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Published byConor Berwick Modified over 9 years ago
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Group Meeting November 26 th, 2012 Derek Hernandez
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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
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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
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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
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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
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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
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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)
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Effect of scan speed
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Future work Focus on limited power range (0-70 mW) Test the effects of: – BP-biotin concentration – BSA structure density
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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
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Effect of immobilization on surface topography Average roughness of BSA structure is ~ 100 nm
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Laser-induced shrinking Trying to quantify modulus changes
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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
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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
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Video 4
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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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