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(CMEMS2008-P276) TEACHER: Cheng-Hsien Liu professor TEAM MEMBER: Yu-Shih Chen 陳育詩

MULTILAYER PARYLENE-C STENCILS FOR DYNAMICALLYCONTROLLING CELL INTERACTIONS. (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. INTRODUCTION.

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(CMEMS2008-P276) TEACHER: Cheng-Hsien Liu professor TEAM MEMBER: Yu-Shih Chen 陳育詩

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  1. MULTILAYER PARYLENE-C STENCILS FOR DYNAMICALLYCONTROLLING CELL INTERACTIONS (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

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

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

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

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

  8. Dynamic co-culture process I

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

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

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

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

  13. 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.

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