Hello Everyone,
We are inexperienced in culture of MSCs trying to grow a sample from BM of a mare. It is the second time that after preparation of new medium (DMEM-F12 with high glucose - 10% Hy clone - 1x gentamicyn) and complete renewal, the cells suddenly stop growing and start to dye. Any possible cause? Same lot number of all the reactives. Any advices?
Thank you all in advance!
Enkhtuya has hit the nail on the head. For details please read the attached document. It is phenomenal. Article Characterization and Expression of Senescence Marker in Prol...
How many passages have you done with your MSCs? Usually in our hand, MSCs will not continue to divide after 2 passages since they go through senescence. We usually keep isolating primary MSCs every time for each experiment, and use P1 for real experiments. If your MSCs become bigger, transparent and slow down the growth, then it indicates the senescence. Then they will start to die.
Enkhtuya has hit the nail on the head. For details please read the attached document. It is phenomenal. Article Characterization and Expression of Senescence Marker in Prol...
I would suggest using Iscove's modified Dulbecco's medium (IMDM) for MSC culture. It is actually virtually identical to the DMEM/F12 mixture - I know this because I did the comparison of the components. That saves you the time of preparation of the DMEM/F12 medium.
Cell senescence is certainly a problem, but that usually occurs after ~50 cell divisions. What is more likely the problem is contact inhibition. This is an old phenomenon that was well known back in the '60s - '80s, but tends not to be talked about in current literature.
Many normal adherent cells lose the ability to proliferate if they are allowed to grow to confluence. If you subculture them at confluence, they will not grow. But if you subculture them at ~ 50% confluence, they will continue to grow until they reach senescence.
I found this to be true when I was examining human MSCs derived from bone marrow back in the '90s. I was interested in establishing stromal layers to support human HSC culture. Initially, they grew quite well. If I allowed the cells to reach confluence, I was completely unable to subculture them. But if I passed them before they became confluent, I could.
Try adding bFGF (2 - 5 ng/mL) in your medium just before addding it to the cells. Your MSCs will keep on growing for more passages without being senescent or losing their differentiation capacity. I assume by "10% Hyclone" you mean the Hyclone serum right? If not, then you should def also add serum in your medium.
Panagiota Moutsatou raises an issue that I forgot to mention about serum. FBS can vary a lot, particularly as it relates to culture of bone marrow derived cells. I have no experience with HyClone for bone marrow cells - I used it a lot with cell lines - but typically it is recommended that you test various sources of serum to see which is best for your purposes.
The second point is in regards to whether you are heat-inactivating the serum or not. Heat inactivation of the complement complex in serum is essential for culturing myelomas and hybridomas that produce antibodies, but it can be very detrimental for culturing hematopoietic and other bone marrow derived cells. I have not heat inactivated FBS for hematopoietic or MSC cultures. I do not know if it affects MSC cultures, but I do know it can adversely affect HSC cultures.
Thank you Maurice Ekpenyong for the document!
Gary Lee Gilmore we buy DMEM-F12 already mixtured and it is the same that we use for embryo culture. Regarding FBS, unfortunately we are unable to test different sources but we do have inactivated (the Hyclone one that we used is actually heat inactivated) and not inactivated. We certainly will try with the other one!
bFGF would be useful for sure. Unfortunately, we are unable to afford it due to the amount that we would need.
Thank you all!
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