12 presentation

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

Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures 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 Photo Courtesy: Yuli Wang

Outline: 

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

Project Overview: 

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

Micro Pallet Array Fabrication: 

Micro Pallet Array Fabrication Micro Pallet Array Fabrication SPIN COAT UV EXPOSURE POST-EXPOSURE BAKE DEVELOP Glass slide SU-8 coating Mask Mask (removed)

Slide5: 

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

Slide6: 

Cell Growth and Release Cell Growth and Release 3. Laser Pallet Release System 4. Cell/Pallet Collection 2 µJ, 10 ns pulse Laser Release System Force causes pallet lift-off Unreleased pallets are not collected

Slide7: 

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

Slide8: 

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

Slide9: 

Effect of Silanization on Cells Effect of Silanization on Cells

Slide10: 

Protein Coating Stability Protein Coating Stability

Slide11: 

Protein Coating Stability Protein Coating Stability We Don’t Know. Cause and Effect?

Slide12: 

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

Slide13: 

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

Slide14: 

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

Acknowledgements: 

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