HyStem-HP

  • Hydrogels are suitable for animal implantation (angiogenesis applications), culturing of primary cells, stem cells, and cell lines in the presence of growth factors.
  • Cells can be encapsulated or grown on the hydrogel surface in 6- to 384-well plates or tissue culture inserts.
  • Hydrogels can be varied by the user to possess the desired compliance and contain the desired growth factors.
  • Tested for bacteria, endotoxins, and lactate dehydrogenase-elevating virus (LDEV).
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Figure legend: Endothelial progenitor cells cultivated in HyStem-HP. Blue = mouse cell nuclei, brown = human cell nuclei, red = CD31 protein (courtesy of Robert Grove, Endgenitor Technologies, Inc., Indianapolis, IN)

General

HyStem-HP® Hydrogel Kit

HyStem-HP® is recommended for stem cell researchers whose applications require slow release of growth factors in a cell’s microenvironment. HyStem-HP contains small amounts of thiolated heparin which ionically binds a wide variety of growth factors and slowly releases them over time1,2,3.

HyStem-HP is ideal for stem cell applications where slowly released growth factors are crucial in recreating a stem cell niche.  HyStem-HP® hydrogels contains thiol-modified heparin which allows the slow release of growth factors (GFs) within an easily customizable, environment. HyStem-HP® is a synthetic extracellular matrix (ECM) that can be injected and crosslinked in situ. Unlike an animal-derived extracellular matrix (ECM), HyStem-HP® is chemically defined and nonimmunogenic. The HyStem-HP® Hydrogel Kit contains HyStem-HP® (a combination of thiol-modified hyaluronan, HA, and thiol-modified heparin), Gelin-S® (thiol-modified gelatin), and Extralink® (a thiol-reactive crosslinker, polyethylene glycol diacrylate, PEGDA).

The immobilized heparin in the hydrogel mimics the heparan sulfate proteoglycans normally present in the extracellular matrix (ECM). It also helps protect GFs from proteolysis and slows their release to attached cells1. This reduces the amount of GF required to achieve stimulation of cell growth or differentiation when compared to the use of free GF in media. All GFs tested to date (bFGF, VEGF, Ang-1, PDGF, TGFβ1, KGF) are released at different rates, but over a period of several weeks1,2,3.

Gelation

Reconstituted HyStem-HP® components remain liquid at 15 to 37°C. The hydrogel is formed when Extralink is mixed with HyStem-HP® and Gelin-S®. Gelation occurs about twenty minutes after all three components are mixed. No steps depend on low temperature or low pH. Diluting the components with phosphate-buffered saline (PBS) or cell-culture medium can increase gelation time.

Flexibility

  • HyStem-HP® ensures complete control over:
  • amount and type of GF incorporated
  • cell encapsulation or plating on top of hydrogel
  • cell-growth format (96- to 6-well plates and/or tissue-culture inserts)
  • amount and type of ECM proteins incorporated
  • hydrogel stiffness

3D Cell Recovery Matrix

The HyStem-HP 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.

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

Printable PDF Version

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-HP® 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.

2-D cell growth on Extracel® hydrogels

Printable PDF Version

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-HP® 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.

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 Stem Cell Growth

In addition to stem cell culture on top of the hydrogel, HyStem-HP® provides the basic scaffold for 3-D stem cell growth. Cells can be encapsulated during crosslinking4, where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo 3-D growth5. Cells are recovered from the hydrogel either by enzyme digestion for cells encapsulated in the hydrogel5,6 or by trypsinization for cells grown on the surface.

Gelin-S® provides basic cell-attachment sites for stem cell lines. Several stem cell types depend on specific ECM components to grow and differentiate. To affect specific cell performance, other factors such as growth factors or ECM proteins may be added to the HyStem-HP® hydrogel. ECM proteins are easily incorporated noncovalently into the hydrogel prior to gel formation as are growth factors..

3-D Stem Cell Growth Using GF-Supplemented Media

For stem cells cultured with GFs in the media, GFs may be removed from the media and added to the HyStem-HP™ hydrogel. The hydrogel is used to coat a culture flask and cells are cultured on top of the hydrogel using medium without GFs. Note, however, that the GFs are released at different rates. Therefore, we recommend an in vitro test to determine the proper concentrations for GF addition to the hydrogel. See growth-factor release for more information about specific GFs and their retention.

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

HyStem-HP® Hydrogel Kits comes in two size:

  • 7.5 ml total hydrogel with three sets of vials that make 2.5 ml each
    • 3x 1.0 ml of HyStem-HP
    • 3X 1.0 ml of Gelin-S
    • 3x 0.5 ml vials of Extralink
  • Trial 2.5 ml total hydrogel with one sets of vials
    • 1.0 ml of HyStem-HP
    • 1.0 ml of Gelin-S
    • 0.5 ml of Extralink

HyStem-HP 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

HyStem-HP 7.5mL Kit Datasheet

HyStem-HP 7.5mL Kit with PEGSSDA Datasheet

HyStem-HP Trial Kit Datasheet

HyStem-HP Trial Kit with PEGSSDA Datasheet

 

 

HyStem®-HP Hydrogel Kit (7.5 mL) Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The HyStem®-HP Hydrogel Kit is composed of HyStem®-HP (thiol-modified sodium hyaluronate with thiol-modified heparin), Gelin-S® (thiol modified gelatin), Extralink® (PEGDA, polyethylene glycol diacrylate), and degassed, deionized water (DG Water). Solutions of HyStem-HP and Gelin-S form a transparent hydrogel when mixed with Extralink. All lyophilized solids are blanketed by argon and under a slight vacuum.

STORAGE

HyStem-HP and Gelin-S
  • Store HyStem-HP and Gelin-S in original vials unopened at -20 °C for up to one year.
  • Do not uncap the HyStem-HP 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

HyStem-HP, 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. HyStem-HP and Gelin-S vials contain 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.

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

  1. Allow the HyStem-HP, 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 HyStem-HP 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 HyStem-HP 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 HyStem-HP + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add Extralink to the HyStem-HP + Gelin-S mix in a 1:4 volume ratio (0.5 mL Extralink® to 2.0 mL HyStem-HP+ 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.

 

HyStem™-HP Hydrogel Kit (7.5 mL) with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The HyStem™-HP Hydrogel Kit with PEGSSDA is composed of HyStem™-HP (thiol-modified sodium hyaluronate with thiol-modified heparin), Gelin-S™ (thiol modified gelatin), PEGSSDA™ (disulfide-containing polyethylene glycol diacry-late), and degassed, deionized water (DG Water). Solutions of HyStem-HP and Gelin-S form a transparent hydrogel when mixed with PEGSSDA. All lyophilized solids are blanketed by argon and under a slight vacuum.

STORAGE

HyStem-HP and Gelin-S
  • Store HyStem-HP and Gelin-S in original vials unopened at -20 °C for up to one year.
  • Do not uncap the HyStem-HP 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

HyStem-HP, 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. HyStem-HP and Gelin-S vials contain 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.

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

  1. Allow the HyStem-HP, 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 HyStem-HP 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 vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of HyStem-HP 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 HyStem-HP + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the HyStem-HP + Gelin-S mix in a 1:4 volume ratio (0.5 mL PEGSSDA to 2.0 mL HyStem-HP + 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.

 

HyStem®-HP Hydrogel Trial Kit Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The HyStem®-HP Hydrogel Trial Kit is composed of HyStem®-HP (thiol-modified sodium hyaluronate with thiol-modified heparin), Gelin-S® (thiol modified gelatin), Extralink® (PEGDA, polyethylene glycol diacrylate), and degassed, deionized water (DG Water). Solutions of HyStem-HP and Gelin-S form a transparent hydrogel when mixed with Extralink. All lyophilized solids are blanketed by argon and under a slight vacuum.

STORAGE

HyStem-HP and Gelin-S
  • Store HyStem-HP and Gelin-S in original vials unopened at -20 °C for up to one year.
  • Do not uncap the HyStem-HP 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

HyStem-HP, 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. HyStem-HP and Gelin-S vials contain 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.

HyStem-HP hydrogels  (2.5 mL) should be prepared in the following manner:

  1. Allow the HyStem-HP, 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 HyStem-HP 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 HyStem-HP 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 HyStem-HP + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add Extralink to the HyStem-HP + Gelin-S mix in a 1:4 volume ratio (0.5 mL Extralink® to 2.0 mL HyStem-HP+ 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.

 

HyStem™-HP Trial Kit (2.5 mL) with PEGSSDA Product Data Sheet

Printable PDF Version

For research use only

PRODUCT DESCRIPTION

The HyStem™-HP Hydrogel Kit with PEGSSDA is composed of HyStem™-HP (thiol-modified sodium hyaluronate with thiol-modified heparin), Gelin-S™ (thiol modified gelatin), PEGSSDA™ (disulfide-containing polyethylene glycol diacry-late), and degassed, deionized water (DG Water). Solutions of HyStem-HP and Gelin-S form a transparent hydrogel when mixed with PEGSSDA. All lyophilized solids are blanketed by argon and under a slight vacuum.

STORAGE

HyStem-HP and Gelin-S
  • Store HyStem-HP and Gelin-S in original vials unopened at -20 °C for up to one year.
  • Do not uncap the HyStem-HP 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

HyStem-HP, 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. HyStem-HP and Gelin-S vials contain 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.

HyStem-HP hydrogels (2.5 mL) should be prepared in the following manner:

  1. Allow the HyStem-HP, 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 HyStem-HP 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 vial. Invert several times to dissolve.
  5. As soon as possible, but within 2 hours of making the solutions, aseptically mix equal volumes of HyStem-HP 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 HyStem-HP + Gelin-S. Pipette back and forth to mix.
  7. To form the hydrogel, add PEGSSDA to the HyStem-HP + Gelin-S mix in a 1:4 volume ratio (0.5 mL PEGSSDA to 2.0 mL HyStem-HP + 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. S. Cai, Y. Liu, X. Z. Shu, G. D. Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor,” Biomaterials, 26, 6054-6067 (2005).
  2. D. B. Pike, S. Cai, K. R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, “Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF,” Biomaterials, 27, 5242–5251 (2006).
  3. Unpublished data from G. D. Prestwich, et al, University of Utah, and R. Peattie, et al, University of Oregon.
  4. 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).
  5. X. Z. Shu, S. Ahmad, Y. Liu, and G. D. Prestwich, “Synthesis and Evaluation of Injectable, In Situ Crosslinkable Synthetic Extracellular Matrices (sECMs) for Tissue Engineering,” J. Biomed Mater. Res. A, 79A(4), 901-912 (2006).