Human dermal fibroblasts grown on the surface of an Extracel™ hydrogel. The cells were fixed and stained by DAPI (nuclear, blue), Alexa 488-conjugated phalloidin (F-actin, green), and primary antibody directed against vinculin and secondary antibody (goat anti-mouse IgG1, Alexa 568) (vinculin, red).

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Protocols & MSDSs

Extracel™ Hydrogel Kit

Extracel™ Hydrogel Kits have proven optimal for culturing primary cells and cell lines in 3-D. Extracel™ hydrogels provide maximum versatility for research. Unlike an animal-derived extracellular matrix (ECM), Extracel™ is fully chemically defined. The hydrogels are based on three biocompatible components: thiol-modified hyaluronan (a major constituent of native ECM), thiol-modified gelatin (denatured collagen), and a thiol-reactive crosslinker, polyethylene glycol diacrylate (PEGDA). Extracel™ hydrogels can be customized by adding ECM proteins and by varying the hydrogel compliance to match the stiffness of native tissues.

Gelation

Reconstituted Extracel™ components remain liquid at 15 to 37°C. The hydrogel is formed when the crosslinking agent, Extralink™ (PEGDA) is added to a mixture of Glycosil™ (thiol-modified hyaluronan) and Gelin-S™ (thiol-modified gelatin). Gelation occurs in about twenty minutes after all three components are mixed. No steps depend on low temperatures or low pH. Diluting the components with phosphate-buffered saline (PBS) or cell-culture medium can increase the gelation time.

Volume and Composition

The Extracel™ Hydrogel Kits come in three sizes:

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

Flexibility

Extracel™ allows customization of experiments:

• dimensionality (3-D encapsulation or 2-D plating on top of hydrogel)
• cell-growth format (96- to 6-well plates and/or tissue-culture inserts)
• amount and type of ECM protein incorporated
• hydrogel stiffness

Applications

3-D Cell Culture

Extracel™ provides the basic scaffold for 3-D cell growth. Cells can be encapsulated during crosslinking¹, where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth². Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel^2,3 or by trypsinization for cells grown on the surface.

Gelin-S™ provides basic cell-attachment sites for cell lines and primary cells^2,3. Several cell types depend on specific ECM components, such as the natural ECM proteins laminin, collagen, fibronectin, and vitronectin, to grow and differentiate–all of which may be added to the Extracel™ hydrogel. These proteins are easily incorporated noncovalently into the hydrogel prior to gel formation. We recommend HyStem™ for incorporating ECMs.

The compliance of the Extracel™ hydrogel can be varied either by changing the amount of Extralink™ used for crosslinking⁴ or by diluting the Glycosil and Gelin-S™ solutions using PBS or cell-culture medium.

The following cells have been cultured in Extracel™:

Choosing an Extracel™ Hydrogel Kit

The Extracel™ Hydrogel Kit is designed to make hydrogels with 50 wt% Glycosil and 50 wt% Gelin-S™ and is optimal for researchers who need a large number of generalized cell attachment signals for their cultures. The HyStem Hydrogel Kit is appropriate for researchers who will either add ECM proteins or who require a minimal number of cell attachment sites. If growth factors will be used, the Extracel-HP™ hydrogel kit is recommended. For in vivo experimentation, we recommend the Extracel-X™ or Extracel-HP™ Hydrogel Kits.

References

1. 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).
2. 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).
3. 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).
4. K. Ghosh, Z. Pan, E. Guan, S. Ge, Y. Liu, T. Nakamura, X. Ren, M. Rafailovich, R. Clark, “Cell Adaptation to a Physiologically Relevant ECM Mimic with Different Viscoelastic Properties,” Biomaterials 28, 671-679 (2007).
5. Y. Liu, Z. X. Shu, S. D. Gray, G. D. Prestwich, “Disulfide-Crosslinked Hyaluronan-Gelatin Sponge: Growth of Fibrous Tissue In Vivo,” J Biomed Mat Res, 68A, 142-149 (2004).
6. X. Z. Shu, Y. Liu, F. Palumbo, Y. Luo, G. D. Prestwich, “In Situ Crosslinkable Hyaluronan Hydrogels for Tissue Engineering,” Biomaterials, 25, 1339-1348 (2004).
7. Unpublished data from G. D. Prestwich, et al, University of Utah.
8. G. D. Prestwich, X. Z. Shu, Y. Liu, S. Cai, J. F. Walsh, C.W. Hughes, K. R. Kirker, R. R. Orlandi, A. H. Park, S. L. Thibeault, M. E. Smith, “Injectable Synthetic Extracellular Matrices for Tissue Engineering and Repair,” Adv. Exp. Med. Biol., 585, 125-133 (2006).
9. Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University, and Medical University of South Carolina.
10. Y. Liu, X. Z. Shu, G. D. Prestwich, “Biocompatibility and Stability of Disulfide-Crosslinked Hyaluronan Films,” Biomaterials, 26, 4737-4746 (2005).
11. X. Z. Shu, Y. Liu, Y. Luo, M. C. Roberts, and G. D. Prestwich, “Disulfide-crosslinked Hyaluronan hydrogels,” Biomacromolecules, 3, 1304-1311 (2002).
12. Unpublished data from C. Scaife, et al, University of Utah.
13. Y. Liu, X. Z. Shu, and G. D. Prestwich, “Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Crosslinked Hyaluronan-Derived Hydrogels,” Tissue Engineering 13(5), 1091-1101(2007).