Hi.. We have tried to culture these cells with keratinocytes serum free media. We also coated the plate with collagen type IV. But the cells are not growing more than 2 days in the same plate. Please kindly tel me what kind of media and coating of plate I should use.
Please suggest me some protocol to grow these cells.
Hello
Cell Culture with DMEM/Ham’s F12 (1:1,vol/vol) with 10% fetal calf serum (for human cells, human serum used instead of fetal calf serum), 2% L-glutamine,100 IU/mL penicillin, 100 μg/mL streptomycin, and 1% amphotericin . then place in 37°C , 5% CO2 humidified incubator.
Good Luck
Hi Narasimhan,
I use DMEM-high glucose with 10% FBS, 1% glutamine, 100IU/ml penicillin.
if you are establishing a primary culture, collect the pancreatic tissue in serum free DMEM. Mince the pancreatic tissue in enzyme mixture (0.1% trypsin and 300U/ml Collagenase II) and incubate for 1 1/2 hr at 37 C shaker. Wash with Wash buffer(1% FBS+1x PBS) , spin at 1000 rpm for 10 mins.
Plate the tissue chunks in the flask with minimal amount of DMEM media such that the tissue doesnot float. Incubatefor 48 hours and observe for any explant culture. discard if unadhered.change media after 48 hrs and grow till confluency.
If u already have PSC line after thawing or while plating make sure you add minimal amount of media so that it adheres to the plate. you can also give FBS coating for an hour (incubate at 37C in 5%CO2)to the flask before you seed the cells. once it has adhered you can increase the media volume
once you trypinise the cells the collagen coating is also lost. hence your cells might not adhere to the same plate.it is better if u can change the flask atleast after the 2nd trypinisation.
Hi.. Thanks a lot for all your comments. I wil try to culture the cells with all your recommended conditions.
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