Those are the 3rd passage of human bone marrow MSC cell lines. The original cells ordered from company. I searched that frozen those cells under liquid nirtogen would decrease their differentiation potential, also that may caused by the eld passages ( ＞8-10 passages). We need about one month to complete one round of test and required low passage cells. So I kept and seeded the cells in low density for decreasing the growth speed of cells ( try to keep them in lower passage as longer as possibe). That's what them looks like now. I also found they begin to grow and divide very slow ( super slow).

1. Can anybody help to check if those cells in senescence now? Do they still have differentiation potential? I researched some MSC is about in senescence by showing that flat round structure. But some other publications said those cells look fine like that. I am really confused.

2. How serious the fronze ( liquid nitrogen) could affect the differentiation potential of human bone marrow MSC? Our project requird the good differentiation potential cells ( 2-6 passages). If froze cells are unacceptable, that means we need to order MSC again and again. That would cost too much.

3. If those cells in senescence now in pictures, can I rescue them back by subculturing and seeding in high density ?

Any advice would be appreciated. Thanks a lot.