A micromethod for long-term in vitro culture of bone marrow cells

A micromethod to study long-term culture of bone marrow cells in vitro has been developed. Using Linbro wells one-fourteenth of the cells and medium usually required for culture in a flask are plated. Thus 40-50 cultures from a single mouse can be studied at any one time. Adherent-layer formation and supernatant cell recovery were very similar when Linbro -well cultures were compared with standard large-flask cultures. In the microculture system, supernatant and adherent-layer cell numbers increased following medium change reaching a maximum 3-4 days later and decreasing by day 7. Supernatant cells were in equilibrium with those of the adherent layer as cell numbers in both compartments prior to and following medium change fluctuated identically. Following medium change, a significant increase in myeloblasts occurred at 24 h and promyelocytes and myelocytes increased 48-72 h later. In contrast no discernible pattern in daily CFU-C production was detected. These findings provide insight into the cellular kinetics of long-term marrow culture and highlight the usefulness of this method to study hematopoiesis at frequent intervals.