High yields from microcarrier cultures by medium perfusion

M. BUTLER 0 1 T. IMAMURAf 0 1 J. THOMAS 0 1 W. G. THILLY 0 1 0 Present address: Department of Biological Sciences , Manchester Polytechnic, Manchester M15 GD , U.K. ( "Present address: Department of Entomology, University of California , Riverside, CA 92521 , U.S.A 1 Department of Nutrition and Food Science, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, U.SA Significant progress has been made in the culture of anchorage-dependent animal cells (van Wezel, 1967, 1973) by the discovery of low-charge microcarriers, which serve as an anchor for cell growth in suspension (Levine, Wong, Wang & Thilly, 1977; Levine, Wang & Thilly, 1979; Thilly & Levine, 1979). Microcarrier cultures have been used to obtain relatively high yields of cells, which can be used for viral production (Giard, Thilly, Wang & Levine, 1977) or interferon production (Giard et al. 1979). In order to improve cell yields even further, and to study the causes for cell growth or inhibition, we have investigated the use of a perfusion system to provide a continuous supply of medium through the microcarrier cultures. This approach was used successfully by Kruse and coworkers (Kruse, Myhr, Johnson & White, 1963; Kruse & Miedema, 1965; Kruse, Miedema & Carter, 1967; Kruse, Whittle & Miedema, 1969; Kruse, Keen & Whittle, 1970) for the growth of various cell lines in T-flasks and roller bottles. In this work we use the word perfusion as defined by Kruse et al. (1963, 1965, 1967, 1969, 1970) to mean a constant supply of medium through our cultures. However, various other terms have been used in the literature for similar processes: superfusion (Smith & Vale, 1980), perifusion (Gebhardt & Mecke, 1979) and circumfusion (Rose et al. 1970). The value of such a perfusion system is to allow a constant supply of nutrients and CULTURES BY SUMMARY A culture perfusion system is described for the growth of anchorage-dependent cells on microcarriers. The critical component of this system is a column separator, which removes medium while allowing the microcarriers to remain in the culture. Amino acids and ammonia were analysed during cell growth of the perfusion culture. None of the amino acids was completely utilized. The accumulation of ammonia to 2-3 mM was observed and may be responsible for, or coincident with, events limiting further cell growth. It is suggested that oxygen deprivation and growth inhibitor accumulation, rather than nutrient depletion, are the major factors in limiting even higher cell yields. constant removal of inhibitors that may accumulate in the culture medium. In this manner a perfusion system more closely resembles an in vivo situation, in which nutrients and inhibitors remain at more constant levels instead of undergoing respective depletion and accumulation, as in a batch culture system. In one set of experiments, amino acid consumption and ammonia production were monitored. In another set of experiments, perfusion of cultures with diluted medium was used to distinguish between the effects of nutrient depletion and inhibitor accumulation on cell yield. By diluting the perfusing medium, the rate of nutrient supply to the culture was lowered while the rate of removal of inhibitors was kept constant. Madin-Darby canine kidney (MDCK) cells were originally obtained as an established cell line derived from the epithelium of a male Cocker Spaniel in 1958 (American Type Culture Collection). We obtained MDCK cells from Flow Laboratories, McLean, VA. Routine tests by fluorescence staining (Russell, Newman & Williamson, 1975) indicated that these cells were free of mycoplasma contamination (Bioassay Systems Corp., Woburn, MA). The cells were maintained on 150 cm2 T-flasks in 50 ml medium or on 490 cm2 roller bottles in 100 ml medium. Cells required for inoculation into spinner bottle cultures were harvested at the day of confluence. The medium from each T-flask or roller bottle was removed, the cells were washed in phosphate-buffered saline (PBS), and removal of cells from the plastic surface was effected by treatment with S ml of 1% trypsin (Flow Labs) plus 0-2% EDTA (Aldrich Chem. Co. Inc., Milwaukee, MI) in PBS. After treatment for lOmin at 37 C, trypsinization was halted by addition of 8 ml of medium. The cells were centrifuged and resuspended in fresh medium. Approximately 2 x 107 cells were obtained from each 150 cm2 T-flask and 8 X 107 cells from each roller bottle. The medium used for all cultures was Dulbecco's modified Eagle's medium (DMEM) containing 4mM-glutamine (Flow Labs). This was stored at 20C until required so that the decomposition of glutamine was minimized. The glucose was replaced by 20 mM-fructose (Sigma Chemical Co., St Louis, MO) in order to maintain a stable pH in cell cultures (Imamura, Crespi, Thilly & Brunengraber, 1982). The medium was supplemented with 10% horse serum (Flow Labs), and the same lot of undialysed horse serum was used in all experiments. In one set of experiments the DMEM was diluted with PBS or with a 'DMEM salt solution'. The DMEM salt solution contained the major inorganic salts of DMEM: CaCl2-2H2O (l-8mM); KC1 (5-4 miw); MgSCy7H2O (0-8HIM); NaCl (109mia); NaH2PO4-2H2O (1-OITIM); and NaHCO3 (44miw). An independently prepared solution of NaHCCb was filter-sterilized and added to an autoclaved mixture of the other salts. The pH of the combined solution was then adjusted to pH7-5. Spinner cultures and microcarriers Cells were grown in microcarrier cultures at 37 C in a 10% CCh/air overlay. Two-litre spinner flasks (Wheaton Scientific Co., Millville, NJ) were used for 500ml perfusion cultures. Smaller spinner flasks (Wilbur Scientific, Boston, MA), 250 ml and 500 ml, were used for 100 ml and 200 ml cultures, respectively. All spinner flasks were siliconized (Prosil-28, PRC Research Chemicals, Inc., Gainesville, FL). Each culture was stirred continuously by a teflon-coated suspended magnetic stir bar (4-5 cm) maintained at 60rev./min by means of a rotating magnetic base (Bellco Glass, Vineland, NJ). The microcarriers (Superbeads from Flow Labs) were washed in the culture medium for lOmin and added to the cultures at a concentration of 7-5 mg/ml. The system used is shown in Fig. 1. The inflow of medium to the culture was maintained by a peristaltic pump (model 7014, Cole-Palmer Instrument Co., Chicago, IL) at a rate of 1 ml/min for 500 ml cultures or 0-4 ml/min for 200 ml cultures. A silicon inflow tube (0-2 cm inner diam.; 0-5 cm outer diam.) passed from the medium reservoir through the peristaltic pump and into the culture. The outflow from the culture was controlled by a column separator at the liquid surface. This separator consisted of a thick piece of silicon tubing, the opening of which was held at the surface of the culture. The dimensions of the separator were 10 cm length and 2-5 cm i.d. for 500 ml cultures and 10 cm X 1-3 cm for 200 ml cultures. The separator was connected to a narrow tube (0'2cm i.d.), which carried (...truncated)