HyStem

  • Hyaluronan hydrogel for stem cell culture
  • Lyophilized solid for reconstitution in DG water
  • Vials are blanketed in argon and under a slight vacuum Tested for bacteria, endotoxins, and lactate dehydrogenase-elevating virus (LDEV)
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H9 human embryonic stem cells plated on HyStem hydrogels containing CVFL and grown for 3 days.

H9 human embryonic stem cells plated on HyStem hydrogels containing CVFL and grown for 3 days.

General

HyStem® Hydrogel Kit

HyStem is recommended for applications requiring attachment factor optimization. Extracellular matrix proteins can be mixed into the hydrogel and incorporated non-covalently before gelation. Alternatively, attachment peptides having an N-terminal cysteine can also be covalently linked to the matrix3.

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 (Novozymes). 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.

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)

3D Cell Recovery Matrix

The HyStem Hydrogel is also available with a novel crosslinker which enables the gel to be broken down for easy encapsulated cell recovery. At checkout simply select PEGSSDA as your crosslinker.

Application

Stem cell expansion

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

  • composition2,3
  • 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 or porcine 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 encapsulated8,9. 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 cells15,16

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.

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)

3D Cell Recovery Matrix

The HyStem Hydrogel is also available with a novel crosslinker which enables the gel to be broken down for easy encapsulated cell recovery. At checkout simply select PEGSSDA as your crosslinker.

Composition

The HyStem Hydrogel Kit comes in two 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
  • Trial 2.5 ml total hydrogel with one sets of vials that make 2.5 ml
    • 2x 1.0 ml of HyStem
    • 0.5 ml of Extralink

HyStem Hydrogel with PEGSSDA

Extracel with PEGSSDA replaces the Extralink Vials with PEGSSDA Vials. Simply specify which crosslinker you would like in the shopping cart

Data Sheets

 

HyStem® Hydrogel Kit (7.5 mL) Product Data Sheet

Printable PDF Version

For research use only

Product Description

The HyStem Hydrogel Kit is composed of HyStem® (thiol-modified hyaluronic acid), Extralink® (PEGDA, polyethylene glycol diacrylate), and degassed, deionized water (DG Water). A solution of HyStem forms a transparent hydrogel when mixed with Extralink. HyStem and Extralink are packaged as lyophilized solids that are blanketed by argon and under a slight vacuum.

Cell Attachment

The HyStem hydrogel system provides a viscoelastic matrix of variable rigidity that supports the expansion of stem cells (human embryonic, CD34+, and hepatic progenitors have been tested to date). HyStem hydrogels do not support surface cell attachment. Cells must be either encapsulated within the hydrogel, or extracellular matrix (ECM) proteins or peptides may be mixed with the HyStem prior to crosslinking to provide attachment signals and allow for cells to be plated on the hydrogel surface. However, the type of ECM protein added depends upon the cell type and the desired outcome (expansion without differentiation or with differentiation). Protocols for ECM protein and peptide incorporation are given in our Protocol/Technical section.

Storage

HyStem
  • Store HyStem in original vials at -20 °C for up to one year.
  • Do not uncap the HyStem vials since they will crosslink in the presence of oxygen. Use a syringe to add DG Water and remove product from the vials.
Extralink
  • Store Extralink in the original vial at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20˚C for ~ one month.

Note: Glycosan recommends reconstituting each vial in its entirety.

Instructions for Use

HyStem and Extralink solutions are prepared by dissolving the lyophilized solids in DG Water. When reconstituted, they will be in phosphate buffered saline (PBS) buffer, pH ~7.4. HyStem vials contain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 10 mg of material and when reconstituted according to instructions will produce a 1% (w/v) solution.

HyStem hydrogels (6 x 1.25 mL = 7.5 mL) should be prepared in the following manner:

  1. Allow the HyStem, Extralink, and DG Water to come to room temperature.
  2. Under aseptic conditions add 1.0 mL of DG Water to the HyStem vial.
  3. Place the vial horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution. The solution will be clear and slightly viscous.
  4. Under aseptic conditions add 0.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  5. Encapsulate cells by resuspending the cell pellet in 1.0 mL of HyStem. Pipette back and forth to mix.
  6. To form the hydrogel, add Extralink to the HyStem in a 1:4 volume ratio (0.25 mL Extralink to 1.0 mL HyStem). The total hydrogel volume with cells is 1.25 mL.
  7. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  8. Gelation will occur within ~20 minutes.
  9. Freeze unused Extralink at -20 °C for use with another HyStem vial.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

HyStem® Hydrogel Trial Kit (2.5 mL) Product Data Sheet

Printable PDF Version

For research use only

Product Description

The HyStem Hydrogel Trial Kit is composed of HyStem® (thiol-modified hyaluronic acid), Extralink® (PEGDA, polyethylene glycol diacrylate), and degassed, deionized water (DG Water). A solution of HyStem forms a transparent hydrogel when mixed with Extralink. HyStem and Extralink are packaged as lyophilized solids that are blanketed by argon and under a slight vacuum.

Cell Attachment

The HyStem hydrogel system provides a viscoelastic matrix of variable rigidity that supports the expansion of stem cells (human embryonic, CD34+, and hepatic progenitors have been tested to date). HyStem hydrogels do not support surface cell attachment. Cells must be either encapsulated within the hydrogel, or extracellular matrix (ECM) proteins or peptides may be mixed with the HyStem prior to crosslinking to provide attachment signals and allow for cells to be plated on the hydrogel surface. However, the type of ECM protein added depends upon the cell type and the desired outcome (expansion without differentiation or with differentiation). Protocols for ECM protein and peptide incorporation are given in our Protocol/Technical section.

Storage

HyStem
  • Store HyStem in original vials at -20 °C for up to one year.
  • Do not uncap the HyStem vials since they will crosslink in the presence of oxygen. Use a syringe to add DG Water and remove product from the vials.
Extralink
  • Store Extralink in the original vial at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20˚C for ~ one month.

Instructions for Use

HyStem and Extralink solutions are prepared by dissolving the lyophilized solids in DG Water. When reconstituted, they will be in phosphate buffered saline (PBS) buffer, pH ~7.4.

HyStem hydrogels (2 x 1.25 mL = 2.5 mL) should be prepared in the following manner:

  1. Allow the HyStem, Extralink, and DG Water to come to room temperature.
  2. Under aseptic conditions add 1.0 mL of DG Water to the HyStem vial.
  3. Place the vial horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution. The solution will be clear and slightly viscous.
  4. Under aseptic conditions add 0.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  5. Encapsulate cells by resuspending the cell pellet in 1.0 mL of HyStem. Pipette back and forth to mix.
  6. To form the hydrogel, add Extralink to the HyStem in a 1:4 volume ratio (0.25 mL Extralink to 1.0 mL HyStem). The total hydrogel volume with cells is 1.25 mL.
  7. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  8. Gelation will occur within ~20 minutes.
  9. Freeze unused Extralink at -20 °C for use with another HyStem vial.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

HyStem™ Hydrogel Kit (7.5 mL) with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

Product Description

The HyStem Hydrogel Kit with PEGSSDA is composed of HyStem™ (thiol-modified hyaluronic acid), PEGSSDA™ (disulfide-containing polyethylene glycol diacrylate), and degassed, deionized water (DG Water). A solution of HyStem forms a transparent hydrogel when mixed with Extralink. HyStem and Extralink are packaged as lyophilized solids that are blanketed by argon and under a slight vacuum.

Cell Attachment

The HyStem hydrogel system provides a viscoelastic matrix of variable rigidity that supports the expansion of stem cells (human embryonic, CD34+, and hepatic progenitors have been tested to date). HyStem hydrogels do not support surface cell attachment. Cells must be either encapsulated within the hydrogel, or extracellular matrix (ECM) proteins or peptides may be mixed with the HyStem prior to crosslinking to provide attachment signals and allow for cells to be plated on the hydrogel surface. However, the type of ECM protein added depends upon the cell type and the desired outcome (expansion without differentiation or with differentiation). Protocols for ECM protein and peptide incorporation are given in our Protocol/Technical section.

Storage

HyStem
  • Store HyStem in original vials at -20 °C for up to one year.
  • Do not uncap the HyStem vials since they will crosslink in the presence of oxygen. Use a syringe to add DG Water and remove product from the vials.
PEGSSDA
  • Store PEGSSDA in the original vial at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20˚C for ~ one month.

Note: Glycosan recommends reconstituting each vial in its entirety.

Instructions for Use

HyStem and PEGSSDA solutions are prepared by dissolving the lyophilized solids in DG Water. When reconstituted, they will be in 1X phosphate buffered saline (PBS) buffer, pH ~7.4. HyStem vials contain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 10 mg of material and when reconstituted according to instructions will produce a 1% (w/v) solution. PEGSSDA vials contain 5 mg of materi-al and when reconstituted according to instructions will produce a 2% (w/v) solution.

HyStem hydrogels (6 x 1.25 mL = 7.5 mL) should be prepared in the following manner:

  1. Allow the HyStem, PEGSSDA, and DG Water to come to room temperature.
  2. Under aseptic conditions add 1.0 mL of DG Water to the HyStem vial.
  3. Place the vial horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution. The solution will be clear and slightly viscous.
  4. Under aseptic conditions and using a syringe add 0.25 mL of DG Water to each of the two PEGSSDA vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, mix equal volumes of HyStem. To mix, pipette back and forth to mix.
  6. Encapsulate cells by  resuspending the cell pellet in 1.0 mL of HyStem. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the HyStem mix in a 1:4 volume ratio (0.25 mL PEGSSDA to 1.0 mL HyStem) mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Hydrogel Dissolution:

Dissolution of gels with cells on top and encapsulated (gel volume of 0.6 mL) in a 24 well plate. Note: The following procedure was optimized particularly for the aforementioned gel geometry. Dissolution of gels with alternate geometry and/or volumes may require adjustments to the protocol. In general, for dissolution add at least twice the gel volume of N-Acetyl-L-Cysteine.

  1. Make up the appropriate amount of 40mM N-Acetyl-L-Cysteine in 1X PBS or media and pH to 7.4.
  2. Add 2 mL of 40mM N-Acetyl-Cysteine to the top of each gel and let sit at 37°C for 1 hour. Agitation by orbital shaking will help decrease dissolution time.
  3. Confirm dissolution by pipetting solution in well up and down, observing any remain-ing gel. If needed, allow another 30 minutes for complete gel dissolution.
  4. Remove liquid from well and place in conical centrifuge tube. If necessary, add PBS to a total of 5mL of liquid.
  5. Centrifuge at 1000 RPM for 5 minutes.
  6. Aspirate off liquid and process cells as desired.

Note: Each kit  component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

HyStem™ Trial Kit (2.5 mL) with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

Product Description

The HyStem Hydrogel Kit with PEGSSDA is composed of HyStem™ (thiol-modified hyaluronic acid), PEGSSDA™ (disulfide-containing polyethylene glycol diacrylate), and degassed, deionized water (DG Water). A solution of HyStem forms a transparent hydrogel when mixed with Extralink. HyStem and Extralink are packaged as lyophilized solids that are blanketed by argon and under a slight vacuum.

Cell Attachment

The HyStem hydrogel system provides a viscoelastic matrix of variable rigidity that supports the expansion of stem cells (human embryonic, CD34+, and hepatic progenitors have been tested to date). HyStem hydrogels do not support surface cell attachment. Cells must be either encapsulated within the hydrogel, or extracellular matrix (ECM) proteins or peptides may be mixed with the HyStem prior to crosslinking to provide attachment signals and allow for cells to be plated on the hydrogel surface. However, the type of ECM protein added depends upon the cell type and the desired outcome (expansion without differentiation or with differentiation). Protocols for ECM protein and peptide incorporation are given are given in our Protocol/Technical section.

Storage

HyStem
  • Store HyStem in original vials at -20 °C for up to one year.
  • Do not uncap the HyStem vials since they will crosslink in the presence of oxygen. Use a syringe to add DG Water and remove product from the vials.
PEGSSDA
  • Store PEGSSDA in the original vial at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20˚C for ~ one month.

Note: Glycosan recommends reconstituting each vial in its entirety.

Instructions for Use

HyStem and PEGSSDA solutions are prepared by dissolving the lyophilized solids in DG Water. When reconstituted, they will be in 1X phosphate buffered saline (PBS) buffer, pH ~7.4. HyStem vials contain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 10 mg of material and when reconstituted according to instructions will produce a 1% (w/v) solution. PEGSSDA vials contain 5 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. Note: Glycosan recommends reconstituting each vial in its entirety.

HyStem hydrogels (2 x 1.25 mL = 2.5 mL) should be prepared in the following manner:

  1. Allow the HyStem, PEGSSDA, and DG Water to come to room temperature.
  2. Under aseptic conditions add 1.0 mL of DG Water to the HyStem vial.
  3. Place the vial horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution. The solution will be clear and slightly viscous.
  4. Under aseptic conditions and using a syringe add 0.25 mL of DG Water to each of the two PEGSSDA vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, mix equal volumes of HyStem. To mix, pipette back and forth to mix.
  6. Encapsulate cells by  resuspending the cell pellet in 1.0 mL of HyStem. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the HyStem mix in a 1:4 volume ratio (0.25 mL PEGSSDA to 1.0 mL HyStem) mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Hydrogel Dissolution:

Dissolution of gels with cells on top and encapsulated (gel volume of 0.6 mL) in a 24 well plate. Note: The following procedure was optimized particularly for the aforementioned gel geometry. Dissolution of gels with alternate geometry and/or volumes may require adjustments to the protocol. In general, for dissolution add at least twice the gel volume of N-Acetyl-L-Cysteine.

  1. Make up the appropriate amount of 40mM N-Acetyl-L-Cysteine in 1X PBS or media and pH to 7.4.
  2. Add 2 mL of 40mM N-Acetyl-Cysteine to the top of each gel and let sit at 37°C for 1 hour. Agitation by orbital shaking will help decrease dissolution time.
  3. Confirm dissolution by pipetting solution in well up and down, observing any remain-ing gel. If needed, allow another 30 minutes for complete gel dissolution.
  4. Remove liquid from well and place in conical centrifuge tube. If necessary, add PBS to a total of 5mL of liquid.
  5. Centrifuge at 1000 RPM for 5 minutes.
  6. Aspirate off liquid and process cells as desired.

Note: Each kit  component has been manufactured under aseptic conditions and tested for bacteria and fungus.

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