Production of Human Natural Killer Cells for Adoptive Immunotherapy Using a Computer-Controlled Stirred-Tank Bioreactor

Abstract

Large-scale ex vivo expansion of human natural killer (NK) cells for adoptive immunotherapy requires assurance of good manufacturing practices. However, maximal expansion of NK is also desired to facilitate clinical trials with large numbers of IL-2-activated NK (ANK). A closed-system stirred-tank bioreactor is amenable to computer control of culture variables, thereby reducing the risk of contamination. We demonstrate that NK cultured in 250-ml spinner flasks expand 2.5-fold more than NK cultured in stationary tissue culture wells. We further show that during 33 days of culture, it is feasible to control the pH between 7.0 and 7.2 and the dissolved oxygen concentration at 40% of air saturation via direct on-line computer control in a 750-ml stirred-tank bioreactor. On-line measurement of optical density by a laser turbidity sensor, as a measure of cell concentration, correlated well with actual cell count data. NK expansion in the 750-ml bioreactor was 7-fold greater than in stationary tissue culture controls and 3-fold greater than in spinner flask controls. Consumption rates of glucose and oxygen and the production rate of lactate were measured and will be used to develop a nutrient feeding strategy to convert the batch reactor experiment into closed-system fed-batch or continuous flow modes. Computer-controlled stirred-tank bioreactors may facilitate clinical trials with high-purity ANK populations for adoptive immunotherapy.

Expansion of mouse embryonic stem cells on microcarriers

“…Due to their characteristics and wide use, stirred culture systems appear as promising candidates for the expansion of stem cells. For instance, several reports have been published using this system to expand bone marrow mononuclear cells (Zandstra et al, 1994), human natural killer cells (Pierson et al, 1996), hematopoietic progenitors (Collins et al, 1998a,b), neural stem cells (Sen et al, 2002b), mammary epithelial stem cells (Youn et al, 2005), primary brain astrocytes (Santos et al, 2005), and EBs Wartenberg et al, 2001). For the expansion of ES cells, so far, a single report has been published using a stirred system (Fok and Zandstra, 2005); therefore, research should be pursued in order to achieve a better understanding of the optimum culture conditions.…”

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: Shear, proliferation, and the IL-2 receptor

“…Bone marrow-derived mononuclear cells were successfully expanded in spinner flask cultures employing agitation rates of 40 and 45 rpm (Sardonini and Wu, 1993;Zandstra et al, 1994). Natural killer cells have been grown successfully in both 250-mL spinner flasks and in a 750-mL computer-controlled stirred tank bioreactor at 60 rpm (Pierson et al, 1996). Culture of T cells in a stirred bioreactor system is described by Swartz et al (1988), although no detail as to the agitation levels or degrees of expansion are given.…”

State-of-the-Art Review: Immunologic Methods of Purging in Autologous Stem Cell Transplantation

“…In spite of work to improve large-scale expansion of LAK and NK cells (58)(59)(60), it remains difficult to obtain sufficient cell numbers for clinical use. As an alternative, other groups have used cytotoxic cell lines, which are amenable to ex vivo expansion.…”