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Group Meeting. November 26 th , 2012 Derek Hernandez. Motivation. Method to control topography and chemistry in 3D Derive a better understanding of how these cues can be used to improve migration and alignment in 3D. Chemical Matrix composition Growth factors. Cell behavior Migration

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Group meeting

Group Meeting

November 26th, 2012

Derek Hernandez


Motivation

Motivation

  • Method to control topography and chemistry in 3D

  • Derive a better understanding of how these cues can be used to improve migration and alignment in 3D

  • Chemical

  • Matrix composition

  • Growth factors

  • Cell behavior

  • Migration

  • Adhesion

  • Differentiation

  • Proliferation

  • Contact

  • Matrix stiffness

  • Topography

  • Compliance

  • Cellular

  • Junctions

  • Paracrine signals

Lust, JR. University of Rochester, Institute of Optics. Scale bar = 2 µm


Project goals

Project goals


Current projects

Current projects


Current projects1

Current projects


Protocol to immobilize cues on protein structures

Protocol to immobilize cues on protein structures

  • 1) Fabricate protein structure

  • Concentrated protein solution

  • Photosensitizer

  • High laser intensity

  • 2) Immobilize BP-biotin

  • 2 mg/mL BP-biotin solution

  • Reduced laser intensity

Remove fabrication solution

Protein structure

Benzophenone-biotin

Neutravidin

Biotinylated peptide with PEG linker

3) Bind peptide using neutravidin-biotin chemistry

Remove BP-biotin solution


Effect of laser power

Effect of laser power

Functionalization Scans

2 4 6

Scan conditions

2 mg/mL BP-Biotin 10% DMSO

40 mW, 40X objective

0.1 Hz (~30 µm/s)


Effect of scan speed

Effect of scan speed


Future work

Future work

  • Focus on limited power range (0-70 mW)

  • Test the effects of:

    • BP-biotin concentration

    • BSA structure density


Current projects2

Current projects


Effect of immobilization on surface topography

Effect of immobilization on surface topography

  • Average roughness of BSA structure is ~100 nm


Laser induced shrinking

Laser-induced shrinking

Trying to quantify modulus changes


Current projects3

Current projects


Improving cell interaction with rgd peptide immobilization

Improving cell interaction with RGD peptide immobilization

Cells adhere strongly to and flatten on RGD-functionalized BSA structures

Cells have negative adhesion preferences for unmodified BSA structures


Video 4

Video 4


Conclusion

Conclusion

  • Cell interaction with structure confined mostly to RGD-functionalized regions

    Future Work:

  • Establish a quantifiable metric for cell interaction

  • Use UV excitation to determine target RGD concentration range

  • Use professionally manufactured biotin-RGD-FITC


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