS: micro total analysis system  Save work and space Lab on chip

16. Applications in Cell Biology  Patterned cell culture  Study single cell  Cell-cell interaction  Cell migration 2 kinds of cells are patterned on concentric squares

17. PARTCELL  Partial treatment of cell using laminar flow Trypsin is only treated over the left side of the cell

18. Cells in Microwells BCE cells in microwells

19. Next…  Use soft lithography to pattern single cell  Study the effect of cell shape on cell survival

20. GEOMETRIC CONTROL OF CELL LIFE AND DEATH Christopher S. Chen et al. 1997 Science

21. By using micropatterned, ECM coated islands, we studied the effect of restricted cell extension on capillary endothelial cells survival. Cell spreading was found to govern individual cells growth or death. Abstract of abstract

22. Outline  Background  Experiments  Conclusions

23. Background  Binding to ECM controls local differentiation in capillary  Disruption of ECM results in cell death  Integrin α ν β 3 antagonists induce apoptosis  Death of suspended cells can be prevented by  Antibodies binding to integrin  Inhibiting tyrosine phosphatases  Assumption1: Adhesion-dependent control of apoptosis is mediated by integrin signaling

24. Background  Dying cells remain in contact with ECM fragments  Cells grow on large beads (>100 μ m) while die on small beads (4.5 μ m)  Assumption2: Cell shape might also affect cells growth and death Q: Whether integrin binding or cell shape governs cell growth and death?

25. Cells Attached to Beads of Different Size  Apoptosis  (60%) for small beads  Cells became more rounded on smaller beads Prob: Spreading is coupled with ECM curvature, small beads internalization

26. Cells Attached to Micropatterned Planar Islands  Apoptosis  for 20 μ m circle  Apoptosis  from 20 μ m to 10 μ m circle Q: Precise spreading requirement?

27. Cells Attached to Square Islands of Different Sizes  Cell shape matched to square islands  When area , apoptosis , DNA synthesis   Spreading leads to cell survival Prob: Integrin binding, focal adhesion formation, accessibility to matrix-bound formation also  when area 

28. Single Cells Spread on Multiple Small Islands  When Projected area  (same ECM contact area), growth , apoptosis   Cell spreading is critical to cell survival Q: Roles of integrin ligands remain unclear

29. Different Integrin Ligand in Cell-Shape Regulated Apoptosis  Apoptosis is much higher for cells on 20 μ m circle (gray) than those on unpatterned substrate (black)  Contact to β 1 is more sensitive than to α ν β 3 Black: unpatterned Gray: on small circles  Integrin binding determines the sensitivity to shape

30. Conclusions Experiments:  Cell shape governs cells survival  Adhesion to different ligands results in different sensitivity to cell size Further Q:  How can cells sense geometrical change in surrounding  Directly linked to mechanical stress  Focal adhesion orients signal transduction In real tissue:  Cell can feel local mechanical deformation and change its activity ex. morphogenesis

31. Thanks for Your Attention!