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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.



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