| Apart from hematopoitic stem cells, there is a kind of non-hematopoietic stem cell, namely mesenchymal stem cells (MSCs), in bone marrow. It is also called bone marrow stromal cells(BMSC), because of its resulting from marrow matrix which is sustaining for the hematopoietic function of the marrow. Under some certain conditions, MSCs have potential ability of mitosis, proliferation and differentiate to kinds of mesenchymal cells. MSCs can differentiate into lipocytes, chondrocytes, osteogenic precursor cells, astrocytes and neuron cell. Latterly, the relation between MSCs and hematopoiesis function has been researched widely in experiment and clinic. The research on MSCs has been the focus in medicine. But in adult bone marrow, the content of MSCs is very low. As a result, it is necessary to culture, proliferate and purificate the MSCs in vitro in order to obtain large quantities of MSCs which is competent for clinical using. In long term of culture, MSCs can be contaminated by microbe or mutated in genotype. To protect stem cells from lose and obtain stem cells which is acquired expediently for effective stem cell transplantation, it is necessary to collect stem cells with cryopresevation. The object of the present study is to create a stable system for culture and proliferation of MSCs in vitro and an optimized method to cryopresevation of MSCs.In part I research, a bone marrow aspirate was collected and processed using density gradientcentrifugation, from which light-density cells were taken. In our study, it was observed that mononuclear cells have adhered to culture flask after 3 to 4-hour culture period. The quantity of adhered cells increased after 24 hrs and no further increased hereafter. After a 2 to 3-day dormant period, the adhered cells mitosised and proliferated and cluster shaped. After a 14-day primary expansion period, MSC nearly reached confluence, and these cells had a fibroblast-like morphology. In the primary culture, some cycloidal hematopoietic cells or endothelial cells grew adhesively. After passage, these cells were not any more adhesive. All adhesive cells were spindle-shaped and uniformly MSCs. After several passage, the number of MSCs at passage 8 reached (2-3) 10 10. The vast majority of MSCs (more than 85%) was at cell cycle of Go/Gl phase and the minority of MSCs was at S, G2 or M phase. It suggested that MSCs have powerful proliferation potential. It was demonstrated with transmission electron microscope that the MSCs was abundant in organelle such as ribosome, mitochondria, rough endoplasma and Golgiosome. It suggested that the cells were powerful in synthesis of protein. It was the structure base of its excretion of cytokine, sustaining of differentiation itself and multiple differentiate potential.Analyzed by flow cytometry, MSC were demonstrated that they were uniformly positive for CD29, CD44 and CD 166, negative for CD34, CD45 and HLA-DR. No significant difference presented after five passages. The result demonstrated that there were no hematopoietic cells in these cells isolated and cultured in vitro and cell surface marker expressed stably even after several passage.To demonstrate the multiple differentiate potential of MSCs, we induced successfully differentiation of MSCs into osteogenic precursor cells, lipocytes and neuron cell. MSCs treated with osteogenic medium (DMEM containing 10% FBS supplemented with 10-8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM B - glycerol phosphate) formed several scattered nodules and then the nodules were calcipectic which was demonstrated by Von Kossa staining. A larger mount of orange lipocytes were observed after MSCs were treated with lipogenic medium and stained with oil red O. After induction into neron cell treated with monothioglycerol, the cells expressed nestin, NSE and NF-M while negative for GFAP.In the part II research, the PI passage cells cultured in vitro were cryopreseved with two-step freezing procedure or program-controlled freezing procedure , using a DMEM media containing 5% DMSO and 30% FBS. The...
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