The HyStem Hydrogel Kits are optimal for culturing stem cells whose natural environment is rich in hyaluronic acid (HA). It is xeno-free since its two components are thiol-modified hyaluronan (HyStem™) and a thiol-reactive crosslinker (polyethylene glycol diacrylate, Extralink™)¹. HyStem can be customized by adding extracellular matrix (ECM) proteins² or cell attachment peptides³ into the hydrogel to provide attachment site and/or differentiation signals. They can also be varied by changing the hydrogel rigidity⁴ to match that of the native cell environment.

Animal-free HyStem

The hyaluronic acid used to produce HyStem™ is made by a proprietary bacterial-fermentation process using bacillus subtilis. It is 100% free of animal-derived raw materials and no animal-derived ingredients are used in its production. Extralink (polyethylene glycol diacrylate) is made by adding acrylate groups to both ends of a polyethylene glycol (PEG) polymer. PEG is derived from petroleum and inorganic sources and contains no animal source materials.

Gelation

The reconstituted HyStem components are liquids at 15-37 ˚C. The hydrogel is formed when the crosslinking agent, Extralink™, is added to the HyStem liquid. Once the two components are mixed, gelation occurs in less than 20 minutes. There are no low temperature or low pH steps. The gelation time can be increased by diluting the components with phosphate buffered saline (PBS) or cell culture media.

Volume and composition

The HyStem Hydrogel Kit comes in one size:

• 7.5 ml total hydrogel with three sets of vials that make 2.5 ml each
  • 6x 1.0 ml of HyStem, 3x 0.5 ml vials of Extralink

Flexibility

HyStem gives the researcher complete control over:

• hydrogel rigidity
• amount and type of ECM protein incorporated
• cell attachment peptide incorporation
• cell encapsulation or plating on top of hydrogel
• cell growth format (96- to 6-well plates and/or tissue culture inserts)

Applications

Stem cell expansion

HyStem provides a basic, viscoelastic matrix for stem cell growth. This matrix can be manipulated by the user by changing its:

• composition^2,3
• rigidity⁴

The rigidity is changed by either diluting the hydrogel components or by adjusting the amount of crosslinker used to form the hydrogel⁴. The rigidity of the matrix in which the cells are grown can influence differentiation, making it an important experimental parameter⁵. Standard preparation of HyStem hydrogels results in a rigidity of ~300 Pa⁶.

Since HyStem hydrogels are composed of only HyStem and Extralink, they do not support cell attachment¹. Glycosan BioSystems’ other hydrogel kits all contain Gelin-S (thiol-modified denatured collagen), which promotes cell attachment but in a non-specific fashion⁷. Since the collagen in Gelin-S is denatured and animal sourced (bovine derived), it has limited utility for stem cell cultivation where attachment sites can also signal cells to differentiate and where animal sourcing is a concern. The lack of attachment sites is less of a concern for stem cells that are encapsulated^8,9. However, if the researcher plans to plate cells on the surface of the hydrogels, then additional components need to be incorporated into the hydrogel in order to promote cell attachment. ECM proteins can be non-covalently added to HyStem prior to crosslinking or cell attachment peptides can be covalently linked into the matrix. Because the ECM proteins and peptides can also provide differentiation signals as well as attachment signals, the researcher must determine the appropriate type to use.

Encapsulated cells are recovered from the HyStem hydrogel by enzyme digestion using hyaluronidase. If cells are plated on the surface, then they are passaged using either trypsin, dispase, collagenase or other gentler detachment solutions.

The following stem cells have been cultured in HyStem:

• human embryonic stem cells (H9s)^10,11
• umbilical cord blood CD34+ stem cells¹²
• hepatic stem cells¹³
• hepatic progenitor cells¹³
• adipose derived stem cells¹⁴
• mesenchymal stem cells^15,16

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. 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).X.Z. Shu, Y. Liu, F. Palumbo, Y, Luo, and G.D. Prestwich,
2. T.D. Mehra, K. Ghosh, X.Z. Shu, G.D. Prestwich, and R.A.F. Clark, “Molecular Stenting with a Crosslinked Hyaluronan Derivative Inhibits Collagen Gel Contraction,” J. Invest. Dermatol., 126, 2202-2209 (2006). *
3. X.Z. Shu, K. Ghosh, Y. Liu, F.S. Palumbo, Y. Luo, R.A. Clark, and G.D. Prestwich, “Attachment and Spreading of Fibroblasts on an RGD Peptide-Modified Injectable Hyaluronan Hydrogel,” J. Biomed. Mat. Res. 68A, 365-375 (2004).
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. A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, ”Matrix elasticity directs stem cell lineage specification,” Cell, 126(4): 677-89 (2006)
6. J. Vanderhooft and G.D. Prestwich, manuscript in preparation
7. 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).
8. W.S. Turner, E. Scmelzer, R. McClelland, E. Wauthier, W. Chen, L.M. Reid, “Human hepatoblast phenotype maintained by hyaluronan hydrogels, “ J Biomed Mater Red B Appl Biomater 82(1): 156-68 (2007). [Note: this material was not provided by Glycosan, but is a precursor technology].
9. S. Gerecht, J.A. Burdick, L.S. Ferreira, S.A. Twonsend, R. Langer, G. Vunjak-Novakovic, “Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells”, PNAs 104(27):11298-303 (2007). [Note: This is a not Glycosan material.]
10. Unpublished data from Xuejun Wen’s lab, Clemson University and Medical University of South Carolina.
11. Unpublished data from Liisa Kuhn’s lab, University of Connecticut
12. Unpublished date from Linda Kelley’s lab, University of Utah, and Glycosan
13. W.S. Turner, L.M Reid, University of North Carolina, manuscript submitted
14. Flynn, G.D. Prestwich, J.L Semple, and K.A. Woodhouse, “Adipose Tissue Engineering with Naturally-derived Scaffolds and Adipose-derived Stem Cells,” Biomaterials, 28, 3834‑3842 (2007)
15. Y. Liu, X.Z. Shu, and G.D. Prestwich, “Osteochondral Defect Repair with Autologous Bone Marrow-Derived Mesenchymal Stem Cells in an Injectable, in situ Crosslinked Synthetic Extracellular Matrix,” Tissue Eng., 12, 3405-3416 (2006).
16. Unpublished data from Glycosan