I would like to test the gelation conditions before I do 3D culture in collagen. Here is my setup (everything on ice before plating on 24-well plate):
90uL of 3.5mg/mL rat tail type I collagen
10uL of 10X PBS
0.5uL of 1M NaOH
(I have actually tried different volumes of NaOH and 0.5uL seems to give a pH closer to 7. )
After incubation at 37oC for 30min, the middle of the gel looked cloudy and the surrounding remained colorless and somewhat aqueous. Same for 2hr incubation. The main problem is that after I overlaid culture medium on the gel and put it at 37oC overnight, the gel seemed to dissolve in the culture medium.
The collagen I used have been frozen at -20oC for quite a while before I thawed it for use. Can it be the reason? Or do you have any suggestions for the gelation of collagen?
If type I collagen doesn't gel when at neutral pH and 37C, that implies that the protein has been either denatured or otherwise degraded in a way that prevents self-assembly. If it was stored in solution at 20*C for a prolonged time, that may have caused some degradation. Storage in lyophilized form until ready to use may be a better method. Also, I have seen significant lot-to-lot variability that results in variable gel formation. Testing several lots may help identify a superior lot.
Type I collagen stock solutions should be stored above freezing. You might try very slow thawing of your collagen prep in the ‘fridge.
Did you adjust the pH to 7 at 4 C or room temperature or 37 C? pH substantially decreases by increasing the temperature. You would expect a pH around ~5.5 at 37 C if you had adjusted it 7 at room temperature.
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