Studies On Cultures Of Clony-derived Human Bone Marrow Mesenchymal Stem Cells And Their Biological Properties

Object:1 To explore the feasibility of built a technique way of ex vivo culture method of mesenchymal stem cells (MSCs )on colony level.2 To further study their biological characteristics of proliferation and differentiation, and to lay a foundation for clinical application of cell transplantation and hematopoiesis support affects.Methods:1 To make use of health volunteer' s bone marrow as experimental object, get Mononuclear cells by density gradient centrifugation and culture MSCs according to their ahesion characteristics to plastic. MSCs are detached with 0.5% trypsin when confluent and a clone circle is used to get clony MSCs.2 Biological characteristics assay of MSCs :(1) Phenotypes assay of MSCs. (2) Growth curves assay of MSCs. (3) CFU-forming ability . (4) Refrigeration and revivification of MSCs.3 Committing induce differentiation of MSCs in vitro. Results:1 The clony human MSCs in vitro cultured under this condition in our laboratory can be expanded efficiently. Doubling time of MSCs is 48h~72h. They can be expanded for 20 passages in vitro. The number of the cultured cells after 15 passages can mount to (1-3) X 1010, MSCs can be expanded in vitro for 20 passages.2 Flow cytometer phenotypes assay show that the cultured cells express CD13, CD29, CD59 , CD44, CD71, not CD11, CD14, CD31, CD34, CD38, CD45, CD80, CD86, CD117. this means the cultured cells are MSCs other than hematopoietic cells. And they can be regard as colony-derived MSCs for their purity is 97% by flow cytometer .3 cultured cells' growth curves shown MSCs grows well before 17passages, and the cells grow slower after 17 passages, their forms become larger and irregular, granules can be detected in cells and culture media. Clony culture assay show different passages before 17 possess similar CFU forming capability (P>0.05) . But different passages after 17 declined with the passages accumulating.Compared with the passage before 17,they have significant difference (P < 0.05)4 Clony culture cells can be refrigerated efficiently by routine freezing method and keep excellent vigor after thawing.5 In vitro induced differentiation capability assay show that the cultured MSCs keep their differentiation potential and can differentiate into nervous stem cells. The induced proportions are 25.6% .Conclusions:1 the culture method used in this experiment can proliferate clony hMSCs efficiently. MSCs can be expanded in vitro for 20 passages. Their phenotypes assay demonstrate that they are hMSCs and they keep potential of differentiating into nervous stem cells when induced by Retinoic acid in vitro. Their growth curves and CFU-forming capability show that MSCs are a kind of cells with limited life span.2 The culture method used in this experiment is simple and reliable. Proliferative cultured cells grows well, their mumber reaches (1-3) X 1010, and posses the abilities of differentiation .They might differentiate into neural stem cells.They can not only be used in experimental investigation, but also meet a demand of clinic cell ransplantation. This methods have some precede.