Dear all,
I am isolating mesenchymal stromal cells (MSCs) from the bone marrow of adult C57BL/6 mice.
The culture conditions are:
-IMDM + L-glut, 20% of 50:50 FBS:HS, 1% P/S
-Medium replaced each 3-4 days
-When confluent, adherent cells were trypsinized and subcultured (process for MSCs purification)
When I obtain a pure MSCs population, I seed 7000 cells par well in a 96-well plate and I induce osteogenesis in the following way:
-osteogenic medium (50 µg/ml ascorbic acid, 10 mM β-glycerophosphate) in IMDM + L-glut, 20% of 50:50 FBS:HS, 1% P/S. )
-The medium is changed every 3 days
-Alizarin Red staining is performed 21-25 days upon induction of differentiation (after PFA fixation) to evaluate mineralization
The experiment seems to work as I obtain a nice Alizarin Red staining (see attached Picture 1). However, I am curious in the morphology of the cells I obtain. As you can see both from Picture 2, 3, 4 and 5 I have plenty of these round-shape cells that stain for DAPI (Picture 4). Does anyone have experience observing this kind of cells? Are these osteoblasts? They are also weakly stained for Alizarin Red, as you can see from picture 5, or is this aspecific?
I thank you in advance for your help
Dear Giulia,
It is clear that your MSCs differentiated well into osteogenic direction (Figure 1) based on the bright Alisarin Red staining. However, you are right that your cultures contain those round cells unlike MSCs, most probably cells of hematopoietic origin, which can occur until passage 6-7 in the newly isolated mouse bone MSC culture. Best option is to immunophenotype your culture for MSC and non-MSC markers in a FACS experiment. Around passage 10, the ratio of hematopoietic cells in MSC culture should be negligible. A good example for such characterization is this article:
Identical, similar or different? Learning about immunomodulatory function of mesenchymal stem cells isolated from various mouse tissues: bone marrow, spleen, thymus and aorta wall - PubMed (nih.gov)
(DOI: 10.1093/intimm/dxq039)
Congratulations, Giulia Trimaglio, on your success in obtaining nice Alizarin Red staining (Picture 1).
It seems there is some background (Picture 4) - DAPI staining.
I agree with Eniko Szabo.
It looks like too many round cells, and they are too tiny as well. So observe the culture for any yeast contamination.
Dear Chandra Somasundaram and Eniko Szabo thanks for your useful answers! To reply to your comment htat these cells could be HSCs, I actually always phenotype by flow-cytometry the cells before starting the diffferentiation to avoid indeed HSCs coontamination. I always start the differentiation when I have a 90% purity of cultured MSCs..so it cannot be this...but maybe contamination as Chandra Somasundaram suggested?
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining