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Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials. Stephanie Seidlits, Jason Shear, Christine Schmidt University of Texas at Austin. ECM Components. 3D Architecture. Alberts, MBOC, 4th ed. Bochaton-Piallat, Ophtamol Vis Sci, 2000. Engineered in vivo-like Scaffolds.

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direct write of 3d biomolecule microstructures into hydrogel materials

Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials

Stephanie Seidlits, Jason Shear, Christine Schmidt

University of Texas at Austin

slide2
ECM Components

3D Architecture

Alberts, MBOC, 4th ed.

Bochaton-Piallat, Ophtamol Vis Sci, 2000

Engineered in vivo-like Scaffolds

Submicron Features

Spatial Control

Esch, J. Neurosci, 1999

Teixeira, JCS, 2003

scaffold design
Neuronal Cell Body

Extending Neurite

3D Hydrogel Support

Crosslinked Protein Structures

Scaffold design
hyaluronic acid
Hyaluronic acid
  • Nonimmunogenic
  • Angiogenesis and --wound healing
  • Enzymatically biodegradable
  • Photocrosslinkable --------------hydrogels (Leach, 2003, Biotech Bioeng)
  • Not cell adhesive

GMHA Reaction

ring opening product

hyaluronic acid

glycidylmethacrylate

transesterification product

Adapted from Derouet, 2002, Eur Poly J

multiphoton excitation
www.fsu.edu/microscopyprimerMultiphoton excitation
  • Advantages of MPE photocrosslinking:
  • Inherently 3D
  • Subcellular resolution
  • Complex geometries --possible
  • Heating effects minor ----and localized
  • Can fabricate---------------structures of natural -----materials

www.cellscience.biorad.com

mpe crosslinking in 3d
Crosslink protein structures directly into saturated GMHA hydrogels using multiphoton excitation

Make GMHA hydrogels and soak in photosensitizer and protein solution

Wash to remove unreacted solution

MPE crosslinking in 3D
3d protein structures in ha hydrogels
Z=40 µm

Z=20 µm

50 µm

50 µm

Z=0 µm

50 µm

50 µm

50 µm

50 µm

3D protein structures in HA hydrogels

Z=40 µm

Z=20 µm

Z=0 µm

avidin biotin functionalization
F

F

F

F

+

+

+

+

+

+

+

B

B

avidin

avidin

bsa

Avidin-biotin functionalization

Strategy 1:

Strategy 2:

strategy 1 avidin structures
Strategy 1: Avidin structures

Avidin

BSA

150 µm

150 µm

~30 ms exposure time

strategy 2 bsa structures modified with avidin
50 µm

50 µm

Strategy 2: BSA structures modified with avidin

BSA + Avidin-fluorescein

BSA + unlabeled avidin

~3 ms exposure time

mpe crosslinking within hydrogels provides
50 µmMPE crosslinking within hydrogels provides:
  • Flexibility to incorporate multiple bioactive molecules in arbitrary patterns
  • Inherent 3D geometry and biomolecule presentation
  • Submicron sized features
acknowledgements
Acknowledgements

Principle Investigators:

  • Dr. Christine Schmidt
  • Dr. Jason Shear

Undergraduates:

  • Rebecca Rosenberger
  • Kat McCarty
  • Jill Heisler

Lab Members:

  • Curt Deister
  • Bryan Kaehr
  • Rex Nielson
  • Rest of Schmidt and Shear Lab Members

Funding:

NSF-IGERT

NSF BES-0500969 and BES-021744

Welch Foundation

slide17
IGERT: Cellular and Molecular Imaging for

Diagnostics and Therapeutics

The University of Texas at Austin

Novel Contrast Agent Development

Novel Imaging and Spectroscopy

System Development

Diagnostic and Vital Imaging

Spectroscopy

Imaging to Monitor Therapeutics

Integrative Graduate Education & Research Training Program

Nicholas Peppas, Sc.D., Director

Jennifer Brodbelt, Ph.D., Co-Principal InvestigatorChristine Schmidt, Ph.D., Co-Principal Investigator

http://www.bme.utexas.edu/igert/

slide18
A)

B)

Figure 4. 3D MPE crosslinked BSA structures within hydrogels. A) Optical transmission images of 200 mg/mL BSA (2% BSA-FITC) photocrosslinked in a 2 wt% GMHA hydrogel with 6 mM FAD using a pulsed TiS laser at 740 nm. Each image was taken in the same xy plane and a different z plane (the first image is focused at the top of the pyramid and the last at the bottom). C) Confocal microscope image of 200 mg/mL BSA (2% BSA-FITC) crosslinked using 6 mM FAD within a 100 mg/mL PEGDA hydrogel. Images are from different planes spanning a total depth of 80 m. Note: concentrations given are those in the uncrosslinked solution. Scale bars = 20 µm.

crosslinking with fad
Crosslinking with FAD
  • Two Mechanisms:
    • Type I: triplet state abstract hydrogen to create radicals
    • Type II: creates singlet oxygen

(Spikes 1999 Photochem Photobio)

  • Photocrosslinkable amino acids: Tyr, Trp, His, Cys…
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