Micropatterning Thin Polystyrene Films for Single Cell Culture Biological Microsystems Lab Dr. David Eddington Elly Sinkala Krina Gandhi

Objective Pattern 500 um wells and plot graphs for : Well depth vs. RPM Well depth vs. # of layers of polystyrene Pattern smaller diameter wells (10 um – 25 um) Culture cells in the wells Bovine capillary endothelial (BCE) cells deposited in an array of wells 50 um in diameter

Motivation Show that we can culture a single cell in one well Control cell-to-cell interactions Control cell shape Applications: Study cell characteristics Drug testing Tissue engineering

Materials Su-8 photoresist Epoxy based photoresist Sensitive to near UV radiation Thermally stable Suited for permanent use applications su-8 molecule

Materials Polydimethylsiloxane (PDMS) Non-toxic Hydrophobic Transparent Readily available Elastomer base Curing agent

Materials Polystyrene Clear, colorless polymer Easily available Cost effective

Processes Photolithography Process of transferring geometric shapes on a mask onto silicon wafer It involves:  Cleaning  Application of photoresist (su-8)  Soft bake  UV Exposure  Post bake  Development

Processes Silanization Wafer placed in a vacuum along with 30 uL of tricholorosilane for 2 hours Produces thin silane coating on the wafer Reduces adhesion strength between PDMS and silicon Facilitates easy separation of PDMS Vacuum chamber Tridecafluoro-1,2,2-Tetrahydrooctyl-1-Trichlorosilane

Processes Soft Lithography Mix PDMS (10:1) Remove bubbles Pour PDMS on the silicon master Cure for 2 hours at 80°C Peel off the PDMS mold Elastomer base Curing agent PDMS in a vacuum chamber

Processes Embossing Spin polystyrene on glass cover slip Place PDMS mold over it Place it on a hot plate for 10 min at 180°C Peel it off once cooled PDMS mold Glass cover slips coated with polystyrene

Processes Height vs. Diameter plot generated using data from profilometer Tencore Profilometer Well Depth Measurement

Processes Cell Culture Sterilized the wells Seeded cells (MDCK-Madin-Darby Canine Kidney Cells) Allowed cells to adhere (3-4 hours) Washed excess cells with PBS Filled the wells with media

Results

Results

Results Smallest wells patterned: 68 um 4 cells in 68 um well 4 MDCK cells in 68 um wells

Conclusion Collected data and plotted them for: Well depth vs. RPM Well depth vs. # of layers Smallest wells patterned: 68 um Was able to culture cells Future Work Continue to work on smaller diameter wells Try to culture one cell in one well using the smaller wells

Acknowledgments Dr. David Eddington Elly Sinkala Dr. Takoudis Dr. Jursich National Science Foundation (NSF) Department of Defense (DOD) EEC-NSF Grant #

References "Single Cell Localization and Patterning." Rapid Prototyping Laboratory for energy and biology Stanford University. 29 July Wang, Chun, Fung Ling Yap, and Yong Zhang. "Micropatterning of polystyrene nanoparticles and its bioapplications." Colloids and Surfaces B: Biointerfaces 46 (2005): Nakanishi, Jun, Tohru Takarada, Kazuo Kamaguchi, and Mizuo Maeda. "Recent Advances in Cell Micropatterning Techniques for." The Japan Society for Analytical Chemistry 24 (2008): Whitesides, George M., Emanuele Ostuni, Shuichi Takayama, Xingyu Jiang, and Donald E. Ingber. "Soft lithography in biology and biochemistry." Annu. Rev. Biomed. Engineering 3 (2001): Yang, Gloria Y., Vasudev J. Bailey, Yu-Hsin Wen, Gisela Lin, William C. Tang, and Joyce H. Keyak. "Fabrication and Characterization of Microscale Sensors for." IEEE Xplore (2004): Questions??