Improving the production of human placental chorionic-derived mesenchymal stem cells by modified primary culture method / 国际生物医学工程杂志

ABSTRACT

Objective To establish a cultivating method for obtaining a large number of P0 generation human placental chorionic-derived mesenchymal stem cells (hpcMSCs).Methods The hpcMSCs were isolated from human placental chorion.After primary culturing and culturing for seven days,the culture medium,the non-adherent tissue and the douching normal saline of the primary culture were centrifuged and re-cultured twice.Cell morphology was observed by an inverted microscope.CCK-8 was used to measure the cell growth curve.Flow cytometry was used to detect cell surface markers.Adipogenic and osteogenic differentiation kits were used to assess the cell differentiation potential.Results The obtained hpcMSCs were fibroblast-like adherent cells and (25.54±3.38)×106 cells were obtained per placenta.The total yield of the primary culture,secondary culture and tertiary culture were (11.73±2.09)×106,(11.12±1.42)×106 and (2.69±0.71)×106,respectively,and the incubation time were (12.00±0.64) d,(8.87±0.63) d and (12.33±0.80) d.There was significant differences in incubation time between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the incubation time of the tertiary culture had an increasing trend (P＞0.05).The yield per culture flask of the primary culture,secondary culture and tertiary culture were (1.12±0.15) × 106,(2.10±0.16)×106 and (1.04±0.16)×106,respectively.There was significant differences in the yield per culture flask between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the yield per culture flask of the tertiary culture had a decreasing trend (P＞0.05).There was no difference among the three cultures in the growth curve and the expression of surface markers,and the osteogenic and adipogenic differentiation were all positive.Conclusions The P0 generation hpcMSCs isolated from a choriocarcinoma sample can be doubled by the three cultures compared with the primary culture,which can provide plenty stem cell source for the regenerative medicine.