Multi-batch production of primary human umbilical cord mesenchymal stem cells derived from the same donor

HUANG Si-rui; KUANG Mei-na; YUAN Yin; LU Xin; CHEN Dan-liang

doi:10.3969/j.issn.1001-9448.2019.05.003

Abstract：Objective To establish a multi-batch separation technique for the same donor derived hUC-MSCs and to improve the utilizing degree of umbilical specimens and cell yield. Methods hUC-MSCs were isolated by the method of tissue block adherence. Before being plated onto culture dishes, UC tissue was pre-soaked with FBS. When harvesting the first batch of hUC-MSCs, the UC tissue that was used only once was recovered and re-plated. The UC tissue derived from the same donor was cultured three times, i.e., three batches of hUC-MSCs were isolated from the same UC sample. At the same time, supernatants in the first and second batches of UC primary culture system were collected, incubated separately in culture flasks, and hUC-MSCs were harvested therefrom. The earliest appearance time, the first passage time and the cell number, etc. of different batches of hUC-MSCs were compared, and their cell morphology, immunophenotype, proliferation and differentiation ability were identified.Results Pre-treating UC tissue blocks with serum significantly shortened the harvest time of primary cells, increased the success rate of UC culture, and increased the cell yield. Compared with the same UC tissue blocks cultured for the first time, both of the UC tissue blocks and their culture supernatants could produce a larger number of primary cells in a shorter time when they were recycled and cultured repeatedly. The total number of primary cells, which were obtained from 3 cycles of repeated culture of the same UC tissue blocks and independent incubation of 2 batches of UC culture supernatant, was (27.48±3.98) times that of the conventional separation method under the same experimental conditions. Each batch of the primary cells shared the same morphology, immunophenotype as well as normal proliferation and differentiation capabilities typical of mesenchymal stem cells. Conclusion The UC tissue blocks are able to produce hUC-MSCs continuously and repeatedly, and their culture supernatant is also an important source of hUC-MSCs. The multi-batch separation method of hUC-MSCs based on the properties aforementioned elevates the separation efficiency and cell yield of hUC-MSCs greatly, which can ensure the stable and high-quality production of hUC-MSCs derived from the same donor.