Technical

3-D Cell Encapsulation in Extracel™ Hydrogels in 96-Well Plates

For research use only

Amount Material Supplier

  • 1 Extracel™ Hydrogel Kit
  • 1 96-well plate

This protocol describes how to encapsulate cells in Extracel™ hydrogels in a 96-well plate format. The protocol can easily be adapted for use with Extracel-LG™, Extracel-HP™, Extracel-HPG™, and Extracel-X™ Hydrogel Kits.

Steps

  1. Prepare Extracel™ 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 5.0 mL of Glycosil™ and 5.0 mL of Gelin-S™. Note: Leftover solutions can be frozen at -20 °C and are viable for ~2 weeks.
  5. Add 1.0 mL cells to Glycosil™ + Gelin-S™ such that the proper cell density is reached when the 100 µL of the total solution volume of 13.5 mL (5.0 mL Glycosil™ + 5.0 mL Gelin-S™ + 2.5 mL Extralink™ + 1.0 mL cells). Pipette up and down to mix.
  6. When you are ready to pour the hydrogels, add 2.5 mL of Extralink™ to Glycosil™ + Gelin-S™ with cells. 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 Extracel™ 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 Extracel™ into each insert. Note: 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 Extracel™ 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.
  11. Change the media as required using the following steps:
    •  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.