A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow.
Studies on the effect of the microenvironment on hematopoiesis would benefit from the availability of pure populations of nontransformed cells of each of the stromal cell types. The adherent murine bone marrow stromal cell population in this study consisted of fibroblasts, endothelial cells, and macrophages. Fibroblasts were segregated from the phagocytic endothelial cells and macrophages by allowing the phagocytic cells to ingest magnetic beads, with subsequent exposure to a magnetic field, effecting cell separation. Pure colony cultures of fibroblasts and endothelial cells were formed by varying the bead-to-cell ratio and incubation period of the cells. For complete purification of the fibroblasts, subsequent passaging was also necessary. Near confluent growth of each type was obtained with subsequent passages and sustained culture. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance endothelial cell growth. We were not able to obtain pure populations of bone marrow macrophages in near confluent culture. The three cell types were identified by cellular morphology, acid and alkaline phosphatase staining, binding with the lectins Ulex europaeus and Bandeiraea simplicifolia, and the capacity to stain for the factor VIII-related antigen (von Willebrand's Factor).
Department of Hematology, Changhai Hospital, Shanghai, People's Republic of China.
R G
RG
Fei
P E
PE
Penn
N S
NS
Wolf
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D001854:Bone Marrow Cells; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D004727:Endothelium / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology; D007150:Immunohistochemistry; D037102:Lectins / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000502:physiology; D008280:Magnetics; D008297:Male; D051379:Mice; D008863:Microspheres; D010587:Phagocytosis; D037121:Plant Lectins; D014841:von Willebrand Factor / Q000032:analysis
D003115:Colony-Stimulating Factors; C045475:Griffonia simplicifolia lectins; D006133:Growth Substances; D037102:Lectins; D037121:Plant Lectins; C036850:Ulex europaeus lectins; D014841:von Willebrand Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000469:Alkaline Phosphatase