LONG-TERM CULTURE-INITIATING CELL EXPANSION IS DEPENDENT ON FREQUENT MEDIUM EXCHANGE COMBINED WITH STROMAL AND OTHER ACCESSORY CELL EFFECTS

Despite considerable effort, the expansion of long-term culture-initia ting cells (LTC-ICs) in cultures of purified hematopoietic cells has n ot yet been achieved. In contrast, LTC-IC expansion has been attained in cultures of bone marrow mononuclear cells (MNC) using frequent medi um exchange. The use of frequent medium exchange was, therefore, exami ned in cultures of CD34-enriched cells. In stromal-free, CD34-enriched cell cultures, medium exchange intervals ranging from 2 days to no fe eding for 14 days gave similar results. Six different growth factor co mbinations, reported by other groups to give optimal expansion of CD34 -enriched cells, were tested in comparison with the control combinatio n of IL-3/GM-CSF/Epo/SCF. None of the combinations resulted in improve d colony-forming unit-granulocyte macrophage (CFU-GM) expansion or LTC -IC maintenance, although two were equivalent. All stromal-free cultur es resulted in loss of LTC-IC to half of input. Because of the limited effect of medium exchange and growth factor variations on CD34-enrich ed cell cultures, the effect of preformed stroma was next examined. Pr eformed stroma increased cell (3-fold), CFU-GM (5-fold), and LTC-IC (3 -fold) output, but only when the medium was exchanged every other day. Under these conditions, the number of LTC-IC was maintained near inpu t level. The lack of LTC-IC expansion in CD34-enriched cell cultures p rompted experiments to examine the effect of cell purification. Parall el cultures were performed at CD34(+)lin(-) cell purities of 20%, 40%, 70%, and 95%, with each well containing exactly 4,000 CD34(+)lin(-) c ells in addition to the CD34(-) accessory cells required to give the d esired percentage. Also, MNC from the same source (similar to 2% CD34( +)lin(-)) were cultured at a concentration to give 4,000 CD34(+)lin(-) cells per well. As CD34(+)lin(-) cell purity was decreased from 95% t o 2%, the output of cells, CFU-GM, and LTC-IC increased by threefold t o fivefold. The loss of culture performance with purification was like ly due to the removal of important accessory cells, because the levels of endogenously produced leukemia inhibitory factor and IL-6 were fou nd to decline significantly with increasing CD34(+)lin(-) cell purity. In summary, preformed stroma abrogated the decrease in cell and CFU-G M output from cultured CD34-enriched cells and led to LTC-IC maintenan ce. In contrast, MNC inocula resulting in a growing stromal layer duri ng the culture led to LTC-IC expansion (3.2-fold). The results suggest that LTC-IC expansion is dependent on accessory cells that are presen t in MNC, whose function is, in turn, dependent on frequent medium exc hange. (C) 1995 by The American Society of Hematology.