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3-D Cell Encapsulation in Hydrogels using 96-Well Plates


This protocol describes how to encapsulate cells in HyStem®-C hydrogels in a 96-well plate format. This protocol can easily be adapted for use with HyStem®-HP and HyStem® hydrogel kits.


Required Materials
  • HyStem-C, HyStem-HP, or HyStem hydrogel kit
  • Sterile 96-well plate


  • 1. Prepare HyStem-C Hydrogel Kit components under aseptic conditions as directed by the instructions.
  • 2. Prepare cells for use in 3-D cell culture, as per standard procedures.
    Seeding density varies with cell type, but a typical range is 5,000 to 20,000 cells per insert.
  • 3. Prepare a 96-well plate by removing it from its sterile packaging.
  • 4. Mix equal volumes of Glycosil® and Gelin-S® (Typically 1.0 mL of each for small vials and 5.0 mL each for large vials).

    Leftover solutions can be frozen at -20° C under a nitrogen or argon head and are viable for ~2 weeks. However, gel property can still change due to slow autocrosslinking, we recommend always using freshly reconstituted material when possible.
  • 5. Add cells to Glycosil + Gelin-S such that the proper cell density will be reached in the  total solution volume, typically 13.5 mL (5.0 mL Glycosil + 5.0 mL Gelin-S + 2.5 mL Extralink® + 1.0 mL cells) or 2.7 mL (1.0 mL Glycosil + 1.0 mL Gelin-S + 0.5 mL Extralink® + 0.2 mL cells). Pipette up and down to mix. More or less cell volume will respectively prolong or shorten gelation.

    Note: Media can impact gelation properties, so where possible it is advised to wash your cell pellet with PBS or dilute media.
  • 6. When you are ready to pour the hydrogels, add Extralink to Glycosil + Gelin-S with cells.
    Extralink volume should be one-fourth the volume of the Glycosil + Gelin-S mixture (typically 2.5 mL or 1.0 mL). Once the Extralink is added, you have < 20 minutes before the hydrogel forms.

    Note: The gelation time is very dependent upon the pH of the HyStem-C solution with the cells. The higher the pH, the faster the gelation time. Different media will have different effects on the final pH and gelation time.
  • 7. Quickly pipette 100 μL of HyStem-C into each insert.

    Do not add media at this point, since this will dilute the hydrogel and prevent it from gelling.
  • 8. Place the plates in the 37° C incubator with 5% CO2.
    Allow HyStem-C to gel for one hour.
  • 9. Remove the plates from incubator.
    Verify that the hydrogel is solid. If so, add 100 μL of media to each well.
  • 10. Place in the 37° C incubator with 5% CO2.


Changing Media

  • Carefully aspirate off the media. The hydrogel can easily be removed by the vacuum as well, so this must be done gently and carefully.
  • Pipette 100 μL media into each well. Try to avoid disrupting the gel.
  • Return the plate to the 37°C incubator with 5% CO2.

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