Purification and characterization of rat bone marrow endothelial cells
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A method is described to obtain endothelial cells from rat bone marrow with high purity and viability. Marrow cell suspensions were prepared by collagenase and subjected to discontinuous gradient centrifugation on Percoll (densities 1.04 and 1.06). Endothelial cells were concentrated in the middle layer as demonstrated by electron microscopy and flow cytometry as well as fluorescent microscopy after staining for factor VIII antigen. Cells of this layer were then subjected to centrifugal elutriation and highly purified endothelial cell preparations were obtained with flow rates of 15-20 ml/min. By fluorescent microscopy, 49%-51% of these cells were factor VIII positive. Identification by means of electron microscopy indicated a much higher purity of endothelium ranging from 63% to 90% with a yield in the range of 10(6) cells and a viability exceeding 90%. Some technical considerations in the development of this method are discussed. This method permits in vitro experiments on relatively high purity, high viability preparations of marrow endothelium.
A method is described to obtain endothelial cells from rat bone marrow with high purity and viability. Marrow cell suspensions were prepared by collagenase and subjected to discontinuous gradient centrifugation on Percoll (densities 1.04 and 1.06). Endothelial cells were concentrated in the middle layer as demonstrated by electron microscopy and flow cytometry as well as fluorescent microscopy after staining for factor VIII antigen. Cells of this layer were then subjected to centrifugal elutriation and highly purified endothelial cell preparations were obtained with flow rates of 15-20 ml/min. By fluorescent microscopy, 49%-51% of these cells were factor VIII positive. Identification by means of electron microscopy indicated a much higher purity of endothelium ranging from 63% to 90% with a yield in the range of 10(6) cells and a viability exceeding 90%. Some technical considerations in the development of this method are discussed. This method permits in vitro experiments on relatively high purity, high viability preparations of marrow endothelium.
