Hi Seonggyu Kwon

It basically relies on which TYPE of primary cells you are isolating and what is the SOURCE, and in which cells, they are getting DIFFERENTIATED. If the source or isolated cells contain a MIXED cell population, then you can wait for unwanted to go off, but that will depend on the life span in in-vitro culture. It will be best to sort the desired cells.

Even in your case, It is very important to note, that which MEDIA and what COMPONENTS, which you are using for culturing your desired cells. Probably, the components which you might be using are pushing the cells for differentiation.

Well, differentiation is not any superficial process, here a cell leaves one state and going into another. There will be a phenomenal change in genes & protein expression, lots of epigenetic modifications, etc.

To happen all of this, there must be an inducer, so you have to look into it, possibly components you are using for culturing.

And how you are finding that cells are differentiating, I mean by cell surface markers, gene or protein expression or what??

Regards

Saurabh

Hi Dear

Primary cells are mature cells of a specific tissue type that are harvested from explant material removed by surgical procedure. An example is primary human osteoblasts that are harvested from the femoral heads removed during total hip replacement operations. Primary cells are the most desirable with regard to immunological compatibility but, in general, they are differentiated, post-mitotic cells. This means that they are no longer able to divide and their proliferation potential is low. This might be compounded by the tendency of some cell types to de-differentiate during ex vivo cultivation and express an inappropriate phenotype, e.g. articular chondrocytes in culture often produce fibrocartilage as opposed to hyaline cartilage. This has stimulated studies to find and develop alternative cell sources for tissue engineering strategies, and stem cells might represent a solution to the limitations of primary cells obtained from explanted tissues.

Saurabh Mandal

Thank you for your answer.

For my experiments, there is no time for checking cell differentiation with surface markers or genes, so I watch the morphology of the cells

I am not sure which cell type are you used.

My idea is to reduce the culture time. Because for my fibroblasts, it will get transdifferentiation with the time increase. When you do the cell isolation, try to get more cells to culture , if you cannot, change the dish to a smaller one at the1st generation. When you do the passage, do not passage them into a too large task. For me, I use 35mm dish to T25. They will grow fast and you can passage them quickly.

Another reason may come from when you do the passage from 1 to 2, you may damage or stimulate the cell to differentiation during that process. Please check your protocol.

How many cells are you required to your experiment? Are there any limitation for the passage times?

Dear colleague, some others mentioned a couple of good points. Depending on the nature of the cells, a specific primary cell media would prevent the differentiation of the cells. There are also ready-to-use media kits available which promote the differentiation to one or the other primary cells, just one example: https://bioscience.lonza.com/lonza_bs/US/en/Primary-and-Stem-Cells/p/000000000000188630/Human-Mesenchymal-Stem-Cell-%28hMSC%29-Osteogenic-Differentiation-Medium-BulletKit%3Csup%3ETM%3C-sup%3E

I use primary keratinocytes (KC) from the oral cavity, and experience that several factors can start early differentiation. Saurabh Mandal and Yakun Wu mentioned several factors:

- area (ours KC do well in T25 but not bigger flasks)

- number of cells per area (to few KC will start early differentiation)

- medium (to much calcium starts differentiation)

- splitting (never grow to confluence; check media used to split, return quick back to right medium; our KCs do well for 6-7 passages but start to differentiate after)

- age of the donor (we have bad experience with elderly donors)

- site of biopsy (KCs from some sites grow better then other sites)

Olaf J.F. Schreurs

It was the mouse cell... and the remaining lifetime is about 3 months.

I think that was one problem. Thank you~