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 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 cell alignment 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 chemistry Remove BP- biotin solution

Effect of laser power Functionalization Scans 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 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