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General Protocol 3:
3-D Cell Culture with 3-D Life ToGro Hydrogel

1. Introductory Notes

 

2. Protocol

2.1. Embed cells in ToGro Hydrogel

Reagents and materials

3-D Life products:

 

Additional Reagents and materials:

 

Preparations

If not already done for previous gel preparations, reconstitute the RGD-Dextran and CD-Link lyophilisates as described in the 3-D Life ToGro Product Data sheet.

Note: Do not expose CD-Link to air longer than necessary to avoid oxidation of the thiol groups. Close
cap after each use. If CD-Link is used for longer than one hour, keep it on ice or at 4°C.

Experimental procedure:

If not indicated otherwise all steps below are performed in a sterile hood:

 

2.2. Dissolving ToGro Hydrogels with Dextranase

ToGro Hydrogels containing live or chemically fixed cells can be dissolved by adding dextranase to the culture medium of the hydrogel culture. For example, a 25 µl gel can be dissolved with 300 µl of a 1:20 dilution of dextranase in medium incubated for 30-60 minutes at 37°C. Gels can be dissolved faster, if they are cut in pieces.

After dissolution of the gel, centrifuge the cell suspension and resuspend the pelleted cells in fresh medium or physiological buffer as required. Repeat this washing procedure once or twice to more effectively remove remains of dextranase and dissolved gel components. The removal of dextranase is important when cells are being embedded again in dextran hydrogels to continue culture. If dextranase is not removed completely, it can destabilize the newly set up hydrogel.