Technical

ECM Protein Incorporation into Extracel™ Hydrogels

For research use only

INSTRUCTIONS FOR LAMININ INCORPORATION

Glycosil™, Gelin-S™, and Extralink™ solutions are prepared by dissolving the lyophilized solids in DG Water™. When reconstituted, they will be in phosophate-buffered saline (PBS) pH ~7.6. Extracel™ hydrogels (12.5 mL) with laminin can be prepared in the following manner:

  1. Remove Glycosil™, Gelin-S™, and Extralink™ vials from the -20 °C freezer and heat them to 37 °C (~30 minutes).
  2. Under aseptic conditions and using a syringe with the exact amount of liquid, add 5.0 mL of DG Water™ to the Glycosil™ vial. Repeat for the Gelin-S™ vial.
  3. Place both vials horizontally on a rocker at 37 °C (for maximum mixing). It will take <30 minutes for the solids to fully dissolve. Solutions will be clear and slightly viscous. Note: Vigorous shaking will speed up dissolving time.
  4. Under aseptic conditions and using a syringe with the exact amount of liquid, add 2.5 mL of DG Water™ to the Extralink™ vial. Invert several times to dissolve.
  5. As soon as possible, but within four hours of making the solutions, mix equal volumes of Glycosil™ and Gelin-S™. Pipette or invert to mix.
  6. Add 1.1 mL of laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane to 10 mL of Glycosil™ + Gelin-S™. Mix thoroughly.
  7. If encapsulating cells, add 1.0 mL of cells to 11.1 mL of Glycosil™ + Gelin-S™ + laminin. Mix thoroughly.
  8. To form the hydrogel, add Extralink™ to the Glycosil™ + Gelin-S™ + laminin mix in a 1:4 volume ratio (2.5 mL Extralink™ to 10.0 mL Glycosil™ + Gelin-S™).
  9. Gelation will occur in ~20 minutes.

Note: All kit components have been tested and show no bacteria growth.

VARIATIONS ON THESE INSTRUCTIONS

The protocol given above can be varied in several ways:

  1. The amount of laminin can be increased or decreased.
  2. The laminin source can be varied.
  3. Other extracellular matrix (ECM) components (e.g., collagen, fibronectin, vitronectin, etc.) can be used in place of or in conjunction with laminin.
  4. The amount of cells encapsulated can be increased or decreased.
  5. Cells can be plated on top of the hydrogels instead of being encapsulated.
  6. Gelin-S™ can be left out entirely and hydrogels can be made with Glycosil™, Extralink™, and the desired ECM. Note that cells do not attach to Glycosil™ alone, so if no Gelin-S™ is used, then an ECM must be added in order for cells to attach.
  7. Other kits (Extracel-HP™, Extracel-HPG™, Extracel™-LG) can be used instead of the Extracel™ Hydrogel Kit.