GROWTH OF ANIMAL CELLS AT HIGH OXYGEN CONCENTRATION

Abstract: A method of culturing mammalian cells to high density is disclosed. The method is based on the discovery that exponential growth of cultured cells can be achieved at oxygen concentrations above physiological levels. According to the method described, cells are cultured under conditions which include introduction into the medium of sufficient oxygen to result in homogeneous dissolved oxygen concentrations of between about 180 mu M and 500 mu M. Themethod provides for growth of animal cells to high density in fermentor without the need for expensive and failure-prone means to mesure and adjust oxygen concentration.

Description: GROWTH OF ANIMAL CELLS AT HIGH OXYGEN CONCENTRATION Growth of human and other animal cells using in XΪ.£_L£ culture techniques has a very important role in research and commerce contexts. However, use of cell culture for production of biological materials in larger quantities has been limited by diffi¬ culties in producing adequate yields and in pro- ducirig biologically active materials. Cell culture technology has typically been used for producing viral vaccines, which is now a major area in which large-scale cell culture technology is used. In recent years, large-scale cell culture techniques have been used to grow cells that produce alpha and beta interferon; hybridomas that produce monoclonal antibodies and genetically engineered cells that express proteins. Development of genetic engineering methods which make it possible to introduce foreign genes into cultured animal cells makes it possible to produce biologically active proteins not previously obtainable using prokaryotic cells or yeast cells. Cultured animal cells are either grown in suspension or attached to solid surfaces for suc¬ cessful growth; the latter are called anchorage- dependent cells. In culturing either type of cell, environmental control is an important consideration. That is, there are several characteristics of the culture system which must be carefully controlled for successful growth to occur. Typically, culture methods are designed to provide the cells with a similar environment or conditions under which they would grow in the intact animal. Important culture conditions which need to be controlled include temperature, pH, and concentration of several gases, including oxygen and carbon dioxide. Dissolved oxygen is a key nutrient in cell metabolism. Its availability can affect cell and product yield. At very low oxygen tensions, for example, cells tend to grow at a low rate; at oxygen tensions which are too high, the cells die. The optimum level of dissolved oxygen for cells to grow appears to depend on the cell line and varies widely among cell lines. Oxygen transfer to cells in culture medium is a critical factor and is often the growth-limiting factor in animal cell culture. Many approaches have been used to aerate cells being grown in culture. For example, oxygen transfer has been achieved by sparging air through a perforated ring or tube, which can be carried out in combination with stir¬ ring (e.g. , by mean of a magnetic stirring bar in a small-scale system or by means of an impeller in a large-scale system) . Other examples include oxygen transfer directly to the culture surface or through oxygen permeable tubing immersed in the culture medium. These ap¬ proaches in general have proved adequate for animal cell cultures of 1-5 million cells/ml. However, commerical applications based on cell densities as high as 10-50 million cells/ml are potentially possible if a means existed to provide an adequate consistent supply of oxygen to growing cells. Numerous studies exist detailing adverse effects such as lower growth rates or cell death when excessive or insufficient oxygen concentrations are provided. Fleischaker, R. J. et a].. Euro£ean j £urnai_£f_A££ii d_M^£r£bi£^o_^2_ £_.__^i __ _l_l£2. iaZ» 12:193-197 (1981) ; Joenje, H. et al. , Lab£rat£ry_ ISZ !tig tio , 5_2.:420-428 (1985) ; Sturrock, J. E. and J. F. Nunn, Mutation_Research , 57 : 27-33 (1978) ; "Standard engineering approaches involving oxygen sensors within feedback control systems have not been successful in large scale applications." Fleischaker e_t_ ; _a 1 . , in Mamma].i.a _ l.l_T hn£l£gγ_, e d. by W.G. Thilly, Butterworth Publ. Co. , Stoneham, MA (1986) . Oxygen sensor instability is one known reason for this lack of success. Development of a technique which would make it possible to meet the oxygen needs of cultured cells without the use of oxygen sensors would provide a means of growing animal cells Ln vi.tr£ to higher numbers (e.g. , greater density) than presently possible. This invention pertains to a method of cultur- ing animal cells to high density by employing a high and constant oxygen concentration in the gas phase throughout the period of cell culture. The inven¬ tion is based upon the discovery that exponential cell growth of a variety of cell types can be maintained at oxygen concentrations significantly above arterial oxygen concentration, for instance, from 180 micromolar (μM) up to 500 μM. Because of this, an initial concentration of dissolved oxygen can be established in an animal cell culture medium at higher than physiological concentrations and this high oxygen concentration can be maintained through¬ out the culture period (without necessary further adjustment) to supply sufficient oxygen for cell growth to a high density. According to the method of this invention, a culture vessel is provided having means for trans - fering oxygen into a cell culture medium. A medium supportive of growth of the animal cells to be cultured is added and an inoculum of cells is introduced therein. Oxygen is introduced into the medium by the oxygen transfering means under con¬ ditions that provide a dissolved oxygen concentra- tion at the desired initial level above about 180 μM and as high as about 500 μM (levels which are well above the physiological concentrations such as 90-180 μM used in conventional processes for cell culture). A high (e.g. , 20% to 55%) level of oxygen in the gas phase ensures a high initial oxygen transfer rate into the medium. As the cell density increases, the oxygen concentration in the liquid will decrease. However, this will cause the rate of oxygen transfer to increase. Therefore, as the cell density of the culture increases the oxygen demand of the cells is continuously satisfied. With knowledge of the oxygen uptake rate of the cultured cells, the desired final cell density and the oxygen tolerance of the cells, the oxygen concentration of the gas phase and K τ a are chosen so that the level of oxygen in the liquid never decreases below a certain predetermined level. In this way, an initial concentration of oxygen in a gas phase can be predetermined which, without further change or monitoring, can supply a sufficient amount of oxygen to the medium to support exponential cell growth to high density. The gas can be introduced by slow sparging into the medium or via oxygen permeable tubing immersed in the medium. Examples of suitable oxygen per eable tubing include silicone rubber tubing and ® stretched polytetrafluoroethylene (TEFLON ) tubing. Use of such tubing is believed to provide a large surface area for gas transfer into solution (i.e. , into the culture medium) . In preferred embodiments , medium containing cells to be grown is agitated to establish and maintain a uniform oxygen concentra¬ tion throughout and to eliminate local buildup of toxins (e.g. , oxygen related) around the cells. Such cultures are also operated in the perfused liquid mode to supply nutrients and to remove water-soluble toxins. The transfer rate of oxygen to cells achieved with high oxygen content of the introduced gas, in combination with agitation of the cell-containing medium, allows high dissolved oxygen concentrations to be maintained throughout the medium, thus providing sufficient ox gen for the requirements of cells grown to high density. The method of the present invention provides means to maintain sufficiently high concentration of dissolved oxygen to supply the oxygen demand for animal cell growth to high density without the need for continuous monitoring of oxygen concentration in the medium. The method can be used to grow cells for use themselves (e.g. artificial skin) or for production of cell products (e.g. , hormones, immuno - globulins and vaccines). These findings have substantial implications for process control. The previously unrecognized ability of cells such as hybridoma cells to grow exponentially in the presence of high levels of dissolved oxygen enables the operation of culture vessels with either oxygen-enriched gas mixtures or with air under pressure. Maximum attainable cell density and/or maximum culture size thus can be increased several-fold without the expense and complication of oxygen control feedback. BrjL f_D £ri££i£n_£f_ h _Fi 1 gur Figure 1 shows the relationship between several different ways of expressing oxygen concentra ion in a cell culture medium. Figure 2 shows a gasification system useful for maintaining a uniform oxygen concentration through¬ out the volume of a cell culture. Figure 3 shows a gasification useful for maintaining a uniform oxygen concentration, useful for microcarrier culture of anchorage-dependent cells (as well as for suspension cells). Figure 4 shows growth curves of TK6 at various oxygen concentra ions. Figure 5 shows growth curves of Vero cells at various oxygen concentrations. Figure 6 shows growth curves of the Sp2/0 - derived cell line at various oxygen concentrations: no oxygen control, θ 90μM, Al80μM, Δ 360μM, 540μM. Detailed Description of the Invention According to one embodiment of this invention animal cells are placed at low cell concentration (3 to 15 x 10 cells/ml) in a fermentor with the intention that they should increase by continuous cell division to at least about 1 to 5 x 10 cells/ ml and, at this high cell density, be used to produce desired cell products. In conventional cell culture technology the aim is to maintain oxygen concentration above approximately 20 μM to provide oxygen for cellular oxidative metabolism and below approximately 150 μM, depending on the type of cell grown, to avoid toxic effects oxygen on cells. However, Applicants experiments revealed that, surprisingly, human B cells, myeloma cells transfected with immunoglobulin genes (i.e. transfectomas) and primate Vero cells maintain growth at oxygen concentrations as high as 500 μM. This invention is based on the discovery that several types of cells in fermentors can tolerate "unphysiologically" high oxygen concentrations for a period of greater than several days. This phe¬ nomenon has been observed surprisingly in cells which have not been previously mutated or adapted to tolerate high levels of 0_ . As a result, a cell culture can be initiated at much higher oxygen concentration than previously employed and main¬ tained at that concentration. As the cell concen¬ tration rises in culture the utilization rate of oxygen also rises, since the oxygen uptake is generally proportional to cell number. Consequent¬ ly, the actual oxygen concentration is reduced below a level which might (if continued) be toxic to cells. This decrease, however, will occasion an increase in the rate of oxygen transfer from the gas phase to the liquid phase (medium) according to the relationship : d0 2 /dt <C - C i> (1) Where K τ a - mass transfer coefficient of oxygen for the system C = concentration of dissolved oxygen in equilibrium with oxygen in gas phase C. =» actual dissolved oxygen concentr tion in the culture medium d0_/dt = oxygen transfer rate A direct consequence of the high (and un¬ changing) oxygen concentrations in the gas phase is that, as the oxygen concentrations in the fermentor drops, the rate of oxygen transfer to the fermentor from the gas phase increases as a function of (C C.) . Thus, even as cell densities rise to usefully 1 7 7 high levels (e.g. , 1 x 10 to 5 x 10 cells/ml) , sufficient oxygen transfer occurs to prevent liquid dissolved oxygen concentration from declining below approximately 20 μM, the level believed necessary to maintain cell growth and productivity. In a preferred embodiment of this invention, a single constant concentration of oxygen is employed in the gas phase to supply the oxygen demand of the cell culture during the entire course of culture. One can determine by routine experimentation, the rate of oxygen uptake per cell for the during exponential growth for the animal cell to be cul¬ tured, the K a for the oxygen transfer apparatus to be used, and the maximum oxygen concentration which would permit approximately normal cell doubling times for several days of growth. Based upon these factors a single, predetermined, initial oxygen concentration in the gas phase can be used without further change or measurement as the fermentor cell density increases from an initial low value e.g. , 3 to 15 x 10 cells/ml, to a final high of from 1 to 5 x 10 cells/ml, or higher. Alternatively, oxygen concentration can be changed over the culture period. For example, an initial 0- concentration, e.g. from 180 or 200 μM to about 450 μM can be used which later can be raised to 360-500 μM as cell density (and 0„ utilization) increases. This strategy could maximize cell growth rates at early stages of culture by utilizing a somewhat lower initial and better tolerated 0„ concentration, in the case of 0 intolerant cells. The method of this invention is applicable to a wide variety of animal cells capable of being grown in cell culture. Animal cells include, but are not limited to, mammalian cells such as human B cells, human and/or murine myeloma cells, myeloma cells that have been transfected with foreign genetic material, hybridoma cells, primate kidney cells, TK6 , Vero, WI-38, and FS-4 cells, among others. The following examples illustrate the calcula- tion of initial dissolved oxygen concentration for several animal cells. Some of the cell oxygen uptake rates are taken from the literature. See e.g. , Fleischaker e_t a1 ^ . (1981) sujra. T 6__cell At a of 6 hr (obtainable, e.g. , with silicone tubing) and a dissolved 0_ concentration of 180 μM , the maximum oxygen transfer rate. (OT ) = _. ma K τ a(C -C.) - 6 hr (180 μM 0. - 20 μM 0.) - 960 μM L o i 2 2 0_/l-hr. The 0„ uptake rate of TK6 cells was -6 measured to be 0.24 x 10 μM 0 /cell-hr so a c ^ maximum of 4 x 10 cells/ml could be obtained before 0„ concentration dropped below 20 μ . If the C is 2. o increased to 500 μM 0„ then the OTR is 2800 μM ^ 2 max ^ 0„/cell-hr which will support a cell density of 1.2 7 x 10 cells/ml (a three-fold increase over that obtained at a dissolved 0„ of 180 μM) . Vero_cells The 0„ uptake rate of Vero cells was measured -6 to be approximately 0.25 x 10 μM 0-/cell-hr. -1 Where K τ a - 10 hr (obtainable e.g. , with caged aeration) and C. — 20 μM and the maximum dissolved 0. is 180 μM, OTR - K τ a(C -C.) - 1600 μM 0,,/1-hr 2. max i_ o l . z which will support the growth of 6.2 x 10 cells/ml. A C of 420 μM, where C. - 20 μM, provides an oxygen transfer rate of 4000 μM 0„/l-hr which could support 7 z the growth of 1.5 x 10 cells/ml (a 2. -fold increase) . WI-38 cells The human diploid cell line WI-38 is reported to have an oxygen uptake rate of 0.15 x 10 μM 0-/cell-hr. Fleischaker, R.J. t^ al^_, J. Annl. i£ϊ_.££Ϊ£.__• * ϊ£._= £]l- ' ϋ£_E.£- ^o S^ ow these cells to a concentration of 3 x 10 cells/ml, without causing a drop in 0„ concentration below 20 μM, the oxygen uptake rate to be provided for is 4500 μM 0_/l-hr. -1 z Assuming K τ a - 10 hr and if C - 180 μM and C. - " L o ^ l 20 μM, the oxygen transfer rate is 1600 μ 0„/l-hr which is too low to support growth at this density. A C value of 470 μM gives an oxygen transfer rate of 4500 μM 0„/l-hr which is sufficient for cell growth to the prescribed density. FS-4 cells FS-4 cells are reported to have an 0„ uptake -6 rate of 0.05 x 10 μM 0„/cell-hr (oxygen uptake rate taken from Fleischaker, R. J. j _ ; _ al^, .J. ££l Mi£ £^£_l . Bj.£_t £h. , £££ra. In a system where K τ a is 10 hr-1 C is 360 μM and C. - 20 μM, the OTR 0 ^ i max is 3400 μM O./l-hr which will support FS-4 cells at 1 7 a density of 6.8 x 10 cells/ml. In the examples for WI-38 and FS-4, rates of 0„ uptake were taken from the literature. For TK-6 and Vero cells 0„ uptake rates are based on direct measurements made by Applicants. In the above Examples, the maximum K τ a was -1 approximately 10 hr . With the use of stretched ® poly etrafluoroethylene (Teflon ) tubing (e.g. ® Goretex tubing; 1 mm I. D. /thin wall, with no external matrix; W.L. Gore, Inc.) a K a of about -1 15-40 hr can be obtained. This allows the crowth of animal cells to even higher densities For example, at a K_ a of 30 hr and a dissolved 0„ oi 500 μM, an OTR /l-hr can be ob- tained. This will support TK6 cells to a density of 6 x 10 cells/ml (before C. drops below 20 μM 0 ) . ® With Gortex tubing as the 0„ transfer means, Vero cells could be grown to a density of 4.1 x 10 cells/ml when Co is 360 μM 0„ (before C. drops under 20 μM 0 2 ) . The concentration of dissolved oxygen in a culture medium is a function of its intrinsic solubility in the medium, the oxygen concentration in the gas phase in equilibrium with the medium, and the pressure of the system. The concentration of oxygen in pure water at a given temperature, in equilibrium with air was obtained from Lange^_s tt≤Ξdbook_£f_Chemϊ.strγ (ed J.A. Dean, 1979). At 37°C the dissolved oxygen concentration in pure water in equilibrium with air saturated with water vapor is 198 μtt 0, (Lange^s_Handbook_ of_Chemistry) . The solubility of oxygen in RPMI 1640 was estimated using the value for dissolved oxygen in pure water and the data by Schumpe e£ al ^ . (1978) . The estimate obtained for the dissolved oxygen concentration in the RPMl 1640 medium at 37 β C, 180 μM, agrees with that obtained by Fleischacker (MIT Thesis, 1982) . The estimate of the dissolved oxygen in the medium represents a lower limit, as organic substrates and products present In the medium may increase the oxygen solubility slightly (Schumpe e al. 1978). The concentration of oxygen in medium obeys Henry's Law, CO. π . . , - CO- x H, where H = J 2 liquid 2 gas Henry's constant. Therefore, we have made the assumption that if we, for example, double the concentration of oxygen in the gas phase from 20 (atmospheric) to 40%, then the dissolved oxygen concentration in the medium in equilibrium with the gas phase will double also, so that, for example, at 40% oxygen in the gas phase the dissolved oxygen in the medium, at equilibrium, will be approximately 360 μM 0-. The relationship between the concen¬ tration of 0 „ in the gas phase and medium, at equilibrium, is summarized in Figure 1. The relative level of dissolved oxygen in the liquid is commonly measured using a galvanic oxygen probe. The dissolved oxygen is commonly expressed as a percentage where 0% is zero dissolved oxygen in the medium (for example, in liquid in equilibrium with pure nitrogen gas) and where 100% is the dissolved oxygen level in the liquid in equilibrium with air at one atmosphere. This scale is also shown on Figure 1. According to the method of this invention, cells can be grown freely in suspension or, for anchorage dependent cells, anchored to an appro¬ priate support. For anchorage dependent cells, the preferred support is microcarriers , such as those described by Thilly and Levine . See U.S. Patent 4,189,534 (1980) and U.S. Patent 4,293,654 (1981) . Microcarriers provide a large surface area or c ll attachment and growth. The culture medium can be any of the conven¬ tional media for cell growth such as RPMl 1640 medium, Eagle's basal medium, Modified Eagle's medium (MEM) and Dulbecco's modified Eagle's medium (DMEM) . Selection of the appropriate media depends on the particular requirements of the type of cells being grown. The optimal medium for a particular cell type can be ascertained readily by persons of ordinary skill in the art. Culture vessels (fermentors) can be those conventionally used in the art. Such vessels can consist of a container of sufficient capacity to hold the cell-containing medium, a gasification or mass transfer means capable of delivering oxygen or other gases to the medium, a means to evenly suspend the culture and a means for maintaining the ste- rility of the culture. A modified spinner fermentor for growth of anchorage-dependent cells on microcarriers at constant oxygen concentration is shown in Figure 3. According to the method of this invention oxygen is provided to the medium as a gas phase at initial concentrations and maintained at these concentrations during all phases of growth. The system for transfer of oxygen into the medium can be any conventional method for gasifying of cell culture vessels. For example, oxygen permeable tubing can be used. An example system is silicone rubber tubing. The tubing can be immersed and colled in the cell culture medium to provide a larger gas/liquid interface for oxygen transfer. Gas is passed through the immersed silicone rubber tubing and transferred into solution. The dissolved oxygen concentration is a function of the oxygen tension of the perfusing gas. While the oxygen is being pumped through the silicon tubing, the cell- containing medium is agitated (e.g. , by stirring) in order to result In a homogeneous oxygen concentra¬ tion throughout the medium. As described in detail in the example, a mixture of oxygen (60%) and air (40%) can be pumped through tubing to produce an approximate oxygen concentration In the medium of 540 μM . The oxygen is pumped into the tube and passes into the medium, which is being stirred at ® the same time. As described, a stretched Teflon ® tubing, such as Goretex , can be used. The present invention is further described in the following examples, which is not to be con¬ sidered limiting in any way. EXAMPLES EXAMPLE_1 ϋi-ii : _£_■_Human_B_l^m h£ JLa j: jLd_£ l. ls__at_o_ v^en S££Sϋ £i B^_ £Z _£li_ i£i _ii _:_.__._.X-.i-. C ].l._l.i.n _and_£ul._tur£_medium The cell line employed was the diploi human 5 lymphoblastoid cell line TK-6 (See U.S. Patent No. 4,302,535) . Cells were grown in suspension in E.PMI 1640 medium supplemented with glutamine and bi¬ carbonate and with 10% horse serum (Gibco) . C_ ].l._C_u] 1 _tu£ _S ^ y; _t£m A culture system was designed to grow human B lymphoblastoid cells under constant oxygen concen¬ tration. To study the toxic and mutagenic effects of various oxygen tensions, it is necessary to devise a culture system that permits the maintain- ence of fixed oxygen concentration for long periods of time (for long term/low dose studies) . Such a system should: a) have the capacity to sustain the growth of 200-500 ml of cultured cells in suspen¬ sion; b) contain a mass transfer system that maintains a constant oxygen concentration in the medium during the daily growth of the cells (from 3 x 10 5 to 9 x 105) ; and c) remain sterile during the daily operations of sampling and diluting. A system that meets the above criteria is shown in Figure 2. The j.ars used (e.g. , Ball canning jars) have a maximum volume of 1 liter; a silicone rubbing tubing (1/8" I.D. , 3/8" O.D. Cole Parmer) was coiled inside the jar and against the jar walls. The desired oxygen mixture was passed through about 80 cm of silicon tubing. The gas permeability of the tubing allows rapid transfer of oxygen into the medium. After a short period of time, the concen¬ tration of oxygen in the medium reaches equilibrium with the oxygen concentration in the gas mixture; the oxygen concentration in the medium remained constant with constant gas flow. The mixture of gases and the flow through the tubing were adjusted and regulated using a mass flow eter, substantially as shown In Figure 2. A Pasteur pipette and two sections of tubing permitted sterile sampling and diluting via siphoning. Cells were diluted daily to 3 x 10 cells/ml; thus, Table 1 and figure 4 show cumulative cell numbers. A dissolved oxygen (D.O.) probe (Ingold) was used to measure the oxygen concentration in the medium for various oxygen mixtures , confirming the accuracy of the gas flow regulation. Using a flow rate of 500 ml/min and fast stirring, 90% saturation with pure oxygen was achieved after one hour. Cell counts were made with a model B Coulter Counter (Coulter Electronics, Hialeah, FL) . The proportion of 0„ in the gas mixture was adjusted so that oxygen concentrations in the culture could be maintained at approximately 70,250,500 or 600 μM. The results of the experimental work are shown in the Table 1 and Figure 4. TABLE 1 Gr£wth_at_varjL£U£_££n tan£_£xy^££_££££ £_=£ £A££ Days Relative Cell Density cells/ml x 10 70 uM 0 -°* 250 uM -°* 500 uM O 600 uM 2.7 -°* 2.7 2.7 2.7 1 8.0 9.7 7.3 5.8 2 22.0 22.7 14.1 7.5 3 63.0 58.3 30.5 9.2 4 156.3 52.8 10.0 5 468.9 88.0 Cells grew at 70 μM and 250 μM 0„ with a doubling time of approximately 16 h. Cell growth slowed at 500 μM 0„ and there was marked inhibition of cell growth at 600 μM 0„ . These results show that cells are able to grow at 250 μM 0 up to 500 μM 0 „ contrary to reports in the literature that cell growth would be Inhibited at oxygen concentra¬ tions higher than 180 μM (40% oxygen) . See e.g. , Kilburn and Webb (1968) Biotech. Bioeng. 10:801-814; Ruckert and Mueller, Cancer Res . (1960) 20 : 9^44-949 ; Balin et al. (1976) J. Cell. Biol.. T&_ : 390 " 48 ° • Taylor et al. (1978) J. Cell. PhV Ϊ£l.. 9J5 : 33 - 40 - Results of this experimental work show that cells grow at higher concentrations of dissolved oxygen (higher oxygen tensions) than previously thought. The high transfer rate which results when higher than physiological oxygen concentrations in the medium are used makes it possible to grow cells in culture to higher density than is possible with presently available methods; the levels used, contrary to expectation, were not toxic to cells under the conditions described. To distinguish between cytostatic and cytotoxic effects of high oxygen concentrations, we measured survival at different times during exposure to a variety of oxygen concentrations, either by a cloning assay or by back extrapolation of growth curves after ceasing treatment. Table 2 shows the relative survival (viability) of TK6 cells at various times during growth at various constant oxygen levels . Table_2 Percent relative survival of TK6 cells at different oxygen concentrations Oxygen concentrations in μM Days of Treatment 75 a .95 a 265 b 390 b 635 a 905 b 100 90 119 91 n.d. 60 90 95 107 106 50 33 n.d. 101 96 96 n.d. n.d. n.d. n.d. n.d. n.d. 40 n.d. a. Relative survival measured by extrapola ion of growth curves after ceasing treatment. b. Relative survival measured by plating efficiency relative to control cultures. The absolute plating efficiency of control cultures was between 60-80%. c. No data No adverse effects of oxygen on relative survival were detected for oxygen concentrations of 75 μM, 95 μM, 265 μM, and 390 μM. Thus , the inhibition of growth seen at 390 μM (doubling time of 18 hours) was not due to cell toxicity. However, at constant oxygen levels of 535 μM, survival of the cells decreased to 50% after two days of exposure. This cytotoxic effect thus contributed to an apparent doubling time of 20 hours. Interes ingly, for oxygen levels of 600 to 720 μ , a partial recovery from the toxic effect was observed after 6 days of continuous growth at those concentrations (data not shown) . During continuous exposure to 905 μM oxygen, a monotonic decrease in survival was detected. After 24 hours, 60% of the cells were viable. After 48 hours, 30% of the cells were viable. These toxic effects were irreversible as cell survival continued to decrease exponentially as a function of exposure time.. E MPLE_2 __£i_H._- _£.___Y ££__£ __i ____.___£2£____ £_££££____-_2_-_-__i_£Σ_ -_£_£_£_!Σ£i£.i£.EΪ_£ i___. X __ C ]_i_L.in _and_C_ul :ur _M di£m The cell line employed was Vero cells. Vero cells are a stable, diploid anchorage - dependent cell line derived from African Green Monkey Kidney. 2 Stock Vero cells were grown In 490 cm polystyrene roller bottles (Corning Glass Works, Corning, NY). Cells were harvested from roller bottles by removing the spent medium, washing once with 100 ml phosphate buffered saline PBS, and then treating with 2 ml of a 0.1% trypsin (Flow Laboratories) in PBS solution for 3 minutes at 37 β C. The cells were resuspended In medium and counted using a Coulter Counter (as in example 1). Spinners (Bellco or Wilbur Scientific, Inc.) were inoculated with 150 ml of medium with cells at approximately 2.4 X 10 cells/ml and with 4 g/1 microcarriers (Superbeads, Flow Laboratories, Inc.) . The medium was RPMl 1640 supplemented with glutamine and bicarbonate (Gibco Laboratories, Inc.) and 10% horse serum (HyClone) . Cell._Cu].ture_^ys.teπ A culture system was designed to grown an¬ chorage dependent cells , such as Vero cells , on microcarriers in spinner culture under constant oxygen tension. This system is shown in Figure 3. The spinner flasks (Bellco) have a working volume of 100-300 ml. Silicone rubber rublng (0.058" O.D. x 0.077" O.D.) was glued in to a coil and placed Inside the spinner with one end venting to the headspace. A gas mixture with a desired percentage of oxygen was passed through about 65" of coiled silicone tubing. The gas permeability of the tubing combined with the surface aeration to give a K a of .1 about 10 hr with 150ml liquid. Mixed gas cylinders contained 5% C0_ , the desired percentage oxygen, and the balance nitrogen were purchased from Granite State. A dissolved oxygen probe (Ingold) was used to measure the oxygen concentration in the medium for various oxygen mixtures . Cell counts were done each day using the method of Sanford et al. , J. Natn. Cancer Inst. 6_.3: 773 -795 (1951) , centrifuged samples were incubated with a 0.1% crystal violet -0.1M citrate solution 1-2 hours at 37°C, and then the stained nuclei wre counted isomg a hemocytometer . The results of the experimental work are shown in the Table 3 and Figure 5. Table 3 Gr£ th_£f_Ver£_Ce^l _at_Var^ U _C£nc£n£ra£i£n _ f_Ox^£ n Time Relative Cell density (cells/ml x 10 5 ) Controls (no oxygen control-.0 Λ (£X∑.g££_££££££.£__- .ti£ ) _. Hours Days between 0 + 180 μM) 180 μM 360 μM 540 μM 720 μM A B 0 0 2.4 2.4 2.4 2.4 2.4 2.4 2 0 2.6 2.3 2.5 2.0 - - 19 1 3.0 2.2 3.1 2.9 2.0 2.3 40 2 4.9 5.6 5.0 5.1 3.5 2.8 65 3 7.3 9.1 7.2 7.4 4.6 2.6 88 4 8.6 13.9 9.0 8.3 4.0 2.7 114 5 13.5 18.6 18.4 16.4 4.6 2.6 140 6 - 21.1 - - 5.4 2.4 160 7 15.6 26.1 20.3 21.7 5.1 2.5 It can be seen that the doubling times and final cell density are similar when cells are grown without oxygen control (0-180 cells μM 0_) , at 180 μM 0„ and at 360 μM 0- . Significant slowing of growth was seen at 540 μM 0-. At 720 μM (80% 0 ) , the Vero cells failed to grow. Oxygen levels up to at least 360 μM were not toxic, while oxygen levels above 540 μM were toxic and slow growth. Therefore, we believe that the maximally tolerated 0„ concen¬ tration for Vero cells lies between 360 μM and 540 μM (e.g. , 420 μM) and that, as is the case for the TK-6 cells, oxygen concentrations of up to 420 μM 0 „ were not toxic to the Vero cells and that oxygen levels below 520 μM and at least 360 μM can be used to allow the growth of Vero cells to high cell densi ies . EXAMPLE... 3 Culture_of_Sp2/0_D£r.ived -d£il.-_._ -ϊ_2i__i__ Il__i£_l£ £-_£ -ii£-l__-__£Σ£ Z_iΣ£i£i£≤i£_._:_L Y ._. Cell_^ine_and_culture_medium A murine transfec oma , derived from the myeloma Sp2/0 cell line, was obtained from Centocor, Inc. (Malvern, PA) . This cell line was obtained by transfection of expression vectors containing immunoglobulin genes into mouse myeloma Sp2/0 cells. §e_e Sun, L.K. et. al Proc^ Nat_^l_-_ Acad^ Sci . U3A, 84, 214-218 (1987); Shaw.D.R. et^ al^, J^ Bi£l Res£. M£d. , 7_, 204-211 (1988). This cell line secretes IgG. Cells were grown in suspension in DMEM, supplemented with 2mM glutamine, 2mM pyruvate, 50 uM Beta-mercaptoethanol, and 2.5% each of horse serum and heat-treated fetal bovine serum. Cell Culture System Sp2/0- derived cells were grown In the same spinners described for Vero cells (see Figure 3) . Cells were inoculated from exponentially growing stock cultures at 3 x 10 cells/ml and diluted tto .5 maintain the cell density between 3 and 12 x 10 ' cells/ml. Spinners were sampled and the cells counted daily. Control cultures were grown in an open air incubator with 5% CO.. Experimental culture oxygenation was as described as for Vero cells . Growth of cells was examined at constant oxygen concentrations ranging from 90 μM to 540 μM. Oxygenation of unregulated control cultures was provided by surface aeration from the headspace, whichwas open to the 5% C0„ incubator. The doubling times of control cultures was 14 to 15 hours. Figure -_. 6 presents the growth curves of the Sp2/0-derived cells at various dissolved oxygen concentrations, shown as relative cumulative cell number. It can be seen that growth at 360 μM was slowed slightly relative to the control cultures. Doubling times were nearly constant over a 5 to 7 day period for all cultures and were calculated from growth data between days 1 and 5. Doubling times for cultures at constant 90 μM oxygen were 14 to 15 hours, identical to the control cultures. Doubling times at 180 μM, 360 μM and 540 μM oxygen were 15.8 to 16.2, 19.5, and 31 hours, respectively Growth of Sp2/0-derived cells at 360μM was slowed slightly relative to control cultures but the change in doubling time parallels results seen for TK6 and Vero cells. Conclusion Doubling times are considered good indicators of the overall health of the cultures. These cell lines could grow at or near their maximum rates at concentrations well in excess of air saturation (200 μM) . Moreover, for all the cell lines, exponential growth was seen at concentrations up to 2.5 times air saturation. The cell lines exemplified herein are commercially useful. Vero cells are used in production of viral vaccines, Sp2/0 cells are used as fusion partners for hybridoma production, or as recipient cells for plasmid(s) containing the genes for products of interest, such as imraunoglobulins (e.g. production of transfeetomas ) , and TK6 cells are used in genetic toxicology. Nevertheless, these cell lines are not known to be unusually tolerant of high levels of oxygen. Thus, it is unexpected that these three different cell types are capable of exponential growth at near-maximum rates in the presence of oxygen at twice the level of atmospheric saturation. ££__.iZ£i I_-Ξ. Those skilled in the art will recognize, or be able to ascertain using no more than routine experi¬ mentation, many equivalents to the specific embodi¬ ments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims: CLAIMS A method of culturing animal cells, comprising the steps of: a. introducing the cells into a culture medium into which oxygen is transferred under conditions which provide an initial dissolved oxygen concentration of from about 180 μM to about 500 μM; and b. maintaining the conditions of oxygen transfer into the medium to sustain the desired oxygen concentration and to supply sufficient dissolved oxygen for cell growth to a desired density. A method of Claim 1, wherein the animal cells are mammalian cells. 3. A method of Claim 2, wherein the mammalian cells are selected from the group consisting TK6 cells, Vero cells, WI-38 cells and FS-4 cells . 4. A method of Claim 2, wherein the mammalian cells are hybridoma or transfectoma cells. A method of Claim 4, wherein the hybridoma or transfectoma cells are Sp2/0 -derived murine cells . 6. A method of Claim 1, wherein oxygen is transferred into the culture medium by means of an oxygen permeable , silicone tubing immersed in the culture medium. 7. A method of Claim 6, wherein a gas mixture of oxygen, air and carbon dioxide is passed through the tubing, the partial pressure of oxygen in the gas mixture and the rate of flow of the gas mixture throughout the tubing being sufficient to provide a concentration of dissolved oxygen in the medium of from about 180 μM to about 500 μM. 8. A method of Claim 1, wherein oxygen is trans¬ ferred into the culture medium by means of an oxygen permeable, stretched tetrofluoroethylene tubing immersed in the culture medium. 9. A method of Claim 8, wherein a gas mixture of oxygen, air and carbon dioxide Is passed through the tubing, the partial pressure of oxygen in the gas mixture and the rate of flow of the gas mixture throughout the tubing being sufficient to provide a concentration of dissolved oxygen in the mediam of from about 180 μM to about 500 μM. 10. A method of Claim 1, wherein the cells are grown in suspension in the culture medium. 11. A method of Claim 1, wherein the cells are grown attached to microcarriers in the culture medium. 12. A method of culturing animal cells, comprising the steps of: a. providing a fermentor containing a culture medium supportive of animal cell growth, the fermentor having means for transfer¬ ring oxygen gas into the culture medium; b. introducing animal cells into the culture medium; c. transfering oxygen gas into the culture medium under conditions which provide an initial dissolved oxygen concentration ranging from about 180 μM to about 500 μM ; and d. maintaining conditions of transfer of oxygen into the medium to sustain the desired oxygen concentration and to supply sufficient dissolved oxygen for cell growth to a desired density. 13. A method of Claim 12, wherein the animal cells are mammalian cells. 14. A method of Claim 13, wherein the mammalian cells are selected from the group consisting of TK6 cells, Vero cells, WI-38 cells and FS-4 cells . 15. A method of Claim 13, wherein the mammalian cells are hybridoma or transfectoma cells. 16. A method of Claim 15 , wherein the hybridoma or transfectoma cells are derived from Sp2/0 murine myeloma cells. 17. A method of Claim 12, wherein oxygen is trans¬ ferred into the culture medium by means of an oxygen permeable silicone tubing immersed in the culture medium. 18. A method of Claim 6, wherein a gas mixture of oxygen, air and carbon dioxide is passed through the tubing, the partial pressure of oxygen in the gas mixture and the rate of flow of the gas mixture throughout the tubing being sufficient to provide a concentration of dissolved oxygen in the medium of from about 180 μM to about 500 μM. 19. A method of Claim 12, wherein oxygen is trans¬ ferred into the culture medium by means of an oxygen permeable, stretched tetrafluoroethylene tubing immersed in the culture medium. 20. A method of Claim 19, wherein a gas mixture of oxygen, air and carbon dioxide is passed through the tubing, the partial pressure of oxygen in the gas mixture and the rate of flow of the gas mixture throught the tubing being sufficient to provide a concentration of dissolved oxygen in the medium of from about 180 μM to about 500 μM. 21. A method of Claim 12, wherein the cells are grown in suspension in the culture medium. 22. A method of Claim 12, wherein the cells are grown attached to microcarriers in the culture medium.

Inventors: BUSER CLAUDIA W, OLLER ADRIANA, THILLY WILLIAM C, BUSER, CLAUDIA, W., OLLER, ADRIANA, THILLY, WILLIAM, C.
Applicants: MASSACHUSETTS INST TECHNOLOGY, MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Application year: 1989
Main classification: C12N5
IPC codes: C12N5