Hi everybody,
I've tried multiple times to make some fibrin gels, but they always seem to gel on me. What's strange is that part of the solution seems to gel instantly, while the rest gel slower as expected after incubation at 37C.
I've tried creating the gel at room temp, as well as with everything on ice but the same thing seems to happen!
This is my gel formulation: 3 mg/ml fibrinogen, 2.1 NIH U/ml thrombin, 3 mM CaCl2. The gel also contains serum containing cell culture media and 15 million cells/ml.
Could it be that my fibrinogen solution is old? It was purchased 6 months ago.
Or am I not warming up my frozen aliquots quickly enough (I've let it thaw at RT, I also ended up storing it on ice when I tried to do everything on ice)?
Maybe I'm not properly dissolving fibrinogen - I mix it the lyophillized powder (>95% clottable protein) with 37C 0.9% saline, occasionally inverting and then 0.22 um filtering after ~ 1 hour.
I've attached a picture of what happens!
Hi John,
I have two suggestions to avoid this (which is not too surprising I think). Can you (a) use a higher concentration of fibrinogen, and (b) centrifuge the cells to remove the cell culture medium? Once you pelletize the cells, they could be resuspended by the fibrinogen solution (with or without CaCl2) and after mixing you add the thrombin at equal volume. You will get a fibrin gel seeded with cells, if this is what you wish to get. Kind regards, Thierry
Increasing fibrinogen concentration will give you a fibrin clot with a stronger strength, more dense honeycomb structure. However, as you mention this may not be suited for all the cells. So maybe you can use the same concentration as you selected before, and only remove the cell growth medium to avoid the dilution and the inhibition of fibrinogen conversion into fibrin. Good luck :) Thierry
Hi Thierry and Everyone,
I ended up addressing the problem by reducing my thrombin:fibrinogen concentrations. Previously I was using 0.6 NIH units thrombin per mg of fibrinogen in my sample. Now I use 0.2 NIH units per mg of fibrinogen and perform everything on ice.
I also make sure that my 20 mg/ml fibrinogen stock solution is completely dissolved prior to use, because sometimes precipitates form. (They eventually dissolve if you wait a bit after thawing)
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