[Method and conditions of isolation and proliferation of multipotent mesenchymal stem cells]

To investigate the method and conditions of isolation, proliferation of multipotent mesenchymal stem cells (MSCs) from human umbilical cord blood in vitro, and to induce osteogenic and adipogenic differentiation directly for identification.

Human umbilical cord blood was collected in asepsis condition, isolated by density gradient centrifugation, or sedimented red cell with methylcellulose, and then the same centrifugation was done, or obtained by negative immunodepletion of CD34+. These isolated mononuclear cells were used to carry on plastic adherent culture. To obtain single cell-derived colonies, these cells were proliferated clonally in medium which consists of L-DMEM or Mesencult medium and 10% fetal calf serum(FCS) respectively, then their differentiation potentiality to osteoblasts and lipoblasts was tested.

The mononuclear cells isolated by sedimented and centrifugated way cultured in Mesencult medium and 10%FCS were most available. These adhesive cells could become obviously short rod-shape or shuttle-shape cells after 5-7 days. The colonies form well in 3rd-passage cells. The mononuclear cells obtained by only centrifugalized in density gradient were hard to form colony, isolated by immunomagnetic beads were hard to culture. The surface antigens of these colonies cells presented CD29, CD59, CD71 but not CD34, CD45 and HLA-DR etc. The colony cells differentiating into osteoblasts that produce mineralized matrices, stained by alizarin red, and differentiating into adipocytes that accumulate lipid vacuoles, stained by oil red.

MSCs can be isolated from human umbilical cord blood and proliferate it in vitro. The way that mononuclear cells are sedimented red cell by methylcellulose and cultured by Mesencult medium and 10% FCS is the valid method of isolation. Proliferation colonies cells present matrix cell immunophenotypes, and can differentiate into osteoblasts and adipocytes.