Hello;
I isolated Mesenchymal Stem Cells (MSCs) from Bone Marrow (BM). My very first 3-4 isolation was all right but then I started to have some round different cells in my culture. Microbial test results are all right so I know that it is not a microbial contamination.
Here is my protocol;
**MSC ISOLATION FROM BONE MARROW
1. Dilute bone marrow with PBS (1:1)
2. Add 2.5X ml diluted BM onto Xml histopaque very slowly (histopaque a density gardient medium like ficoll)
3. Diluted BM is layered carefuly on the top of the histopaque
4. Centrifuged 430g for 30 min
5. Aspirate the buffy coat and collect in a 15ml falcon tube
6. Complete the tube with PBS and wash at 430g for 15 min
7. Discard the supernatant
8. Re-suspend the pellet with DMEM, count mono nuclear cells
9. Seed 16x104 mononuclear cells/cm2 into ... T175 flask for the patient ...T25 flask for mycoplasma test ... 6 well plates for differentiation tests.
10. Leave the cells in the incubator for 4-5 days , let the MSCs adhere onto flask surface
11. Collect the medium and change the medium once in 3days **Grow the cells around 3 weeks for the patient **Mycoplasma medium is without antbio/antimyco, test after 2-3 weeks **After 10 days in poliferation medium, add differentiation medium into 6well plates and do the differantiation tests after 10 days
ALSO seed MSC into 6 well plate in proliferation medium for CFU-F test.
1st well 1x103 cells/cm2 2nd well 2x103 cells/cm2
3rd well 5x103 cells/cm2
Grow them for 1 week and check the collonies
If you could check my protocol and help me I would appreciate it.
Thank you.
Hi Berrin.
How many passages has the cultured cells shown in the images?.
Hello Luis;
it is the primary culture, directly from the BM. I thought there might be some bone cells but still there are some other round cells. Do you think that my protocole is all right?.
and thank you.
Hi Berrin. In my opinion, it seems that your protocol is OK, but you will only be 100% sure when you perform a flow cytometry test with the ISCT positive and negative markers.
In the passage 0, so many cell types attach to the bottom of the flask, not only MSCs. It will take some days for this "contaminant" cells to enter in apoptosis, detach from the bottom and acquire that round shape.
When you tripsinize the cultures and seed again in the next culture passages, many of this contaminant cells will not attach again to the bottom and will be discarded. You will not have a really MSC enriched culture probably up to passage 3.
With the info provided, this could be the "easy" explanation of the images.
Hope it helps!
but according to protocols that I checked, in 4-5 days just MSCs attach to the bottom. Also my very first 3-4 isolation did not have these problems.
My flowcytometry results are not really good too.
But thank you very much Luis I was suspicious about my protocol but it seems it is all right.
Hi Berrin!
I use the same protocol with ficoll to isolate MSC from Bone Marrow. The cells images remember me fibroblasts induced to senescence by oxidative stress. What kind of medium do you use for growing? DMEM supplemented with FBS, antibiotics?
Hi Sara;
Ficoll wouldn't make a big difference, well.... I guess...
I use DMEM low-glucose supplemented by antibio/antimycotics and FBS.
Do you think is it all right? and other supplement suggestion?
Hace you changed the FBS batch?. Sometimes with diferent lots of the manufactuter the cells shows diferent growth patterns.
ok, I use the same with 10% of FBS. Sometimes I prefer increased the FBS at 20% especially during the first passages after isolation for better cell growin.
For these cells I add also 1% of L-glutamin.
You can try to prepare a new fresh medium with fresh aliquote of each supplement to evaluate if is a problem of your reagent and not of the protocol.
Hi Berrin,
Just for "science" take a look at these papers from us.
Good luck
Simone
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