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