1.Endothelial cell isolation from murine tissues
A rapid, reproducible method for the isolation of murine tissue endothelial cells (EC) has been developed by Dong et al (1) using Dynabeads M-450 Sheep anti-Rat IgG coupled with a monoclonal rat anti-mouse CD31 antibody. After releasing the beads with trypsin/EDTA, the released cells were centrifuged and resuspended in growth medium for cell culture and further cloning.
Of 300 cells plated, 29 clones were obtained after two weeks and all clones were positive for CD31. To evaluate the specificity and recovery efficiency of this method, the CD31+ murine endothelial cell line H5V was mixed with a melanoma cell line and/or mixed with L929 fibroblasts. In both cases the percentage of isolated cells was > 98% and the recovery was 65% of the original CD31+ murine endothelial cells.
2.Primary mouse lung endothelial cell isolation from cultures
Hartwell et al (2) used lung tissue as plated cells. Dynabeads M-450 Sheep anti-Rat IgG were coated with rat anti-mouse intercellular adhesion molecule-2 IgG and added to the plated cells. After incubation, the cells were trypsinized to release all cells before isolation with the Dynabeads. Endothelial cells were stained for P-selectin, an adhesion receptor for leucocytes.
3.Pure fibroblast and endothelial cell colony cultures from murine bone marrow
The adherent stromal cells of bone marrow consist of endothelial cells, fibroblasts and macrophages. Endothelial cells and macrophages are phagocytic but fibroblasts are not. These differences can be used to isolate the different cell populations. Fei et al (3) used magnetic separation to obtain cultures of EC and fibroblasts (3). Bone marrow cells were cultured and non-adherent cells poured off. The adherent cells were subsequently used in two ways:
i) To obtain endothelial cells, adherent cells were released from the culture plates with trypsin and Dynabeads M-450 Epoxy were added at the ratio 3 beads to 1 cell and incubated for 2 hours at 37°C. At this temperature, phagocytic cells in culture engulf the beads. The bead-captured cells were collected with the magnet and non-phagocytic fibroblasts discarded. The bead/cell complexes were then cultured in low concentration for 10 days to reduce macrophage growth. Endothelial cell clones (> 20 cells) were then picked and cultured again to confluent growth.
ii) To achieve pure fibroblasts more magnetic beads were used. A 40 bead to 1 cell ratio was added to trypsinized adherent cells to ensure all phagocytic cells (endothelial cells and macrophages) engulf beads. The bead-captured cells were collected and the non-phagocytic fibroblasts were collected and cultured. The fibroblasts were re-passaged to ensure there were no contaminating cells and then grown to confluence.
1.Q. G. Dong, et al., A general strategy for isolation of endothelial cells from murine tissues -Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler.Thromb.Vasc.Biol. 1997; 17:1599-1604.
2.D. W. Hartwell, et al., Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses. Journal of Cell Biology 1998; 143 4:1129-1141.
3.R-G. Fei, et al., A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow. Exp.Hematol. 1990, 18:953-957.