Does anyone know of a protocol to generate primary fibroblast cell cultures from frozen tissue? I have both tissue that was placed directly into cryotubes and frozen with no cryoprotection and I have a few samples that were placed into cryoprotectant media before freezing.
Most of the protocols I am seeing, and all of the people I've contacted in person, have only cultured from fresh tissue.
Thanks in advance!
Hello Megan,
I might be totally wrong but if there is no cryoprotectant e.g. DMSO in those tubes, and kept frozen then cells would have shocked and damaged. depending on freezing techniques used the cell damage might vary. in particular, if they are frozen fast using liquid N2 then I believe it is waste of time to play with. also if larger than about 2cm then probably no viable cell left.
so if you have samples not snap frozen, have cryoprotectant and are not larger than 2-3 cm then it should be possible to obtain viable cells.
Regards
Amin
Thanks, all.
I should add that I've had some luck getting cells from the tissue frozen without cryoprotectant-- these samples were collected before I joined the lab and were all I initially had available. But they grew very slowly, did not look like healthy fibroblasts, and cultures all failed after splitting. Also, Agata-- you're 100% right, I got nothing from the samples I tried to dissociate.
Eventually, I got the pathologist to put the samples into vials of cryoprotectant I'd prepared before freezing. But I have very little of this newer tissue, so not a lot of room for trial and error.
From reading your responses, I think my plan is to 1. thaw the cryoprotected specimens, 2. cut into 0.5mm square, 3. plate onto adherent plates (I'm still debating whether to use gelatin, matrigel, or collagen...)
Whether or not cells grow from this round of plating, I'll probably then enzymatically dissociate and replate the tissue sections to try to coax a few more cells from the tissue, similar to the protocol in the linked paper-- thanks for sending.
Difficult project... fingers crossed I get something viable.
Thanks again!
Hello Megan,
Finally, did you get any cell from your samples??
Thanks!
Ricardo
Hi Ricardo. Although I did get cell growth from my samples, the cells grew very sluggishly, despite the addition of many growth factors. Although they survived the thaw, they unfortunately did not survive passaging. Unfortunately, I was not permitted to be present in the medical examiner's lab at the time of the tissue collection and freeze-- I provided tubes with DMSO + freeze media for the pathologist. Many of the samples the pathologist put in the tube were just too large. So I ultimately did not have luck with this, but I do think it's possible. The above suggestions were very helpful. Certainly it's better to culture fresh if you have the option, though.
Thank you very much for yor answer. Nowadays, i'm culturing from fresh samples, but i have some old tissue samples in the freezer and i was wondering to use them.
Best regards,
Ricardo
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