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Optimization of Efficient Method for the Isolation of Mesenchymal Stem Cells from Human Umbilical Cord

Author: DongMin
Tutor: NiuBo
School: Shanxi Medical
Course: Biochemistry and Molecular Biology
Keywords: mesenchymal stem cells umbilical cord collagenase trypsin hyaluronidase DNAase cell culture method
CLC: R329
Type: Master's thesis
Year: 2012
Downloads: 57
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Background: Recent evidence has suggested that umbilical cord is the ideal source ofmesenchymal stem cells (MSCs), umbilical cord is high in the mesenchymal stem cells and hasextensive sources, and low cost. HUCMSC are of great value in Cellular therapy and TissueEngineering. Currently, different protocols have been developed to isolate MSCs from umbilicalcord including explant culture method, such as single enzyme digestion method (collagenase,collagenase IV, or collagenase I), double enzyme digestion method using collagenase and trypsin,and three enzyme digestion method using collagenase, hyaluronidase and trypsin. but the yield ofMSCs is low and the operation is time-consuming. It goes against the cells to be isolated andcultured in large-scale.Therefor, to optimize the protocol for the isolation of HUCMSC willpromote the application of MSCs.Objective: However, the yield and efficiency of isolating MSCs from umbilical cord are limitedby current protocols. This study aimed to optimize and develop a new efficient method for theisolation of MSCs from human umbilical cord.Methods: Experiment was divided into four groups, the traditional enzyme digestion method(trypsin and collagenase II) was set as the control group, on this basis, added in collagenase IV,hyaluronidase, and DNase singly according to the umbilical cord structure. Each experimentalgroup were set a group of different digestion time,than recorded the cell number, cell activity,primary adherent cells , the integration time ,the proliferation capacity of HUCMSC, surfacemarkers and differentiation capacity in the 3rd generation by different separation methods, atdifferent time.Results:To comparision among the experimental groups in the same time,with the increase inthe types of enzymes, cell production, cell activity showed an increasing trend, and thedifference was significant. Comparision within the single experimental group, with the extend oftime ,cell production increased, and reached a plateau in 4 ~ 6h and than declined , the differencewas significant; cell activity reached to maximum at 4h, than followed a downward trend, andthe difference was significant statistically. The cooperation of Collagenase type II, type IVcollagenase, trypsin, hyaluronidase, and DNAase which was used to digeste the umbilical cord,cell count reaches up to 12.47±0.16×10~6/cm (P <0.01) in 4h; cell activity was 75.00±5.07%(P <0.01); harvest cells of CD105-positive rate was 41.1%; and of CD29 positive rate was 83.1%,these measurement indicators were higher than the control group; the obtained cells got adherentat 3.25±0.46d ((P <0.01); and than associated fusion after 6.25±0.46d ((P <0.01); cells gradually got uniform shape, the cell morphology of the third-passage achieved unity:spindle-shaped, swirling. Then assayed the cell immunophenotypes by flow cytometry.The cellsisolated from the group of four enzymes were high in CD44, CD105 and CD29,and lowlyexpressed CD34; CD105 positive rate of P3HUCMSCs from four enzymes experimental groupwas 85.3%; CD29 positive rate was 97.6%; CD44 was 95.8%; all exhibited higher positive ratethan the control,on the contrary, CD34 positive rate was 2.6% lower than the control group. Afterthe induction to fat cells, differentiation results were assayed by oil red O staining, showing thata large number of lipid droplets were observed in cells; differentiation capacity to osteoblast wasmeasured by alizarin red staining, visible positive calcium deposition was observed. Therefore,the combined effects of the four enzymes in 4h, improved the cell activity and cell production,shorten the culturation time of HUCMSC, and without significant difference in the proliferativecapacity, surface marker and multi-differentiation ability compared with traditional methods.Conclusion: The present study demonstrated that the combined use of collagenase II and IV,hyaluronidase, trypsin and DNAase to digest umbilical cord in 4h could significantly improvethe yield, viability and colony-forming ability of MSCs isolated from umbilical cord. In addition,the time for MSCs to adhere and grow to confluence was less by using the combined digestionmethod than common digestion method. Further characterization of MSCs demonstrated that thepurity and differentiaion potential of MSCs isolated using combined digestion method weremuch better than those of MSCs isolated using common digeston method. Taken together, weestablished a more efficient and less time-consuming method for the isolation of MSCs fromumbilical cord, facilitating the large-scale in vitro expansion of MSCs and their application inregenerative medicine.

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