Cell-Environment Interaction (outside-in)

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Cell-Environment Interaction (outside-in)

• Yuan Liu

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1st Soft lithography 2nd the effect of cell
shape on cell survival
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Soft lithography in biology and biochemsitry

• George M. Whitesides et al.Annu Rev Biomed Eng
  2001

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Abstract of abstract

• 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.

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Outline

• Photolithography to soft lithography
• Features of soft lithography
• Applications

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Phtolithography

• Photolithography
• Disadvantages
• Expensive
• Poor surface control
• Time consuming

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Soft Lithography

• Soft LithographySoft polymer stamps for
  microfabrication
• Advantages
• Inexpensive
• Fine surface control
• Convenient
• Lot of applications

KU Jayhawks stamp
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Surface Engineering

• Self-Assembled Monolayers (SAMs)
• Alkanethiolates HS(CH2)nX on gold
• Surface properties determined by X
• Inert Surface
• No absorption and adsorption

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Surface Engineering

• Attaching Ligand
• Bind to specific protein
• Promote adhesion

• Homogeneous and Heterogeneous

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PDMS Stamps

• Poly-dimethylsiloxane
• Elastomeric
• Nontoxic
• Commercially available
• Replica molding

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µCP

• Micro-contact printing
• Use PDMS stamp
• Small feature ? Contact ? Print
• Sub-micron
• SAM on gold
• Other materials
• AFM writing

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Dry Lift-Off

• Lift-off
• Wet traditionally
• Elastomeric membrane for dry lift-off

Membrane, method, and patterned cells
Image from Biotherm
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Microfluid System

• By soft lithography
• Laminar flow
• Not turbulent
• Contact at the interface
• Use for patterning

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Microfluid System

• Generate gradient
• Mimic biological system

Gradient produced by microfluid system
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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
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Applications in Cell Biology

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

2 kinds of cells are patterned on concentric
squares
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PARTCELL

• Partial treatment of cell using laminar flow

Trypsin is only treated over the left side of the
cell
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Cells in Microwells
BCE cells in microwells
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Next

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

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Geometric control of cell life and death

• Christopher S. Chen et al. 1997 Science

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Abstract of abstract

• 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.

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Outline

• Background
• Experiments
• Conclusions

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Background

• Binding to ECM controls local differentiation in
  capillary
• Disruption of ECM results in cell death
• Integrin a?ß3 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

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Background

• Dying cells remain in contact with ECM fragments
• Cells grow on large beads (gt100µm) 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?
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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
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Cells Attached to Micropatterned Planar Islands

• Apoptosis ? for 20 µm circle
• Apoptosis ? from 20 µm to 10 µm circle

Q Precise spreading requirement?
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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 ?
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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
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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 ß1 is more sensitive than to a?ß3

Black unpatterned Gray on small circles
? Integrin binding determines the sensitivity to
shape
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Conclusions

• Experiments
• Cell shape governs cells survival
• Adhesion to different ligands results in
  different sensitivity to cell size
• Further 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

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• Thanks for Your Attention!