CELL-ENVIRONMENT INTERACTION (OUTSIDE-IN)

Yuan Liu

1ST SOFT LITHOGRAPHY 2ND THE EFFECT OF CELL SHAPE ON CELL SURVIVAL

SOFT LITHOGRAPHY IN BIOLOGY AND BIOCHEMSITRYGeorge M. Whitesides et al.Annu Rev Biomed Eng 2001

Soft lithography is a set of techniques for microfabrication using soft polymer stamps. It offers fine structural control of surfaces and has wide applications in biological research.

Abstract of abstract

Outline

Photolithography to soft lithography Features of soft lithography Applications

Phtolithography

Photolithography Disadvantages:

Expensive Poor surface

control Time consuming

Soft Lithography

Soft LithographySoft polymer stamps for microfabrication

Advantages: Inexpensive Fine surface control Convenient

Lot of applications KU Jayhawks stamp

Surface Engineering

Self-Assembled Monolayers (SAMs) Alkanethiolates

HS(CH2)nX on gold Surface properties

determined by X Inert Surface

No absorption and adsorption

Surface Engineering

Attaching Ligand Bind to specific

protein Promote adhesion

Homogeneous and Heterogeneous

PDMS Stamps

Poly-dimethylsiloxane Elastomeric Nontoxic Commercially

available Replica molding

μCP

Micro-contact printing Use PDMS stamp Small feature

Contact Print Sub-micron

SAM on gold Other materials

AFM writing

Dry Lift-Off

Lift-off Wet

traditionally Elastomeric

membrane for dry lift-off

Membrane, method, and patterned cellsImage from

Biotherm

Microfluid System

By soft lithography Laminar flow

Not turbulent Contact at the

interface Use for patterning

Microfluid System

Generate gradient Mimic biological

system

Gradient produced by microfluid system

Applications in Biochemical Assays Miniaturize

biochemical assays Increase # of assays

in parallel Microfluid structures

Easily done by μCP Lab on chip

μTAS: micro total analysis system

Save work and space

Lab on chip

Applications in Cell Biology

Patterned cell culture Study single cell Cell-cell interaction Cell migration

2 kinds of cells are patterned on concentric squares

PARTCELL

Partial treatment of cell using laminar flow

Trypsin is only treated over the left side of the cell

Cells in Microwells

BCE cells in microwells

Next…

Use soft lithography to pattern single cell Study the effect of cell shape on cell

survival

GEOMETRIC CONTROL OF CELL LIFE AND DEATHChristopher S. Chen et al. 1997 Science

By using micropatterned, ECM coated islands, we studied the effect of restricted cell extension on capillary endothelial cells survival. Cell spreading was found to govern individual cells growth or death.

Abstract of abstract

Outline

Background Experiments Conclusions

Background

Binding to ECM controls local differentiation in capillary

Disruption of ECM results in cell death Integrin ανβ3 antagonists induce apoptosis Death of suspended cells can be prevented

by Antibodies binding to integrin Inhibiting tyrosine phosphatases

Assumption1: Adhesion-dependent control of apoptosis is mediated by integrin signaling

Background

Dying cells remain in contact with ECM fragments

Cells grow on large beads (>100μm) while die on small beads (4.5μm)

Assumption2: Cell shape might also affect cells growth and death

Q: Whether integrin binding or cell shape governs cell growth and death?

Cells Attached to Beads of Different Size Apoptosis (60%) for small beads Cells became more rounded on smaller

beads

Prob: Spreading is coupled with ECM curvature, small beads internalization

Cells Attached to Micropatterned Planar Islands Apoptosis for 20 μm circle Apoptosis from 20 μm to 10 μm circle

Q: Precise spreading requirement?

Cells Attached to Square Islands of Different Sizes Cell shape matched to square islands When area , apoptosis , DNA

synthesis Spreading leads to cell survival

Prob: Integrin binding, focal adhesion formation, accessibility to matrix-bound formation also when area

Single Cells Spread onMultiple Small Islands When Projected area (same ECM

contact area) , growth , apoptosis Cell spreading is critical to cell survival

Q: Roles of integrin ligands remain unclear

Different Integrin Ligandin Cell-Shape Regulated Apoptosis Apoptosis is much

higher for cells on 20 μm circle (gray) than those on unpatterned substrate (black)

Contact to β1 is more sensitive than to ανβ3

Black: unpatternedGray: on small circles Integrin binding determines the

sensitivity to shape

Conclusions

Experiments: Cell shape governs cells survival Adhesion to different ligands results in

different sensitivity to cell sizeFurther Q: How can cells sense geometrical change in

surrounding Directly linked to mechanical stress Focal adhesion orients signal transduction

In real tissue: Cell can feel local mechanical deformation

and change its activity ex. morphogenesis

Thanks for Your Attention!