Biological characteristics of human umbilical cord mesenchymal stem cells:cold trypsin digestion versus tissue explant method in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND:Cold trypsin digestion is rarely reported to culture umbilical cord mesenchymal stem cells. OBJECTIVE:To compare the biological characteristics of umbilical cord mesenchymal stem cells cultured by cold trypsin digestion and tissue explant method. METHODS:Human umbilical cord mesenchymal stem cells were isolated, purified and passaged using cold trypsin digestion and tissue explant method, and then the first adhesion time and cellcycle were recorded. Morphology of umbilical cord mesenchymal stem cells was observed under inverted phase contrast microscope to draw growth curve of cells at passage 3. Flow cytometry was used to detect the surface markers of passage 3 umbilical cord mesenchymal stem cells, and Nestin expression was detected in passage 3 cells after 3 days culture in neural induction medium by fluorescence immunochemistry staining. RESULTS AND CONCLUSION:These two methods were both successful to harvest umbilical cord mesenchymal stem cells, but the first adhesion time was earlier in cells cultured by cold trypsin digestion than tissue explant method (P0.05). Under the inverted microscope, cells grew adherently and presented fibroblast-like shape. However, the minority of primary cells under induction of cold trypsin digestion was polygonal, irregular, and had larger cellvolume than those cultured by tissue explant method. Passage 3 cells cultured by tissue explant method showed faster proliferation rate than those cultured by cold trypsin digestion, and at logarithmic growth phase, cells cultured by these two methods were significant different in cellnumber (P<0.05). Two types of cells had a uniform cellphenotype, both of which expressed CD29, CD105, but did not express CD34, CD45. Under induction, passage 3 cells cultured by these two methods were both positive for nestin. These findings indicate that these two methods can both be used to culture umbilical cord mesenchymal stem cells, but the tissue explant method is more suitable for culture of umbilical cord mesenchymal stem cells and exhibits less damage to cells.