Extracel-HP™

Extracel-HP™ Hydrogel Kit

Extracel-HP™ hydrogels facilitate the use of growth factors (GFs) in 3-D cell culture and in vivo angiogenesis studies within an easily customizable, heparin-containing environment. Extracel-HP™ is a synthetic extracellular matrix (ECM) that can be injected and crosslinked in situ. Unlike an animal-derived extracellular matrix (ECM), Extracel-HP™ is fully chemically defined and nonimmunogenic. The Extracel-HP™ Hydrogel Kit contains Heprasil™ (a combination of thiol-modified hyaluronan, HA, and thiol-modified heparin), Gelin-S™ (thiol-modified gelatin), and Extralink™ (a thiol-reactive crosslinker, polyethylene glycol diacrylate, PEGDA).

The immobilized heparin in the hydrogel mimics the heparan sulfate proteoglycans normally present in the extracellular matrix (ECM). It also helps protect GFs from proteolysis and slows their release to attached cells1. This reduces the amount of GF required to achieve stimulation of cell growth or differentiation when compared to the use of free GF in media. All GFs tested to date (bFGF, VEGF, Ang-1, PDGF, TGFβ1, KGF) are released at different rates, but over a period of several weeks1,2,3.

Gelation

Reconstituted Extracel-HP™ components remain liquid at 15 to 37°C. The hydrogel is formed when Extralink is mixed with Heprasil™ and Gelin-S™. Gelation occurs about twenty minutes after all three components are mixed. No steps depend on low temperature or low pH. Diluting the components with phosphate-buffered saline (PBS) or cell-culture medium can increase gelation time.

Volume and Composition

Extracel-HP™ Hydrogel Kits come in two sizes:

  • 12.5 ml with the components in three vials (for large-volume applications)
    • 5.0 ml of Heprasil, 5.0 ml of Gelin-S™, 2.5 ml of Extralink
  • 7.5 ml with three sets of vials that make 2.5 ml each (for small-volume applications)
    • 3x 1.0 ml of Heprasil, 3x 1.0 ml of Gelin-S™, 3x 0.5 ml of Extralink

Flexibility

Extracel-HP™ ensures complete control over:

Applications

3-D Cell Growth

Extracel-HP™ provides the basic scaffold for 3-D cell growth. Cells can be encapsulated during crosslinking4, where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth5. Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel5,6 or by trypsinization for cells grown on the surface.

Gelin-S™ provides basic cell-attachment sites for cell lines and some primary cell types5,6. Several cell types depend on specific ECM components to grow and differentiate. To affect specific cell performance, other factors such as growth factors or ECM proteins may be added to the Extracel-HP™ hydrogel. ECM proteins are easily incorporated noncovalently into the hydrogel prior to gel formation as are growth factors. If the user plans to incorporate ECM proteins, we recommend using Extracel-HPG™, which contains more Heprasil™ relative to the Gelin-S™.

3-D Cell Growth Using GF-Supplemented Media

For cells cultured with GFs in the media, GFs may be removed from the media and added to the Extracel-HP™ hydrogel. The hydrogel is used to coat a culture flask and cells are cultured on top of the hydrogel using medium without GFs. Note, however, that the GFs are released at different rates. Therefore, we recommend an in vitro test to determine the proper concentrations for GF addition to the hydrogel. See growth-factor release for more information about specific GFs and their retention.

Angiogenesis

Growth factors (angiopoietin 1, Ang-1, and vascular endothelial growth factor, or VEGF) delivered in hydrogel films composed of Extracel-HP™ improve neovascularization and microvessel formation in vivo and allow formation of intact microvessel beds with well-defined borders7. The heparin helps regulate the in vivo GF release, which makes development of a functional microvessel network possible2,7,8.

Choosing an Extracel™ Hydrogel Kit

The Extracel-HP™ Hydrogel Kit is designed to make hydrogels with 50 wt% Heprasil™ and 50 wt% Gelin-S™. The Extracel-HPG™ Hydrogel Kit makes hydrogels with 70-99 wt% Heprasil™ and 1-30 wt% Gelin-S™. If no ECM proteins are added, then Gelin-S™ provides the only attachment sites for cells, making the Extracel-HP™ Hydrogel Kit the best choice. However, less Gelin-S™ is needed if ECM components will be added, or if fewer attachment sites are required; in these cases, we recommend the Extracel-HPG™ Hydrogel Kit. If no GFs will be used, then we recommend the Extracel™ or Extracel-LG™ hydrogel kits.

References

  1. S. Cai, Y. Liu, X. Z. Shu, G. D. Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor,” Biomaterials, 26, 6054-6067 (2005).
  2. D. B. Pike, S. Cai, K. R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, “Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF,” Biomaterials, 27, 5242–5251 (2006).
  3. Unpublished data from G. D. Prestwich, et al, University of Utah, and R. Peattie, et al, University of Oregon.
  4. G. D. Prestwich, Y. Liu, M. Serban, B. Yu, X. Z. Shu, and A. Scott, “3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery,” invited, Adv. Enz. Res., in press (2007).
  5. X. Z. Shu, S. Ahmad, Y. Liu, and G. D. Prestwich, “Synthesis and Evaluation of Injectable, In Situ Crosslinkable Synthetic Extracellular Matrices (sECMs) for Tissue Engineering,” J. Biomed Mater. Res. A, 79A(4), 901-912 (2006).
  6. X. Z. Shu, Y. Liu, F. Palumbo, G. D. Prestwich, “Disulfide-Crosslinked Hyaluronan-Gelatin Hydrogel Films: A Covalent Mimic of the Extracellular Matrix for In Vitro Cell Growth,” Biomaterials, 24, 3825-3834 (2003).
  7. C. M. Riley, P. W. Fuegy, M. A. Firpo, X. Z. Shu, G. D. Prestwich, R. A. Peattie, “Stimulation of in vivo angiogenesis using dual growth factor-loaded crosslinking glycosaminoglycan hydrogels,“ Biomaterials, 27, 5935-5943 (2006).
  8. R. A. Peattie, E. Rieke, E. Hewett, R. J. Fisher, X. Z. Shu, and G. D. Prestwich, “Dual growth-factor-induced angiogenesis in vivo using hyaluronan hydrogel implants,” Biomaterials, 27, 1868-1875 (2006).