1. C onfidential (CMEMS2008-P276) TEACHER: Cheng-Hsien Liu professor TEAM MEMBER: Yu-Shih Chen 陳育詩 Kun-Chih Pan 潘昆志 2008 年 11 月 4 日 MULTILAYER PARYLENE-C STENCILS FOR DYNAMICALLYCONTROLLING CELL INTERACTIONS

2. C onfidential INTRODUCTION Patterned co-cultures of two or more cell types have been generated using some approaches. In this study, they develop a multilayer parylene-C stencil for patterning extracellular matrix (ECM) proteins and cells. Using the multilayer parylene-C stencil, they describe a novel, rapid, and convenient method for the generation of dynamic co- cultures of 5 different cell types. Ali K. et al. Biomaterials 2004 Hui and Bhatia, PNAS 2007

3. C onfidential Fabrication process for multilayer parylene-C stencil d) Plasma etching of parylene a) Parylene-C was first Deposited on a silicon wafer b) Coating anti-stiction layer (detergent, micro 90) c) Three layers of Parylene-C with Al mask on top e) Remove Al hard mask f) Peel off and place it on top of PDMS for dynamic co-culture

4. C onfidential SEM image of multilayer paralene-C stencils A) top view, B) cross-sectional view5-5-5um, C) cross- sectional view10-10-10um, D) cross-sectional view5-5-10um.

5. C onfidential FN Fibronectin ( 纖維糖連蛋白 ) Biocompatible Materials Parylene ：聚對二甲苯 (Poly-para-xylylene HA Hyaluronic Acid ：玻尿酸 ; 琉璃醣碳基酸 3D model of the Collagen structure 膠原蛋白

6. C onfidential Cell adhesion with ECM factors Collagen On HA 1000 900 800 700 600 500 400 300 200 100 0 Cells/ mm2 Uncoated Collagen FN HA without detergent with detergent Collagen on HA Cell adhesion on untreated and detergenttreated parylene-C stencils coated with ECM factors.

7. C onfidential Five cells ES cells Embryonic stem cell 3T3 cells NIH-3T3 HUVECs Human Umbilical Vein Endothelial Cells HL-1 cells ACLs Ameloblast-Lineage Cells 造牙釉質 - 世系細胞

8. C onfidential Dynamic co-culture process I

9. C onfidential Dynamic co-culture process II Collagen coating HUVECs ALCs 3T3 cells FN coating HL-1 cells A B C D

10. C onfidential Patterning of fluorescently labeled proteins using a multilayer stencil, scale bar is 200 μm

11. C onfidential Fluorescent images of the cells Fluorescent images of the cells during formation of dynamic co-cultures, scale bar is 200 μm.

12. C onfidential ES cells remaining rate The number of remaining ES cells inside the microwells for different stencil thicknesses. 5-5-5 um 5-5-10 um 10-10-10 um 100 80 60 40 20 0 Co-Cultured HL1 Peeled 1st layer Co-Cultured ALC Peeled 2nd layer Co-Cultured 3T3 Peeled 3rd layer Co-Cultured HUVEC Percentage of retained ES cells(%)

13. C onfidential CONCLUSIONS In this study, they have developed a multilayer parylene-C stencil technology for creating microscale patterns of proteins and cells. They demonstrate that the multilayer parylene-C stencils can be used to generate dynamic co-cultures of at least 5 different cell types. That technique is simple, versatile, and inexpensive, and it may find potential application in studying stem cell differentiation, developmental biology, and regenerative medicine.