Micro Patterning for Biological Applications

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Micro Patterning for Biological Applications

• IGERT/HHMI Journal Club
• April 23, 2007
• Matt Keuss
• ZiQiu Tong

2
History

• patterning of species on surfaces with micron
  scale resolution
• 1978, MacAlear from semiconductor industry
• Integration of protein molecules into
  bioelectronic microcircuits

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Potential Applications

• Celluar studies
• Tissue engineering
• Development of biosensors
• Immunoassay

Delamarche et al, Advanced Materials. 2005
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Micropatterning Requirements

• Selective attachment of protein at desired
  regions
• High protein resistivity by other regions
• Protein orientation immunodiagnostic devices
• Retaining protein functionality

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Protein Attachment

• (Direct) Non-covalent hydrophobic, van der
  Waals, hydrogen bonding, electrostatic forces,
  etc
• Advantage no chemical modification is required
• Disadvantage may get denatured
• (Indirect) Covalent bifunctional crosslinker
• Thiol (-SH3) on gold substrate
• Silane on glass and silicon substrate (SiO2)

Mrksich, Annu. Rev. Biophys. Biomol. Struct. 1996
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Outline

• Techniques --Tommy
• Photolithography
• Soft lithography
• Microcontact printing
• Microfluidic network
• Applications -- Matt
• Cell growth
• Cell migration
• 3D Mcirofluidic network

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Photolithography
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Photolithography

• Dominant technique for patterning solid state
  devices
• Requires clean room facilities
• Chemicals used can denature biomolecule

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Soft Lithography

• Whitesides and colleagues 98
• A soft elastic stamp, Polydimethylsiloxane (PDMS)
• Common Techniques
• Microcontact printing (µCP)
• Microfluidic patterning
• Stencil patterning

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MicroContact Printing

• Replica molding
• Transfer by conformal contact
• Simple and inexpensive method
• Compatible with many molecules
• Nonplanar surfaces
• Multilayers

Whitesides, Annu. Rev. Mater. Sci 1998 Yap,
Biosensors and Bioelectronics. 2007 Bernard,
Advanced Materials, 2000
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Micro Fluidic Network

• Characteristics 10-100 micron, 0.1cm/s,
  milisecond for diffusion and reaction times
• Capillary force
• Pressure assisted flow
• Suitable for patterning delicate materials
  (protein and cells)

• Yap, Biosensors and Bioelectronics. 2007

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Drawbacks

• Lost normal functionality upon attachment to
  substrate
• Limitation of the number of proteins can be
  patterned

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Outline

• Techniques --Tommy
• Photolithography
• Soft lithography
• Microcontact printing
• Microfluidic network
• Applications -- Matt
• Cell growth
• Cell migration
• Other Applications

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Cell Growth and Viability

• Binding to the extracellular matrix (ECM)
  controls local differentiation in capillaries
• Disruption in ECM leads to cell death
• Allowing suspended cells to bind antibodies to
  integrins prevents apoptosis
• However in vivo studies have shown that dying
  capillary cells remain in contact with the ECM
• Cells grow and spread on large beads (gt100mm) and
  die on smaller beads (4.5mm)

Chen, CS et al. Science v276 p1425
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Cell Growth and Viability Cont.

• However small beads can be engulfed into the
  cells complicated the analysis
• Micropatterned square fibronectin adhesion
  islands onto gold
• Vary the island size and measure growth and
  apoptosis

Chen, CS et al. Science v276 p1425
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Geometric Control of Cell Life and Death

• Increase the area of patterned square from 75 to
  300 um2
• Apoptosis declines
• DNA synthesis increases
• However this maybe due to more integrin binding,
  focal adhesion formation, or greater
  accessibility to growth factors in the matrix

Chen, CS et al. Science v276 p1425
17

Geometric Control

• Modify experiment
• Arrange closely spaced islands of 3 to 5 um in
  diameter.
• Keep the area of contact the same but vary the
  spacing
• As projected area increases
• DNA synthesis increases
• Apoptosis decreases

Chen, CS et al. Science v276 p1425
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Experimental Conditions

• Cell type
• Human capillary endothelial cells
• Proteins patterned
• Fibronectin
• Antibodies to integrin b1 and integrin avb3
• Collagen (binds b1)
• Vitronectin (binds avb3)
• Observation
• Contacts specific to b1 lead to apoptosis more
  than contacts to avb3

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Conclusion

• Findings
• Cell size can control whether a cell grows or
  dies regardless of ligand
• However adhesion of different receptors
  determines how sensitive the cell is to size
• Possible Explanations
• Focal adhesions may orient the signal
  transduction machinery of the cell
• Growth and survival might be directly linked to
  the mechanical stress

Chen, CS et al. Science v276 p1425
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Cell Migration

• It is known that chemoattractants and integrin
  receptors can contribute to cell migration
• Do the mechanical forces within a cell alter the
  mechanism of cell migration?

Parker K.K. et al. 2002, FASEB J v16 p1196
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Experiment

• Micropattern different ECM components in
  different shapes with microcontact printing
• Fibronectin, collagen, thrombospondin-1
• squares and circles
• Treat cells with growth factors
• PDFG and FGF
• Observe how the cells extend lamellipodia

Parker K.K. et al. 2002, FASEB J v16 p1196
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Finding

• Circle random extension
• Square extension from the corners
• Occurs for endothelial cells on fibronectin and
  myoblasts on thrombospondin-1 each patterned on
  glass
• Growth factor receptors are still homogenously
  distributed

Parker K.K. et al. 2002, FASEB J v16 p1196
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More observations

• Stress fibers on square islands orient themselves
  with the diagonal of the cell
• Vinculin concentrated at corners indicated focal
  adhesions are at the corners
• Do physical constraints cause cells to focus
  traction forces at the corners?

Parker K.K. et al. 2002, FASEB J v16 p1196
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Traction Force Microscopy

• Used to measure tension in the cell
• Collagen islands on polyacrylamide gel with
  imbedded 2mm fluorescent beads
• Culture human airway smooth muscle cells
• Greatest displacement was at the corners

Parker K.K. et al. 2002, FASEB J v16 p1196
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Possible Explanation

• Local tension transfer locally activate Rho
  GTPases or CDC42
• Known previously Rac can be active globally but
  needs effectors that colocalize with integrins
• Cell generates mechanical forces which causes the
  arrangement of stress fibers and focal adhesions
  which allow for the local activation of
  lamellipodia extension by Rho Rac and CDC42
• Mechanical forces are important for migration
  direction

Parker K.K. et al. 2002, FASEB J v16 p1196
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Other Application

• Use microfluidics to create stable and easily
  producible concentration gradients
• Cell-cell interactions in cocultures
• Cell shape can control stem cell differentiation
• High through put assays
• Optimize culture conditions
• Yield information about cell fate decisions
• Microfluidics and layer approaches to develop 3D
  environments for tissue engineering

Khademhosseini, A. Langer R, Borenstein J.
Vacanti, J.P. 2006 PNAS v.103 p.2480
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• Thus, the continued merger of engineering,
  medicine, materials, and biological sciences as
  mediated by microscale approaches in tissue
  engineering and biology will enhance our ability
  to create in vivo like physiological models that
  can be used for fabricating tissues or for
  understanding fundamental biology

Khademhosseini, A. Langer R, Borenstein J.
Vacanti, J.P. 2006 PNAS v.103 p.2480
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Conclusion and Outlook

• Softlithography can be carried out conveniently,
  rapidly, and inexpensively.
• Pattern delicate biological matter and is
  applicable to wide range of materials
• Can be applied to many biological aspects such as
  
• Immunoassay
• Tissue Engineering
• Biosensors

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References

• Delamarche et al, Advanced Materials. 2005
• Whitesides et al, Annu. Rev. Mater. Sci 1998
• Yap et al, Biosensors and Bioelectronics. 2007
• Bernard et al, Advanced Materials, 2000
• Mrksich et al, Annu. Rev. Biophys. Biomol.
  Struct. 1996
• Takayama et al. 1999. Proc. Natl. Acad. Sci. USA