Can anyone suggest how to fix my explant for isolation of fibroblasts? actually after I add media to the tissue culture plate having explants, they start floating and there is no growth of fibroblasts.
just slightly slide the biopsy on the plastic, leave 15-20 min then add a drop of medium. After 24 hours, add medium as usual
Dear Monika,
explantation of fibroblasts from skin is not usually so hard, but requires some practice. I the sample in very small fragments (
I have placed the tissue on the dish up to 30 minutes without media, closed tissue culture dish in the incubator, otherwise just like Véronique said, and cells will migrate out. Maybe the smaller pieces can't withstand 30 minutes but the pieces I use are usually 2mm squared.
If you cover the explants with a coverslip, that will hold them down. Some fibroblasts will grow on the dish and some on the coverslip. If you need all of them it is a simple matter to trypsinize them off of the coverslip.
May I ask you all, do you coat your culture dishes with anything (e.g. collagen, Matrigel) or do you use them naked? I will be attempting fibroblast culture from intestinal explants soon.
Emily, for fibroblasts I have always used regular TC treated dishes (static-charge), no need for special extracellular matrices. This has worked with lung and skin tissues. I have not attempted harvesting fibroblasts from other connective tissue sources though. Good luck!
@ Emily
As Melville does I don't use any special matrix protein(s). Uncoated culture dishes have been working with skin and heart valves.
Same here--no substrate needed. In fact in one instance I know it to be detrimental. Gelatin works well and is cheap when used as a substrate for fibroblasts used as feeders; but you don't want to try to expand them on it. I used to work with someone who regularly made that mistake and they are much more difficult to trypsinize when they are happily attached via gelatin.
Healthy fibroblasts tend to be quite robust and need nothing but the charged surface of tissue culture plates.
you have to gently move the biopsy on the plastic to dry it . Wait several minutes and add only one drop of medium. After 24h you can add more medium. Hope this help
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