Technical

Extracel-X™ (12.5 mL) Hydrogel Kit Product Data Sheet

For research use only

PRODUCT DESCRIPTION

The Extracel-X™ Hydrogel Kit is composed of Glycosil™ (CMHA-S, thiol-modified hyaluronic acid), Gelin-S™ (Gtn- DTPH, or thiol-modified gelatin), Extralink™ (PEGDA, polyethylene glycol diacrylate), and degassed deionized  water (DG Water™). Solutions of Glycosil™ and Gelin-S™ form a transparent hydrogel when mixed with Extralink™. All lyophilized solids are blanketed by argon and under a slight vacuum.

STORAGE

Glycosil™ and Gelin-S™

  • Store Glycosil™ and Gelin-S™ in original vials at -20 °C for up to one year.
  • Solutions can be stored at -20 °C for ~1 month if they are frozen immediately after reconstitution in their original argon-blanketed vials.
  • Do not mix Glycosil™ and Gelin-S™ prior to freezing.
  • 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 Glycosil™ and Gelin-S™ vials since both materials 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 ~1 month.

INSTRUCTIONS FOR USE

Glycosil™, Gelin-S™, and Extralink™ solutions are prepared by dissolving the lyophilized solids in the DG Water™. When reconstituted, they will be in phosophate-buffered saline (PBS) pH ~7.4. Glycosil™, Gelin-S™, and Extralink™ solutions can be used in a variety of ways, depending on the desired final gel characteristics. The standard Extracel-X™ formulation with a gelation time of ~20 minutes is:

  • 1.0 mL Glycosil™
  • 1.0 mL Gelin-S™
  • 0.5 mL Extralink™
  • 0.2 mL of cells in medium (serum-free)

However, the Glycosil™ + Gelin-S™ solutions can be further diluted with medium to either increase the gelation time or to give cells more nutrients prior to formation of the hydrogel. Glycosan recommends performing a test run prior to animal experimentation to determine the optimal medium dilution for your application (see “Guidelines for 3-D Cell Encapsulation in Extracel-X™ Hydrogels for Xenograft Applications”).

STANDARD EXTRACEL-X™ FORMULATION

Extracel-X™ hydrogels (12.5 mL) should 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. 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 5.0 mL of DG Water™ to the Glycosil™ vial. Repeat for the Gelin-S™ vial.
  4. 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.
  5. 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.
  6. As soon as possible, but within four hours of making the solutions, mix equal volumes of Glycosil™ and Gelin-S™. To mix, pipette back and forth gently or invert the vial.
  7. If encapsulating cells, add 1.0 mL of cells to 10.0 mL of Glycosil™ + Gelin-S™. Pipette back and forth to mix.
  8. To form the hydrogel, add Extralink™ to the Glycosil™ + Gelin-S™ mix in a 1:4 volume ratio (2.5 mL Extralink™ to 10.0 mL Glycosil™ + Gelin-S™). The total hydrogel volume with cells is 13.5 mL.
  9. Gelation will occur within ~20 minutes.

Extracel-X™ Gelation Time

Diluting Extracel-X™ with Medium

Extracel-X™ can be further diluted with medium to either increase the gelation time or to give cells more nutrients prior to formation of the hydrogel. As Extracel-X™ is diluted, the hydrogel characteristics change based on the medium. Representative data is given for RPMI 1640 and DMEM (see the gelation-time graph above). Note that if another medium is used, the final gelation times may be different than stated here. Contact Glycosan Technical Services (801-583-8212) if you have questions about a specific medium.

Diluted Extracel-X™ hydrogels (12.5 mL) 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. 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 5.0 mL of DG Water™ to the Glycosil™ vial. Repeat for the Gelin-S™ vial.
  4. 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.
  5. 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.
  6. As soon as possible, but within four hours of making the solutions, mix equal volumes of Glycosil™ and Gelin-S™. To mix, pipette back and forth or invert the vial.
  7. Based on the desired dilution, add the following volumes of medium + cells to the 10.0 mL of Glycosil™/Gelin-S™ mix:
    Dilution, % 10 20 30 40 50
    Medium + cells, mL 1.11 2.50 4.30 6.67 10.00
  8. Pipette back and forth to mix.
  9. To form the hydrogel, add Extralink™ to the Glycosil™ + Gelin-S™ mix in a 1:4 volume ratio (2.5 mL Extralink™ to 10.0 mL Glycosil™ + Gelin-S™).
  10. See see the gelation-time graph above for the expected gelation time.

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