Technical

2-D cell growth on Extracel™ hydrogels

For research use only

Amount Material Supplier

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

This protocol describes how to make Extracel™ hydrogels in a 96-well plate format for cell growth on the surface of the hydrogels. The protocol can easily be adapted for use with 6-, 12-, 24-, and 48-well plates. It can also 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. Mix 5.0 mL of Glycosil™ and 5.0 mL of Gelin-S™. Pipette up and down thoroughly to mix. Note: If the Extracel™ solutions are not well mixed, then the hydrogel surface may not be uniform. This can cause variation in how the cells attach and grow on the hydrogel.
  3. When you are ready to pour the hydrogels, add 2.5 mL of Extralink™ to the Glycosil™ + Gelin-S™ mix. Pipette up and down thoroughly to mix. Note: Once the Extralink™ is added you have <20 minutes before the hydrogel forms and it becomes impossible to pipette the solution.
  4. Pipette 100 µL of Extracel™ into each well. Place the lid on the 96-well plate. Note: Leftover solutions can be frozen at -20 °C and are viable for ~2 weeks.
  5. Allow Extracel™ to gel by placing the plate on a rocker for at least one hour with the lid on. Note: If the hydrogel is left for an extended period of time, it will dry out and form a film.
  6. Prepare cells for use in 2-D cell culture as per standard procedures. Seeding density varies with cell type, but a typical range is 5,000 to 50,000 cells per well.
  7. Once the hydrogels are solid, add 100 µL of cell slurry in media to each well on top of the hydrogel.
  8. Place in the 37 °C incubator with 5% CO2.
  9. 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.