Hi, I am trying to get the dermal human fibroblasts (isolated from skin biopsies) to secrete collagen and I want to harvest this collagen and look at them under AFM.
I was wondering if anyone has a working protocol to share with me.
I appreciate your help,
You should add vitamin C (ascorbic acid) to the culture medium. In the range of 65- 180 µg/ml (or 10-3 - 10-4M) and culture for at least 2 weeks. You will see a wavy pattern appearing on the surface of the culture flask.
see also Effect of vitamin C and its derivatives on collagen synthesis and cross-linking by normal human fibroblasts.
Boyera, Galey, Bernard in 1998 Jun;20(3):151-8.Int J Cosmet Sci
doi: 10.1046/j.1467-2494.1998.171747.x..
@Bouke Boekema
Thank you. Should starve the cell before adding vitamin C . Also do you think adding TGFb helps?
You can also seed the cells so that they are fully confluent the next day, incubate in the presence of vitamin C (TGF-B should also help) and then remove the cells, leaving behind the ECM so you don't interfere with the structure. I have previously got a mature ECM after 21 days in culture
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