Hi everyone,
I am trying to produce extracellular matrix using NIH-3T3 cells. According to previous literature the cells need to be quite old, around 20 passages before you use them to produce matrix. So, I have cultured them until they reach passage 20 and then I feed them for 9 days with ascorbic acid. I lyse them and then I stain for collagen I fibrils. There is one problem though: my fibrils are too thin!
I have seeded the cells in high densities trying to get thicker fibrils but either the lysis is incomplete or the medium turns yellow quite quickly. Also, if I feed the cells with higher volume of medium, they overgrow and again don't lyse properly!
Any ideas what could be the reason for the low collagen I secretion? There is one thing though: In order to use the cells quicker, I split them 3 even 4 times per week.
Hi Despoina, I do not have much experience with 3T3 cells, but ECM production by cells that I have worked with requires 1-2 weeks of continuous culture of sparse populations (~5-10% confluence).
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