Hi, I'm facing a lot of difficulties in the culture of adipose derived stem cells (ADSCs). I use tissue explant method for the isolation of ADSCs; generally the cells start migrating out of the tissue within 7-10 days. But the cells do not survive the culture conditions after 2 or 3 passages. I have used alpha minimum essential medium supplemented with 10% fbs; dmem + 10% fbs; and dmem/f12 supplemented with 100ng dexamethasone + 10% fbs but they don't seem to work. I'm attaching the microscopic images of the same. Can anyone please tell me the reason for this and suggest me any changes to improve their viability?
Do you use tripsin 0.05% or tryple? Mesenchymal are sensible to that. Tripsin 0.25% usually stress these cells.
I have just started to use 0.05% trypsin (on a revived stock) but i think its too early to say anything because the cells did not survive after the first subculture.
Hi
Enzyme extraction (collagenase type I) would be the best method for isolation of these cells. For the zero passage DMEM-f12 accompanied with 15% would be the best medium than alpha. Your cells morphology is getting flatten at 3rd passage that is caused by high trypsin exposure time. If your are using 0.25% the time must not be overridden (about 1 min). But if the cells are not getting through the first passage, change your medium to F12 and collagenase isolation as your medium contained to many debrids that could induce apoptosis by themselves.
Best
We have produced AD-SC in large quantities by using MEM alpha and 10% in house prepared platelet lysate, and they grow well in our lab. For passage we used TripLE Select, and reculture without washing step, just deactivate the enzyme by equal volume of complete medium, and after they attached the next day, we change the medium, so avoiding cell lost due to centrifugation.
We have used FBS 10% and trypsin for harvest before, but the cells tend to age faster, so now we did not use FBS and trypsin anymore.
Perhaps the cells that are growing out of your tissue explants are just ordinary fibroblasts with limited proliferative capabilities- why would you expect that stem cells would grow out of unfractionated tissues given the rarity of this population? I'd strongly advise trying to isolate based on known cell surface markers of adMSC populations rather than hoping to establish bulk cultures.
Brenton, I have characterized these cells based on their cell surface markers (as per ISCT guidelines). These are indeed mesenchymal stem cells. I have no doubt about the identity of these cells; however the problem I'm currently facing is their limited profileration with subsequent necrosis in vitro.
Cultured cells all end up looking alike phenotypically, there's nothing like a good FACS session to get a known population of cells. If you're having issues growing them I'd suggest trying a combination of: higher serum (20%), batch tested serum screened on MSCS, pre-coat wells with an appropriate ECM protein (Fibronectin is good, Laminin 10/11 is better but expensive), switch to a chemically defined or serum free MSC media (the one from Stem Cell technologies is great, I made it), dissociate with collagenase 1 instead of trypsin (remember to wash it off). Good luck.
Hi Aditi, try to use 0.1 % collagenase Type 1 (we have best results with Collagenase from Gibco; cat. No. 17100017). Digest adipose tissue for 60 min at 37°C with gentle agitation. Obtained ADSC may be expanded in D-MEM (low glucose) supplemented with 10% FBS and antibiotics.
We use in our lab DMEM medium low glucose supplemented with 10% FBS, 2.5 g of free acid HEPES and 3.7 g of sodium bicarbonate. We cultivate the cells very well here, we have already reached the thirteenth passage without problems or appearance of cellular suffering.
Thanks Lubos and Laynna. But can you please tell me which brand of FBS do you use? I think its probably because of serum that the cells are becoming proliferation arrested.
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