Patent Publication Number: US-2021171904-A1

Title: Cell culture method and cell culture apparatus

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 2018-155328, filed on Aug. 22, 2018, and International Patent Application No. PCT/JP2019/032069, filed on Aug. 15, 2019, the entire content of each of which is incorporated herein by reference. 
    
    
     BACKGROUND 
     Field of the Invention 
     The present invention relates to a cell culture method and a cell culture apparatus. 
     Description of the Related Art 
     In recent years, in fields such as pharmaceutical manufacturing and regenerative medicine, it has been required to artificially and efficiently mass-culture cells and microorganisms. Examples of cells for which mass culture is required include antibody-producing cells such as Chinese hamster ovary cells (CHO cells); and pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). These cells, if stably cultured in large quantities for a long period of time, make it possible to efficiently produce biological substances such as monoclonal antibodies, and differentiation-inducing tissues derived from pluripotent stem cells. 
     A possible way of industrial mass production of cells may be suspension stirred culture with use of a culture tank such as a spinner flask. The suspension stirred culture, however, tends to need large scale of equipment. Increase of cell density during culture will therefore be effective for cost reduction. Increase of the cell density has, however, been known to suppress proliferation of cells. This is because concentration levels of wastes (metabolites) in the culture solution (liquid medium) will increase due to densification of cells, and thereby the proliferative activity of cells will decline. Lactic acid and ammonia are known as representative wastes that can affect the cells. 
     It is therefore preferred to remove lactic acid and ammonia accumulated in the culture solution, in order to allow the cells to stably proliferate in a dense state. On the other hand, Patent Literature 1 for example discloses a cell culture apparatus in which a cell culture tank and a component adjusting solution tank are connected by a liquid feeding line provided with a culture solution component adjustment membrane that allows components to permeate depending on concentration difference. In this cell culture device, the waste products accumulated in the culture solution move to the component adjusting solution side, so that the concentration in the culture solution decreases. At the same time, the nutrients whose concentration has decreased during the culture are transferred from the component adjusting solution to the culture solution, and are replenished. The environment in the culture solution is thus maintained in a condition suitable for cell culture. 
     PATENT LITERATURE 
     
         
         Patent Literature 1: WO2015/122528 
       
    
     The cell culture apparatus disclosed in Patent Literature 1 has removed waste products from the culture solution by use of the principle of dialysis. In order to attain sufficient removal of waste products, the capacity of the component adjusting solution tank has therefore been set to 10 times or more the capacity of the cell culture tank. In addition, the concentration levels of lactic acid and ammonia in the culture solution have not been monitored. That is, there have been no extensive investigations on timing, volume and so forth of the culture solution fed from the cell culture tank to the ingredient conditioning solution tank, in view of regulating the lactic acid concentration and the ammonia concentration. There is therefore a problem that the required liquid amounts huge and becomes costly. In particular, in a case where the culture solution itself is used as the component adjusting solution, a large amount of expensive medium is consumed, which further increases the cost. 
     SUMMARY OF THE INVENTION 
     The present invention was conceived in consideration of such circumstances, wherein one object of which is to provide a technique for mass-culture of cells at low cost. 
     Aimed at solving the aforementioned problem, one aspect of the present invention relates to a cell culture method. The cell culture method includes culturing a cell in a culture solution that satisfies at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution is lower than 7.5 mM. 
     Condition (II): ammonia concentration in the culture solution is lower than 2.5 mM. 
     Another aspect of the present invention relates to a cell culture apparatus. The cell culture apparatus includes a culture vessel structured to house a cell and a culture solution; and a conditioner structured to condition the culture solution so as to satisfy at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution is lower than 7.5 mM. 
     Condition (II): ammonia concentration in the culture solution is lower than 2.5 mM. 
     Note that also free combinations of these constituents, and also any of the constituents and expressions exchanged among the method, device and system, are valid as the aspects of the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments will now be described, by way of example only, with reference to the accompanying drawings which are meant to be exemplary, not limiting, and wherein like elements are numbered alike in several Figures, in which: 
         FIG. 1  is a schematic drawing illustrating a cell culture apparatus of Embodiment 1. 
         FIG. 2  is a schematic drawing illustrating a cell culture apparatus of Embodiment 2. 
         FIG. 3  is a schematic drawing illustrating a cell culture apparatus of Embodiment 3. 
         FIG. 4  is a schematic drawing illustrating a cell culture apparatus of Embodiment 4. 
         FIG. 5  is a graph illustrating relation between lactic acid concentration and cell density. 
         FIG. 6  presents optical microphotographs of cells cultured in a lactic acid-added medium. 
         FIG. 7  is a chart summarizing relation between lactic acid concentration and gene expression levels. 
         FIG. 8  is a graph illustrating relation between ammonia concentration and cell density. 
         FIG. 9  presents optical microphotographs of cells cultured in an ammonia-added medium. 
         FIG. 10  is a chart summarizing relation between ammonia concentration and gene expression levels. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     One aspect of the present invention relates to a cell culture method. The cell culture method includes culturing a cell in a culture solution that satisfies at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution is lower than 7.5 mM. 
     Condition (II): ammonia concentration in the culture solution is lower than 2.5 mM. 
     This aspect enables mass-culture of a cell at low cost. 
     In this aspect, the cell may be a pluripotent stem cell. The lactic acid concentration in condition (I) may be 5 mM or lower. The ammonia concentration in condition (II) may be 1 mM or lower. 
     Another aspect of the present invention relates to a cell culture apparatus. The cell culture apparatus includes a culture vessel structured to house a cell and a culture solution; and a conditioner structured to condition the culture solution so as to satisfy at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution is lower than 7.5 mM. 
     Condition (II): ammonia concentration in the culture solution is lower than 2.5 mM. 
     The conditioner in this aspect may include: a storage tank structured to store a replenishing solution; a feed pump structured to fees the replenishing solution from the storage tank to the culture vessel; a concentration sensor structured to detect at least one of lactic acid concentration or ammonia concentration in the culture solution; and a controller structured to drive the feed pump in response to result of detection made by the concentration sensor. The conditioner may include: a substance exchanger structured to house a substance exchange membrane and an ingredient conditioning solution, and, upon feeding of the culture solution, allows substance exchange to proceed through the substance exchange membrane between the ingredient conditioning solution and the culture solution; a circulation pump structured to circulate the culture solution between the culture vessel and the substance exchanger; a concentration sensor structured to detect at least one of lactic acid concentration or ammonia concentration in the culture solution; and a controller structured to drive the circulation pump in response to result of detection made by the concentration sensor. The conditioner may have an adsorbent structured to adsorb at least one of lactic acid or ammonia in the culture solution. 
     The present invention will be explained below on the basis of preferred embodiments, referring to the attached drawings. The embodiments are merely illustrative, and are not restrictive about the invention. All features and combinations thereof described in the embodiments are not always necessarily essential to the present invention. All constituents, members and processes illustrated in the individual drawings will be given same reference numerals, so as to properly avoid redundant explanations. Reduced scales and shapes of the individual parts in the individual drawings are properly given for simplicity of explanation, and should not be interpreted restrictively unless otherwise specifically noted. Also note that ordinal terms “first”, “second” and so on used in this patent specification and in claims do not represent any sequential order or importance, and are used only for discrimination of one structure from the other. Further, in each drawing, some of the members that are not important for explaining the embodiment will be omitted. 
     The present inventors went through extensive investigations on effects of lactic acid and ammonia, which are representative wastes, exerted on cells, and found lactic acid concentration and ammonia concentration at which cells are adversely affected. The present inventors also found from our further investigations that lactic acid and ammonia can affect each state of proliferation and undifferentiation of the cell, at different concentration levels, when the cell is a pluripotent stem cell. 
     Embodiment 1 
     The cell culture method according to this embodiment includes culturing a cell in a culture solution that satisfies at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution is lower than 7.5 mM (7.5×10 −3  mol/L). 
     Condition (II): ammonia concentration in the culture solution is lower than 2.5 mM. 
     The cell proliferation may be suppressed from being inhibited, when the culture solution contains lactic acid, with the lactic acid concentration kept lower than 7.5 mM. The cell proliferation may also be suppressed from being inhibited by lowered pH of the culture solution due to lactic acid. The cell may be retained in an undifferentiated state, when the lactic acid concentration is kept lower than 10 mM. Hence, the cell when cultured in the culture solution that satisfies condition (I) may be less affected by lactic acid, in both states of cell proliferation and undifferentiation. 
     The cell proliferation may be suppressed from being inhibited, when the culture solution contains ammonia, with the ammonia concentration kept lower than 10 mM. Meanwhile, the cell may be kept in the undifferentiation state, with the ammonia concentration kept lower than 2.5 mM. Hence, the cell when cultured in the culture solution that satisfies condition (II) may be less affected by ammonia, in both states of cell proliferation and undifferentiation. 
     The lactic acid concentration in condition (I) is preferably 5 mM or lower. This enables to further suppress the cell proliferation from being inhibited by lactic acid. Meanwhile, the ammonia concentration in condition (II) may be 1 mM or lower. This enables to further retain the cell in the undifferentiated state. 
     The cell possibly cultured in the culture solution is exemplified by pluripotent stem cells such as human iPS cell, human ES cell, and human Muse cell; somatic stem cells such as mesenchymal stem cell (MSC) and nephron progenitor cell; tissue cells such as human renal proximal tubule epithelial cell, human distal tubule epithelial cell, and human collecting duct epithelial cell; antibody-producing cell lines such as human fetal renal cell (HEK293 cell); and antibody-producing cell lines derived from animals other than human such as Chinese hamster ovary cell (CHO cell) and insect cell (SF9 cell). The cell is preferably the pluripotent stem cell. 
     The cell is preferably cultured by suspension culture. As the culture vessel for suspension culture, employable is a spinner flask, large stainless steel culture tank, cell culture bottle, cell culture bag and so forth. The culture solution is properly selectable depending on cells to be cultured. 
     Cell Culture Apparatus 
     Next, the cell culture apparatus according to this embodiment will be explained.  FIG. 1  is a schematic drawing illustrating a cell culture apparatus of Embodiment 1. A cell culture apparatus  1  has a culture vessel  2  and a conditioner  6 , as the principal components. The culture vessel  2  is a bioreactor that houses a cell  8  and a culture solution  10 . The conditioner  6  conditions the culture solution  10 , so as to satisfy at least one of the aforementioned condition (I) and condition (II). The conditioner  6  of this embodiment has a storage tank  12 , a feed pump  14 , a concentration sensor  4 , and a controller  16 . 
     The storage tank  12  stores a replenishing solution  18 . The replenishing solution  18  may be same as the culture solution  10  in the culture vessel  2 , or may be high-concentration culture solution densified from the culture solution  10 . The replenishing solution  18  may be a solution other than the medium, whose ingredients are blended depending on types of the cell  8 . 
     The storage tank  12  and the culture vessel  2  are connected with a feed pipe  20 . A feed pump  14  is connected in the middle of the feed pipe  20 . The feed pump  14  may be any of known pumps such as Peristaltic pump, diaphragm pump or the like. 
     The concentration sensor  4  detects at least one of lactic acid concentration or ammonia concentration in the culture solution  10 . As the concentration sensor  4 , employable is any of known sensors such as a medium ingredient analyzer or the like. The lactic acid concentration and the ammonia concentration are detectable by employing any of known methods, such as colorimetry with use of predetermined measurement reagent, enzyme electrode method utilizing substrate specificity of enzyme, high performance liquid chromatography (HPLC), and so forth. The concentration sensor  4  and the culture vessel  2  are connected with a sampling tube  5 . The culture solution  10  in the culture vessel  2  is sampled through the sampling tube  5  into the concentration sensor  4 . The sampling tube  5  has, at the end connected to the culture vessel  2 , an unillustrated filter attached thereto. This successfully suppresses the cell  8  from being sucked into the sampling tube  5 . The concentration sensor  4  samples the culture solution  10  at regular temporal intervals for ingredient analysis. The sampling tube  5  is typically provided with a solenoid valve  5   a . With open/close operation of the solenoid valve  5   a  controlled by the controller  16 , flow of the culture solution  10  into the concentration sensor  4  is switched between allowed and blocked. This enables the concentration sensor  4  to repeat the ingredient analysis of the culture solution  10  in a certain timing. 
     The controller  16  drives the feed pump  14  in response to result of detection made by the concentration sensor  4 . More specifically, the feed pump  14  is controlled so as to satisfy condition (I), when the concentration sensor  4  detects the lactic acid concentration. The feed pump  14  is controlled so as to satisfy condition (II), when the concentration sensor  4  detects the ammonia concentration. The feed pump  14  is controlled so as to satisfy condition (I) and condition (II), when the concentration sensor  4  detects both concentration levels. Note that the controller  16  may control the feed pump  14  so as to satisfy only one of conditions, even when the concentration sensor  4  is used for detecting both concentration levels. 
     The controller  16  in terms of hardware configuration is embodied by computer elements and circuits represented by CPU and memory, and the one in terms of software configuration is embodied by computer program product and so forth, all of which being properly depicted in  FIG. 1  as functional blocks embodied by cooperation of these components. Those skilled in the art will easily understand that these functional blocks may be embodied in various ways, depending on combinations of the hardware and the software. 
     When the feed pump  14  is driven by the controller  16 , the replenishing solution  18  is fed from the storage tank  12  through the feed pipe  20  to the culture vessel  2 . The replenishing solution  18  in the storage tank  12  is thus filled in the culture vessel  2 . The culture solution  10  in the culture vessel  2  has accumulated therein lactic acid and ammonia excreted from the cell  8 . Meanwhile, the replenishing solution  18  has the lactic acid concentration and the ammonia concentration lower than those of the culture solution  10 . The replenishing solution  18  is preferably free of lactic acid and ammonia. Hence, upon addition of the replenishing solution  18  to the culture solution  10 , the lactic acid concentration and the ammonia concentration in the culture solution  10  decrease. Also medium ingredients such as glucose and protein, necessary for culturing the cell  8 , are replenished to the culture solution  10 . 
     The controller  16  puts the feed pump  14  under feedback control in response to result of detection made by the concentration sensor  4 , so that the culture solution  10  satisfies condition (I) and/or condition (II). More specifically, the controller  16  accepts result of detection made by the concentration sensor  4 , repetitively at certain times. When conditioning the culture solution  10  so as to satisfy condition (I), the controller  16  determines whether the lactic acid concentration in the culture solution  10  is kept lower than 7.5 mM or not, on the basis of result of detection made by the concentration sensor  4 . If the lactic acid concentration is found to be 7.5 mM or higher, the controller  16  drives the feed pump  14 . Upon falling of the lactic acid concentration below 7.5 mM, the feed pump  14  is stopped operating. 
     A concentration range is preferably preset with a certain margin added to 7.5 mM, which is a threshold value. The controller  16  drives the feed pump  14  if the lactic acid concentration exceeds the concentration range, meanwhile stops the feed pump  14  if the concentration falls below the concentration range. The concentration range is more preferably determined so as to have an upper limit value of 7.5 mM. That is, the controller  16  stops the feed pump  14 , when the lactic acid concentration falls down to a level lower just by a predetermined value than 7.5 mM. Note that the upper limit value of the concentration range may alternatively be determined to a level lower than 7.5 mM, so as to more reliably suppress the lactic acid concentration in the culture solution  10  from reaching 7.5 mM and above. 
     When conditioning the culture solution  10  so as to satisfy condition (II), the controller  16  determines whether the ammonia concentration in the culture solution  10  is kept lower than 2.5 mM or not, in response to result of detection made by the concentration sensor  4 . If the ammonia concentration is found to be 2.5 mM or higher, the controller  16  drives the feed pump  14 . Upon falling of the ammonia concentration below 2.5 mM, the feed pump  14  is stopped operating. 
     A concentration range is preferably preset with a certain margin added to 2.5 mM, which is a threshold value. The controller  16  drives the feed pump  14  if the lactic acid concentration exceeds the concentration range, meanwhile stops the feed pump  14  if the concentration falls below the concentration range. The concentration range is more preferably determined so as to have an upper limit value of 2.5 mM. That is, the controller  16  stops the feed pump  14 , when the ammonia concentration falls down to a level lower just by a predetermined value than 2.5 mM. Note that the upper limit value of the concentration range may alternatively be determined to a level lower than 2.5 mM, so as to more reliably suppress the ammonia concentration in the culture solution  10  from reaching 2.5 mM and above. 
     In a case where the culture solution  10  is conditioned so as to satisfy both of condition (I) and condition (II), the controller  16  stops the feed pump  14  if both conditions are satisfied, meanwhile drives the feed pump  14  if either or both conditions are not satisfied. 
     The culture vessel  2  is connected through a drain pipe  22  to an unillustrated waste tank. In the middle of the drain pipe  22 , connected is a drain pump  24 . The drain pump  24  employable here may be any of known pumps, just like the feed pump  14 . The controller  16  also controls drive of the drain pump  24 . The controller  16  drives the drain pump  24 , accompanied by drive of the feed pump  14 . With the drain pump  24  thus driven, the culture solution  10  in the culture vessel  2  is fed through the drain pipe  22  to the waste tank. This makes it possible to keep liquid level in the culture vessel  2  at constant. The drain pipe  22  is provided, at the end thereof connected to the culture vessel  2 , with an unillustrated filter. This successfully suppresses the cell  8  from being sucked into the drain pipe  22 . The drain pipe  22  may also be used as the sampling tube  5 . In this case, the concentration sensor  4  samples the culture solution  10  through the drain pipe  22 . Note that the drain pump  24  may be driven independently from the feed pump  14 . 
     As explained above, the cell culture method of this embodiment includes culturing the cell in the culture solution  10  that satisfies at least one of condition (I) or condition (II) below. 
     Condition (I): lactic acid concentration in the culture solution  10  is lower than 7.5 mM. 
     Condition (II): ammonia concentration in the culture solution  10  is lower than 2.5 mM. 
     Moreover, the cell culture apparatus  1  of this embodiment includes the culture vessel  2  that houses the cell  8  and the culture solution  10 ; and the conditioner  6  that conditions the culture solution  10  so as to satisfy at least one of the aforementioned condition (I) or condition (II). 
     With the culture solution  10  conditioned so as to satisfy at least one of condition (I) or condition (II), it becomes possible to moderate any adverse effect possibly exerted by at least one of lactic acid or ammonia. This enables mass-culture of the cell  8  at high density. Alternatively, with the culture solution  10  conditioned so as to satisfy both of condition (I) and condition (II), it becomes possible to moderate any adverse effect possibly exerted by both of lactic acid and ammonia. This enables mass-culture of the cell  8  at high density in a more reliable manner. 
     Also note this embodiment specifies the concentration of each of lactic acid and ammonia, at which the cell  8  may be affected, and conditions the culture solution  10  by monitoring the concentration values of lactic acid and ammonia. This successfully reduces consumption of the ingredient conditioning solution and the culture solution, as compared with a known cell culture apparatus in which the culture solution is dialyzed to an unnecessarily excessive degree. This successfully enables cost reduction. This embodiment therefore enables mass-culture of the cell  8  at low cost. 
     In a case where the cell  8  is a pluripotent stem cell, reduction of lactic acid concentration and/or ammonia concentration enables not only high-density, mass-culture of the cell  8 , but also enables the cell  8  to retain an undifferentiated state, that is, the cell  8  can remain pluripotent (can keep pluripotency). This therefore enables to obtain a large amount of the cell  8  suitable for producing differentiation-inducing tissues. Since a cell population with a uniform state is thus obtainable, and the efficiency of differentiation induction can be thus suppressed from degrading, so that costs for production of cellular medicine and regenerative medicine may be reduced. 
     In the cell culture apparatus  1  of this embodiment, the conditioner  6  has a storage tank  12  that stores a replenishing solution  18 ; the feed pump  14  that feeds the replenishing solution  18  from the storage tank  12  to the culture vessel  2 ; a concentration sensor  4  that detects at least one of lactic acid concentration or ammonia concentration in the culture solution  10 ; and a controller  16  that drives the feed pump  14  in response to result of detection made by the concentration sensor  4 . The lactic acid concentration and the ammonia concentration in the culture solution  10  may be decreased by adding the replenishing solution  18  to the culture solution  10 . The controller  16  also drives the drain pump  24  together with the feed pump  14 , to discharge the culture solution  10  from the culture vessel  2 . That is, this embodiment suppresses the waste concentration from increasing, by employing continuous culture in which an old medium is removed while replenishing a new medium. 
     Embodiment 2 
     Embodiment 2 has a common structure with Embodiment 1, except for structures of the cell culture apparatus. The explanation below will deal with this embodiment, emphasizing the structures different from those in Embodiment 1, while briefing or skipping explanation of the common structure.  FIG. 2  is a schematic drawing illustrating a cell culture apparatus of Embodiment 2. 
     The cell culture apparatus  1  of this embodiment has the culture vessel  2  and the conditioner  6 , as the principal components. The cell  8  and the culture solution  10  are housed in the culture vessel  2 . The conditioner  6  conditions the culture solution  10 , so as to satisfy at least one of the aforementioned condition (I) and condition (II). The conditioner  6  of this embodiment has a storage tank  12 , a feed pump  14 , a concentration sensor  4 , and a controller  16 . 
     The storage tank  12  stores a replenishing solution  18 . The storage tank  12  and the culture vessel  2  are connected with a feed pipe  20 . A feed pump  14  is connected in the middle of the feed pipe  20 . The concentration sensor  4  detects at least one of lactic acid concentration or ammonia concentration in the culture solution  10 . The controller  16  drives the feed pump  14  in response to result of detection made by the concentration sensor  4 . When the feed pump  14  is driven by the controller  16 , the replenishing solution  18  is fed from the storage tank  12  through the feed pipe  20  to the culture vessel  2 . The replenishing solution  18  in the storage tank  12  is thus filled in the culture vessel  2 . Timing of driving of the feed pump  14  by the controller  16  is same as in Embodiment 1. 
     Unlike Embodiment 1, the cell culture apparatus  1  of this embodiment has neither the drain pipe  22  nor the drain pump  24 . The volume of liquid in the culture vessel  2  therefore gradually increases. That is, this embodiment suppresses the waste concentration from elevating, by employing fed-batch culture in which the replenishing solution  18  is continuously fed to the culture vessel  2 . In this structure, both of the number of cells and the volume of the culture solution in the culture vessel  2  increase with the elapse of culture period. Cell density in the culture solution  10  may thus be kept constant. Note that the size (capacity) of the culture vessel  2  is determined considering typically the culture period of the cell  8 , the volume of the culture solution  10  housed in the culture vessel  2  at the start of culture, and the amount of addition of the replenishing solution  18 . Also this embodiment can demonstrate effects same as in Embodiment 1. 
     Embodiment 3 
     Embodiment 3 has a common structure with Embodiment 1, except for the structure of the cell culture apparatus. The explanation below will deal with this embodiment, emphasizing the structures different from those in Embodiment 1, while briefing or skipping explanation of the common structure.  FIG. 3  is a schematic drawing illustrating a cell culture apparatus of Embodiment 3. 
     The cell culture apparatus  1  of this embodiment has the culture vessel  2  and the conditioner  6 , as the principal components. The cell  8  and the culture solution  10  are housed in the culture vessel  2 . The conditioner  6  conditions the culture solution  10 , so as to satisfy at least one of the aforementioned condition (I) or condition (II). The conditioner  6  of this embodiment has a substance exchanger  26 , a circulation pump  28 , the concentration sensor  4 , and the controller  16 . 
     The substance exchanger  26  houses a substance exchange membrane  30  and an ingredient conditioning solution  32 . Upon being fed with the culture solution  10  in the culture vessel  2 , the substance exchanger  26  takes part in substance exchange through the substance exchange membrane  30 , between the ingredient conditioning solution  32  and the culture solution  10 . The substance exchange membrane  30  may employ any of known substance exchange membranes made of hollow fiber or the like. Like the replenishing solution  18 , the ingredient conditioning solution  32  may be same as the culture solution  10  in the culture vessel  2 , or may be high-concentration culture solution condensed from the culture solution  10 . Alternatively, any solution other than the medium, whose ingredients are blended depending on types of the cell  8 , is employable. 
     The culture vessel  2  and the substance exchanger  26  are connected with a circulation path  34 . The circulation path  34  includes a forward path  34   a  having one end connected to the culture vessel  2  and the other end connected to the substance exchanger  26 , and a return path  34   b  having one end connected to the substance exchanger  26  and the other end connected to the culture vessel  2 . The ends of the forward path  34   a  and the return path  34   b , on the side closer to the substance exchanger  26 , are connected through the substance exchange membrane  30 . The substance exchange membrane  30  is immersed in the ingredient conditioning solution  32 . Hence a flow path of the culture solution  10 , composed of the forward path  34   a , the substance exchange membrane  30  and the return path  34   b , is provided so as to bridge the culture vessel  2  and the substance exchanger  26 . 
     The circulation pump  28  circulates the culture solution  10  between the culture vessel  2  and the substance exchanger  26 . More specifically, the circulation pump  28  has a first pump  28   a  and a second pump  28   b . The first pump  28   a  is connected in the middle of the forward path  34   a , and feeds the culture solution  10  from the culture vessel  2  to the substance exchange membrane  30  of the substance exchanger  26 . The second pump  28   b  is connected in the middle of the return path  34   b , and feeds the culture solution  10  from the substance exchange membrane  30  to the culture vessel  2 . 
     The concentration sensor  4  detects at least one of lactic acid concentration or ammonia concentration in the culture solution  10 . The controller  16  drives the circulation pump  28  in response to result of detection made by the concentration sensor  4 . Upon start of driving of the circulation pump  28  by the controller  16 , the culture solution  10  circulates through the circulation path  34 , between the culture vessel  2  and the substance exchanger  26 . During the process, substance exchange proceeds through the substance exchange membrane  30 , between the culture solution  10  and the ingredient conditioning solution  32 . As a consequence, lactic acid and ammonia in the culture solution  10  move to the ingredient conditioning solution  32 , meanwhile medium ingredients such as glucose and protein in the ingredient conditioning solution  32  move to the culture solution  10 . Timing of driving of the circulation pump  28  by the controller  16  is same as the timing of driving of the feed pump  14  in Embodiment 1. 
     The lactic acid concentration and ammonia concentration in the culture solution  10  thus decrease. Also medium ingredients such as glucose and protein, necessary for culturing the cell  8 , are replenished to the culture solution  10 . Note that unillustrated filter is provided at the end of the forward path  34   a  on the side connected to the culture vessel  2 . This successfully suppresses the cell  8  from being sucked into the drain pipe  22 . The substance exchanger  26  may have an adsorbent for waste placed therein. This successfully lowers the concentration of the waste dissolved in the ingredient conditioning solution  32 , and reduces the amount of ingredient conditioning solution  32  necessary for the substance exchange. The adsorbent employable here may be any of known adsorbents such as silica, activated carbon and so forth, capable of adsorbing at least one of lactic acid or ammonia. 
     That is, this embodiment suppresses the waste concentration in the culture solution  10  from elevating, by allowing the culture solution  10  to flow from the culture vessel  2  through the substance exchange membrane  30  in the substance exchanger  26  to perform substance exchange, and then by discharging the waste into the ingredient conditioning solution  32  in the substance exchanger  26 . Also this embodiment can demonstrate effects same as in Embodiment 1. 
     Embodiment 4 
     Embodiment 4 has a common structure with Embodiment 1, except for the structure of the cell culture apparatus. The explanation below will deal with this embodiment, emphasizing the structures different from those in Embodiment 1, while briefing or skipping explanation of the common structure.  FIG. 4  is a schematic drawing illustrating a cell culture apparatus of Embodiment 4. 
     The cell culture apparatus  1  of this embodiment has the culture vessel  2  and the conditioner  6 . The cell  8  and the culture solution  10  are housed in the culture vessel  2 . The conditioner  6  conditions the culture solution  10 , so as to satisfy at least one of the aforementioned condition (I) or condition (II). The conditioner  6  in this embodiment has an adsorbent  36 . 
     The adsorbent  36  employable here may be any of known adsorbents such as silica, activated carbon and so forth. The adsorbent  36  may be a single adsorbent that adsorbs both of lactic acid and ammonia, or may be a mixture of a first adsorbent that adsorbs lactic acid and a second adsorbent that adsorbs ammonia. The culture solution  10  is conditioned so as to satisfy at least one of condition (I) or condition (II) described previously, by bringing the culture solution  10  into contact with the adsorbent  36 . Amount of the adsorbent  36  necessary for satisfying condition (I) and/or condition (II) may be properly determined by a skilled person in the art. The adsorbent  36  preferably chosen here is less toxic to the cell  8 . 
     The adsorbent  36  is typically granular, and is housed in the culture vessel  2 . The adsorbent  36  disperses, sediments or suspends in the culture solution  10 , in the culture vessel  2 . The adsorbent  36  in this case preferably has a predetermined value or larger size, which is typically 10 μm or larger, in view of preventing phagocytosis by the cells  8 . Upon bringing the culture solution  10  into contact with the adsorbent  36 , lactic acid and/or ammonia in the culture solution  10  are adsorbed to the adsorbent  36 . The lactic acid concentration and/or the ammonia concentration in the culture solution  10  may thus be reduced, and thereby condition (I) and/or condition (II) may be satisfied. Hence, also this embodiment can demonstrate effects same as in Embodiment 1. 
     The conditioner  6  may alternatively have a structure in which the adsorbent  36  is packed in an unillustrated adsorption column connected to the culture vessel  2 . The culture solution  10  in the culture vessel  2  in this case is fed to the adsorption column, and is brought into contact with the adsorbent  36 . The conditioner  6  may alternatively have a structure in which the adsorbent  36  is supported on the inner wall of the culture vessel  2 . Methods for supporting the adsorbent  36  on the inner wall of the culture vessel  2  are exemplified by a method of adhering the adsorbent  36  to the inner wall of the culture vessel  2 ; and, when the culture vessel  2  is made of a resin, a method of molding the culture vessel  2  by using the resin preliminarily mixed with the adsorbent  36 . 
     Again alternatively, the conditioner  6  may have a structure in which the inside of the culture vessel  2  is partitioned with a diaphragm typically made of a porous membrane, into an upper compartment and a lower compartment, with the adsorbent  36  housed in the lower compartment. The cell  8  in this structure is housed in the upper compartment. The culture solution  10  can move through the diaphragm between the upper compartment and the lower compartment. In contrast, the cell  8  and the adsorbent  36  cannot pass through the diaphragm. Upon bringing the culture solution  10  into contact with the adsorbent  36  housed in the lower compartment, lactic acid and/or ammonia in the culture solution  10  may be adsorbed to the adsorbent  36 . 
     The adsorbent  36  is preferably coated with a resin such as polyvinyl alcohol; and a biological gel such as collagen, alginic acid, or gelatin, or the like. This successfully suppresses outflow of fine particles that may affect the cell  8  and the like, out from the adsorbent  36  into the culture solution. The adsorbent  36  may alternatively be formed, after kneading an adsorbent ingredient with ceramic binder, resin binder, biological gel or the like. This also makes it possible to suppress the outflow of fine particles. Examples of the ceramic binder include alumina binder and colloidal silica. Examples of the resin binder include polyvinyl alcohol, and carboxymethyl cellulose. Examples of the biological gel include collagen, alginic acid, and gelatin. 
     The embodiments of the present invention have been described in detail above. The above-described embodiments merely illustrate specific examples in carrying out the present invention. Contents of the embodiments do not limit the technical scope of the present invention, instead allowing numerous design changes such as modification, addition, and deletion of constituents, without departing from the spirit of the present invention specified in the claims. Any new embodiment with design change will have effects derived both from an embodiment and modification to be combined. In the above-described embodiments, all contents possibly subject to such design change have been emphasized with a notation stating “ . . . of the present embodiment” or “in the present embodiment”. Also any content without such notation is, however, acceptable for the design change. Free combination of the above constituents is also valid as an aspect of the present invention. 
     Examples 
     Examples of the present invention will be explained below. Examples are merely illustrative, and by no means limit the present invention. 
     Determination of Lactic Acid Concentration Influential to Cell Proliferation 
     Onto a 24-well plate (Corning cellBIND Surface: from Corning Incorporated), seeded were 1×10 3  human pluripotent stem cells, to which a medium for pluripotent stem cell (StemFit: from Ajinomoto Co. Inc.) was added, and then cultured. To the medium before 24 hours from the start of culture, a culture substrate (iMatrix-511; from Nippi, Inc.) was added so as to adjust the concentration to 0.5 μg/ml, and also ROCK inhibitor (Y-27632: from FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the concentration to 10 μM. Meanwhile to the medium at 24 hours or after from the start of culture, lactic acid (FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the lactic acid concentration in various levels. The lactic acid concentration was varied among 0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM and 15.0 mM. 
     As lactic acid is added, pH of the medium will decrease. Hence, the inventors prepared an experimental group with pH of the medium corrected to 7.2, and an uncorrected experimental group. In the uncorrected experimental group, the medium with a lactic acid concentration of 5.0 mM was found to have pH 6.9, the medium with a lactic acid concentration of 7.5 mM was found to have pH 6.8, medium with a lactic acid concentration of 10 mM was found to have pH 6.7, and the medium with a lactic acid concentration of 15 mM was fond to have pH 6.5. 
     The culture was continued four days while refreshing the medium every day (hence the culture period totals 5 days). After the elapse of 5 days, the number of cells in each well was measured by using TC20 full-automatic cell counter (from Bio-Rad Laboratories, Inc.), to calculate cell density. Results are illustrated in  FIG. 5 .  FIG. 5  is a graph illustrating relation between the lactic acid concentration and the cell density. 
     As can be seen in  FIG. 5 , the cell density was found to sharply decrease at a lactic acid concentration of 7.5 mM or higher, irrespective of pH correction. The pH-corrected experimental group showed the cell density at a lactic acid concentration of 15 mM, reduced by half or more from the value at a lactic acid concentration of 0 mM. In contrast, the pH-uncorrected group showed the cell density at a lactic acid concentration of 10 mM or above, sharply reduced down below the cell density observed at a lactic acid concentration of 15 mM in the pH-corrected group. This confirms that lactic acid per se inhibits cell proliferation, and also inhibits cell proliferation by inducing pH reduction. 
     It was thus confirmed from the results that the cell proliferation is effectively suppressed from degrading, by keeping the lactic acid concentration to lower than 7.5 mM. It was also confirmed that the lactic acid-induced inhibition of cell proliferation may be suppressed more reliably, by keeping the lactic acid concentration to 5 mM or below. 
     Determination of Lactic Acid Concentration Influential to Undifferentiated State of Cell 
     Onto a 24-well plate (Corning cellBIND Surface: from Corning Incorporated), seeded were 1×10 3  human pluripotent stem cells, to which a medium for pluripotent stem cell (StemFit: from Ajinomoto Co. Inc.) was added, and then cultured. To the medium before 24 hours from the start of culture, a culture substrate (iMatrix-511; from Nippi, Inc.) was added so as to adjust the concentration to 0.5 μg/ml, and also ROCK inhibitor (Y-27632: from FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the concentration to 10 μM. Meanwhile to the medium at 24 hours or after from the start of culture, lactic acid (FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the lactic acid concentration in various levels. The lactic acid concentration was varied among 0 mM, 1.0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM and 15.0 mM. 
     The culture was continued four days while refreshing the medium every day (hence the culture period totals 5 days). The individual cells on the 5th day of culture were observed under an optical microscope.  FIG. 6  presents optical microphotographs of the cells cultured in the lactic acid-added medium. As can be confirmed from  FIG. 6 , colonies of the cells markedly decreased at a lactic acid concentration of 10 mM or higher, indicating inhibition of cell proliferation. 
     mRNA was then extracted from each cell on the 6th day of culture, using RNeasy Mini Kit (from QIAGEN N.V.), and then purified. Next, cDNA was synthesized from the thus purified mRNA, by using QuantiTect Reverse Transcription Kit (from QIAGEN N.V.). By using each cDNA as a template, and by using Thermal Cycler Dice Real Time System I (from Takara Bio Inc.), expression levels of Oct4, Nanog, T, Pax6 and Fox2 genes were measured by real-time PCR. 
     Oct4 and Nanog are undifferentiation markers indicating that the pluripotent stem cell retains its property. T, Pax6 and Fox2 are differentiation markers indicating that the pluripotent stem cell no longer retains its property, that is, the pluripotent stem cell has differentiated. More specifically, expression of T gene indicates that the cell has differentiated into mesoderm, expression of Pax6 gene indicates that the cell has differentiated into ectoderm, and expression of Fox2 indicates that the cell has differentiated into endoderm. 
     Proportion of expression levels of the individual genes at the individual concentration levels of lactic acid were calculated, on the basis of the expression level at a lactic acid concentration of 0 mM. Results are summarized in  FIG. 7 .  FIG. 7  is a chart summarizing relation between the lactic acid concentration and the gene expression levels. As can be seen in  FIG. 7 , the expression level of T gene, which is a mesoderm differentiation marker, was found to increase at a lactic acid concentration of 10 mM, to a level ten times or more of the level at a lactic acid concentration of 0 mM. This confirms that keeping of the lactic acid concentration at lower than 10 mM is effective to keep the cell in the undifferentiated state. 
     Determination of Ammonia Concentration Influential to Cell Proliferation 
     Onto a 24-well plate (Corning cellBIND Surface: from Corning Incorporated), seeded were 1×10 3  human pluripotent stem cells, to which a medium for pluripotent stem cell (StemFit: from Ajinomoto Co. Inc.) was added, and then cultured. To the medium before 24 hours from the start of culture, a culture substrate (iMatrix-511; from Nippi, Inc.) was added so as to adjust the concentration to 0.5 μg/ml, and also ROCK inhibitor (Y-27632: from FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the concentration to 10 μM. To the medium at 24 hours or after from the start of culture, ammonium chloride (FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the ammonia (ammonium ion) concentration to various levels. The ammonia concentration was varied among 0 mM, 7.5 mM, 10.0 mM, 15.0 mM and 25.0 mM. 
     The culture was continued four days while refreshing the medium every day (hence the culture period totals 5 days). After the elapse of 5 days, the number of cells in each well was measured by using TC20 full-automatic cell counter (from Bio-Rad Laboratories, Inc.), to calculate cell density. Results are summarized in  FIG. 8 .  FIG. 8  is a graph illustrating relation between the ammonia concentration and the cell density. 
     As can be seen in  FIG. 8 , the cell density was found to sharply decrease at an ammonia concentration of 10.0 mM or higher. This confirms that keeping of the ammonia concentration at lower than 10 mM is effective to suppress inhibition of cell proliferation. It was also confirmed that the ammonia-induced inhibition of cell proliferation may be suppressed more reliably, by keeping the ammonia concentration at 7.5 mM or below. 
     Determination of Ammonia Concentration Influential to Undifferentiated State of Cell 
     Onto a 24-well plate (Corning cellBIND Surface: from Corning Incorporated), seeded were 1×10 3  human pluripotent stem cells, to which a medium for pluripotent stem cell (StemFit: from Ajinomoto Co. Inc.) was added, and then cultured. To the medium before 24 hours from the start of culture, a culture substrate (iMatrix-511; from Nippi, Inc.) was added so as to adjust the concentration to 0.5 μg/ml, and also ROCK inhibitor (Y-27632: from FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the concentration to 10 μM. To the medium at 24 hours or after from the start of culture, ammonium chloride (FUJIFILM Wako Pure Chemical Corporation) was added so as to adjust the ammonia concentration to various levels. The ammonia concentration was varied among 0 mM, 0.1 mM, 1.0 mM, 2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM and 20.0 mM. 
     The culture was continued four days while refreshing the medium every day (hence the culture period totals 5 days). The individual cells on the 5th day of culture were observed under an optical microscope.  FIG. 9  presents optical microphotographs of the cells cultured in the ammonia-added medium. As can be seen in  FIG. 9 , morphological changes of the cell were observed at an ammonia concentration of 2.5 mM or above. 
     mRNA was then extracted from each cell on the 6th day of culture, using RNeasy Mini Kit (from QIAGEN N.V.), and then purified. Next, cDNA was synthesized from the thus purified mRNA, by using QuantiTect Reverse Transcription Kit (from QIAGEN N.V.). By using each cDNA as a template, and by using Thermal Cycler Dice Real Time System I (from Takara Bio Inc.), expression levels of Oct4, Nanog, T, Pax6 and Fox2 genes were measured by real-time PCR. 
     Proportion of expression levels of the individual genes at the individual concentration levels of ammonia were calculated, on the basis of the expression level at an ammonia concentration of 0 mM. Results are summarized in  FIG. 10 .  FIG. 10  is a chart summarizing relation between the ammonia concentration and the gene expression levels. As can be seen in  FIG. 10 , the expression level of T gene, which is a mesoderm differentiation marker, and Pax6 gene, which is an ectoderm differentiation marker, were found to increase at an ammonia concentration of 2.5 mM, to a level nearly ten times as large as the level at an ammonia concentration of 0 mM. This confirms that keeping of the ammonia concentration at lower than 2.5 mM is effective to keep the cell in the undifferentiated state. It was also confirmed that the ammonia-induced promotion of cell differentiation may be suppressed more reliably, by keeping the ammonia concentration at 1 mM or below. 
     The findings confirm that the lactic acid concentration and the ammonia concentration in the medium are necessarily kept lower than 7.5 mM and lower than 2.5 mM, respectively, in order to allow the pluripotent stem cell to proliferate in the undifferentiated state.