Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures

1. Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures Photo Courtesy: Yuli Wang Julius Oatts Emory University – Neuroscience and Behavioral Biology August 25, 2006 Dr. Mark Bachman, Department of Electrical Engineering and Computer Science Dr. Nancy Allbritton & Dr. Chris Sims, Department of Physiology and Biophysics

2. Outline Outline • Project Overview – Long term goals • Micro Pallet Array Fabrication • Cell Growth on Pallets and Laser Release • Virtual Wall Discussion • Effect of silanization on cell growth • Fluorescent Protein Coating Stability • Density’s effect on cell growth • Acknowledgements

3. Project Overview Project Overview Separate single cells from a heterogeneous population or isolate monoclonal colonies Expand this technology to multiple cell lines Select Collect Expand Photos Courtesy: Grace Young Mock Transfection: HeLa cells on 175um base, 50um high pallets

4. UV Radiation Unwanted SU-8 dissolved Patterned, cross-linked SU-8 Micro Pallet Array Fabrication Micro Pallet Array Fabrication • SPIN COAT • UV EXPOSURE • POST-EXPOSURE BAKE • DEVELOP SU-8 coating Glass slide Mask Mask (removed)

5. Cell Growth and Release SU-8 (epoxy groups) Cell Growth and Release Cell Growth and Release Surface Protein (amine groups) 1. Protein Attachment 2. Adherent cell attachment Adherent cells Protein-SU-8 bond

6. Cell Growth and Release Collection Well Formation of Cavitation Bubble Inverted Collection Well Released Adherent Cell on Pallet Collected Adherent Cell on Pallet Cell Growth and Release Force causes pallet lift-off 3. Laser Pallet Release System 4. Cell/Pallet Collection 2 µJ, 10 ns pulse Laser Release System Unreleased pallets are not collected

7. “Virtual Wall” Discussion 3. Formation of “Virtual Air Wall” Formation of Water Droplets Cell Growth and Release “Virtual Wall” Discussion 1. Deposition of Hydrophobic Silane Layer on Glass between pallets Protein Coating: Selective to Pallets 2. Adding aqueous solution containing surface coating proteins

8. “Virtual Wall” Discussion “Virtual Wall” Discussion Top view Side view Water Droplets on Silanized Glass Cell attached to pallet Dermal Fibroblasts on 175m base pallets

9. Effect of Silanization on Cells Silanized SU-8 Silanized Glass Glass (control) Effect of Silanization on Cells

10. Protein Coating Stability Protein Coating Stability

11. Protein Coating Stability Same Pallet Array: Cells attached well Poor cell adhesion Inconsistency: HeLa cells growing on collagen coated 175 µm pallets Protein Coating Stability Cause and Effect? We Don’t Know.

12. Cell Density’s Effect on Growth Cell Density’s Effect on Growth Calculating Doubling Time with a Growth Curve Number of cells at time = t y(t) = y(0) * 2nt Time (hours) Cell Cycles/Hour Initial number of cells Solve for “n” to find Doubling Time (Doubling time = hours to complete 1 cell cycle)

13. Cell Density’s Effect on Growth Cell Density’s Effect on Growth A-172’s

14. Cell Density’s Effect on Growth Cell Density’s Effect on Growth Dermal Fibroblasts

15. Acknowledgements Acknowledgements Professors Mark Bachman, G.P. Li (project direction) Professors Nancy Allbritton, Chris Sims (laboratory technique, project direction) Grace Young (laboratory technique, tissue culture, mentoring) Yuli Wang (mentoring, pallet fabrication) University of California, Irvine and the Undergraduate Research Opportunities Program National Institute of Health National Science Foundation