How to isolate and purify placental stem cells, can they be cultured under sterile conditions in the laboratory?
The placenta tissues were dissected, minced, and dissociated in trypsin and DNAse I. The dissociation was performed in three stages of incubation at 180 r/min for 20 minutes at 37 ℃. The digesting suspension was filtered using a 200 mesh strainer before separated by Percoll gradients. The cytotrophoblast cells and pMSCs fractions were collected respectively. The pMSCs were seeds on 75-cm2 flask directly for culture.
ate mesenchymal stem cells from placental tissue, placental tissue is firstly collected under sterile conditions in the operating room and after delivery. Then the decidua tissue is separated from it and after repeated washing in phosphate buffered saline (PBS), it is transferred to the transfer medium containing PBS with 1 unit/ml of penicillin and 1 mg/ml of streptomycin. to be To isolate cells, first, under a laminar hood and in a 10 mm plate, all blood vessels and blood clots are mechanically separated from placenta tissue and divided into small pieces.
Isolation of stem cells In this step, cells are washed in PBS solution at 1250 RPM for 5 minutes. After that, in order to separate the cells, 30 ml of collagenase solution with a concentration of 1 mg/ml is added to the precipitate and incubated in an incubator at 37°C and CO2 with a concentration of 5% for 1 hour. To isolate the mesenchymal cells, the resulting suspension is centrifuged at 1250 RPM for 5 minutes and 0.25% trypsin solution containing 1 mM EDTA is added to the cell sediment and incubated in an incubator for 30 minutes.
RBC filtration and lysis The sediment obtained from cell separation is washed twice and centrifuged and passed through a filter with 70 micron pores. It should be noted that during cell separation in order to lyse the remaining RBCs in the sample, 2 ml of hypotonic ammonium chloride solution is added to the sample and washed again after 10 minutes. The cell sediment resulting from placental tissue cell separation is transferred to a T75 flask and cultured in Dulbecco's Modified Eagle Medium-Low Glucose (DMEM-LG) medium.
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