, Volume 28, Issue 1–3, pp 127–138 | Cite as

Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

  • Antonio De león
  • Héctor Mayani
  • Octavio T. Ramírez


The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells.

expansion hematopoiesis monitoring oxygen uptake rate redox potential umbilical cord blood 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bradley TR, Hodgson GS and Rosendaal M (1978) The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro. J Cell Physiol 97: 517-522.CrossRefGoogle Scholar
  2. Collins PC, Papoutsakis ET and Miller WM (1996) Adaptation of hematopoietic cell culture to stirred systems. Paper 57a. American Institute of Chemical Engineers Annual Meeting, Chicago IL, U.S.A.Google Scholar
  3. Collins PC, Nielsen LK, Wong CK, Papoutsakis ET and Miller WM (1997) Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures. Biotechnol Bioeng 55: 693-700.CrossRefGoogle Scholar
  4. Collins PC, Nielsen LK, Patel SD, Papoutsakis ET and Miller WM (1998) Characterization of hematopoietic cell expansion, oxygen uptake, and glycolysis in a controlled, stirred-tank bioreactor system. Biotechnol Prog 14: 466-472.CrossRefGoogle Scholar
  5. Court JR (1988) Computers in fermentation control: laboratory applications. In: Bushell ME (ed.) Computers in Fermentation Technology. Progress in Industrial Microbiology, Vol. 25, Elsevier, Amsterdam, The Netherlands, pp. 1-45.Google Scholar
  6. Eyer K and Heinzle E (1996) On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement. Biotechnol Bioeng 49: 277-283.CrossRefGoogle Scholar
  7. Gluckman E, Broxmeyer HE, Auerbarch AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J and Boyse EA (1989) Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from a HLA-identical sibling. The New England J Med 321: 1174-1178.CrossRefGoogle Scholar
  8. Higareda AE, Possani LD and Ramírez OT (1997) The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures. Biotechnol Bioeng 56: 555-563.CrossRefGoogle Scholar
  9. Hu WS, Meier J and Wang DIC (1986) Use of surface aerator to improve oxygen transfer in cell culture. Biotechnol Bioeng 28: 122-125.CrossRefGoogle Scholar
  10. Hwang, C and Sinskey AJ (1991) The role of oxidation-reduction potential in monitoring growth of cultured mammalian cells. In: Spier RE, Griffiths JG and Meignier B (eds.) Production of biologicals from animal cells in culture, Butterworth-Heinemann, Oxford, U.K., pp. 548-568.Google Scholar
  11. Kimura T, Lijima S and Kobayashi T (1987) Effects of lactate and ammonium on the oxygen uptake rate of human cells. J Ferm Technol 65: 341-344.CrossRefGoogle Scholar
  12. Kohli-Kumar M, Shahidi NT, Broxmeyer HE, Masterson M, Delaat C, Sambrano J, Morris C, Auerbach AD and Harris RE (1993) Haemopoietic stem/progenitor cell transplant in Fanconi anemia using HLA-matched sibling umbilical cord blood cells. British J Haematol 84: 419-422.Google Scholar
  13. Koller MR, Bebder JG, Miller WM and Papoutsakis ET (1992) Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow. Exp Hematol 20: 264-270.Google Scholar
  14. Koller MR, Emerson SG and Palsson BO (1993) Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion culture. Blood 82: 378-384.Google Scholar
  15. Lavery M and Nienow AW (1987) Oxygen transfer in animal cell culture medium. Biotechnol Bioeng 30: 368-373.CrossRefGoogle Scholar
  16. Levee MG, Lee GM, Paek SH and Palsson BO (1994) Microencapsulated human bone marrow cultures: a potential culture system for the clonal outgrowth of hematopoietic progenitor cells. Biotechnol Bioeng 43: 734-739.CrossRefGoogle Scholar
  17. Mayani H, Guilbert LJ and Janowska-Wieczorek A (1990) Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures. Exp Hematol 18: 174-179.Google Scholar
  18. Mayani H, Dragowska W and Lansdorp PM (1993) Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines. Blood 82: 2664-2672.Google Scholar
  19. Mayani H, Guitiérrez-Rodríguez M, Espinoza L, López-Chalini E, Huerta-Zepeda A, Flores E, Sánchez-Valle E, Luna-Bautista F, Valencia I and Ramírez OT (1998) Kinetics of hematopoiesis in Dexter-type long-term cultures established from human umbilical cord blood cells. Stem Cells 16: 127-135.CrossRefGoogle Scholar
  20. Mayani H and Lansdorp PM (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells (in press).Google Scholar
  21. McAdams TA, Miller WM and Papoutsakis ET (1996a) Hematopoietic cell culture therapies (Part I): cell culture considerations. TIBTECH 14: 341-349.Google Scholar
  22. McAdams TA, Winter JN, Miller WM and Papoutsakis ET (1996b) Hematopoietic cell culture therapies (Part II): clinical aspects and applications. TIBTECH 14: 388-396.Google Scholar
  23. Palomares LA and Ramírez OT (1996) The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture. Cytotechnol 22: 225-237.CrossRefGoogle Scholar
  24. Pierson BA, Europa AF, Hu WS and Miller JS (1996) Production of human natural killer cells for adoptive immunotherapy using a computer-controlled stirred-tank bioreactor. J Hematother 5: 475-483.Google Scholar
  25. Peng CA and Palsson BO (1996) Determination of specific oxygen uptake rates in human hematopoietic cultures and implications for bioreactor design. Annals Biomed Eng 24: 373-381.Google Scholar
  26. Sandstrom CE, Bender JG, Miller WM and Papoutsakis, ET (1996) Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human ‘mobilized’ peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma. Biotechnol Bioeng 50: 493-504.CrossRefGoogle Scholar
  27. Sardonini CA and Wu YJ (1993) Expansion and differentiation of human hematopoietic cells from static cultures through small-scale bioreactors. Biotechnol Prog 9: 131-137.CrossRefGoogle Scholar
  28. Saito H, Ebisawa M, Tachimoto H, Scichijo M, Fukagawa K, Matsumoto K, Likura Y, Awaji T, Tsujimoto G, Yanagida M, Uzumaki H, Takahashi G, Tsuji K and Nakahata T (1996) Selective growth of human mast cells induced by steelfactor, Il-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol 157: 343-350.Google Scholar
  29. Zandstra PW, Eaves CJ and Piret JM (1994) Expansion of hematopoietic progenitor cell population in stirred suspension bioreactors of normal human bone marrow cells. Biotechnol 12: 909-914.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Antonio De león
    • 1
  • Héctor Mayani
    • 2
  • Octavio T. Ramírez
    • 1
  1. 1.Departamento de Bioingeniería, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoMorelosMéxico
  2. 2.Centro Médico Nacional Siglo XXI, MéxicoHospital de OncologíaD.F.México

Personalised recommendations