Schwartz R M, Palsson B O, Emerson S G
Department of Chemical Engineering, University of Michigan, Ann Arbor 48109.
Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6760-4. doi: 10.1073/pnas.88.15.6760.
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.
长期体外人造血（德克斯特）培养在其寿命（8 - 12周）以及随着时间推移的细胞生成方面都受到限制。体内造血在一生中持续不断，这表明在体外未能获得持续造血可能是由于培养条件未能准确重现体内状态。由于体内骨髓细胞以高密度存在并被血浆成分快速灌注，我们研究了培养基灌注速率和接种密度对人骨髓培养物寿命和生产力的影响。培养效率和寿命相对独立于接种密度从每毫升10⁶到5×10⁶个细胞的变化，但会因培养基灌注速率而显著改变。增加培养灌注优于传统的德克斯特方案，每天进行0.5倍培养基体积交换（每周3.5倍体积）是最佳的。在这些条件下的培养物在第4周到第10周显示出细胞生成速率增加，细胞生成速率大约每2周翻倍。在培养10 - 12周后，所有培养物的细胞生成速率下降。在每周进行3.5倍培养基体积交换灌注的培养物中，非贴壁祖细胞的生成也最高，在第6周到第18周祖细胞生成稳定。到第19周时收集的非贴壁细胞主要是巨噬细胞，除了每周以3.5倍体积速率灌注并以每毫升5×10⁶个细胞接种的培养物，其中粒细胞生成在第19周时仍保持较高水平。建立更优化的灌注条件支持在培养5个月以上持续稳定地生成祖细胞，这表明原始干细胞在这些培养物中存活并增殖。
