FREQUENT HARVESTING FROM PERFUSED BANE MARROW CULTURES RESULTS IN INCREASED OVERALL CELL AND PROGENITOR EXPANSION

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of col ony forming units-granulocyte/macrophage (CFU-GMs), and a four- to ten fold increase in the number of long-term culture initiating cells (LTC -ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading t o stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based biore actor systems. One possible constraint is the size of the growth surfa ce area required. This constraint can be overcome by harvesting half t he cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the C FU-GM expansion exceeded 30-fold over a 27-day period. These calculate d values are based on growth that could be obtained from the harvested cell population. Growth of the ad here nt cell layer was stable, wher eas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols publishe d to date are suboptimal for long-term cultivation, and that further d efinition and refinement is likely to lead to even greater expansion o f hematopoietic cell populations obtained from bone marrow. More impor tantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realize d by frequent harvesting. Finally, the present culture conditions prov ide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo. (C) 1994 John Wiley and Sons, Inc.