冻存人脐带间充质干细胞的分离培养.

BACKGROUND: Current studies have addressed the mesenchymal stem cells (MSCs) mostly from fresh human umbilical cord, but rarely from cryopreserved human umbilical cord. OBJECTIVE: To isolate MSCs from cryopreserved human umbilical cord, and to compare the biological characteristics of human umbilical cord MSCs (hUC-MSCs) harvested using several culture methods. METHODS: hUC-MSCs isolated from cryopreserved umbilical cord tissues by explants culture method were cultured in serum-containing and serum-free media. Cell morphology was observed to draw the growth curve of cells in different media. Passage 3 hUC-MSCs cultured in serum-free medium and passage 14 hUC-MSCs in serum-containing medium were selected to detect the expression of surface biomarkers by flow cytometry. Passage 3 cells in the serum-free medium were induced for adipogenic, osteogenic and chondrogenic differentiation, and differentiation marker genes were identified by qPCR. Expression of cytokines in the passage 7 cells cultured in the serum-free medium and in the culture supernatant was detected. RESULTS AND CONCLUSION: The hUC-MSCs isolated from cryopreserved human umbilical cord exhibited a spindle-shaped appearance and exuberant growth, and moreover, the cells could be expanded in serum-free medium but the cell proliferation was certainly slower than that in the serum-containing medium. Flow cytometry analysis revealed that the hUC-MSCs were positive for CD73, CD90, CD103, but negative for CD45, CD34, CD14/CD11b, CD79a/CD19, and HLA-DR. Adipogenic (PPARγ), osteogenic (Osteonectin) and chondrogenic (Collagen II) biomarker genes were highly expressed at 14-28 days of induction. Passage 7 hUC-MSC and its supernatant secreted several cytokines including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon α2, chemokine IP-10, interleukin 4, and interleukin-6. In conclusion, we could successfully isolate and expand hUC-MSCs from cryopreserved human umbilical cord tissuesBACKGROUND: Current studies have addressed the mesenchymal stem cells (MSCs) mostly from fresh human umbilical cord, but rarely from cryopreserved human umbilical cord. OBJECTIVE: To isolate MSCs from cryopreserved human umbilical cord, and to compare the biological characteristics of human umbilical cord MSCs (hUC-MSCs) harvested using several culture methods. METHODS: hUC-MSCs isolated from cryopreserved umbilical cord tissues by explants culture method were cultured in serum-containing and serum-free media. Cell morphology was observed to draw the growth curve of cells in different media. Passage 3 hUC-MSCs cultured in serum-free medium and passage 14 hUC-MSCs in serum-containing medium were selected to detect the expression of surface biomarkers by flow cytometry. Passage 3 cells in the serum-free medium were induced for adipogenic, osteogenic and chondrogenic differentiation, and differentiation marker genes were identified by qPCR. Expression of cytokines in the passage 7 cells cultured in the serum-free medium and in the culture supernatant was detected. RESULTS AND CONCLUSION: The hUC-MSCs isolated from cryopreserved human umbilical cord exhibited a spindle-shaped appearance and exuberant growth, and moreover, the cells could be expanded in serum-free medium but the cell proliferation was certainly slower than that in the serum-containing medium. Flow cytometry analysis revealed that the hUC-MSCs were positive for CD73, CD90, CD103, but negative for CD45, CD34, CD14/CD11b, CD79a/CD19, and HLA-DR. Adipogenic (PPARγ), osteogenic (Osteonectin) and chondrogenic (Collagen II) biomarker genes were highly expressed at 14-28 days of induction. Passage 7 hUC-MSC and its supernatant secreted several cytokines including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon α2, chemokine IP-10, interleukin 4, and interleukin-6. In conclusion, we could successfully isolate and expand hUC-MSCs from cryopreserved human umbilical cord tissues [ABSTRACT FROM AUTHOR]