Slide chamber culture system for the in vitro study of humoral regulation of granulocyte and monocyte-macrophage proliferation and differentiation

A liquid culture system employing slide chambers was developed to facilitate the study of proliferation and differentiation of mouse neutrophilic granulocytes and cells of the monocyte-macrophage series. Cultures were initiated with 1–2.5 X 10(4) light density marrow cells which had been fractionated on Ficoll-Hypaque. In the presence of colony-stimulating activity (CSAHU), two types of clusters were observed. One was tight, spheroidal, and composed of neutrophilic granulocytes, while the other was a loose grouping of flattened cells of the monocyte-macrophage series. The slide chamber culture is adaptable to microscopic assay techniques (e.g., cluster size, autoradiography, immunofluorescence) as well as quantitative biochemical methods (e.g., rate of 3H-thymidine incorporation, DNA quantitation). We have demonstrated both a shortening of the generation time of granulocytes in tight clusters and increasing rates of 3H- thymidine incorporation into DNA, with a corresponding increase in total culture DNA as a function of CSAHU concentration. Granulocytic differentiation in the tight spheroidal clusters has been demonstrated by histochemical stains and immunofluorescence of an antibody to a marker protein specific for the secondary granule of the neutrophil (lactoferrin).