I have currently been trying to isolate pancreatic mesenchyme. We have been mincing the pancreas, digesting with collagenase, centrifuging and seeding the entire digest on tissue-cultured dishes. The entire digest has been cultured in DMEM + 10 FBS and 1% penicillin/streptomycin.
The protocol is perfect.All plastic adherant fibroblast like cells will be mostly mesenchymal stromal cells.You have to discard all floating cells containing acinar fraction and islets. Once monolayer is complete you can subculture.the MSCs and characterize by surface CD markers and adipo,chondro,osteogenic differentiation.
We have been trying to do that using pancreas from one wister rat, but we still fail to find any adhering cells. Could this be due to the fact that there is very little MSCs and thus we should consider using more pancreatic tissue?.
You have to take entire splenic as well as mesenteric pancreas from Wistar rat.After 20-30 minutes collagenase digestion yoy have to take pellet containing all cellular components. You may use DMEM KO + 10% FBS as culture medium. You willstart getting MSC colonies from 48hrs post seeding.
Is there a specific reseason why knockout DMEM is used?. I see that most protocols are using that.
YES. DMEM KO is an ideal medium for the growth of MSCs. We have tried it for mouse pancreaticMSCs. We have found that it works efficiently even with human detal pulp mesenchymal stem cells.It has high glucose concentration and yet it is not toxic to cell attchment and proliferation .
Ohk i will keep that in mind. We have been making use of "starndard" DMEM. Just for curiosity how much pancreatic tissue do you seed onto the culture plate. Tissue from one or two rats?
i am actually using the article that was published by your lab as a protocol. It was written by Mr Gopurappilly
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