I have had isolated bone marrow mesenchymal stem cells from both C57BL and BALB/c strains. The cells obtained from BALB/c mouse are growing normally without any problems but the C57BL cells are not in the same way. Their growth rate is extremely low. The cells are very sensitive and I miss a lot of them during sub-culture. I use low glucose DMEM supplemented with 15% FBS and 1% pen/str for my culture. I have used female animals in the case of both strains and I have tried several C57BL mice with the outcomes almost similar. The mice were weighting 20 g.
My sub-culture protocol is 2-3 times of PBS wash followed by 1 ml of 1X trypsin/EDTA for a T25 flask and 5 minutes of incubation at 37.
Can anyone help me with troubleshooting?
Hello Alireza.
It is very unlikely that both strains of mice have different proportions of BM cell phenotypes in their marrow. It may be the inexperienced and inconsistent operations.
They include the way of cutting the ends of the bones, flushing the bone marrow, T-E treatment including immediate stopping further digestion with serum and complete removal of remaining trace of T-E solution by enough washings, the first length of the cells plated to allow the cell attachment to the bottom of the dish (3~4 days), and re-feeding the cells with no disturbance (change of medium alone). After maximum proliferation and de-selection of the cells with no fitting in the culture condition, the cells are ready for MSC culture condition for subsequent proliferation.
Select male mice instead of female mice who show estrus-cycle-dependent hormonal activities to eliminate any possible individual differences. The juvenile females get first heat under good condition. The female mice start the first estrus cycle around 20g body weight.
Regards.
Dear Sang,
Thank you very much for your comprehensive answer. I really enjoyed reading your comments.
Thanks!
Hello
Yes, I agree with Sang Ho Lee, its a great answer.
For your experiment, the males mice are better than female additionally, the age is very important and must be kept your experiment in standard conditions.
mMSC primary culture consisted of ( LG-Dulbecco’s modified Eagle’s medium (LG- DMEM; containing 10% fetal bovine serum ; and 0–5Æ96 g/l HEPES buffer and antibiotics p/s 10% )
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Good Luck
We use C57BL female mice routinely and we get good cultures of MSCs. However we use IMDM with 20% MSC specific serum (GIBCO). MSCs are very sensitive to serum and so using good quality MSC specific serum in imperative. We use TPVG for trypsinization but it has to be quick as these cells succumb to excessive trypsinization. What is the age of the mice that you are using? Usually it should be between 6 to 8 weeks to get good MSCs. With age the quality of MSCs may deteriorate. Also do not seed very high/less numbers of cells in plate after dissection. Very high numbers may lead to HSC contamination of MSCs (we faced this issue in our lab recently). Very low numbers on the contrary may challenge the growth of the MSCs. When do you change the medium of the cells at passage 0? Try doing full medium change after 48 hours of culture at passage 0 and before splitting them.
Special thanks to dear Saleh and Anuradha. I finally got appropriate cells from C57BL mice by changing the medium only 72 hours after bone marrow flushing. I noticed that the cells from C57BL mice attached with some delay compared to BALB/c cells. Actually, sufficient initial cell numbers seems critical for suitable cell expansion.
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