Group Meeting

November 26th, 2012Derek 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

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

Chemical• Matrix composition• Growth factors

Contact• Matrix stiffness• Topography• Compliance

Cell behavior• Migration• Adhesion• Differentiation• Proliferation

Cellular• Junctions• Paracrine signals

•Produce 3D immobilized, chemical gradients

•Evaluate the effect of gradients on cell migration•Cue,

concentration, slope

Chemical cues

•What feature sizes and

geometries promote cellalignment and migration?

•How does a cell respond to topographical changes? (Eric)

Topographical cues

Project goals

Current projects

•Further characterization of BP-biotin immobilization•Step size,

concentration, scan speed

•Cell interaction with RGD-functionalized BSA microstructures

Chemical cues

•Quantification of SC alignment on ridged, methacrylated gelatin hydrogels

•Evaluate changes in structure mechanical properties from laser-induced shrinking (Eric)

Topographical cues

Current projects

•Further characterization of BP-biotin immobilization•Concentration

, scan speed, scan power

•Cell interaction with RGD-functionalized BSA microstructures

Chemical cues

•Quantification of SC alignment on ridged, methacrylated gelatin hydrogels

•Evaluate changes in structure mechanical properties from laser-induced shrinking (Eric)

Topographical cues

Protocol to immobilize cues on protein structures

Benzophenone-biotin

Neutravidin

Biotinylated peptide with PEG linker

Protein structure

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

3) Bind peptide using neutravidin-biotin chemistryRemove BP-

biotin solution

Effect of laser power

30 40 50 60 70 80 90 100 110 120 1300

500

1000

1500

2000

2500

2 scans/plane 4 scans/plane 6 scans/plane

Laser Power (mW)

Fluo

resc

ence

inte

nsit

y

Functionalization Scans2 4 6Scan conditions

2 mg/mL BP-Biotin 10% DMSO

40 mW, 40X objective0.1 Hz (~30 µm/s)

Effect of scan speed

2 4 60

200

400

600

800

1000

1200

40 mW, 0.05 Hz 40 mW, 0.1 Hz 40 mW, 0.2 Hz

Functionalization Scans

Fluo

resc

ence

Inte

nsit

y

Future work

• Focus on limited power range (0-70 mW)• Test the effects of:– BP-biotin concentration– BSA structure density

Current projects

•Further characterization of BP-biotin immobilization•Concentration

, scan speed, scan power

•Cell interaction with RGD-functionalized BSA microstructures

Chemical cues

•Quantification of SC alignment on ridged, methacrylated gelatin hydrogels

•Evaluate changes in structure mechanical properties from laser-induced shrinking (Eric)

Topographical cues

Effect of immobilization on surface topography

• Average roughness of BSA structure is ~100 nm

Laser-induced shrinking

Trying to quantify modulus changes

Current projects

•Further characterization of BP-biotin immobilization•Concentration

, scan speed, scan power

•Cell interaction with RGD-functionalized BSA microstructures

Chemical cues

•Quantification of SC alignment on ridged, methacrylated gelatin hydrogels

•Evaluate changes in structure mechanical properties from laser-induced shrinking (Eric)

Topographical cues

Improving cell interaction with RGD peptide immobilization

Cells have negative adhesion preferences for unmodified

BSA structures

Cells adhere strongly to and flatten on RGD-functionalized

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