Troubleshooting Hydrogel Kits

1. The components in my Extracel® Hydrogel Kit are not going into solution. What should I do? If the Glycosil®, Heprasil®, or Gelin-S® are not going into solution that means that they have started to disulfide crosslink as a lyophilized solid. Most likely this is because they are past their useful shelf life. Alternatively, the vials could have been exposed to high temperatures (>30 °C) for several days. Please contact Technical Service at Glycosan (801-583-8212) if you are having problems.

2. A hydrogel is not forming after I add Extralink®. What happened? If you are encapsulating cells in the Extracel hydrogel, there are several possibilities:

• The pH of the hydrogel is <7.4. This could result from using cell culture media to resuspend the Glycosil, Gelin-S and Extralink. Use sterile 0.1 N NaOH to increase the solution pH.
• The number of cells that you are encapsulating is so high that the hydrogel cannot form.
• The volume of the cells added to hydrogel solution was more than 50% of the total volume. This would dilute the solutions past the point where a hydrogel can form. Try spinning down your cells, aspirating off all the media and resuspending the cells directly in Extracel.

3. My cells are not adhering to the surface of the hydrogel. What happened?  If you are using Glycosil-only hydrogels there won’t be any cell adherence on the surface. The Gelin-S in Glycosan’s standard hydrogel kits provides the cellular attachment sites. If Gelin-S is not used, then either ECM proteins or attachment peptides (like RGD) need to be incorporated into the hydrogel. If you used Gelin-S and are still not getting cell attachment, then there are two possibilities:

• At Gelin-S concentrations less than 25% of the hydrogel, some cells may not attach. Use more Gelin-S.
• The mixing of the Glycosil and Gelin-S was poor, so that the hydrogel that formed is not uniform. Try vortexing the solution together.

4. Why do I see cell growth in some areas of the hydrogels that I have made, but not in others? Most likely there is no cell growth in some areas because of poor mixing of the Gelin-S/Glycosil/Extralink. Glycosil only will not support cell attachment. Try vortexing the Extracel components before plating.  This is especially important if you are making Glycosil-only hydrogels with non-covalently incorporated extracellular matrix proteins.

Analysis Techniques for Cells Encapsulated in Extracel Hydrogels

5. Can I do immunostaining with cells encapsulated in Extracel hydrogels? No. Antibodies are too big to permeate the Extracel hydrogels. Antibody staining can only be done by treating the “hydrogel + encapsulated cells” as tissue. The “hydrogel + cells” can be paraffin embedded, sectioned and stained. The other alternative is to recover the cells from the hydrogel and then stain them normally.  

6. Can I use vital dyes on cells encapsulated in Extracel? Yes. The fluorescent dyes work very well for this purpose. Vital dyes are small and can penetrate the hydrogel easily.

7. Can I stain my cells for live/dead? Yes. The fluorescent dyes work particularly well for this purpose.  Vital dyes are small and can penetrate the hydrogel easily.

8. Can I do an MTS type assay on cells grown in Extracel hydrogels? Yes. Most cell proliferation assays, including MTS, work well with the Extracel hydrogels and sponges. However, the proper controls using hydrogels and sponges without cells need to be included.

9. What size molecules can freely diffuse through the hydrogels? Globular particles less than 75 kDa should freely diffuse.

How to use Extracel Hydrogel Kits

10. Can I reconstitute Glycosil, Gelin-S or Extralink in my cell culture media instead of DG Water? Yes, but it may affect the gelation time. The gelation time of the Extracel hydrogels is very pH sensitive – a change of just a few tenths in pH can alter the gelation time by several minutes. For, example if the cell culture media decreases the pH by 0.2, it will increase the gelation time by ~ 10 minutes.

11. What is the pH of Extracel when reconstituted? 7.6

12. Do I have to use DG Water to reconstitute Glycosil, Gelin-S or Extralink? No. Any sterile, deionized, degassed water will work. However, for the dissolution and gelation times to match the instructions, we recommend using the DG Water. This water is degassed and blanketed in argon and has been tested with the hydrogel kit components.

13. Do I have to encapsulate my cells or can I grow them on the surface of the hydrogel?  It is also possible to grow cells on the surface of the hydrogels in a monolayer. See our HA Technical page for protocols.

14. When do I use a specific extracellular (ECM) protein instead of Gelin-S? If you are cultivating stem or progenitor cells on Extracel hydrogels, you should consider using specific ECM proteins in place of Gelin-S. Gelin-S is thiol-modified, denatured bovine collagen I. It is possible that the Gelin-S (especially at lower concentrations, <25 vol%) will function primarily to provide cell attachment sites without signaling differentiation. However, it is also possible that it will cause your cells to differentiate in undesirable ways. If you understand the native extracellular matrix for your cell type, then adding the appropriate ECM protein may provide better results than using Gelin-S.

15. What is providing cellular attachment sites in the hydrogel? Gelin-S, which is thiol-modified, denatured, Collagen I derived from either bovine or porcine sources.

16. Can I use peptides in place of Gelin-S for cell attachment? Yes. The peptides must have a cysteine so they will be covalently incorporated into the hydrogel. See our HA Technical page for instructions.

17. Can I use extracellular matrix (ECM) proteins in place of Gelin-S? Yes. ECM proteins like laminin, collagen, fibronectin or vitronectin can be non-covalently incorporated into the hydrogel prior to crosslinking with Extralink. However, the best ECM protein(s) to use depends upon the cell type. See our Technical page for details on ECM incorporation.

18. Does Gelin-S covalently link with the rest of the hydrogel? Gelin-S has been thiol-modified in the same manner as the hyaluronan in Glycosil so that it covalently crosslinks with the Extralink in the Extracel hydrogels.

19. Can I non-covalently incorporate gelatin into the hydrogel? You can incorporate normal gelatin in the same way that extracellular matrix proteins are incorporated. See our HA Technical page for details.

20. How much Gelin-S should I use? It depends on your experiment. Gelin-S provides the cell attachment sites. If you want lots of attachment sites, use at least 50 vol% Gelin-S with 50 vol% Glycosil or Heprasil. However, for stem cells lower Gelin-S concentrations may work better.

21. How do I change the crosslinking time for my application? Crosslinking time is affected by the amount of crosslinker (Extralink) used in solution.  Decreasing the Extralink amount increases the gelation time slightly, while increasing it decreases the gelation time. However, the gelation time is most dramatically affected by changing the Glycosil and Gelin-S concentrations. Reconstituting these in less DG Water than stated on the vial, will make more concentrated solutions with much shorter gelation times. Alternatively, diluting them in large volumes will dramatically increase the time to form the hydrogel.

22. How much Extracel should I use in my tissue culture dishes? The amount of hydrogel you use is up to you and your application. However, to culture cells normally on the surface of an Extracel hydrogel we routinely use a gel thickness of ~1 millimeter. If you want to make thin films, add a greater volume of hydrogel to each well. Rock the plate to cover the entire well and then pipette out the excess hydrogel. Please refer to our Protocol/Technical section under ‘Info’ for more information on how to coat plates with Extracel.

1 mm Extracel hydrogels

Thin coating of Extracel hydrogels

How to recover cells from Extracel hydrogels

23. How can I detach cells grown on the surface of Extracel hydrogels? You can use trypsin, dispase, collagenase or most any common detachment solution to remove cells grown on the surface of Extracel hydrogels. See our HA Technical page for details.

24. Do I have to use trypsin to remove my cells from the surface of Extracel hydrogels? No. You can use dispase, collagenase or most any common detachment solution to remove cells grown on the surface of Extracel hydrogels. See our HA Technical page for details.

25. How do I recover cells that I have encapsulated in Extracel hydrogels? We have a protocol for recovering cells from Extracel hydrogels posted on the HA Technical page. All involve enzymatic digestion by hyaluronidases and/or collagenases.

Encapsulating Cells in Extracel Hydrogels

26. What can I expect to see under the microscope when I encapsulate my cells? Cells will often take on a different morphology when cultured in 3D compared to 2D on tissue-culture treated plastic. Cells like NIH 3T3 fibroblasts will remain rounded instead of flattening out as they do on plastic.  Also, the cells will not all be in the same plane so that when trying to focus on cells, not all the cells will be in focus at the same time.

27. What does the hydrogel look like under the microscope? The hydrogel is virtually transparent.

Tissue Engineering Applications of Extracel hydrogels

28. Is it okay to use Extracel in animals? Extracel has a well established history of use in animals, but should only be used in animals with accepted protocols by either an internal review board or institutional IACUC.  See our ‘Animal Models Tested’ page for a list of animals used.

29. Is Extracel injectable? Yes. It can be injected subcutaneously or orthotopically.

30. Does Extracel crosslink in vivo? Yes. Extracel is injected subcutaneously or orthotopically as a liquid, but the hydrogel will set up within 5 minutes to over 2 hours (depending upon the Extracel solution concentrations and the amount of Extralink used).

31. Does Extracel provoke any immunological or inflammatory response in vivo? Extracel may generate mild inflammation as a part of the body’s healing response to injury.  It does not stimulate an immunological response. 

32. How long does Extracel persist in vivo? Residence time of Extracel in vivo depends on several factors, including the form in which it is implanted, the amount of crosslinker added, and the biological environment or system in which it is used.  Extracel sponges have been shown to persist in a bone regeneration model for up to 8 weeks, while Extracel hydrogels last up to 4 weeks in a cartilage defect model.  Aggressive environments with high mechanical stresses or blood flow will shorten the resorption period.  Additionally, increasing cross-linking density can lengthen the degradation of the material. 

33. What is the mechanism of degradation of Extracel in vivo? Extracel is degraded in vivo by matrix metalloproteinases (collagenases) and hyaluronidases.

34. What types of mechanical loads can Extracel withstand? Extracel can display compliances of between a few hundred to a few thousand Pa, depending on the form in which it is tested, as well as the material and crosslinking density.  Sponges that have been lyophilized from Extracel hydrogels typically display higher stiffness than the hydrogels themselves. Extracel can be mixed with other matrices or biological components, such as demineralized bone matrix, to increase the stiffness of the construct.  The shear stiffness and tensile strength of Extracel has not been measured. 

35. What types of processing can I use with Extracel? Extracel is a very versatile material.  It can be cast, electrospun, molded or injection molded, and freeze-dried into a variety of shapes and sizes.  Use of excessive heat (greater than 37˚C) is not recommended when processing Extracel; however, since temperature boundaries have not been established.

36. Can I terminally sterilize Extracel? Extracel sponges can be terminally sterilized by E-beam. Extracel hydrogels have not been tested for E-beam sterilization; however, they have been tested for gamma irradiation and it was shown that they cannot be terminally sterilized with this method.

37. Is Extracel FDA approved? Extracel is composed of materials that have been FDA approved and are commonly used in medical devices in humans.  However, Glycosan does not have a Device Master File on record with the FDA and therefore Extracel may be used for research only.

38. Is Extracel cGMP compliant? No

39. Does cross-linking in vivo cause any adverse effects to surrounding biological tissue or the construct itself? None that we are aware of.

40. Do the hydrogels change compliance over time since they have been in the cell culture medium for days? No. The compliance is set by the amount of Extralink, the concentration of the Glycosil and Gelin-S, and the ratio of Glycosil to Gelin-S. Adding the media to the hydrogels does not change the compliance since the chemical structure of the hydrogel is fixed.

Which Hydrogel Kit Should I Use?

41. What is the difference between the Extracel and HyStem kit? HyStem does not contain Gelin-S and is used for growing cells when the user wants to control the addition of growth factors and attachment sites. 

Miscellaneous

42. What is the shelf-life of Extracel? Extracel will last for over one year if stored at -20 °C. If an Extracel Hydrogel Kit is reaching the end of its shelf life, the first indication is that the Glycosil will take longer than 30 minutes to go into solution. Even with longer dissolution times, the Extracel components are still usable. In extreme cases, the Glycosil will form a gel before it fully goes into solution. In this case, the materials are no longer usable.

43. What cell types have been grown in Extracel? Please see our ‘Cells Cultivated’ page.

44. What is the rigidity of Extracel, and how can I change it? Please see ‘Compliance (Hydrogel Dilution)’, ‘Compliance (Extralink Variation)’, and ‘Compliance’ for more information.

45.Why culture cells in 3-D?

Cells grown in 3-D closely mimic cellular behavior in biological tissues. Such 3-D cell cultures are critical for accurate and robust results in future pharmaceutical drug discovery and development, recalcitrant cell cultivation, tissue preservation, and tissue engineering. By contrast, cells grown in a 2-D environment can exhibit different behavior from those that are observed in vivo or in a 3-D cell culture.

References:

G. A. Hamilton; S. L. Jolley; D. Gilbert; D. J. Coon; S. Barros; E. L. LeCluyse, “Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions.” Cell Tissue Research 2001, 306, (1), 85-99.

A. Abbott, “Cell culture: biology’s new dimension.” Nature 2003, 424, (6951), 870-2.