group meeting november 26 th, 2012 derek hernandez
TRANSCRIPT
![Page 1: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/1.jpg)
Group Meeting
November 26th, 2012Derek Hernandez
![Page 2: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/2.jpg)
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
![Page 3: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/3.jpg)
•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
![Page 4: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/4.jpg)
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
![Page 5: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/5.jpg)
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
![Page 6: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/6.jpg)
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
![Page 7: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/7.jpg)
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)
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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
![Page 9: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/9.jpg)
Future work
• Focus on limited power range (0-70 mW)• Test the effects of:– BP-biotin concentration– BSA structure density
![Page 10: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/10.jpg)
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
![Page 11: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/11.jpg)
Effect of immobilization on surface topography
• Average roughness of BSA structure is ~100 nm
![Page 12: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/12.jpg)
Laser-induced shrinking
Trying to quantify modulus changes
![Page 13: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/13.jpg)
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
![Page 14: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/14.jpg)
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
![Page 15: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/15.jpg)
Video 4
![Page 16: Group Meeting November 26 th, 2012 Derek Hernandez](https://reader031.vdocuments.site/reader031/viewer/2022032517/56649c815503460f94939ec2/html5/thumbnails/16.jpg)
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