I tried to obtain bone marrow-derived mesenchymal stem cells of mice. However stem cells don't adhere to the culture dish after the first passage. Maybe anybody faced this problem.
Hi Daria
Please give us more info about how long you culture the bone marrow derived cells and about your culture media. How you separate your cells from dish?
Hi Daria,
I have worked with Mouse Stromal Cells that we ordered from ATCC. These cells adhered well when we started the culture in TCPs. But after we cryopreserved some of the flasks and tried restarting the cultures, the cells did not adhere to the bottom of the flask and kept dividing in the medium. We used DMEM medium with 10% FBS and 1% Penstrep both the times.
Regards
Thank you very much for the answers!
I used the MesenCult™ Proliferation Kit Mouse (Stem cell Technologies). To separate the cells from dish I added ACCUTASE™ (Stemcell Technologies) and incubated for 5-10 minutes at room temperature. I used to separate the cells by Trypsin-EDTA (0,25%). But the result was the same. Before the first passage the cells were cultivated approximately for one week.
Hi again
You are using proper media. I suggest to cultivate the bone marrow derived cells (first hand cells) not more than 3 days. you must eliminate leukocytes from your population. passage the cells in a shorter intervals and this will delete the end cells. Add more serum (about 15-20%) after every passage to your cells to reinforcement your cells.And tell us what happen.
good luck.
How sure are you that your cell population are stem/stromal cells? other cell types may weakly interact with the dish surface. It is possible that you have quite a mixed population of cells, which may affect you stromal cells.
Hello Daria,
you can opt two approaches, first to eliminate any blood contaminants and leukocytes, once you put the cells in petriplate with standardized media+10%FBS and change the media after 3 hrs. Second as in case of animal BMSCs, you can change the media after 2-3 days intially and culture obtained after certain days (around 10-15) can be trypsinized and regrown but in that case you obtain a pure culture after 2-3rd passage.
If cells are not adhering then you can try out 6-well culture plates as cells do adhere very well in them.
Hope it helps !
Also it is good to check if the culture flask used is appropriate for adherent cells or if it is a low adherence plastic.
Hi Daria,
most of the suggestione you have already received from our collegiate here are sound and should fix your problem. I can only suggest a prioritized troubleshooting agenda:
check that in fact you are not using low attachment plastic but also different manufactures, the differences are huge in this respect.
Keep the acutase reaction as shorter as possible, I.e the bare minimum required to collect or dissociate the cells the way you wish to have them for replating
quality of serum is another key parameter. Do you use standardized batches?
poly-lysine is a good suggestion and can be improved by incubating the poly-lysine treated dishes for 2 hours in basal medium containing 20 feral calf serum prior to plating the cells.
if that does not work try coating with a laminin/ fibronectin mix (plenty of protocols out there) or if a non standardised matrix does not bother you can do dish coating with materiel.
If push comes to shove try Cultrex and if even that does not work drop me a line and I will try to send you some SAP based adhesion matrix which is not commercially available just yet but that may help with this specific problem. The latter approach was the only one that helped us to fix a similar issue with colon stem cells from humans so it may work for you as well