Full Product Description
Extracel™ is recommended as a starting point for optimization of a cell’s microenvironment since it contains Gelin-S™. Gelin-S, or thiolated gelatin (denatured collagen), allows attachment of a wide variety of cell types and takes the guesswork out of the appropriate attachment factor to use.
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 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.
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:
- 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™
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
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 hydrogel2,3 or by trypsinization for cells grown on the surface.
Gelin-S™ provides basic cell-attachment sites for cell lines and primary cells2,3. 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™:
- human tracheal scar fibroblasts 5,6
- human and rat primary hepatocytes 1,7
- human dermal fibroblasts 8
- human mesenchymal stem cells 9
- MDCK cells 7,9
- L929 fibroblasts 10
- NIH 3T3 fibroblast 11
- Hep G2 and HepG2 C3A cells 1,7
Cancer-Cell Lines 12,13
- MDA-MB-468 (breast)
- MCF-7 (breast)
- MCF-10A (breast)
- SK-Br-3 (breast)
- OVCAR-3 (ovarian)
- SK-OV-3 (ovarian)
- HCT-116 (colon)
- Caco-2 (colon)
- MiaPaCa-2 (pancreatic)
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.
- 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).
- 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).
- 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).
- 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).
- 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).
- 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).
- Unpublished data from G. D. Prestwich, et al, University of Utah.
- 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).
- Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University, and Medical University of South Carolina.
- Y. Liu, X. Z. Shu, G. D. Prestwich, “Biocompatibility and Stability of Disulfide-Crosslinked Hyaluronan Films,” Biomaterials, 26, 4737-4746 (2005).
- X. Z. Shu, Y. Liu, Y. Luo, M. C. Roberts, and G. D. Prestwich, “Disulfide-crosslinked Hyaluronan hydrogels,” Biomacromolecules, 3, 1304-1311 (2002).
- Unpublished data from C. Scaife, et al, University of Utah.
- 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).