Serum-free culture of human hemopoietic progenitors in attenuated culture media

To elucidate the precise mechanisms of molecular and cellular regulation of hemopoiesis, it is necessary to develop a chemically defined culture assay for purified hemopoietic progenitors. To approach this long-term goal, we attempted to develop a serum-free culture system for enriched human progenitors that permits expression of all hemopoietic lineages and stages of development. Preliminary studies indicated that alpha-medium was superior to Iscove's modified Dulbecco's medium (IMDM) and that culture under low (5%) oxygen condition was better than an ambient level of oxygen. We developed an attenuated (modified quarter-strength) alpha-medium and compared the colony-supporting ability of the three media by plating 1,000 bone marrow null cells per dish in the presence of a combination of recombinant human colony-stimulating factors (CSFs). The numbers of colonies supported in alpha-medium and attenuated alpha-medium were approximately 70% of those in serum-containing cultures. IMDM failed to support colony formation. While, in general, the colony sizes were smaller in the serum-free cultures than in the serum-containing cultures, a variety of types of single lineage and multilineage colonies were seen in serum-free culture. A linear relationship between cell number and colony formation was seen in 100-2,000 cells per dish. Serum-free cultures of enriched human progenitors should be an important tool for analysis of the mechanisms of recombinant CSFs.