Extracel

  • Suitable for culturing primary cells, stem cells and cell lines
  • Cells can be encapsulated or grown on the hydrogel surface in 6- to 384-well plates or tissue culture inserts
  • Can be varied by the user to possess the desired compliance
  • Tested for bacteria, endotoxins, and lactate dehydrogenase-elevating virus (LDEV)
Purchase Now
Human dermal fibroblasts grown on the surface of an Extracel™ hydrogel.

Human dermal fibroblasts grown on the surface of an Extracel™ hydrogel.

General

Hydrogel Kit

Extracel® is recommended as a starting point for optimization of a cell’s microenvironment since it contains Gelin-S®. Gelin-S, or thiolated gelatin (denatured collagen), allows attachment of a wide variety of cell types and takes the guesswork out of the appropriate attachment factor to use.

Extracel® Hydrogel Kits have proven optimal for culturing primary cells and cell lines in 3-D. Extracel® hydrogels provide maximum versatility for research. Unlike an animal-derived extracellular matrix (ECM), Extracel® is chemically defined. The hydrogels are based on three biocompatible components: thiol-modified hyaluronan (a major constituent of native ECM), thiol-modified gelatin (denatured collagen), and a thiol-reactive crosslinker, polyethylene glycol diacrylate (PEGDA). Extracel® hydrogels can be customized by adding ECM proteins and by varying the hydrogel compliance to match the stiffness of native tissues.

Gelation

Reconstituted Extracel® components remain liquid at 15 to 37°C. The hydrogel is formed when the crosslinking agent, Extralink® (PEGDA) is added to a mixture of Glycosil® (thiol-modified hyaluronan) and Gelin-S® (thiol-modified gelatin). Gelation occurs in about twenty minutes after all three components are mixed. No steps depend on low temperatures or low pH. Diluting the components with phosphate-buffered saline (PBS) or cell-culture medium can increase the gelation time.

Flexibility

Extracel® allows customization of experiments:

3D Cell Recovery Matrix

The Extracel Hydrogel is also available with a novel crosslinker which enables the gel to be broken down for easy encapsulated cell recovery. At checkout simply select PEGSSDA as your crosslinker.

ECM Protein Incorporation into Extracel® Hydrogels

Printable PDF Version

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 can be used instead of the Extracel® Hydrogel Kit.

Application

3-D Cell Culture

Extracel® provides the basic scaffold for 3-D cell growth. Cells can be encapsulated during crosslinking1, where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth2. Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel2,3 or by trypsinization for cells grown on the surface.

Gelin-S® provides basic cell-attachment sites for cell lines and primary cells2,3. Several cell types depend on specific ECM components, such as the natural ECM proteins laminin, collagen, fibronectin, and vitronectin, to grow and differentiate–all of which may be added to the Extracel® hydrogel. These proteins are easily incorporated noncovalently into the hydrogel prior to gel formation. We recommend HyStem® for incorporating ECMs.

The compliance of the Extracel® hydrogel can be varied either by changing the amount of Extralink® used for crosslinking4 or by diluting the Glycosil and Gelin-S solutions using PBS or cell-culture medium.

The following cells have been cultured in Extracel®:

Primary Cells

  • human tracheal scar fibroblasts5,6
  • human and rat primary hepatocytes1,7
  • human dermal fibroblasts8
  • human mesenchymal stem cells9

Cell Lines

  • MDCK cells7,9
  • L929 fibroblasts10
  • NIH 3T3 fibroblast11
  • Hep G2 and HepG2 C3A cells1,7

Cancer-Cell Lines12,13

  • MDA-MB-468 (breast)
  • MCF-7 (breast)
  • MCF-10A (breast)
  • SK-Br-3 (breast)
  • OVCAR-3 (ovarian)
  • SK-OV-3 (ovarian)
  • HCT-116 (colon)
  • Caco-2 (colon)
  • MiaPaCa-2 (pancreatic)

Enzyme Digestion of Extracel® Hydrogels for Recovery of Encapsulated Cells

Printable PDF Version

For research use only

Material Supplier

  • 10x collagenase/hyaluronidase (Cat # 07912) StemCell Technologies

This protocol is for recovering cells that have been encapsulated in Extracel®, Extracel-HP® and Extracel-X® hydrogels and grown in tissue-culture inserts.

Key Points

  • Be cautious about mechanically breaking up the hydrogel prior to digestion because this can lower cell viability significantly.
  • If the 1:10 dilution of 1x collagenase/hyaluronidase is not satisfactory, try a 1:5 dilution with digestion overnight.

Steps

  1. Dilute the 10X collagenase/hyaluronidase solution 1:10 in the cell culture media (with no FBS) used to cultivate your cells.Note: Do not use undiluted enzyme since this results in low cell viability.
  2. If you are using media that contains FBS, make sure to wash the hydrogels with FBS-free media before starting the digestion process. At a minimum, wash hydrogels two times for one hour to clear FBS.
  3. Remove the tissue culture insert from the 24-well culture plate. Place upside down in a Petri dish.
  4. Run a 200 µL pipette tip around the edge of the membrane, cutting it loose from the insert. The membrane will stay attached to the insert, but usually flips up out of the way.
  5. Turn the insert right side up and, using the back of a 10 µL pipette tip, punch the hydrogel out of the insert into the Petri dish.
  6. Place the hydrogel in a 15 mL conical.
  7. Add 5 mL of the dilute collagenase/hyaluronidase solution to the hydrogel for each 100 µL of hydrogel.
  8. Incubate by shaking gently overnight at 37 °C.
  9. At the end of the incubation, there will still be some hydrogel left in the tube.
  10. Centrifuge the conical at 1500 rpm for five minutes. Aspirate off enzymes in media.
  11. Wash cells in 5 mL PBS.
  12. Centrifuge at 1500 rpm for five minutes. Aspirate off PBS.
  13. Resuspend the cell pellet and remove all the PBS and cells. Note: In the PBS you can see any remaining hydrogel.
  14. When you remove the cells, leave behind any remaining hydrogel.
  15. Wash the cells in media and centrifuge at 1500 rpm for five minutes. Aspirate off all media but ~0.5 mL.
  16. Resuspend the 0.5 mL of remaining media and cells in media.

Trypsin Cell Recovery from the Surface of Extracel® Hydrogels

Printable PDF Version

For research use only

This protocol is for releasing cells from the surface of Extracel®, Extracel-HP®, and Extracel-X® hydrogels.

Steps

  1. Aspirate off media.
  2. Wash the hydrogel surface with PBS.
  3. Add trypsin to the hydrogel surface. Note: Other products (i.e., Accutase, Detachin, TrypLE) that are gentler than trypsin and have greater cell viability can also be used. However, they may also degrade the hydrogel so that recovered cells carry some hydrogel particles with them. If this occurs, then use a 10X collagenase/hyaluronidase solution (like that sold by StemCell Technologies, Cat # 07912) to digest the remaining hydrogel.
  4. Incubate at room temperature until the cells begin to detach.
  5. Gently tap the plate to loosen the cells.
  6. Add media with serum to the hydrogel surface.
  7. Pipette up and down to get a uniform cell suspension.
  8. Transfer cells to a culture tube.
  9. Spin down cells, remove supernatant, and replace with fresh media.
  10. Cell viability will be similar to that of cells grown on plastic and detached with trypsin.

Composition

The Extracel® Hydrogel Kits come in three sizes:

  • 12.5 ml total hydrogel with the components in three vials (for large-volume applications)
    • 5.0 ml of Glycosil®
    • 5.0 ml of Gelin-S®
    • 2.5 ml of Extralink®
  • 7.5 ml total hydrogel with three sets of vials that make 2.5 ml each (for small-volume applications)
    • 3 x 1.0 ml of Glycosil®
    • 3 x 1.0 ml of Gelin-S®
    • 3 x 0.5 ml of Extralink®
  • 2.5 ml total hydrogel with the components in three vials (for small-volume applications)
    • 1.0 ml of Glycosil®
    • 1.0 ml of Gelin-S®
    • 0.5 ml of Extralink®

Extracel™ Hydrogel with PEGSSDA

Extracel with PEGSSDA replaces the Extralink Vials with PEGSSDA Vials. Simply specify which crosslinker you would like in the shopping cart.

Data Sheets

Extracel 12.5mL Kit Datasheet

Extracel 7.5mL Kit Datasheet

Extracel 7.5mL Kit with PEGSSDA Datasheet

Extracel 2.5mL Trial Kit Datasheet

Extracel 2.5mL Trial Kit with PEGSSDA

 

12.5 mL Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The Extracel Hydrogel Kit is composed of Glycosil® (thiol-modified sodium hyaluronate), Gelin-S® (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 unopened at -20 °C for up to one year.
  • Do not uncap the Glycosil and Gelin-S vials, since both materials will crosslink in the presence of oxygen. Use a syringe and needle to add DG Water and remove product from the vials through the septum.
Extralink
  • Store Extralink in the original vial unopened at -20 °C for up to one year.
  • Reconstituted Extralink solutions can be stored at -20 °C for ~one month.

INSTRUCTIONS FOR USE

Glycosil, Gelin-S, and Extralink solutions are prepared by dissolving the lyophilized solids in the DG Water. When reconstituted, the three materials will be in 1x phosphate buffered saline (PBS), pH ~7.4. Glycosil and Gelin-S vials con-tain 50 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 50 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. *Note: Glycosan recommends reconstituting each vial in its entirety.*

Extracel hydrogels (12.5 mL) should be prepared in the following manner:

  1. Allow the Glycosil, Gelin-S, Extralink, and DG Water vials to come to room temperature.
  2. Under aseptic conditions, using a syringe and needle, 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 or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gentle vortexing will speed dissolution. Solutions will be clear and slightly viscous.
  4. Under aseptic conditions, using a syringe and needle, add 2.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of Glycosil and Gelin-S. To mix, pipette back and forth slowly to avoid trapping air bubbles..
  6. If encapsulating cells, resuspend cell pellet in 10.0 mL of Glycosil + Gelin-S. Pipette back and forth to mix.
  7. 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) and mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

7.5 mL Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The Extracel® Hydrogel Kit is composed of Glycosil® (thiol-modified sodium hyaluronate), Gelin-S® (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 unopened at -20 °C for up to one year.
  • Do not uncap the Glycosil and Gelin-S vials since both materials will crosslink in the presence of oxygen. Use a syringe and needle to add DG Water.
Extralink®
  • Store Extralink in the original vial unopened at -20 °C for up to one year.
  • Reconstituted Extralink solutions can be stored at -20 °C for ~ one month.

INSTRUCTIONS FOR USE

Glycosil, Gelin-S and Extralink solutions are prepared by dissolving the lyophilized solids in the DG Water. When reconstituted, the three materials will be in 1x phosphate buffered saline (PBS) pH ~7.4. Glycosil and Gelin-S vials con-tain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 10 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. Note: Glycosan recommends reconstituting each vial in its entirety.

Extracel hydrogels (3 x 2.5 ml = 7.5 mL) should be prepared in the following manner:

  1. Allow the Glycosil, Gelin-S, Extralink, and DG Water vials to come to room temperature.
  2. Under aseptic conditions, using a syringe and needle, add 1.0 mL of DG Water to the Glycosil vial. Repeat for the Gelin-S vial.
  3. Place both vials horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution,   Solutions will be clear and slightly viscous.
  4. Under aseptic conditions, using a syringe and needle, add 0.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of Glycosil and Gelin-S™. To mix, pipette back and forth slowly to avoid trapping air bubbles.
  6. If encapsulating cells, resuspend cell pellet in 2.0 mL of Glycosil + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add Extralink to the Glycosil + Gelin-S mix in a 1:4 volume ratio (0.5 mL Extralink™ to 2.0 mL Glycosil + Gelin-S) and mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

7.5 mL with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The Extracel Hydrogel Kit with PEGSSDA is composed of Glycosil™ (thiol-modified hyaluronic acid), Gelin-S™ (thiol-modified gelatin), PEGSSDA (disulfide-containing polyethylene glycol diacrylate), and degassed, de-ionized water (DG Water). Solutions of Glycosil and Gelin-S form a transpar-ent hydrogel when mixed with PEGSSDA. 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 unopened at -20 °C for up to one year.
  • Do not uncap the Glycosil and Gelin-S vials since both materials will crosslink in the presence of oxygen. Use a syringe and needle to add DG Water.
PEGSSDA™
  • Store PEGSSDA in the original vial unopened at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20 °C for ~ one month.

INSTRUCTIONS FOR USE

Glycosil, Gelin-S and PEGSSDA solutions are prepared by dissolving the lyophilized solids in the DG Water. When reconstituted, the three materials will be in 1x phosphate buffered saline (PBS) pH ~7.4. Glycosil and Gelin-S vials con-tain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. PEGSSDA vials contain 5 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. *Note: Glycosan recommends reconstituting each vial in its entirety.

Extracel hydrogels (3 x 2.5 ml = 7.5 mL) should be prepared in the following manner:

  1. Allow the Glycosil, Gelin-S, PEGSSDA, and DG Water vials to come to room temperature.
  2. Under aseptic conditions, using a syringe and needle, add 1.0 mL of DG Water to the Glycosil vial. Repeat for the Gelin-S vial.
  3. Place both vials horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution,   Solutions will be clear and slightly viscous.
  4. Under aseptic conditions and using a syringe add 0.25 mL of DG Water to each of the two PEGSSDA vials. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of Glycosil and Gelin-S™. To mix, pipette back and forth slowly to avoid trapping air bubbles.
  6. If encapsulating cells, resuspend cell pellet in 2.0 mL of Glycosil + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the Glycosil + Gelin-S mix in a 1:4 volume ratio (0.5 mL PEGSSDA to 2.0 mL Glycosil + Gelin-S) mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Hydrogel Dissolution:

Dissolution of gels with cells on top and encapsulated (gel volume of 0.6 mL) in a 24 well plate. Note: The following procedure was optimized particularly for the aforementioned gel geometry. Dissolution of gels with alternate geometry and/or volumes may require adjustments to the protocol. In general, for dissolution add at least twice the gel volume of N-Acetyl-L-Cysteine.

  1. Make up the appropriate amount of 40mM N-Acetyl-L-Cysteine in 1X PBS or media and pH to 7.4.
  2. Add 2 mL of 40mM N-Acetyl-Cysteine to the top of each gel and let sit at 37°C for 1 hour. Agitation by orbital shaking will help decrease dissolution time.
  3. Confirm dissolution by pipetting solution in well up and down, observing any remain-ing gel. If needed, allow another 30 minutes for complete gel dissolution.
  4. Remove liquid from well and place in conical centrifuge tube. If necessary, add PBS to a total of 5mL of liquid.
  5. Centrifuge at 1000 RPM for 5 minutes.
  6. Aspirate off liquid and process cells as desired.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

Trial Kit (2.5 mL) Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The Extracel Hydrogel Kit is composed of Glycosil® (thiol-modified sodium hyaluronate), Gelin-S® (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 unopened at -20 °C for up to one year.
  • Do not uncap the Glycosil and Gelin-S vials, since both materials will crosslink in the presence of oxygen. Use a syringe and needle to add DG Water.
Extralink
  • Store Extralink in the original vial at -20 °C for up to one year.
  • Reconstituted Extralink solutions can be stored at -20 °C for ~one month.

INSTRUCTIONS FOR USE

Glycosil, Gelin-S, and Extralink solutions are prepared by dissolving the lyophilized solids in the DG Water. When reconstituted, the three materials will be in 1x phosphate buffered saline (PBS), pH ~7.4. Glycosil and Gelin-S vials con-tain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. Extralink vials contain 10 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. *Note: Glycosan recommends reconstituting each vial in its entirety.

Extracel hydrogels (2.5 mL) should be prepared in the following manner:

  1. Allow the Glycosil, Gelin-S, Extralink, and DG Water vials to come to room temperature.
  2. Under aseptic conditions, using a syringe and needle, add 1.0 mL of DG Water to the Glycosil vial. Repeat for the Gelin-S vial.
  3. Place both vials horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gentle vortexing will speed dissolution. Solutions will be clear and slightly viscous.
  4. Under aseptic conditions, using a syringe and needle, add 0.5 mL of DG Water to the Extralink vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of Glycosil and Gelin-S. To mix, pipette back and forth slowly to avoid trapping air bubbles.
  6. If encapsulating cells, resuspend cell pellet in 2.0 mL of Glycosil + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add Extralink to the Glycosil + Gelin-S mix in a 1:4 volume ratio (0.5 mL Extralink to 2.0 mL Glycosil + Gelin-S) and mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

 

Trial Kit (2.5 mL) with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The Extracel Hydrogel Kit with PEGSSDA is composed of Glycosil™ (thiol-modified hyaluronic acid), Gelin-S™ (thiol-modified gelatin), PEGSSDA (disulfide-containing polyethylene glycol diacrylate), and degassed, de-ionized water (DG Water). Solutions of Glycosil and Gelin-S form a transpar-ent hydrogel when mixed with PEGSSDA. 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 unopened at -20 °C for up to one year.
  • Do not uncap the Glycosil and Gelin-S vials since both materials will crosslink in the presence of oxygen. Use a syringe and needle to add DG Water.
PEGSSDA™
  • Store PEGSSDA in the original vial unopened at -20 °C for up to one year.
  • Reconstituted solutions can be stored at -20 °C for ~ one month.

INSTRUCTIONS FOR USE

Glycosil, Gelin-S and PEGSSDA solutions are prepared by dissolving the lyophilized solids in the DG Water. When reconstituted, the three materials will be in 1x phosphate buffered saline (PBS) pH ~7.4. Glycosil and Gelin-S vials con-tain 10 mg of material and when reconstituted according to instruction will produce a 1% (w/v) solution. PEGSSDA vials contain 5 mg of material and when reconstituted according to instructions will produce a 2% (w/v) solution. *Note: Glycosan recommends reconstituting each vial in its entirety.

Extracel hydrogels (2.5 mL) should be prepared in the following manner:

  1. Allow the Glycosil, Gelin-S, PEGSSDA, and DG Water vials to come to room temperature.
  2. Under aseptic conditions, using a syringe and needle, add 1.0 mL of DG Water to the Glycosil vial. Repeat for the Gelin-S vial.
  3. Place both vials horizontally on a rocker or shaker. It will take <30 minutes for the solids to fully dissolve. Warming to not more than 37 °C and/or gently vortexing will speed dissolution,   Solutions will be clear and slightly viscous.
  4. Under aseptic conditions and using a syringe add 0.25 mL of DG Water to each of the two PEGSSDA vials. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of Glycosil and Gelin-S™. To mix, pipette back and forth slowly to avoid trapping air bubbles.
  6. If encapsulating cells, resuspend cell pellet in 2.0 mL of Glycosil + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the Glycosil + Gelin-S mix in a 1:4 volume ratio (0.5 mL PEGSSDA to 2.0 mL Glycosil + Gelin-S) mix by pipette.
  8. If encapsulating cells, allow solution to react for 10 minutes then mix again by pipette to ensure even distribution of cells.
  9. Gelation will occur within ~20 minutes.

Hydrogel Dissolution:

Dissolution of gels with cells on top and encapsulated (gel volume of 0.6 mL) in a 24 well plate. Note: The following procedure was optimized particularly for the aforementioned gel geometry. Dissolution of gels with alternate geometry and/or volumes may require adjustments to the protocol. In general, for dissolution add at least twice the gel volume of N-Acetyl-L-Cysteine.

  1. Make up the appropriate amount of 40mM N-Acetyl-L-Cysteine in 1X PBS or media and pH to 7.4.
  2. Add 2 mL of 40mM N-Acetyl-Cysteine to the top of each gel and let sit at 37°C for 1 hour. Agitation by orbital shaking will help decrease dissolution time.
  3. Confirm dissolution by pipetting solution in well up and down, observing any remain-ing gel. If needed, allow another 30 minutes for complete gel dissolution.
  4. Remove liquid from well and place in conical centrifuge tube. If necessary, add PBS to a total of 5mL of liquid.
  5. Centrifuge at 1000 RPM for 5 minutes.
  6. Aspirate off liquid and process cells as desired.

Note: Each kit component has been manufactured under aseptic conditions and tested for bacteria and fungus.

References

  1. G. D. Prestwich, Y. Liu, M. Serban, B. Yu, X. Z. Shu, and A. Scott, “3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery,” invited, Adv. Enz. Res., in press (2007).
  2. X. Z. Shu, S. Ahmad, Y. Liu, and G. D. Prestwich, “Synthesis and Evaluation of Injectable, In SituCrosslinkable Synthetic Extracellular Matrices (sECMs) for Tissue Engineering,” J. Biomed Mater. Res. A, 79A(4), 901-912 (2006).
  3. X. Z. Shu, Y. Liu, F. Palumbo, G. D. Prestwich, “Disulfide-crosslinked Hyaluronan-Gelatin Hydrogel Films: A Covalent Mimic of the Extracellular Matrix for In Vitro Cell Growth,” Biomaterials, 24, 3825-3834 (2003).
  4. K. Ghosh, Z. Pan, E. Guan, S. Ge, Y. Liu, T. Nakamura, X. Ren, M. Rafailovich, R. Clark, “Cell Adaptation to a Physiologically Relevant ECM Mimic with Different Viscoelastic Properties,” Biomaterials 28, 671-679 (2007).
  5. Y. Liu, Z. X. Shu, S. D. Gray, G. D. Prestwich, “Disulfide-Crosslinked Hyaluronan-Gelatin Sponge: Growth of Fibrous Tissue In Vivo,” J Biomed Mat Res, 68A, 142-149 (2004).
  6. X. Z. Shu, Y. Liu, F. Palumbo, Y. Luo, G. D. Prestwich, “In Situ Crosslinkable Hyaluronan Hydrogels for Tissue Engineering,” Biomaterials, 25, 1339-1348 (2004).
  7. Unpublished data from G. D. Prestwich, et al, University of Utah.
  8. G. D. Prestwich, X. Z. Shu, Y. Liu, S. Cai, J. F. Walsh, C.W. Hughes, K. R. Kirker, R. R. Orlandi, A. H. Park, S. L. Thibeault, M. E. Smith, “Injectable Synthetic Extracellular Matrices for Tissue Engineering and Repair,” Adv. Exp. Med. Biol., 585, 125-133 (2006).
  9. Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University, and Medical University of South Carolina.
  10. Y. Liu, X. Z. Shu, G. D. Prestwich, “Biocompatibility and Stability of Disulfide-Crosslinked Hyaluronan Films,” Biomaterials, 26, 4737-4746 (2005).
  11. X. Z. Shu, Y. Liu, Y. Luo, M. C. Roberts, and G. D. Prestwich, “Disulfide-crosslinked Hyaluronan hydrogels,” Biomacromolecules, 3, 1304-1311 (2002).
  12. Unpublished data from C. Scaife, et al, University of Utah.
  13. Y. Liu, X. Z. Shu, and G. D. Prestwich, “Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Crosslinked Hyaluronan-Derived Hydrogels,” Tissue Engineering 13(5), 1091-1101(2007).