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Product FAQs

HyStem® Hydrogels

ESI BIO Product FAQs


What size molecules can diffuse through a HyStem hydrogel?
Globular particles less than 75 kDa should be able to freely diffuse through a HyStem hydrogel.
What is the pH of the reconstituted HyStem components?
When reconstituted using DG water, the pH of each HyStem component will be approximately 7.6.


What is the shelf-life of HyStem® hydrogel components?
The shelf-life of HyStem hydrogel components is one year from the date of receipt, if stored properly. It is important to be aware of the expiry date for each component, and to use the reagents within the shelf-life timeframe for proper structure and function of the hydrogel.
Is DG Water required to reconstitute Glycosil®, Gelin-S®, and Extralink®?
No. Any sterile, deionized, degassed water can be substituted for reconstitution. However, in order to ensure accurate and predictable dissolution and gelation times, DG Water is highly recommended, as it is degassed, blanketed in argon, and has undergone validation testing with each HyStem component.
Are any cellular attachment sites included in the HyStem® hydrogel?
Gelin-S provides cellular attachment sites when incorporated in the hydrogel. Gelin-S is thiol-modified, denatured collagen I, derived from either bovine or porcine sources. Gelin-S is included in all HyStem-C, HyStem-HP, and HyStem Hydrogel UV QuickSet Kits.
How does Gelin-S® link with the hydrogel?
Gelin-S has been thiol-modified in the same manner as the hyaluronan in Glycosil (or Heprasil), so that it covalently crosslinks with the Extralink in the HyStem hydrogels, and with UVlink in the HyStem Hydrogel UV QuickSet Kit.
Can other cell attachment peptides be used in place of Gelin-S®?
Yes. Peptides that contain a cysteine residue can be used. The cysteine residue must be present for the peptide to be covalently bonded to the hydrogel substrate.
Can extracellular membrane (ECM) proteins be used in place of Gelin-S®?
Yes. ECM proteins, such as laminin, collagen, fibronectin, or vitronectin can be non-covalently incorporated into the hydrogel prior to crosslinking. The appropriate ECM or combination to use will depend upon the cell type and experimental needs. For more information, see the ECM Protein Incorporation Protocol
When should an extracellular membrane (ECM) protein be incorporated into the hydrogel in place of Gelin-S®?
Progenitor cells and some stem cells often require ECM proteins incorporated into the hydrogel to facilitate cell attachment. If the native extracellular matrix for the cell type is known, then adding an appropriate ECM protein may provide enhanced cellular attachment.


What is the difference between a HyStem® hydrogel and a HyStem® sponge?
HyStem hydrogels and sponges differ in hydration and homogeneity. HyStem sponges are typically polymerized hydrogels that are subsequently freeze-dried. The resulting sponge is a fibrous, mesh network with pores and niches that enable cells to infiltrate and adhere. A true HyStem hydrogel is an encapsulating liquid that polymerizes around suspended cells in culture.
What types of mechanical loads can HyStem® hydrogels withstand?
HyStem hydrogels can display compliance to stresses between a few hundred to a few thousand pascal (Pa), depending on the form in which it is tested, the material used, and the crosslinking density of the hydrogel. HyStem sponges that have been lyophilized from HyStem hydrogels typically display higher stiffness than the hydrogel form. HyStem hydrogels can be mixed with other matrices or biological components, such as demineralized bone matrix, to increase stiffness of the construct.
Do HyStem® hydrogels change in compliance to stress over time if they are kept in cell culture medium?
No. The compliance of the hydrogels is set by the amount of Extralink crosslinker added, the concentration of Glycosil (or Heprasil) and Gelin-S used, and the ratio of Glycosil (or Heprasil) to Gelin-S. Once this chemical structure of the hydrogel is fixed, it is not altered by prolonged exposure to cell culture medium.
What type of material processing can be done with HyStem® hydrogels?
HyStem hydrogels are very versatile materials. They can be cast, electrospun, molded, injection molded, and freeze-dried into a variety of shapes and sizes. Use of heat in excess of 37˚C is not recommended, however, as clear temperature boundaries for the hydrogels have not yet been established.
Can HyStem® hydrogels be terminally sterilized?
HyStem sponges can be terminally sterilized by E-beam. HyStem hydrogels have not yet been validated for use with E-beam sterilization methods. HyStem hydrogels are not terminally sterilized by gamma irradiation.


How can the gelation time be varied when making a HyStem® hydrogel?
Gelation time is affected by multiple aspects of the gel’s composition.

One way to change the gelation time of a hydrogel is to vary the amount of crosslinker used. Gels with a lower amount of Extralink crosslinker will have a longer gelation time than those with a higher amount of crosslinker. Changing the amount of crosslinker will produce slight changes in gelation time.

Gelation time can be dramatically changed by varying the Glycosil (or Heprasil) and Gelin-S concentrations. Concentrated solutions of Glycosil (or Heprasil) and Gelin-S will create a solution with a much shorter gelation time. This can easily be done by reconstituting the components in a smaller volume of DG Water. Alternatively, diluting these components in larger volumes of DG Water will dramatically increase the total time to form the hydrogel.

For more information about crosslinking and customizing the properties of a hydrogel, refer to the BenchTip Article Optimizing 3D Cell Culture with Customizable Hydrogels


Are the cells encapsulated within a HyStem® hydrogel easily visible under a microscope?
HyStem Hydrogels are virtually transparent and should not interfere with microscopy.
Are there differences in cell morphology when cells are embedded in a hydrogel?
Cells will often take on a different morphology when cultured in 3D environments as compared to their morphology in 2D culture. Some cells, such as the NIH 3Y3 fibroblast cell line, will have a more spherical morphology in 3D culture, as opposed to a flattened appearance when cultured in 2D.

It is important to remember that cells embedded within a hydrogel will not all be on the same plane when viewed under the microscope, as they will be evenly distributed throughout the three dimensional space of the gel. By adjusting the focal plane of the microscope, different vertical slices of the gel will be brought into focus, allowing for the visualization of cells throughout the entire vertical space of the gel.


Can HyStem® hydrogels be used for in vivo experiments in animal models?
HyStem has a well-documented history of use in animal models. Contact ESI BIO’s Technical Support Team for a list of animal models previously used within the recent HyStem publications. Remember, any experiment or protocol involving the use of live animal models must be approved by an internal review board or Institutional Animal Care and Use Committee (IACUC).
Are HyStem® hydrogels injectable?
Yes. HyStem hydrogels can be injected subcutaneously or orthotopically in vivo. When injected, HyStem hydrogels must be liquid. Crosslinking should then be set to occur between 5 minutes to 2+ hours to form the hydrogel. Gelation time will vary depending on the Glycosil (or Heprasil) and Gelin-S concentrations, as well as the amount of Extralink used. Remember, any experiment or protocol involving the use of live animal models must be approved by an internal review board or Institutional Animal Care and Use Committee (IACUC).
Does injection of HyStem® hydrogels trigger an immunological or inflammatory response in vivo?
HyStem hydrogels may generate mild inflammation as part of the body’s natural healing process in response to injury. HyStem hydrogels do not trigger immune response when used in vivo.
What is the mechanism of degredation of HyStem® in vivo?
HyStem is degraded in vivo by matrix metalloproteinases (collagenases) and hyaluronidases.


Can cells grown in 2D culture be detached from the surface of HyStem® hydrogels?
Yes. Methods of detachment will vary depending on cell type. Trypsin, Dispase, Collagen IV, and most common cell culture detatchment solutions can dissociate cells cultured on the surface of HyStem hydrogels. For more information, please view our Cell Recovery using Trypsin Protocol

PEGSSDA can also be used as an alternative crosslinker in place of Extralink, enabling non-enzymatic dissolution of the HyStem hydrogel to gently detach cells cultured on the hydrogel surface. For more information, please view our Hydrogel dissolultion using PEGSSDA Protocol
How can cells encapsulated within HyStem® hydrogels be recovered?
Enzymatic digestion with collagenase or hyaluronidase solutions can be used to efficiently recover cells encapsulated within a hydrogel. PEGSSDA can also be used as an alternative crosslinker in place of Extralink, enabling non-enzymatic dissolution of the HyStem hydrogel to recover cells embedded within the hydrogel.