Technical

HyStem™ Hydrogel Kit (7.5 mL) Product Data Sheet

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 on our website at www.glycosan.com/technical.

Storage

HyStem

  • Store HyStem in original vials at -20 °C for up to one year.
  • Solutions can be stored at -20 ˚C for ~ one month if they are frozen immediately after reconstitution in their original argon blanketed vials.
    • Freeze/thaw solutions a minimum number of times for best results.
    • Solutions should be thawed at 37 °C on a rocker and used immediately after thawing.
    • During thawing, solutions go through a “gel-like” phase before returning to their liquid state.
  • Do not uncap the HyStem vials since it will disulfide 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 ~7.6. HyStem hydrogels (7.5 mL) should be prepared in the following manner:

  1. Remove one HyStem vial and one Extralink vial from the -20 ˚C freezer. Warm them to 37 ˚C (~30 minutes).
  2. Remove the DG Water from the -20 ˚C freezer and thaw in a 37 ˚C water bath (~15 minutes).
  3. Under aseptic conditions and using a syringe with the exact amount of liquid, add 1.0 mL of DG Water to the HyStem vial.
  4. Place the vial horizontally on a rocker at 37 ˚C (for maximum mixing). It will take <30 minutes for the solids to fully dissolve. The solution will be clear and slightly viscous. Note: vigorous shaking will speed up dissolution.
  5. Under aseptic conditions and using a syringe with the exact amount of liquid, add 0.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  6. Encapsulate cells by adding 0.1 mL of cells to 1.0 mL of HyStem. Pipette back and forth to mix.
  7. 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 1.35 mL.
  8. Quickly pipette the HyStem + Extralink + cells into a well or tissue culture insert. Gelation will occur within ~10 minutes.
  9. Freeze unused Extralink at -20 °C for use with another HyStem vial.

Note: Each kit component has been tested and shows no evidence of bacteria growth.

For more information about the HyStem Hydrogel Kit go to www.glycosan.com/hystem_kit.