HyStem-C™ provide an excellent starting point for optimizing the matrix for stem cell culture.since its composition can be easily tailored to find suitable matrix compositions. Unlike an animal-derived extracellular matrix (ECM), HyStem-C™ 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). HyStem-C™ hydrogels can be customized by adding ECM proteins and by varying the hydrogel compliance to match the stiffness of native tissues.

Gelation

Reconstituted HyStem-C™ 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 HyStem-C™ Hydrogel Kits comes in one size:

• 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 HyStem™, 3x 1.0 ml of Gelin-S™, 3x 0.5 ml of Extralink™

Flexibility

HyStem-C™ 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 Stem Cell Culture

In addition to stem cell culture on top of the hydrogel, HyStem-C™ provides the basic scaffold for 3-D stem cell growth. Stem 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 HyStem-C™ hydrogel. These proteins are easily incorporated noncovalently into the hydrogel prior to gel formation. We recommend HyStem™ for incorporating ECMs.

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

A variety of stem cells (human embryonic, human mesenchymal stem cells⁴) and progenitor cells (neural progenitor, hepatic progenitor) have been cultured in HyStem-C.

Choosing a HyStem™ Hydrogel Kit

The HyStem-C™ 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 HyStem-HP™ hydrogel kit is recommended. For in vivo experimentation, we recommend either the HyStem™ or HyStem-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. Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University, and Medical University of South Carolina.