I want to investigate the role of mitochondria transfer using mtDNA-depleted UC-MSCs (UC-Rho0). For that reason, firstly, UC-Rho0 cells were attempted to be generated by treating them using EtBr (0.1 μg/ml), Uridine (50 μg/ml), and 1mM sodium pyruvate in complete alpha-MEM supplemented with human platelet lysate, sodium pyruvate, and penicillin/streptomycin. We seeded 6000 cells per cm of human UC-MSCs (P2) in a T75 flask. After culturing overnight, the medium was replaced with complete alfa-MEM containing EtBr(0.1ug/ml), Uridine(50ug/ml), and 1 mM sodium pyruvate. The culture medium was changed every two days. The cells were incubated for 22 days in this EtBr cocktail(P2+3 passage). Based on this publication, UC-MSCs should be cultured for 6 weeks to complete mtDNA. Article Delivery of exogenous mitochondria via centrifugation enhanc...
Unfortunately, After 22 days, it was observed that the cells had died. Compared with treated UC-MSCs in the first week, the growth of EtBr-treated UC-MSCs was decreased in the final week. Shortly before they died, the cells had formed a cluster. I would like to restart the experiment, but first, I need to resolve the issue. Please help me sort out the problem. Attached are images of cells taken at the beginning and end of the day or at different times. Please take a look. Many thanks!