Reproducible cloning assays for in vitro growth of canine hematopoietic progenitor cells and their potential applications in investigative hematotoxicity.

A variety of in vitro cloning assays have been used for studying hematopoiesis in mice and human beings. However, these techniques have had limited use in dogs, a species used extensively as a model for hematopoietic research, particularly hematotoxicity. We have adopted cloning assays for in vitro growth of canine colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitor cells, using modified microplasma clot and soft agar culture systems, respectively. Marrow mononuclear cells separated by density-gradient centrifugation were added to the aforementioned culture systems. Erythroid colonies were stimulated with sheep plasma erythropoietin and incubated at 37 C in 5% CO2 for 2 days. The CFU-E colonies were fixed with 5% glutaraldehyde, stained with benzidine, counted, and expressed as a mean of 8 replicates. The CFU-GM colonies were stimulated with pooled serum from endotoxin-treated dogs and incubated for 8 days at 37 C in 10% CO2. Using an inverted microscope, the CFU-GM colonies were counted and expressed as a mean of 6 replicates. The number of colonies was proportional to the plated cell concentrations. The addition of 10% autologous serum to CFU-GM cultures increased the number of colonies by 80 to 100%, but markedly reduced the size and number of CFU-E colonies. The marrow cloning capacity among dogs of comparable age was similar, and little variation was noticed when bone marrow cells from the same dogs were cultured repeatedly over a period of 3 to 4 months. We concluded that these cloning assays are fast, reliable, and reproducible and that they allow quantitative determination of canine hematopoietic progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)