Tissues were stored in DMEM + 1% penicillin/streptomicin + FBS 20% + DMSO 10%
I thawed tissue quickly and treated with collagenase IA 125U 37 °C 1h. I filtered the tissue and centrifuge at 750 g 10'. Then resuspending the pellets with DMEM/HAM F12 + FBS 10%. I did not see any cells.
I do not understand if the problem is the frozen cells or some step/condition of my protocol.
frozen... you killed the cells, do it with fresh tissue´next
Hi Elena,
there are some questions in my mind. How much tissue did you use? What was the filter size? Are you sure you did not lose the pellet? And how much solution did you use to resuspend your pellet?
I have tried an assay from frozen tissue before where the cells were still alright and intact... I haven't tried SVF isolation in particular from frozen tissue but I believe you should still be able to find some cells at the end...
Dear Elena,
I agree with Veronica. We always isolated preadipocytes from fresh tissue because freezing damages the cells and it is not many preadipocytes which you get out of intact adipose tissue anyway. However, when working with stool samples, one can homogenate it adding 10 % glycerol as cryoprotectant, freeze immediately in liquid nitrogen and there will be quite a lot of bacteria alive when thawing the samples. It may be worth trying this prodedure with adipose tissue (homogenating it would be essential so that glycerol also gets in contact with the cells). Skurk et al has published a paper which allows you to passage the preadipocytes from primary culture several times (if you do not have so many cells) and these cells keep the capability to differentiate.
Good luck
Hello everybody, thank you for your interest in answering.
I cut the tissue in small pieces 10mm (n=4). I used 100 um filter and 5 ml to resuspend.
Hi Elena,
Isabelle is right, it is hard to find alive preadipocytes (or progenitors) in the frozen tissue. You need to cryopreserve it appropriately to be able to use it again.
But if you don't have access to fresh tissue, I would give it another try just to be sure.
100um filter is fine, try to chop them more finely and wash them with cold sterile pbs.. Instead of 1 hour, incubate with collagenase maybe 40 minutes.. And be very gentle about washing the pellet.. 500 g is enough for centrifugation.. And 5ml is too much, use maybe 1 ml or even less..
These are my suggestions, good luck :)
Hello Elena,
do you use serum free medium when you treat your tissue with collagenase? Please keep in mind that serum inactivates collagenase. Like Fahrettin said, try to mince your tissue as small as possible, use collagenase in serum free medium for maybe 30-40 minutes, inactivate collagenase with medium+serum. Filter your tissue and centrifuge at 500 g for 10 min. Carefully wash and then resuspend your pellet.
Good luck
I used solution w/o serum for collagenase. I used HBSS with magnesium. Probably I should add also medium that contains zinc ...
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