Hi
Iam making organotypic culture with collagen embedded fibroblast. I took 5ml of rat tail collagen ( 4mg/ml) , added 0.625ml of 10X HBSS with phenol red and few drops of 1M NaOH, the color turned orangish pink, then i added fibroblast ( 10^5 /ml) . The solution was poured in culture inserts (2.5ml) and incubated in cell culture incubator at 37C (5%CO2). After 1 hour I noticed that the gel formed was pale yellow color instead of pinkish color.
Is it due to CO2 during incubation? Since pH is changed my fibroblast will be still
surviving?
Pls advice
Hi Apoorva, if you are using acid extracted collagen, your neutralizing step might have been strong enough. The yellow phenol red means pH~6.0.
You can change the media and see if it helps.
Worse new is if you have heavy bacterial contamination that is changing the pH rapidly.
Another possibility is that you didn't let enough timeto equilibrate pH in the gel.
As Steffansson proposed, you might try to change the medium. Second, your collagen is in an acetic acid solution. So you may calculate how much medium you need to add in order to achieve pH 7.
The color of a collagen solution will sometimes revert over time back to acidic if the buffering capacity is low. I use 10x EMEM for the mix. HBSS itself in the incubator becomes acidic. Additionally, the color should be more red when you add the cells; if the final concentration of the collagen is around 1.5 mg/ml or less, the cells will sink to the bottom before it polymerizes if it is too acidic. If you can get the pH to 7.5 but less than 8, you can quickly mix and plate without such problems. But I agree change from HBSS to a better bufferend formula like MEM or EMEM or DMEM, whatever is available at a 10x concentration. Hope this helps.
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