Alginate Gels (2)

Dominik Haudenschild from C. Kuo, March 2007 at TSRI

Make 2% Protanal LF200 Alginate BATCH 5 with 10mM HEPES and 140ug/ml ascorbate as follows: make as 450ml including 10mM HEPES and ascorbate in water, autoclave 30 minutes on liquid cycle (takes 1 hour), then after autoclaving add rest of filter-sterile buffer solution in 50ml. Final concentrations are: 150mM NaCl=4.01g, 25mM HEPES, 6mg phenol red, 4500mg/L Glucose).

1.  Make gels, worked best with 3600ul Alginate and 300ul each CaCO3/GDL, fewer bubbles, only a little settling, and strong gel.

2.  Put cells in gels

1.  Trypsinize 4 T150 flasks of ~75% confluent P1 chondrocytes. 

2.  For each ml of alginate gel desired, add the following:

3.  Resuspend in 850ul 2% Alginate

4.  Add 80ul 0.6M CaCO 3 , mix well

5.  Add 80ul 1.2M Freshly Filtered (<5minutes old) GDL

6.  Aspirate through 18 guage needle in to 3ml syringe

7.  Place on Microgravity Device to prevent settling of CaCO 3

8.  Gels should be solid enough to cut in 50 minutes

9.  Cut sections pushed out of syringe after cutting luer-lock end with razor blade

10.  Put into incubator "dry" to allow further curing of gel about an hour