Hi all,
I am trying to grow human bone-marrow derived mesenchimal stem cells and they take ages to grow in a T75. When i get confluence after 2-3 weeks and passage them into a T175, they grow much slower and stop at a confluence around 60%.
I was wondering whether the % of FBS should be bigger (perhaps 15 or 20% instead of 10%).
I have also noticed that the number of cells that I achive from a T75 flask near 80% confluence is around 250k, whereas other types of cells yield 1kk+. Is this normal due to the flattening and expansion of MSC?
10% FBS is good. I isolated MSCs from Fresh bone marrow. First isolated MNCs isolated from bone marrow by Ficoll gradient are grown in Bigger dishes. First set of cells attach very slowly and take 2 weeks, after that they grow faster. 1-3% MNCs attach to make stem cells, rest not. I do not recall now exact.
One thing I want to make you clear, I isolated MSCs from around 100 different patients. There is a big variation between patients how successfully patients bone marrow grow.
There is possibility that the batch of stem cells from a bad batch. You might need to get from a different batch. Or isolate from a different patient. Some patients are blessing, I get hundreds of millions after 3rd passage and some patients very very low number and I had to discard them.
You can use my method (reprint enclosed) if you are isolating from patient or volunteers. If you have any question, please let me know. My method works very well.
My media worked perfect. Sometimes FBS batch also has variation. Some FBS batch works better than other.
Subhash C. Juneja thank you a lot for your help!
I've got the cells already isolated, and frozen. I have them on different passages (either p2 or p6). In my case I was using aMEM with 10% FBS, no glutamax no penstrep. Perhaps the difference in medium (you using DMEM, and me aMEM) has also some effect. But it seems that our timings are pretty similar.
For some reason, though, when I passage them from a T75 to a T175, they suddenly stop growing I don't know why.
Could you also give me an approximation of the number of cells you got out of a T75 and a T175 in confluence? So that I can also plan how many cells grow based on my future experiments? I wouldn't like to waste cryovials.
Again, thank you a lot for helping me!!
*1 million MSCs recovered in T-175 flask at 80/90% confluency. Split in 3 flasks from one flask after trypsinization.
*At passage 3, I used to freeze, than thaw when needed. Upon thawing, I used to use for making cartilage by differentiation
*Thawing of vials done at 37C waterbath,
*Glutamax is important to add.
*Pen/step is good to to add to avoid any contamination or any alternative
**In brief, my medium worked very well. Catalog numbers are in the paper. If you can just repeat following my medium, should work.
Best wishes
Yes, proliferation of bone marrow stem cells takes longer than adipose tissue stem cells. So I had increased FBS from 10% to 15% in the bone marrow.
