I am trying to isolate MSCs from intestinal biopsy. I was able to observe individual cells from five days. In tenth day I was able to see cells are group of cells (images attached). However, i have a suspicious that are they MSCs.
I have a few questions
1. Is there any specific medium that we can culture MSCs from intestinal tissue.
2. How to get rid of all other cell types excluding MSCs.
3. Is there any way that we can identify MSCs based on morphology?
4. What can be the basic and initial study to characterize the MSCs.
Looking forward to everyone's suggestions.
Thanks in advance
I have been thinking of this question a lot but on skin. The issue with MSCs are that they are almost similar to fibroblast. There are a few molecules that distinguishes a fibroblast from MSC and may be you can use them to separate the cells by MACS. Also MSCs that I work with have a larger size than the fibroblast of the same organ origin. You can use this feature and sort cells based on size with FACS. I do use a separate media for primary MSCs but it's more of a tailored media to support MSC rather than a selective MSC only media.
hi Murugadas A
Biswajoy suggested correctly about MACS. As we all know, MSCs are fibroblast cells, and identifying them from a particular source needs a rigorous step, MACS cell sorting is one of the best ways. Since such isolation consists of a heterogeneous population.
Well, come to your query, my understanding says
1. Since you have not mentioned your media type, I think MSCs grow nicely in KO/F12 media with glutamax.
2. Subsequent passaging will help you get rid of other cells.
3. MSC is of fibroblastic morphology, so subsequent passage will push or will gain a more fibroblastic look. As of now, in your condition, it seems only some cells have fibroblastic morphology i.e. tapering at the two ends. I am exactly not getting the group of cells thing as you mentioned, but in many times at initial stage cell cluster can be seen from where cells ooze outs.
4. Even if you sort the cells and get the MSC cell type, you will need to go for characterization. The MSCs proliferation is also necessary which you may see after the first passage since it's your freshly isolated and growing and adapting the initial in-vitro environment. Passage 2 to passage 4 will be right stage to characterize. Characterization can include population doubling assay, flow cytometry cell surface CD markers analysis, MSC markers gene, and protein expression. Vimentin immunofluorescence. {Note: Kindly check the intestinal tissue specificity}.
My answers are based on WJMSCs' isolation and characterization experience.
Regards
Saurabh
Agree with most of Saurabh's suggestions, start growing the cells in MSC specific media and over subsequent passages you will get rid of the other cell types. The isolation process / technique used is also critical, here's a paper that discusses some strategies used https://pubmed.ncbi.nlm.nih.gov/28144997/
Characterization via cell surface markers is a must, you can also do tri-lineage differentiation assays and follow ISCT guidelines - Article Dominici M, Blanc KL, Mueller I, Slaper-Cortenbach I, Marini...
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