Isolation and purification of human cytotrophoblasts and placental mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Cytotrophoblasts in placental cell components plays an important role in fetal immunological tolerance.Placental mesenchymal stem cells(pMSCs)have potential of multiple differentiation and inhibition of lymphocyte proliferation.However,conventional methods cannot acquire a large amount of purified human cytotrophoblasts or pMSCs.OBJECTIVE:To establish a method to obtain large placenta tissue,and harvest plenty of cytotrophoblasts and pMSCs with high purity and activity.METHODS:Human 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.Fibroblasts of cytotrophoblast cells fraction were removed by differential adhesion.The pMSCs were seeds on 75-cm2 flask directly for culture.The dissociation of placenta tissue was observed.The number of harvesting cytotrophoblasts was quantified and Cytokeratin 7 expression was tested.The pMSCs primary culture time,cell passage,induced osteoblast differentiation were observed.The cell surface makers were also detected.RESULTS AND CONCLUSION:After digesting in trypsin and DNAse I,there was only little residue left.(5.48±1,98)×10~8 cytotrophoblasts were obtained after differential adhesion.(90±4.36)% of these cells were positive for Cytokeratin 7.At 19-21 days after pMSCs reached approximately 90% confluency,the cell number was(1.96±0.24)×10~6.The subcultre cells could be passaged again in 4 or 5 days.Flow cytometric analysis of pMSCs showed that the cells expressed CD29,CD44 and HLA-ABC intensively and were negative for CD34,CD45,CD14 and HLA-DR.pMSCs differentiated into osteoblast-like cells after induction,which stained bright salmon pink by Alizarin Red.Dissociating the placenta tissue in trypsin and DNAse I in combination with discontinuous Percoll gradient separation obtained a large number of cytotrophoblasts and pMSCs recovered from placenta tissue,with high purity and activity.