Optimal Conditions for Cell Viability on SU-8 Adherent Cell Sorting Microstructures Julius Oatts Emory University – Neuroscience and Behavioral Biology.

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Presentation transcript:

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 1)Project Overview – Long term goals 2)Micro Pallet Array Fabrication 3)Cell Growth on Pallets and Laser Release 4)Virtual Wall Discussion Effect of silanization on cell growth 5)Fluorescent Protein Coating Stability 6)Density’s effect on cell growth 7)Acknowledgements

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 SelectCollectExpand Photos Courtesy: Grace Young

Micro Pallet Array Fabrication 1.SPIN COAT 2.UV EXPOSURE 3.POST-EXPOSURE BAKE 4.DEVELOP Glass slide SU-8 coating UV Radiation Mask Mask (removed) Unwanted SU-8 dissolved Patterned, cross-linked SU-8

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

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

Cell Growth and Release“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 3.Formation of “Virtual Air Wall” Formation of Water Droplets

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

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

Protein Coating Stability

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

Cell Density’s Effect on Growth Calculating Doubling Time with a Growth Curve y (t) = y (0) * 2 nt 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)

Cell Density’s Effect on Growth Cell DensityDoubling Time 1,00082 hours 10,00062 hours A-172’s

Cell Density’s Effect on Growth Cell DensityDoubling Time 1,00069 hours 10,00050 hours Dermal Fibroblasts

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 HealthNational Science Foundation