关键词: 脐带, 脐带间充质干细胞, Ⅱ型胶原酶, Ⅳ型胶原酶, 透明质酸酶, DNAase, 高效

Abstract:

BACKGROUND: The theoretical value of the mesenchymal stem cells from human umbilical cord isolated by the traditional methods is far away from that of the mesenchymal stem cells from human umbilical cord contained in the human umbilical cord tissues, which lead to the great waste of the umbilical cord tissues and prevent the large-scale preparation and culture of mesenchymal stem cells.
OBJECTIVE: To optimize and develop a new efficient method for the isolation of human umbilical cord mesenchymal stem cells in order to provide the technical solutions for the clinical application of mesencymal stem cells.
METHODS: The experiment was divided into four groups. The control group was treated with traditional enzymatic method (trypsin and collagenase type II), on this basis, the collagenase type Ⅳ, hyaluronidase and DNase were added into the umbilical cord tissues for digestion. The indicators obtained through different isolation methods and different operation time were compared, including the amount of the cells, cells activity, time for the occurrence of the primary cell adherent and fusion time, as well as the proliferative capacity, surface markers and differentiation capacity of the passage 3 umbilical cord mesenchymal stem cells.
RESULTS AND CONCLUSION: Collagenase type II, collagenase type IV, trypsin, hyaluronidase, and DNAase were used to digest the umbilical cord, thus cell count reached up to (12.47±0.16)×106/cm (P < 0.01) in 4 hours; cell activity was (75.00±5.07) (P < 0.01); CD105 positive cell rate was 41.1%; and CD29 positive cell rate was 83.1%, these measurement indicators in the experimental groups were higher than those in the control group; the obtained cells got adherent and associated fusion, and the time in the experimental groups was shorter than that in the control group (P < 0.01); the cells gradually got uniform shape, the cell morphology of the third-passage was spindle-shaped and swirling. The positive rates of the specific markers CD44, CD105 and CD29 of passage 3 human umbilical cord mesenchymal stem cells were higher than those in the control group, and the positive rate of CD34 in the experimental groups was lower than that in control group. After induction to fat cells for 4 weeks, differentiation results were assayed by oil red O staining, showing that a large number of lipid droplets were observed in cells; the differentiation capacity to osteoblasts was measured by alizarin red staining, and visible positive calcium deposition was observed. The experiment demonstrated that the combination of collagenase type Ⅱ, collagenase type Ⅳ, hyaluronidase, trypsin and DNAase for digestion of umbilical cord for 4 hours could significantly improve the yield, viability and proliferation ability of mesenchymal stem cells and shorten the time of isolation. In addition, the time for primary mesenchymal stem cells to adhere and grow to confluence was shortened by using the combined digestion method than common digestion method. And the ratio of mesenchymal stem cells in the primary mesenchymal stem cells and the purity of the passage three mesenchymal stem cells obtained using combined digestion method are much better than those isolated using common digestion method.