Confidential

(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

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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

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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-Cwith Al mask on top

e) Remove Al hard mask f) Peel off and place it on top of PDMS for dynamic co-culture

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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.

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FNFibronectin

(纖維糖連蛋白 )

Biocompatible Materials

Parylene：聚對二甲苯 (Poly-para-xylyleneHA

Hyaluronic Acid：玻尿酸 ;琉璃醣碳基酸

3D model of theCollagen structure

膠原蛋白

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Cell adhesion with ECM factors

Collagen

On HA

1000

900

800

700

600

500

400

300

200

100

0

Cel

ls/ m

m2

Uncoated Collagen FN HA

without detergent

with detergent

Collagen on HA

Cell adhesion on untreated and detergenttreatedparylene-C stencils coated with ECM factors.

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Five cells

ES cellsEmbryonic stem cell

3T3 cells NIH-3T3

HUVECsHuman Umbilical Vein Endothelial Cells

HL-1 cells

ACLsAmeloblast-Lineage Cells造牙釉質 -世系細胞

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Dynamic co-culture process I

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Dynamic co-culture process II

Collagen coating

HUVECs

ALCs

3T3 cells

FN coating

HL-1 cells

A

B

C

D

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Patterning of fluorescently labeled proteins

Patterning of fluorescently labeled proteinsusing a multilayer stencil, scale bar is 200 μm

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Fluorescent images of the cells

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

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ES cells remaining rate

The number of remaining ES cells inside themicrowells for different stencil thicknesses.

5-5-5 um

5-5-10 um

10-10-10 um

100

80

60

40

20

0

Co-Cul

ture

dH

L1Peele

d 1s

t la

yer

Co-Cul

ture

dALC

Peele

d 2n

dla

yer

Co-Cul

ture

d3T

3 Peeled

3r

d la

yer

Co-Cul

ture

d

HUVEC

Perc

enta

ge o

f re

tain

ed E

S c

ells

(%)

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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.