Abstract:
The present invention includes a bioreactor support system that provides an improved way to culture large quantities of mammalian cells. Mammalian cells are extremely sensitive to their environment, undergoing a programmed cell death in response to nutrient deprivation, growth factor withdrawal, oxygen starvation and excess shear levels. The present invention includes a bioreactor support system which permits the culture of cells at high density and prevents cell death by providing enhanced oxygen delivery to the cells without excessive shear; maintaining cell secreted or exogenously added low molecular weight products (e.g., growth, differentiation and maintenance factors) in the culture space during perfusion culture; and provides the ability to feed the cultures without dilution of the conditioned media. The improved bioreactor support system features two flow paths emanating from a central integrating chamber. The first flow path is a slow speed loop from a bioreactor containing a cell retention module and a return line for cells. Cell-free media from the cell retention module enters the integrating chamber. The second flow path is a high speed loop containing an oxygenation module and a dialysis module. The return line from the integrating chamber to the bioreactor is a rate controlled perfusion system containing oxygen-saturated, pH adjusted, nutrient-rich, metabolic waste depleted, conditioned media. The support system can be used to enhance the growth and maintenance of mammalian cells in a variety of bioreactor configurations, including fermentors, spinner flasks, flexible bags, rotational devices and hollow fibers.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The present invention generally relates to a system and method for growing or maintaining biological cells in vitro. More specifically, the present invention relates to a bioreactor system and method that are effective to grow or maintain biological cells at a high cell density, while providing adequate oxygen and nutrients to, and removing waste products, from the biological cells.  
           [0002]    Mammalian cells are increasingly used in diagnostic and medical applications. For example, mammalian cells may be used for production of proteins for vaccines, therapeutics and diagnostics. In addition, mammalian cells may be used for adoptive cell therapy or tissue engineering. Furthermore, mammalian cells may be used as part of a medical device that functions as an artificial organ. As a result, there is an increase in demand for mammalian cells and/or mammalian cell-based products. The increase in demand has fueled a need for techniques and/or equipment that promote efficient mammalian cell growth and/or productivity.  
           [0003]    Bioreactors have commonly been employed for the cultivation of living organisms, such as mammalian cells. A bioreactor typically includes a housing that contains cells and nutrients maintained at bioreactor conditions that permit cell growth and/or production of secreted products. Furthermore, bioreactors used for mammalian cell cultures are well known in the art, and are commercially available from a variety of manufacturers (see, Maisenholder, G., The Scientist 13(14):21 1999). As an example, bioreactors may include spinner flasks, roller bottles (see U.S. Pat. No. 8,866,419 issued to Meder), hollow fibers (see U.S. Pat. No. 3,997,396 issued to Knazek), stirred tank fermenters, gas permeable bags (see U.S. Pat. No. 6,190,913 issued to Singh) and porous bed reactors (see U.S. Pat. No. 5,510,262 issued to Stephanopoulos).  
           [0004]    Cell culture efficiency and productivity of mammalian cells are generally related to (1) supply of oxygen, (2) supply of nutrients and removal of waste products, and (3) control of the cell micro-environment.  
           [0005]    Supply of Oxygen  
           [0006]    The growth and culture of mammalian cells typically require a constant supply of adequate oxygen. Oxygen diffusion in culture media is a function of a liquid-to-air surface area when operating the bioreactor. Furthermore, oxygen transfer is limited by the liquid-to-air surface area and any shear forces created by agitation and/or sparging. In addition, as the bioreactor volume increases, the ability to adequately supply oxygen is diminished.  
           [0007]    As a result, several methods have been employed to provide adequate oxygen to bioreactors to permit scale-up for cell culture production. The methods include agitation (see U.S. Pat. No. 6,199,913 issued to Singh), sparging by introducing gas bubbles into airlift fermenter devices or oxygenating by diffusion through gas permeable tubing (see U.S. Pat. No. 5,112,760 issued to Baumgartner and U.S. Pat. No. 5,081,035 issued to Halberstadt). Oxygen diffusion may also be increased through increased pressurization of the bioreactor headspace.  
           [0008]    Furthermore, addition of a draft tube has been used to enhance axial mixing in a bioreactor and reduce bubble coalescence that result in smaller bubbles. Smaller bubbles are desirable in bioreactors since smaller bubbles produce less shear and increase a rate of oxygen transfer and/or oxygen diffusion. Nonetheless, even with these methods, adequate oxygen supply is still a major impediment to scaling-up mammalian cell cultures produced in bioreactors.  
           [0009]    When bioreactor volumes are greater than three liters, air sparging is required for effective oxygen transfer since introducing bubbles into a culture media by sparging results in a dramatic increase in the liquid-to-air surface area. In addition, agitation is used to break up the bubbles to thereby further increase oxygen transfer.  
           [0010]    Unfortunately, both bubbling and agitation typically have a detrimental effect on biological cells, such as mammalian cell cultures. Biological cells may be rendered non-viable through bubble breakup and/or coalescence within the culture media, especially at a surface gas-to-liquid interface. Therefore, maximizing oxygen transfer in the bioreactor must be balanced by maintaining cell viability.  
           [0011]    Supply of Nutrients and Removal of Waste Products  
           [0012]    Mammalian cell cultures typically utilize glucose as an energy source. When glucose is limited, glutamine becomes a predominant energy source. As a result, most culture media include glucose as a primary nutrient component along with glutamine and other types of amino acids. Under typical cell culture conditions, mammalian cells metabolize (1) glucose into lactate, and (2) glutamine into ammonia. Unfortunately, accumulation of metabolic waste products, such as lactate and ammonia in cell cultures greatly restricts cell concentration, cell growth and cell viability.  
           [0013]    When biological cells are grown in the bioreactor, such as a roller bottle, a flask, a bag or a fermentor, the bioreactor may be operated in a batch culture mode. In the batch culture mode, the bioreactor is inoculated with cells at a starting concentration of between about 0.1 million cells per milliliter (cells per mL) and 0.5 million cells per mL of culture media. Next, the cells are grown to confluence or to about 1 million cells per mL to about 2 million cells per mL. After reaching about 1 million cells per mL to about 2 million cells per mL, cell growth is terminated, and contents of the bioreactor are harvested.  
           [0014]    When the number of cells for inoculation is limited, the bioreactor can be operated in a fed-batch culture mode. In the fed-batch culture mode, cells are inoculated at a same concentration as in the batch culture mode, but into a lower volume than a maximum liquid bioreactor volume. Next, culture media is continuously added to the bioreactor until the maximum liquid bioreactor volume is reached. Both batch culture and fed-batch culture modes are the least efficient modes of operating a bioreactor, since both culture modes do not have provision for removal of metabolic waste products like lactate and ammonia.  
           [0015]    However, accumulation of metabolic waste products can be reduced by changing from the batch culture or the fed-batch culture modes to a continuous perfusion culture mode. In the continuous perfusion culture mode, fresh culture media is continuously added to the bioreactor, while culture media already present in the bioreactor that contains cells, secreted products, exogenously added factors, metabolic waste products and/or unused nutrients are continuously removed. As a result, the continuous perfusion culture mode enables (1) continuous removal of metabolic waste products, (2) replenishment of nutrients, and (3) an extended life for cell cultures. In addition, bioreactor productivity, as measured in weight per volume per unit of time, is significantly higher in the continuous perfusion culture mode rather than the batch culture or fed-batch culture modes.  
           [0016]    However, in order to maintain adequate levels of nutrients and avoid accumulation of metabolic waste products, large volumes of culture media must be perfused through the bioreactor. The large volume requirement of culture media makes the continuous perfusion culture mode both expensive and labor-intensive. In addition, beneficial products, such as the biological cells and exogenously added factors are also removed with the metabolic waste products. Removal of beneficial products from the bioreactor decreases the efficiency of the bioreactor operating in the continuous perfusion culture mode.  
           [0017]    As an example, the continuous perfusion culture mode may operate in a chemostat design. A bioreactor operating in a continuous chemostat mode is programmed to maintain a constant volume in the bioreactor by continuously feeding culture media into the bioreactor and continuously removing waste culture media from the bioreactor. Maintaining the constant volume in the bioreactor may be accomplished by using an overflow device that causes excess culture media to overflow when the bioreactor volume rises above the overflow device. In addition, maintaining the constant volume in the bioreactor may also be accomplished with matched pumps for feeding the culture media into the bioreactor and removing the waste media from the bioreactor. Furthermore, the feed rate for the culture media can be varied to adjust to the cell growth rate in order to maintain a constant number of cells in the bioreactor.  
           [0018]    The loss of biological cells when operating the bioreactor in the continuous chemostat mode typically decreases the productivity and increases the expense of operating the bioreactor. As a result, a number of modifications to reduce loss of cells have been attempted. The modifications include the incorporation of methods to retain cells in the bioreactor. Unfortunately, cell retention is most difficult when cells are grown in batch suspension culture mode. Anchorage-dependent cells or adherent cells, on the other hand, can be grown on solid matrixes, such as micro-carriers. Nonetheless, employing micro-carriers to retain cells often creates more technical problems, such as the necessity for maintaining suspension of the micro-carriers.  
           [0019]    In addition, mixing of cells, whether the cells are in suspension or immobilized on micro-carriers, typically subjects the cells to additional mechanical shear stress which may result in cell rupture, since mammalian cells are fragile and very sensitive to shear stress. Solutions for retaining cells and/or micro-carriers in the bioreactor during perfusion, such as in-line cell filters which permit fluid to pass but retain the cells have problems with clogging, especially when high molecular weight proteins are present in the culture media (see U.S. Pat. No. 4,166,768 issued to Tolbert). The in-line cell filters also impede flow in the bioreactors, and thus limit the culture media feed rate that perfuses through the bioreactor.  
           [0020]    Another approach is to incorporate an in-line settling device, which allows cells to return to the bioreactor by gravity. Unfortunately, bioreactors that include in-line settling devices also limit the culture media feed rate that perfuses through the bioreactor. In addition, the in-line setting devices may restrict the scale-up of the bioreactor.  
           [0021]    Cells may also be immobilized by entrapment in solid, non-moving surfaces such as plastic blocks, ceramic matrixes, fibrous material or shavings of plastic or glass wool. Cells may also be immobilized inside micro-capsules, polysaccharide gels, porous beads, or hollow fiber membranes in order to retain them in the bioreactor operating in the continuous perfusion culture mode. Nonetheless, cell immobilization or encapsulation also creates an additional barrier for the diffusion of oxygen to the cells.  
           [0022]    Another technical problem with bioreactors operating under the continuous perfusion culture mode is that the high turn over of culture media or the high culture media flow rate through the bioreactor can dilute cell secreted products, which would require additional processing steps. Additional processing steps typically result in an increase in expense since subsequent purification steps would be necessary.  
           [0023]    Additionally, cells often secrete protein growth factors that support continued cell growth, differentiation, function and viability. Protein growth factors may be exogenously added to the cell culture in order to support cell growth, as is, or as part of a serum. Unfortunately, bioreactors operating under continuous perfusion culture modes typically dilute the protein growth factors in the culture media, and therefore, add to the expense of operating bioreactors since the protein growth factors require replacement.  
           [0024]    Therefore, many prior art bioreactors are limited by problems of nutrient exhaustion, growth factor deprivation and metabolic waste product accumulation. Furthermore, these problems generally increase as the size of the bioreactor increases.  
           [0025]    Control of Cell Micro-environment  
           [0026]    Mammalian cell cultures typically grow to relatively low densities of about 1 million to about 2 million cells per ml when prepared in bioreactors operating under the batch suspension culture mode. Furthermore, some scientists have observed that operating bioreactors under the continuous perfusion culture mode often enables bioreactors to grow the cells to a higher density than is achieved when operating the bioreactor under the batch suspension culture mode. Indeed, bioreactors operating under continuous perfusion culture mode can increase the cell density by about 3 to about 30 fold.  
           [0027]    Unfortunately, growing and maintaining the cell culture at the high density creates additional technical problems. As an example, the amount of nutrients consumed and metabolic waste production produced per unit volume increases in proportion to the increase in cell density. The increase in nutrient consumption and metabolic waste product places a greater requirement for efficient feed and waste removal systems. In addition, increased production of metabolic waste products like increased lactic acid per unit volume fuels a greater requirement for a more sophisticated pH control. Typically, pH is controlled by addition of sodium bicarbonate to the bioreactor. In the presence of high levels of lactate, addition of sodium bicarbonate causes an increase in osmolarity that can be detrimental to cell cultures.  
           [0028]    In another example, cell cultures maintained at the high density also consume more oxygen per unit volume. Increased oxygen demand increases requirements for maintaining oxygen saturation levels around the cells. However, maintaining adequate oxygen saturation levels in the bioreactor typically requires the use of gas sparging and/or higher agitation rates that increase hydrodynamic shear forces.  
           [0029]    In addition, cells also produce significant levels of carbon dioxide at the high density which can induce cell death if carbon dioxide is allowed to accumulate in the bioreactor. Unfortunately, current gas sparging techniques are designed to increase the amount of oxygen available to cell cultures and not the removal of carbon dioxide from the bioreactor.  
           [0030]    Therefore, there exists an urgent need to design a bioreactor system that is effective to deliver adequate oxygen and nutrients to mammalian cells. In addition, an urgent need exists to provide a bioreactor system that efficiently removes metabolic waste products from the culture media without dilution of exogenously added protein growth factors or removal of valuable biological cells. Furthermore, an urgent need exists to control the cellular micro-environment within a bioreactor system so that high cell culture densities may be obtained without concomitant destruction of cell culture viability.  
         BRIEF SUMMARY OF THE INVENTION  
         [0031]    The present invention relates to a bioreactor support system having a cell retention module, an integrating module and a reconditioning media loop. The cell retention module is in communication with a bioreactor which has a cell culturing chamber. The chamber has ingress for oxygenated waste-free cell culturing media and egress for waste-containing, oxygen-depleted media. The integrating module provides to the bioreactor the oxygenated waste-free cell culturing media while accepts the waste-containing, oxygen depleted-media and provides such waste-containing, oxygen-depleted media to the reconditioning media loop. The reconditioning media loop includes a media oxygenator and can also include a dialysis device. The media within the reconditioning loop is circulated through the oxygenator and the dialysis device at a flow rate greater than the flow rate suitable for culturing cells and thereby provides on a continuous basis to the integrating module, and in turn to the bioreactor, oxygenated media with waste removed suitable for culturing cells.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0032]    [0032]FIG. 1 illustrates a bioreactor system in accordance with the present invention.  
         [0033]    [0033]FIG. 2 illustrates an alternate embodiment of a bioreactor system in accordance with the present invention. 
     
    
     DETAILED DESCRIPTION  
       [0034]    The present invention generally relates to a system and method for growing or maintaining biological cells in vitro. More specifically, the present invention relates to a bioreactor system and method that are effective to grow or maintain biological cells at a high cell density, while providing adequate oxygen and nutrients to, and removing waste products, from the biological cells.  
         [0035]    A bioreactor system is generally depicted at  10  in FIG. 1. The bioreactor system includes:  
         [0036]    (a) a bioreactor  11  in communication with a cell retention module  12 ;  
         [0037]    (b) an integrating module  14  for collecting cell-free conditioned culture media;  
         [0038]    (c) a high speed circulation loop containing an artificial lung device  20  (oxygenator) connected to the integrating module  14 ;  
         [0039]    In an alternate embodiment, FIG. 2 illustrates a bioreactor system that includes:  
         [0040]    (a) a microporous hollow-fiber cell retention module  24  connected to a bioreactor  26  where the cell-containing culture media is slowly pumped through the lumen of the hollow fibers  28  of the cell retention module  24 ;  
         [0041]    (b) a pump  30  or other motive force that facilitates movement of culture media from the extra-capillary space of the cell retention module  32  to an integrating module  34 ;  
         [0042]    (c) the integrating module  34  which receives the cell-free culture media from the extra-capillary space  32  of the cell retention module  24 ;  
         [0043]    (d) a high speed pump  36  connected to an artificial lung  38  (oxygenation module);  
         [0044]    (e) a heat exchange device  40 ;  
         [0045]    (f) a return line  42  from the artificial lung  38  to the integrating module  34  that returns oxygen-saturated, pH-adjusted culture media;  
         [0046]    (g) a high speed pump  44  connected to a lumen side of an artificial kidney (dialysis module)  46 ;  
         [0047]    (h) a waste line  48  connected to a shell side  50  of the artificial kidney  46  for removal of metabolic waste products  52 ;  
         [0048]    (i) a return line  54  from the artificial kidney  46  to the integrating module  34  that returns high molecular weight proteins and metabolic waste-depleted media;  
         [0049]    (j) a return line  56  from the integrating module  34  to the bioreactor  26  that provides oxygen-saturated, waste-depleted conditioned culture media.  
         [0050]    Cell Retention Module  
         [0051]    The cell retention module  24  functions to retain and/or separate cells from culture media when using the bioreactor system  10  of the present invention. Preferably, the cell retention module  24  is a membrane device, such as a hollow fiber membrane cartridge that provides a barrier to separate and/or cells from culture media components. The hollow fiber membrane device is selected to have a membrane molecular weight cut-off of less than a size of the cells so that cells will remain on one side of the membrane, while media can freely flow across or through the membrane.  
         [0052]    Selection of membranes with a higher porosity typically improve culture media flow and decrease the possibility of clogging of the hollow fiber pores. In addition, it is desirable to select a hollow fiber membrane cartridge with a higher surface area in order to prevent clogging of the hollow fiber membrane. Preferably, the molecular weight cut-off of the hollow fiber membranes is in the ultrafiltration range of 0.2 to 0.65 microns.  
         [0053]    Cell-containing culture media that is removed from the bioreactor  26  is delivered to the cell retention module  24 , and preferably to the lumen side of the hollow fibers. A wider inner diameter of the hollow fibers is desirable, such as an inner diameter in excess of 250 microns, in order to avoid excessive shear on the cells trafficking through the lumen side of the hollow fibers.  
         [0054]    Though descriptions of the present invention are primarily made in terms of the preferred cell retention module  24  that includes a hollow fiber membrane cartridge, it is to be understood that any other cell retention device, such as settling devices, may be substituted in place of the cell retention module  24  in accordance with the present invention while still realizing benefits of the present invention. Likewise, it is to be understood that any combination of any hollow fiber membrane cartridge and any other cell retention module may be used in accordance with the present invention, while still realizing the benefits of the present invention.  
         [0055]    The cell retention module  24  may be placed at any position proximate the bioreactor  26 , the integrating module  24 , the artificial lung  38  or the artificial kidney  46  when practicing the present invention so long as the cell retention module  24  is effective to retain cells. Preferably, the cell retention module  24  is placed above the bioreactor  26  to create a settling effect on the cells traveling through the cell retention module  24 . The cells, therefore, travel against gravity when passing through the cell retention module  24  to minimize the percentage of cells which travel through the cell retention module  24 , and thus minimize the risk of damage to large numbers of cells trafficking through the cell retention module  24 .  
         [0056]    Cell-free culture media is removed from the cell retention module  24  through the extra-capillary ports  58  and  59  on the shell side of the cell retention module  24  as best depicted in FIG. 2. Preferably, the cell-free culture media is removed by a pump  60  connected to the extra-capillary ports  58 ,  59 . The pump  60  may be operated at pre-determined on-off intervals or continuously. As an example, minimum cell damage is attained when the pump  60  is on for 1 minute and off for the amount of time it takes for media to travel from one end of the cell retention module to the other (not shown). The time maybe varied by adjusting the rate of the pump  30  which delivers cell-containing culture media from the bioreactor  26  to the cell retention module  24  and/or by selecting a shorter hollow fiber membrane cartridge. In addition, the off-cycle permits flushing off cells from the membranes that may have been retained on the fiber walls due to the hydrostatic force caused by the removal of culture media radially across the hollow fiber membrane.  
         [0057]    Integrating Module  
         [0058]    The integrating module  34  generally serves as an interface between the slow speed perfusion loop  62  that delivers culture media to the bioreactor  26  and the high speed loop  64  which delivers culture media to the oxygenation module  38  and the dialysis module  46 . As an example, the integrating module  34  may be a spinner flask with a cover designed to accommodate all entry and exit ports for a fluid path into the bioreactor  26 , or a plastic, disposable vessel. Preferably, a volume of the integrating module  34  ranges between about 25 to about 50% of the volume of the bioreactor  26 .  
         [0059]    High Speed Circulation Loop Containing an Artificial Lung  
         [0060]    Culture media is oxygenated in a high flow rate circuit  61  from the integrating module  34  to the oxygenation module  38  and then back to the integrating module  34 . The oxygenation module  38  or artificial lung  38  maybe a gas-permeable hollow fiber cartridge. As an example, an artificial lung device such as the Capiox SX 10 module manufactured by Terumo may be used to provide oxygen, remove carbon dioxide and control pH. For efficient oxygen transfer to the culture media, the rate of culture media flow through the oxygenation module  38  may range from between about 1 liter per minute (L/min) to about 4 L/min. At about 1 L/min approximately 80 milliliters per minutes (ml/min) of oxygen is transferred into the media. At about 4 L/min, the transfer rate of oxygen increases to over 245 ml/min. In addition, the oxygenation module  38  contains a heat exchange component  40  that enables warming of the fluid path without the need for placing the oxygenation module  38  in an incubator. A circulating water bath  66  is connected to the heat exchanger  40  in order to maintain physiological temperature for the cell cultures. If the volume of the bioreactor  26  is too large and/or the perfusion rate too low, it may be necessary to provide an additional heat source to the bioreactor  26  in order to effectively control the temperature.  
         [0061]    A controlled gas mixture is introduced into the oxygenation module  38  through amass control device  39 . Generally, a mixture of between about 0% and about 10% carbon dioxide in air is adequate for pH control and oxygen saturation. In addition, the gas mixture may be passed through the mass control device  39  at a rate of about 100 ml/min, for example.  
         [0062]    The high culture media flow rates required for adequate oxygen transfer rates have prevented the use of this type of oxygenation method for bioreactor processes in the past. As an example, cell culturing media rates of about 31 ml/minute is required for the bioreactor versus about the 1000 ml/min to about 4000 ml/min that is required to attain good oxygen transfer into the culturing media. Such high flow rates of about 1000 ml/min to about 4000 ml/minute create too much shear and destroy fragile mammalian cells. Therefore, the present invention separates the high speed flow from the bioreactor perfusion loop  62  and provides a reservoir of readily available high oxygen-containing culture media in the integration module  34 . This high oxygen-containing culture media can then be pumped slowly through the bioreactor  26  in loop  62  to provide saturated oxygen without excessive shear of the cells.  
         [0063]    High Speed Circulation Loop Containing an Artificial Kidney  
         [0064]    In order to remove metabolic waste products from the culture media while retaining exogenous and/or endogenous growth factors, a hollow fiber dialysis cartridge  70  may used as the dialysis module  46 . The dialysis cartridge  70  is placed in a high speed loop  64 . The dialysis cartridge  70  functions as an artificial kidney. The artificial kidney  46  may be connected to the integration module  34  independently, as illustrated in FIG. 2, or in series with the oxygenation module  38 . When the artificial kidney  46  is placed in series with the artificial lung  38 , the artificial kidney  46  is preferably placed upstream from the artificial lung  38  in order to increase the back pressure on the artificial kidney  46 . The flow through the porous hollow fibers  70  of the artificial kidney  46  creates a pressure drop axially along the length of the fibers. The pressure drop causes fluid motility due to forces known as the Starling Effect.  
         [0065]    Referring back to FIG. 2, under normal operating conditions, culture media would cross the membranes of the hollow fibers  70  on the front high pressure end  72  of the dialysis cartridge  46  and return to the lumen at the back low pressure end  74  of the dialysis cartridge  46 . In order to achieve flow, and thus waste product removal from the lumen  70  to the extra-capillary ports  76 ,  78 , the low pressure port  78  is occluded and the exit tubing inner diameter is narrowed by restriction  80  in order to create a back pressure motive force.  
         [0066]    The molecular weight cut-off of the hollow fibers  70  may be less than 10,000 daltons, and more preferably is between 5,000 daltons and 6,000 daltons. Combining high speed recirculation rates through the lumen of the hollow fibers  70  and the restriction  80  on the exit port of the dialysis cartridge  70  increases the pressure drop across the dialysis cartridge  70 , and generates sufficient flow for effective waste product removal.  
         [0067]    Metabolic waste products of the cells typically have molecular weights less than 5,000 daltons, while endogenous and exogenous growth factors have molecular weights that generally exceed 10,000 daltons. Thus, the artificial kidney  46  is capable of removing metabolic waste products while retaining exogenous and endogenous growth factors. Media-containing metabolic waste products that is removed from the artificial kidney  46  can be replaced in the fluid circuit by addition to the integrating module  34  from a source  82  using a pump  84 . In this manner, the cells in the bioreactor  26  can be fed and cleared of metabolic waste products without dilution of exogenous and endogenous growth factors. At the same time secreted products from the cells will accumulate and concentrate in the media and will not be diluted by the feeding.  
         [0068]    Overall System  
         [0069]    The bioreactor vessel  26  can be any type of commercially available housing for the culture of mammalian cells, including fermentation vessels, flexible bags and hollow fiber modules. A variable speed pump  30  may comprise a centrifugal pump, positive displacement pump or gear pump. The pump  30  is in fluid communication with the bioreactor  26  and the lumen side of the cell retention module  24 . The pump  30  delivers media containing cells, metabolic waste, cell produced products, exogenously added factors to the cell retention module  24 , preferably entering from the lumen side of the cell retention module  24 . A valve  25  is placed on the lumenal exit side of the cell retention module  24  which is fluidly connected to the bioreactor  26  as a return line  27 . When valve  25  is engaged so as to block the fluid flow from the lumen exit of the cell retention module  24 , the media from the bioreactor will pass from the lumen of the cell retention module  24  to the extra-capillary space  32 . Due to the pore size of the capillaries (hollow fibers) of the cell retention module  24 , the media containing metabolic waste products, cell produced products and exogenously added factors will pass through the fibers and enter the integrating module  34 . The cells will remain on the lumen side of the hollow fibers of the cell retention module  24 .  
         [0070]    Furthermore, valve  25  is operated to intermittently open and close. In the open mode, cells are returned to the bioreactor  26 . In the closed mode, cell-free media is delivered back to the integrating module  34  for reconditioning.  
         [0071]    The integrating module  34  is connected to a high speed circulation loop  64 . The pump  36  may be operated to provide circulation of about 2 liters per minute to about 4 liters per minute. Cell free culture media from the integrating module  34  entering the high speed circulation loop  64  is first delivered to the lumen side of an artificial lung device  38 . The artificial lung  38  is preferably constructed from a hollow fiber cartridge. Controlled gases are introduced into the extra-capillary side of the artificial lung  38  to replenish the oxygen and adjust the pH of the cell-free media by removal of carbon dioxide. The oxygenated and pH adjusted cell-free culture media is then returned back to the integrating module  34 . Waste containing media is drawn from the integrating module  34  by pump  44  and passed through the lumen side of an artificial kidney device  46 . The artificial kidney device is of a hollow fiber cartridge construction having a plurality of hollow fibers extending therethrough within a housing that defines an extra-capillary space between the housing and the hollow fibers. The cartridge contains the ports  76  and  78 , port  76  being the high pressure extra-capillary port while port  78  is the low pressure extra-capillary port. The low pressure extra-capillary port  78  is blocked. The high pressure extra-capillary port  76  is connected to a waste container  52  by line  48  and valve  77 . Opening valve  77  allows waste media to enter the waste container  52 . The low molecular weight cut-off of the hollow fibers within the artificial kidney  46  retain all exogenously added factors and cell produced products while allowing metabolic waste products to be removed from the culture media. The metabolic waste removed media is then returned to the integration module  34 .  
         [0072]    Fresh nutrient culture media may also be delivered to the integrating module  34  from a fresh media container  82  and delivered by pump  84 . Nutrient-replenished, oxygen-replenished, pH-adjusted, waste-depleted media is then returned to the bioreactor  26  by pump  31  which is in fluid communication with the integrating module  34  and the bioreactor  26 .  
         [0073]    The present invention provides an improved method to deliver oxygen to mammalian cells in a bioreactor providing greater oxygen transfer with less shear. In addition, the present invention provides an improved, universal method of operating bioreactors of any type in a continuous perfusion culture mode while retaining cells in the bioreactor, providing efficient removal of metabolic waste products, and providing a means to supply nutrients without dilution of exogenously added serum or proteins or endogenously produced protein factors.  
         [0074]    The present invention further provides an improved control of the cell micro-environment in order to enable efficient high density cell culture in a bioreactor. Specifically, the bioreactor system provides efficiently oxygenated media to a bioreactor, maintains precise control of media pH and osmolarity and efficiently removes carbon dioxide from the media. This invention also provides a method to scale-up bioreactors without the prior limitations of oxygen availability, nutrient availability, removal of metabolic waste and control of cellular microenvironment.  
         [0075]    The following example is intended for illustrative purposes only and does not limit the present invention in anyway.  
       EXAMPLES  
       [0076]    A 1.5 Liter Celligen (New Brunswick Scientific, Edison, N.J.) bioreactor system equipped with two marine impellers and a six-blade Rushton turbine positioned at the gas/liquid interface to increase gas transfer capability (the agitation at the surface increases the liquid/gas surface area) was set at an agitation speed of about 100 revolutions per minutes. Perfusion was accomplished by recirculating the culture media through a external hollow-fiber module (CellFlo, Spectrum, Laguna Hills, Calif.). The total working volume of the system was 1.7 Liters, including the volume of the recirculation loop. The dissolved oxygen was set to be maintained at 40%.  
         [0077]    1×10 7  HL- 60  leukemia cells were inoculated in the system in X-Vivo 15 serum-free media (BioWhittiker) supplemented with 2 mM glutamine, provides an initial cell density of approximately 0.5×10 6  cells/ml. Samples from the bioreactor were taken twice daily and analyzed for total cell number, viability, glucose, lactate and ammonia levels. The culture was perfused at 0.5 volume per volume per day with fresh media.  
         [0078]    After 5 days in culture the cell density reached a peak of 5×10 6  cells/ml. By day 7, the viable cell number had decreased over 60%. By introducing sparging on day 9 at 0.008 volume per volume per minute, the viable cell density increased to 7×10 6  cells per ml on day 14. However, the ratio of alive:dead cells in the reactor steadily decreased from 1:2 on day 14 to 1:4 on day 16.  
         [0079]    When the same experiment was conducted with the oxygen enrichment system of the present invention, the culture density reached 9×10 6  cells/ml on day 7. The alive:dead ratio was 10:1 on day 7, and no sparging was required. By day 14, the culture had reached a steady state at 3×10 7  cells/ml and the alive:dead cell ratio was 9:1. These results demonstrate that the present invention is capable of enhancing the oxygen availability to cells in perfusion culture. The enhanced oxygen delivery for a given rate of perfusion can maintain cells at higher density without the need for sparging, which increases shear and viability of the cells in the culture.  
         [0080]    Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.