HyStem-HP is ideal for stem cell applications where slowly released growth factors are crucial in recreating a stem cell niche. HyStem-HP™ hydrogels contains thiol-modified heparin which allows the slow release of growth factors (GFs) within an easily customizable, environment. HyStem-HP™ is a synthetic extracellular matrix (ECM) that can be injected and crosslinked in situ. Unlike an animal-derived extracellular matrix (ECM), HyStem-HP™ is fully chemically defined and nonimmunogenic. The HyStem-HP™ Hydrogel Kit contains HyStem-HP™ (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 weeks^1,2,3.

Gelation

Reconstituted HyStem-HP™ components remain liquid at 15 to 37°C. The hydrogel is formed when Extralink is mixed with HyStem-HP™ 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

HyStem-HP™ Hydrogel Kits comes in one size:

• 7.5 ml with three sets of vials that make 2.5 ml each (for small-volume applications)
• 3x 1.0 ml of HyStem-HP, 3x 1.0 ml of Gelin-S™, 3x 0.5 ml of Extralink

Flexibility

• HyStem-HP™ ensures complete control over:
• amount and type of GF incorporated
• cell encapsulation or plating on top of hydrogel
• cell-growth format (96- to 6-well plates and/or tissue-culture inserts)
• amount and type of ECM proteins incorporated
• hydrogel stiffness

Applications

3-D Stem Cell Growth

In addition to stem cell culture on top of the hydrogel, HyStem-HP™ provides the basic scaffold for 3-D stem 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 hydrogel^5,6 or by trypsinization for cells grown on the surface.

Gelin-S™ provides basic cell-attachment sites for stem cell lines. Several stem 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 HyStem-HP™ hydrogel. ECM proteins are easily incorporated noncovalently into the hydrogel prior to gel formation as are growth factors..

3-D Stem Cell Growth Using GF-Supplemented Media

For stem cells cultured with GFs in the media, GFs may be removed from the media and added to the HyStem-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.

Choosing a HyStem™ Hydrogel Kit

The HyStem HP™ Hydrogel Kit is designed to make hydrogels with 50 wt% HyStem-HP™ and 50 wt% Gelin-S™. If no GFs will be used, then we recommend the 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. 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).