BACKGROUND: The theoretical value of the mesenchymal stem cel s from human umbilical cord isolated by the traditional methods is far away from that of the mesenchymal stem cel s 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 cel s.nrnOBJECTIVE: To optimize and develop a new efficient method for the isolation of human umbilical cord mesenchymal stem cel s in order to provide the technical solutions for the clinical application of mesencymal stem cel s.nrnMETHODS: The experiment was divided into four groups. The control group was treated with traditional enzymatic method (trypsin and col agenase type II), on this basis, the col agenase 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 cel s, cel s activity, time for the occurrence of the primary cel adherent and fusion time, as wel as the proliferative capacity, surface markers and differentiation capacity of the passage 3 umbilical cord mesenchymal stem cel s.nrnRESULTS AND CONCLUSION: Col agenase type II, col agenase type IV, trypsin, hyaluronidase, and DNAase were used to digest the umbilical cord, thus cel count reached up to (12.47±0.16)×106/cm (P <0.01) in 4 hours; cel activity was (75.00±5.07)(P <0.01); CD105 positive cel rate was 41.1%; and CD29 positive cel rate was 83.1%, these measurement indicators in the experimental groups were higher than those in the control group; the obtained cel s got adherent and associated fusion, and the time in the experimental groups was shorter than that in the control group (P <0.01); the cel s gradual y got uniform shape, the cel 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 cel s 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 cel s for 4 weeks, differentiation results were assayed by oil red O staining, showing that a large number of lipid droplets were observed in cel s; 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 col agenase type Ⅱ, col agenase 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 cel s and shorten the time of isolation. In addition, the time for primary mesenchymal stem cel s to adhere and grow to confluence was shortened by using the combined digestion method than common digestion method. And the ratio of mesenchymal stem cel s in the primary mesenchymal stem cel s and the purity of the passage three mesenchymal stem cel s obtained using combined digestion method are much better than those isolated using common digestion method.