Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures.

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Abstract
Long-term in vitro human hematopoietic (Dexter) cultures are limited both in their longevity (8-12 weeks) and in their cell production over time. Hematopoiesis in vivo continues unabated throughout life, suggesting that failure to obtain continuous hematopoiesis in vitro is probably due to the failure of culture conditions to accurately reproduce the in vivo state. Since bone marrow cells in vivo exist at high density and are rapidly perfused by plasma components, we examined the effects of the culture medium perfusion rate and inoculum density on longevity and productivity of human bone marrow cultures. Culture efficiency and longevity were relatively independent of the variation in inoculum density from 10(6) to 5 x 10(6) cells per ml but were significantly altered by the medium perfusion rate. Increased culture perfusion was superior to traditional Dexter schedules, with 0.5 medium volume exchange per day (3.5 volume per week) being optimal. The cultures under these conditions demonstrated an increasing rate of cell production from weeks 4 to 10, with the cell production rate doubling approximately every 2 weeks. Following weeks 10-12 in culture, the cell production rate of all cultures decayed. Production of nonadherent progenitor cells was also highest in cultures perfused at 3.5 medium volume exchanges per week, where progenitor cell production was stable from weeks 6 to 18. The nonadherent cells collected were predominantly macrophages by week 19, except for the cultures perfused at a rate of 3.5 volumes per week and seeded at 5 x 10(6) cells per ml, in which production of granulocytes remained high through week 19. Establishment of more optimal perfusion conditions supports the continuous stable generation of progenitor cells over 5 months in culture, suggesting that primitive stem cells are surviving and proliferating in these cultures.

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