To improve MSC yield and growth: optimize centrifugation to 300–400 ×g, reduce FBS to 10–15% (preferably MSC-qualified), increase plating density, delay the first media change to 48 hours, and perform regular medium changes afterward. Using high-quality plasticware and careful monitoring of morphology will also support successful culture. If problems persist, you might also consider isolating mononuclear cells with Ficoll or Histopaque gradients as a cleaner starting point.

Thank you very much for your response — the information you shared was very helpful for me.

I have a few short questions, if that’s okay:

• For MSC isolation, is 300 ×g for 7 minutes centrifugation appropriate? Or should I adjust the time?
• Is centrifugation temperature important? I usually do it at 15°C — do you think that’s suitable?
• You suggested doing the first medium change after 48 hours. But I usually change it at 24 hours because I worry about hematopoietic cells attaching. Could this affect MSC attachment or purity?
• Would you recommend a gentle wash before the first change, or is it better to leave the culture undisturbed?

Thanks again — your support means a lot to me.

Hello, Tugba Yalcinkaya .

I collect some suggestions to improve enrichment of MSC from our technical team:

(1) High concentration of FBS (20%) may promote the growth of miscellaneous cells (such as hematopoietic cells) and inhibit MSC adhesion. The concentration is generally 10% (slightly different from the suggestion made by Supti Das );

(2) The culture medium can be replaced with α-MEM.

(3) The obtained cells need to be added with red blood cell lysis buffer, and the steps are as follows: pass through a 70 μm cell sieve and centrifuge at 1500 rpm for 5 minutes. Treat with red blood cell lysis buffer for 5 mins at room temperature and wash with PBS twice.

Reason: Granulocytes, red blood cells and lymphocytes dominate the bone marrow. Although the proportion of MSCs is extremely low, after removing non-target cells by lysing red blood cells, they can be gradually purified from mixed cells by using their adhesion properties.

(4) If the total number of cells collected is not large, all the collected cells from a mouse can be placed in one well of a 6-well plate for culture.

(5) You are currently using direct centrifugation. You can also consider density gradient centrifugation to improve the enrichment rate.

(6) To promote proliferation, add some cytokines, 1-2 ng/mL bFGF (basic fibroblast growth factor).

Tugba Yalcinkaya ,

I noticed that you continued to ask about cell inoculation and culture:

MSC isolation:

We usually filter the cell suspension obtained from the bone marrow sample with a 70 μm filter to remove bone fragments and impurities. Then centrifuge at 1500 rpm for 5 minutes and discard the supernatant.

Initial inoculation:

It is recommended to resuspend the cells with complete culture medium and inoculate them in a T25 culture flask (density: 1×10⁷ cells/flask). After standing in a 37℃, 5% CO₂ incubator for 24 hours, perform the first medium change to remove non-adherent cells.

Frequency of medium change:

In the first few days, change the medium once daily to remove other cells that are not attached to the wall. Change the medium every 2-3 days afterwards and observe the morphology of adherent cells (MSC should be long spindle-shaped and grow in colonies).

Precautions for medium change:

The first medium change should not be too early to avoid loose adhesion. Subsequent medium changes should be gentle.

Of course, you can do appropriate cleaning before the first medium change, but the number of washes should not exceed 2 times to avoid cell loss.

One more important point, the whole bone marrow adherence method may be contaminated with hematopoietic cells, and purity can be improved by multiple medium changes and passaging.

If you have any other questions, please contact me.