I will be isolating MSCs from BM of 6-8 week old mice. I am planing to deplete the hematopoietic cells, endothelial cells and erythrocytes using magnetic beads against CD45, CD31 and Ter119 respectively right after isolating bone marrow cells. What could be the best seeding density for the remaning mesenchymal stem-progenitor cells?
it depends on viability (maximum 5-6 day) and proliferation of BMMSCs.
best seeding density will be 20000 cell per well in 96 well plate.
these answer is based on our study......
Hi Nese. We usually plate whole BM cells at a density of 2x106 cells/cm2.
Be aware that this should be reduced during subcultiring, as your MSCs start to predominate. Also, if you deplete these populations, the ideal density will probably be lower than mine, since we plated whole bone marrow cells (we only eliminated the erythrocytes by incubating in ACK lysis buffer).
The CD31 endothelial cells did not survive in our MSC culture medium (aMEM medium). Most of the hematopoietic cells in BM are not adherent, so they are removed after the first medium change. The problem are the CD45/CD11b+ macrophages, which firmly adhere to cultures. But we get rid of these cells after 4-5 passages.
Dear Madam
If you cut the ends of the Femur bone and wash with DMEM then use the centrifuge after this time use the ficol finally centrifuge and transport to the 25 flask ,you will see after 4 days many BMS cells after 2 media change you should 1 passage .
Honestly after 3 passages you will have best confluent then for labeling you can use the CD 73 .
Sincerely
Hi Nese:
If you are growing in the cells in atmospheric oxygen it is best to plate them at 5000 cells/sq cm. The higher plating density helps protect the cells from oxidative stress but still allows a modest degree of expansion. If you grow the cells in low oxygen than 1000-25000 cells/sq cm works well. Primary MSCs grow slowly and you will not be able to expand them long-term without risk of immortalization. However, you should be able to maintain the cells for several passages. This all depends on how they were isolated and their overall purity.
Thank you everyone for the answers. However, after the magnetic depletion, cells were dead. Can anyone think of a reason for that? For the staining with the Abs against CD45, CD31 and Ter119, I used PBS as staining buffer supplied with 0,5% BSA and 1mM EDTA.To culture the cells I am using mesencult medium from Stem Cell Technologies.
Answer: 1x10.000/mL, but those cells need the Growing Factors, like IGF-I or TGF-ß! Good Luck!
Nese, how did you access the viability of your cells after selection? Also, do you make three rounds of selection (one for each marker) or just one with a simultaneous staining of the 3 markers?
Dear Nese
About the Magnet :
Let me know , which did you use Tesla potential of magnetic field?
I used biotinylated abs against three markers and stained them all together in one round. Afterwards, I used streptavidin beads and columns from MACS to do the depletion then just seeded the flow through. I dont know whether I had stromal cells after the depletion but since nothing attached to the plates, I assumed that even I had stromal cells they were dead. I dont think the growing medium is a problem because I am using mesencult medium
Just a quick explanation why I am using magnetic depletion. I want to get rid of all the other cell types as soon as possible because in the downstream application I need a population composing of both mesenchymal progenitor and stem cells. Since after 14days or so 'real stem cells' start to express other markers and somehow differentiate, I dont want to keep them in the culture so long.
How many cells did you start with? MSCs are such a small portion of BM, if you didn't have a large starting number, there simply might not have been enough left.
10 million which is the limit for MS columns of MACS. However, next time I will try LD columns and start with 100 million cells.
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