Characterization of Hematopoietic Cell Expansion, Oxygen Uptake, and Glycolysis in a Controlled, Stirred-Tank Bioreactor System Paul C. Collins, † Lars K. Nielsen, ‡ Sanjay D. Patel, E. Terry Papoutsakis, and William M. Miller* Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208-3120 Cultures of umbilical cord blood and mobilized peripheral blood mononuclear cells were carried out in a stirred bioreactor with pH and dissolved oxygen control. Expansion of total cells and colony-forming units granulocyte-macrophage was greatly enhanced by the use of a cell-dilution feeding protocol (as compared to a cell-retention feeding protocol). The specific oxygen consumption rate (q O 2 ) for these cultures ranged from 1.7 × 10 -8 to 1.2 × 10 -7 μmol/(cell‚h). The maximum in q O 2 for each culture closely corresponded with the maximum percentage of progenitor or colony-forming cells (CFCs) present in the culture. The maximum q O 2 values are slightly less than those reported for hybridomas, while the lowest q O 2 values are somewhat greater than those reported for mature granulocytes. Examination of the ratio of lactate production to oxygen consumption in these cultures suggests that post-progenitor cells of the granulomonocytic lineage obtain a greater portion of their energy from glycolysis than do CFCs. The different metabolic profiles of CFCs and more mature cells suggest that monitoring the uptake or production of oxygen, lactate, and other metabolites will allow estimation of the content of several cell types in culture. Introduction Recent clinical trials (Williams et al., 1996; Bertolini et al., 1997) have demonstrated that ex vivo expanded hematopoietic cells offer great promise in reconstituting in vivo hematopoiesis in patients who have undergone intensive chemotherapy. Thus, it is likely that the demand for ex vivo expanded hematopoietic cells for transplantation in the treatment of cancer will increase dramatically. It is therefore necessary to develop clinical- scale culture systems that both provide expansion of desired cell types and satisfy FDA requirements (U.S. Food and Drug Administration, 1997). Recently, we described the successful application of spinner flask culture for hematopoietic cells from a variety of sources in both serum-containing and serum-free media (Collins et al., 1998). Spinner flask systems have also been used to culture bone marrow (BM) mononuclear cells (MNCs) (Zandstra et al., 1994; Sardonini and Wu, 1993). Al- though peripheral blood (PB) MNC-derived natural killer (NK) cells have been cultured in a stirred bioreactor (Pierson et al., 1996), controlled, stirred-tank bioreactor systems have not yet been reported for the culture and characterization of myeloid-lineage hematopoietic cells. A well-controlled, closed, and reproducible culture environment, such as that offered by stirred bioreactors, will undoubtedly prove advantageous for clinical applica- tions, especially considering the scale involved for clinical cultures. The culture volume employed for recent clinical trials averaged about 5 L (Zimmerman et al., 1995; Williams et al., 1996). Fifty T-150 flasks each containing 100 mL of culture medium or 20 gas-permeable 300-cm 2 culture bags each containing 250 mL would be necessary to accommodate this volume. These phase I clinical trials were conducted to determine the safety of infusing expanded cells. As trials continue, greater numbers of cells will undoubtedly be transfused in an effort to increase the efficacy of expansion protocols. While bone marrow was the traditional source of hematopoietic cells for transplantation therapies, other blood cell sources are becoming more popular. Mobilized PB progenitor cell transplants have proved effective, and it is likely that mobilized PB MNC (hereafter just PB MNC) will replace BM MNC as the preferred source of hematopoietic cells for transplantation (Korbling and Champlin, 1996). Umbilical cord blood (CB) is both readily available and easily collected. CB stem cells are thought to be more immature than those found in adults. This attribute makes CB stem cells a potential target for the correction of genetic blood diseases (Clapp and Williams, 1995). We demonstrate that both CB and PB MNC can be successfully cultured in a stirred bioreactor configuration. Traditional hematopoietic cell cultures also suffer from an inability to fully characterize cell behavior. We have previously demonstrated that glucose and lactate meta- bolic rates are directly related to the progenitor or colony- forming cell (CFC) content of hematopoietic cultures (Collins et al., 1997). Data regarding oxygen consump- tion rates in human hematopoietic cultures are scarce, and the published reports have not fully examined the effect of various cell populations on glucose and oxygen metabolism. We therefore examined the effect of differ- ent hematopoietic populations (CFCs and more mature cells) on oxygen consumption and on the ratio of glycolytic to oxidative metabolism. We found that the maximum specific oxygen consumption rate (q O2 ) corresponds to the maximum in the percentage of CFCs present. The ratio of the specific lactate production rate (q lac ) to q O2 increased * Corresponding author. Telephone: (847) 491-4828. Fax: (847) 491-3728. E-mail: wmmiller@nwu.edu. † Present address: Merck Research Laboratories, P.O. Box 2000, RY801-107, Rahway, NJ 07065. ‡ Present address: Chemical Engineering Department, Univer- sity of Queensland, Brisbane QLD 4072, Australia. 466 Biotechnol. Prog. 1998, 14, 466-472 S8756-7938(98)00032-0 CCC: $15.00 © 1998 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/07/1998