ECM Protein Incorporation
Introduction
This protocol is outlined for incorporating laminin into a 12.5 mL HyStem®-C kit. However, it can be easily adapted to HyStem® and HyStem®-HP kits of any size, or for other ECM proteins. HyStem-C includes cellular attachment cites in the Gelin-S but additional ECM proteins are needed for some cell types. HyStem hydrogel kits not containing Gelin-S will not support any attachment without additional factors.
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. HyStem®-C hydrogels (12.5 mL) with laminin can be prepared in the following manner:
Required Materials
- One HyStem, HyStem-C, or HyStem-HP hydrogel kit
- ECM Protein
Steps
- 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).
Note: Gelation will occur in ~20 minutes.
Protocol Variations
- The amount of laminin can be increased or decreased.
- The laminin source can be varied.
- Other extracellular matrix (ECM) components (e.g., collagen, fibronectin, vitronectin, etc.) can be used in place of or in conjunction with laminin.
- The amount of cells encapsulated can be increased or decreased.
- Cells can be plated on top of the hydrogels instead of being encapsulated.
- 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.
- Other kits can be used instead of the HyStem-C Hydrogel Kit.
Ordering Information
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DG Water
SKU: Multiple SKUS - View Products OptionsDegassed, deionized water at pH 5.0-8.0. Learn MoreThe HyStem family of products for research and development is now being exclusively manufactured and sold by Advanced BioMatrix.
Please visit their homepage.Starting at: $11.50
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Fibronectin
SKU: 5050Fibronectin is used as an additive in HyStem® hydrogels to customize the cellular attachment surface. Learn More$190.00 -
Nutragen® Bovine Collagen
SKU: 5010-DNutragen® collagen is ideal for coating of surfaces, providing preparation of thin layers for culturing cells, or use as a solid gel. Learn More$365.00 -
PureCol® Collagen
SKU: 5005-BPureCol® collagen is ideal for coating of surfaces, providing preparation of thin layers for culturing cells, or use as a solid gel. Learn More$350.00 -
VitroCol® Collagen
SKU: 5007-AVitroCol® collagen is the first widely available, naturally produced purified human collagen for research purposes. Learn More$270.00
