The Cultivation And Cryopreservation Of Mouse Multipotent Adult Progenitor Cells

PartⅠThe isolation and cultivation of BALB/c mouse- derived multipotent adult progenitor cellsObjective: Establish the isolation and primary culture methods for obtaining large amount of BALB/c mouse-derived multipotent progenitor cells ,that provide the foundation for getting purified MAPC in the following experiment.Methods:Isolate bone marrow cells from tibia and femur bones of BALB/c mouse, then culture them in the low serum medium added EGF,PDGF and LIF by the whole bone marrow adherent method.After two weeks later, passage the culture cells in a certain ratio and continue to culture them.Giemsa staining treat with cells at different times of culture and observe cell morphology. MTT colorimetric assay was used to assess the effect of the MAPC growth in different ages of mouse,different seeding cell density of pimary culture and subculture.Results:Cells derived from 3-weeks old mouse have the best homogenicity and proliferation ability. The best culture conditon for primary MAPC growth is (6～12)×10~5/cm~2 cell density of primary culture and (20-30)×10~3/cm~2 of the subculture. We can observe some spindle and triangular cells after Giemsa staining. Conclusions: Under the above cell conditon,BALB/c mouse-derived primary MAPC can grow and proliferate steadily, that provide large amount of cells in the next sorting experiment.PartⅡThe purification and proliferation of BALB/c mouse-derived multipotent adult progenitor cellsObjective:Obtain purifed MAPC and explore the culture conditon in vitro for stable passaging and proliferation of MAPC,that lay the foundation for the germline induction of MAPC.Methods:Depletion was done with the BMCs after two passgages. Removed the CD45~+ and Ter119~+ cells by magnetic negative cell sorting.Then culture the negative cells at a certain density and observe the cells growth. Also draw up the MAPC growth curve. The cell viability before and afer MACS was determined by trypan blue exclusion.MTT colorimetric assay was applied to assess the effect of different cell seeding density on MAPC growth.Results:There was no significant difference upon viability between the cells before and after separating by MACS(P﹥0.05).The best MAPC seeding density was(6-10)×10~3/cm~2.The cells were arrest of growth in the first and second days afer culture, then entered into the increased logarithmic phase on the third day. The phase last for about 3 days and later cells entered into platform.The purified MAPC are spindle or triangular in shape with a large nucleus and scant cytoplasm.Conclusions: The MAPC with homogenicity and favorable proliferation ability can be obtained by magnetic negative cell sorting. PartⅢThe identification and cryopreservation of BALB/c mouse-derived multipotent adult progenitor cellsObjective: Identify whether the cultured cells were MAPC and assess the effect of the cryopreservation method to provide enough cells for induction experiment.Methods:MAPC cell surface antigen CD13,CD44 and CD34 expression were determined by flow cytometry sorting.The cryopreservation method was changed a little on the basis of the previous article reported.And the cell viablity was compared before and after cryopreservation.Results:The antigens on MAPC surface are CD13~+,CD44~- and CD34~-. There was no significant difference upon viability between the cells before and after cryopreservation (P﹥0.05).Conclusions:Whole bone marrow adherent method added with MACS is an effective method to obtain MAPC.The cryopreservation method could preserve MAPC well and maintain cell growth activity.