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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

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Group Meeting

November 26th, 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

  • 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


Current projects


Current projects


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

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


Future work

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

  • Test the effects of:

    • BP-biotin concentration

    • BSA structure density


Current projects


Effect of immobilization on surface topography

  • Average roughness of BSA structure is ~100 nm


Laser-induced shrinking

Trying to quantify modulus changes


Current projects


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


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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