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)×10⁸ 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)×10⁶. 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.