Patent Publication Number: US-2017369830-A1

Title: Cell culture method and cell culture device

Description:
FIELD 
     The present invention relates to a cell culture method and a cell culture apparatus capable of culturing cells for a long period of time without causing stress or damage to the cells. 
     BACKGROUND 
     As regenerative medicine using stem cells in recent years, for example, treatment of liver cirrhosis, blood disease, and myocardial infraction, construction of blood vessels, regeneration of bones and cornea, securing skin for transplantation are conceivable. In regenerative medicine, desired cells and organs are expanded from stem cells and the like in a culture dish so as to be transplanted to a person. Recently, angiogenesis is performed by stem cells derived from bone marrow, and treatment for angina pectoris, myocardial infarction, etc. is successfully performed. 
     Here, in a conventional cell culture apparatus, a culture liquid in a culture dish is periodically exchanged to grow cultured cells. The cell culture apparatus has a problem that the cells are stressed or damaged because the cells are greatly stimulated in association with exchange of the culture liquid and a waste product is discharged into the culture liquid in association with the metabolic activity of the cells. 
     On the other hand, there is a device that carries out cell culture by feeding a culture liquid to a culture dish without exchanging the culture liquid. For example, Patent Literature 1 describes a cell culture apparatus that monitors the growth rate of cells, predicts a decrease in nutrients in a culture liquid based on the monitored growth rate, and adds a consumed amount of nutrients to the culture liquid. 
     In addition, there is a method in which cell culture is carried out by feeding and discharging a culture liquid without exchanging the culture liquid. For example, Patent Literature 2 describes a cell culture method in which a culture liquid is circulated in a culture tank by a filter module made of hollow fibers to add nutrients in the culture liquid and remove waste product. 
     CITATION LIST 
     Patent Literature 
     Patent Literature 1: Japanese Laid-open Patent Publication No. 2012-170366 
     Patent Literature 2: Japanese Laid-open Patent Publication No. 2012-090632 
     SUMMARY 
     Technical Problem 
     However, because the cell culture apparatus described in Patent Literature 1 adopts a system of collecting the culture liquid in a culture tank although the nutrients are continuously fed to the culture tank, when the concentration of the waste product discharged by metabolic activity of cells in the culture liquid increases, the culture liquid in the culture tank has to be exchanged at one time at arbitrary timing similarly to the conventional cell culture apparatus. Frequency of exchanging all the culture liquid is once in a day, which results in bringing stress or damage to the cells similarly to the conventional cell culture apparatus. 
     On the other hand, in Patent Literature 2, it is configured to continuously feed and discharge an extremely small amount of culture liquid, however, when a culture period is long, the filter module is clogged due to the waste product, so that the method is not suitable for long term culture. Particularly because the culture period until cells required for recent regenerative medicine or the like are obtained is long such as about one month, the failure of long term culture means that cells having a large cell area required for regenerative medicine etc. cannot be cultured. 
     The present invention has been made to solve the conventional problems, and it is an object of the present invention to provide a cell culture method and a cell culture apparatus capable of culturing cells for a long period of time without causing stress or damage to the cells. 
     Solution to Problem 
     To solve the problem described above and to achieve the object, a cell culture method according to the present invention is a cell culture method for arranging cultured cells in a culture dish and continuously culturing the cultured cells by supplying a liquid required to grow or maintain the cultured cells to the culture dish and discharging the liquid from the culture dish. The cell culture method includes: providing a supply port of the liquid at one end of the culture dish and providing a discharge port of the liquid at other end of the culture dish so as to sandwich the cultured cells between the supply port and the discharge port, and discharging the liquid while supplying the liquid to the culture dish so that a moving linear velocity of the liquid from the supply port toward the discharge port is less than a maximum velocity at which shear stress is not applied to the cultured cells. 
     In the cell culture method according to the present invention, the moving linear velocity of the liquid is equal to or less than a diffusion velocity due to molecular motion of the liquid. 
     A cell culture apparatus according to the present invention is a cell culture apparatus configured to arrange cultured cells in a culture dish and continuously culture the cultured cells by supplying a liquid required to grow or maintain the cultured cells to the culture dish and discharging the liquid from the culture dish. The cell culture apparatus includes: a supply port of the liquid provided at one end of the culture dish; a discharge port for the culture dish provided at other end of the culture dish so as to sandwich the cultured cells between the supply port and the discharge port, a reservoir tank configured to store the liquid to be supplied to the culture dish; a waste liquid tank configured to store the liquid to be discharged from the culture dish; a supply-side micro flow rate pump configured to supply the liquid in the reservoir tank to the culture dish through the supply port; a discharge-side micro flow rate pump configured to discharge the liquid from the culture dish through the discharge port; and a flow rate controller configured to perform flow rate control of the supply-side micro flow rate pump and the discharge-side micro flow rate pump so that a moving linear velocity of the liquid from the supply port toward the discharge port is less than a maximum velocity at which shear stress is not applied to the cultured cells. 
     In the cell culture apparatus according to the present invention, the moving linear velocity of the liquid is equal to or less than a diffusion velocity due to molecular motion of the liquid. 
     In the cell culture apparatus according to the present invention, a side face of the culture dish has surface free energy smaller than surface free energy of a bottom face of the culture dish. 
     The cell culture apparatus according to the present invention further includes: a horizontal adjustment mechanism configured to adjust the bottom face of the culture dish horizontally. 
     The cell culture apparatus according to the present invention further includes: an inclination adjustment mechanism configured to adjust the bottom face of the culture dish diagonally. 
     In the cell culture apparatus according to the present invention, the supply port includes a plurality of supply ports which are discretely arranged in linear order, and the discharge port includes a plurality of discharge ports which are discretely arranged in linear order. 
     In the cell culture apparatus according to the present invention, an outlet of the supply-side micro flow rate pump and the supply port are connected by a flexible tube, and the discharge port and an inlet of the discharge-side micro flow rate pump are connected by a flexible tube, and a diaphragm adjustment mechanism configured to adjust an opening of the flexible tube connected between the outlet of the supply-side micro flow rate pump and the supply port is provided between the supply-side micro flow rate pump and the supply port, and a diaphragm adjustment mechanism configured to adjust an opening of the flexible tube connected between the discharge port and the inlet of the discharge-side micro flow rate pump is provided between the discharge-side micro flow rate pump and the discharge port. 
     In the cell culture apparatus according to the present invention, the reservoir tank and an inlet of the supply-side micro flow rate pump are detachably and directly connected to each other, an outlet of the supply-side micro flow rate pump and the supply port are detachably and directly connected to each other, the discharge port and an inlet of the discharge-side micro flow rate pump are detachably and directly connected to each other, and an outlet of the discharge-side micro flow rate pump and the waste liquid tank are detachably and directly connected to each other. 
     In the cell culture apparatus according to the present invention, a multistage configuration in which the reservoir tank is provided on an upper side of a supply-side flow path that connects between the supply port and the outlet of the supply-side micro flow rate pump is disposed horizontally in a manner that one end of the multistage configuration provided the reservoir tank therein is directed to the supply port of the culture dish, and at other end of the multistage configuration provided the reservoir tank therein, the inlet of the supply-side micro flow rate pump is detachably connected to the reservoir tank and the outlet of the supply-side micro flow rate pump is detachably connected to an inlet of the supply-side flow path, and a multistage configuration in which the waste liquid tank is provided on an upper side of a discharge-side flow path that connects between the discharge port and the inlet of the discharge-side micro flow rate pump is disposed horizontally in a manner that one end of the multistage configuration provided the waste liquid tank therein is directed to the discharge port of the culture dish, and at the other end of the multistage configuration provided the waste liquid tank therein, the outlet of the discharge-side micro flow rate pump is detachably connected to the waste liquid tank and the inlet of the discharge-side micro flow rate pump is detachably connected to an outlet of the discharge-side flow path. 
     The cell culture apparatus according to the present invention further includes: a flow path plate in which a first flow path between the reservoir tank and the supply-side micro flow rate pump, a second flow path between the supply-side micro flow rate pump and the culture dish, a third flow path between the culture dish and the discharge-side micro flow rate pump, and a fourth flow path between the discharge-side micro flow rate pump and the waste liquid tank are embedded, the flow path plate being disposed on an upper side of the reservoir tank, the culture dish, and the waste liquid tank. The first flow path and an inlet of the supply-side micro flow rate pump are pin port connected to each other, the second flow path and an outlet of the supply-side micro flow rate pump are pin port connected to each other, the third flow path and an inlet of the discharge-side micro flow rate pump are pin port connected to each other, and the fourth flow path and an outlet pipe of the supply-side micro flow rate pump are pin port connected to each other. 
     The cell culture apparatus according to the present invention further includes: a liquid level detection sensor configured to detect a liquid level in the culture dish. The flow rate controller is configured to perform flow rate control so that the liquid level detected by the liquid level detection sensor becomes constant. 
     In the cell culture apparatus according to the present invention, a supply-side drive source for driving the supply-side micro flow rate pump is configured to be detachably attached to the supply-side micro flow rate pump, and a discharge-side drive source for driving the discharge-side micro flow rate pump is configured to be detachably attached to the discharge-side micro flow rate pump. 
     The cell culture apparatus according to the present invention further includes: a substrate configured to arrange at least the culture dish. The substrate has a hole or a colorless and transparent concave portion in an area of the substrate where a culture state of the cultured cells is observed, the concave portion having an opening formed in the substrate on a side opposite to a side of the substrate on which the culture dish is arranged. 
     The cell culture apparatus according to the present invention further includes: a transparent conductive film heater provided at a bottom face of the culture dish; a temperature sensor configured to detect a temperature of the liquid in the culture dish; and a temperature controller configured to perform control to keep the temperature of the liquid in the culture dish within a predetermined temperature range by energizing the transparent conductive film heater based on detection result of the temperature sensor. 
     Advantageous Effects of Invention 
     According to the present invention, the supply port of the liquid required to grow or maintain cultured cells is provided at one end of the culture dish and the discharge port of the liquid is provided at the other end of the culture dish so that the cultured cells are sandwiched between the supply port and the discharge port, and the liquid is discharged while being supplied to the culture dish so that the moving linear velocity of the liquid from the supply port toward the discharge port is less than the maximum velocity at which shear stress is not applied to the cultured cells. Therefore, it is possible to culture the cells for a long period of time without causing stress or damage to the cells and suppress consumption of the liquid. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a perspective view illustrating an appearance configuration of a cell culture apparatus according to an embodiment of the present invention. 
         FIG. 2  is a block diagram illustrating a configuration of the cell culture apparatus illustrated in  FIG. 1 . 
         FIG. 3  is a cross-sectional view illustrating a configuration of a hole made on a substrate at an area where a culture dish is provided. 
         FIG. 4  is a cross-sectional view illustrating a configuration of a hole made on a substrate at an area where a reservoir tank is provided. 
         FIG. 5  is an explanatory diagram for explaining a function of a diaphragm adjustment mechanism. 
         FIG. 6  is a diagram illustrating an incidence rate of cells affected by shear stress appearing in cultured cells with respect to a moving linear velocity of a culture liquid from a supply port toward a discharge port. 
         FIG. 7  is a diagram illustrating a state image of the culture liquid flowing from a supply-port side face toward a discharge-port side face. 
         FIG. 8  is a diagram illustrating movement of the culture liquid with a passage of time as a change in relative concentration. 
         FIG. 9  is a flowchart illustrating a procedure for controlling a flow rate of the culture liquid by a flow rate controller. 
         FIG. 10  is a diagram illustrating an example of an arrangement configuration of supply port openings. 
         FIG. 11  is a diagram illustrating an example of an arrangement configuration of supply port openings. 
         FIG. 12  is a diagram illustrating a state image of the culture liquid flowing from the supply-port side face toward the discharge-port side face when a fluorine-based water repellent agent is applied to a side face of the culture dish. 
         FIG. 13  is a front view illustrating a configuration near the culture dish of the cell culture apparatus provided with a horizontal adjustment mechanism. 
         FIG. 14  is a partially broken front view illustrating a specific configuration of the horizontal adjustment mechanism. 
         FIG. 15  is a diagram illustrating an example of an arrangement configuration and a connection configuration of a liquid feeding portion and a liquid discharging portion. 
         FIG. 16  is a diagram illustrating an example of an arrangement configuration and a connection configuration of the liquid feeding portion and the liquid discharging portion. 
         FIG. 17  is a perspective view illustrating an arrangement configuration of cell culture apparatuses in a transportation box. 
         FIG. 18  is a plan view illustrating a configuration of a cell culture apparatus using a closed perfusion system flow path. 
         FIG. 19  is an A-A line cross-sectional view of the cell culture apparatus illustrated in  FIG. 18 . 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Some embodiments for implementing the present invention will be explained below with reference to the accompanying drawings. 
     Overall Configuration 
       FIG. 1  is a perspective view illustrating an appearance configuration of a cell culture apparatus  1  according to an embodiment of the present invention. FIG.  2  is a block diagram illustrating a configuration of the cell culture apparatus  1  illustrated in  FIG. 1 . As illustrated in  FIG. 1  and  FIG. 2 , the cell culture apparatus  1  includes a culture dish  3  provided at the center of the upper side of a substrate  2 , and also includes a liquid feeding portion  10  for supplying a culture liquid to the culture dish  3  and a liquid discharging portion  20  for discharging the culture liquid from the culture dish  3  which are provided on the substrate  2  so as to sandwich the culture dish  3  from both ends of the culture dish  3 . 
     In the culture dish  3 , a supply port  4  of the culture liquid is provided on a supply-port side face  3   a , which is one end side of the culture dish  3 , and a discharge port  5  of the culture liquid is provided on a discharge-port side face  3   b , which is the other end side of the culture dish  3  so as to sandwich cultured cells  6  between the supply port  4  and the discharge port  5 . Six supply ports  4  and six discharge ports  5  are provided, and the supply ports and the discharge ports are discretely arranged linearly along the supply-port side face  3   a  and the discharge-port side face  3   b , respectively. The openings of the supply port  4  and the discharge port  5  are provided at positions at any depth less than the depth of the culture liquid in the culture dish  3 . 
     The liquid feeding portion  10  includes a reservoir tank  11 , a supply-side micro flow rate pump  12 , and a diaphragm adjustment mechanism  13 . The reservoir tank  11  stores the culture liquid. The supply-side micro flow rate pump  12  supplies the culture liquid in the reservoir tank  11  to the supply port  4  of the culture dish  3 . The diaphragm adjustment mechanism  13  has a variable diaphragm function of readjusting the flow rate of the culture liquid supplied from the supply-side micro flow rate pump  12 . 
     A culture liquid outlet of the reservoir tank  11  and a culture liquid inlet of the supply-side micro flow rate pump  12  are connected by a flow path L 11  including six flexible tubes. Moreover, a culture liquid outlet of the supply-side micro flow rate pump  12  and the supply port  4  are connected by a flow path L 12  including six flexible tubes. The diaphragm adjustment mechanism  13  can perform variable diaphragm on each of the six flexible tubes of the flow path L 12 . 
     The supply-side micro flow rate pump  12  has a pump group  12   a  including six peristaltic pumps, a speed reducer  12   b , and a motor  12   c . Each peristaltic pump can be connected in multiple stages along its rotation axis. The speed reducer  12   b  reduces the rotation of the motor  12   c  in multiple stages and transmits the reduced rotation to the pump group  12   a . The motor  12   c  can be attached to and detached from the speed reducer  12   b . A flange  2   a  erected on the substrate  2  is formed on the substrate  2  on the liquid feeding portion  10  side. The pump group  12   a  is attached to one end face side of the flange  2   a  and the speed reducer  12   b  is attached to the other end face side of the flange  2   a , so that the speed reducer  12   b  and the pump group  12   a  are connected to each other. The speed reducer  12   b  is configured to be detachably attached to the flange  2   a  and the pump group  12   a.    
     On the other hand, the liquid discharging portion  20  includes a waste liquid tank  21 , a discharge-side micro flow rate pump  22 , and a diaphragm adjustment mechanism  23 . The discharge-side micro flow rate pump  22  discharges the culture liquid in the culture dish from the discharge port  5  of the culture dish  3 . The waste liquid tank  21  stores the culture liquid discharged by the discharge-side micro flow rate pump  22 . The diaphragm adjustment mechanism  23  has a variable diaphragm function of readjusting the flow rate of the culture liquid discharged from the discharge-side micro flow rate pump  22 . 
     The discharge port  5  and a culture liquid inlet of the discharge-side micro flow rate pump  22  are connected by a flow path L 22  including six flexible tubes. Moreover, a culture liquid outlet of the discharge-side micro flow rate pump  22  and a culture liquid inlet of the waste liquid tank  21  are connected by a flow path L 21  including six flexible tubes. The diaphragm adjustment mechanism  23  can perform variable diaphragm on each of the six flexible tubes of the flow path L 22 . 
     The discharge-side micro flow rate pump  22  has a pump group  22   a  including six peristaltic pumps, a speed reducer  22   b , and a motor  22   c . Each peristaltic pump can be connected in multiple stages along its rotation axis. The speed reducer  22   b  reduces the rotation of the motor  22   c  in multiple stages and transmits the reduced rotation to the pump group  22   a . The motor  22   c  can be attached to and detached from the speed reducer  22   b . A flange  2   b  erected on the substrate  2  is formed on the substrate  2  on the liquid discharging portion  20  side. The pump group  22   a  is attached to one end face side of the flange  2   b  and the speed reducer  22   b  is attached to the other end face side of the flange  2   b , so that the speed reducer  22   b  and the pump group  22   a  are connected to each other. The speed reducer  22   b  is configured to be detachably attached to the flange  2   b  and the pump group  22   a.    
     As illustrated in  FIG. 1 , the culture dish  3  is attached to the substrate  2  by holding members  103   a  and  103   b . The reservoir tank  11  is attached to the substrate  2  by holding members  111   a  and  111   b . The waste liquid tank  21  is attached to the substrate  2  by holding members  121   a  and  121   b.    
     As explained above, the motors  12   c  and  22   c  and the speed reducers  12   b  and  22   b  are removable and can be removed upon autoclaving. Because the culture dish  3  is usually formed of polycarbonate, it is exchanged each time the autoclaving is performed. 
     As illustrated in  FIG. 2 , the motor  12   c  and the motor  22   c  are driven by a flow rate controller  30 . The pump groups  12   a  and  22   a  control the flow rate of the culture liquid using the flow rate controller  30 . A transparent conductive film heater  41  and a temperature sensor  42  are provided at the bottom face of the culture dish  3 . A Peltier element  43  and a temperature sensor  44  are provided in the reservoir tank  11 . A temperature controller  40  controls temperature so that a temperature of the culture liquid in the culture dish  3  will reach a desired temperature, for example, 37° C., by controlling energization of the transparent conductive film heater  41  based on the detection result of the temperature sensor  42  and controls temperature so that a temperature of the culture liquid in the reservoir tank  11  will reach a desired temperature, for example, 5 to 20° C., by controlling energization of the Peltier element  43  based on the detection result of the temperature sensor  44 . A power supply  50  supplies power to the flow rate controller  30  and to the temperature controller  40 . The culture liquid in the culture dish  3  needs to be set to a temperature suitable for growth of cells, and the culture liquid in the reservoir tank  11  needs to be set to a temperature at which it can be stored for a long time. Although the culture liquid in the reservoir tank  11  is set to a temperature lower than that of the culture liquid in the culture dish  3 , the culture liquid flowing out of the reservoir tank  11  has a temperature close to ordinary temperature by passing through the flow paths L 11  and L 12  until it reaches the supply port of the culture dish  3 . 
     As illustrated in  FIG. 3 , the substrate  2  includes a hole  2   c  such as a circular hole, which is provided in an area of the substrate  2  where the culture dish  3  is disposed. The reason for providing the hole  2   c  is to shorten the length of the culture dish  3  in the height direction so that the state of the cell during cell culture can be observed by an optical microscope. Therefore, the substrate  2  may include a concave portion hollowed out a lower portion of the substrate  2  at a position where the culture dish  3  is disposed. However, the bottom face of the concave portion is preferably colorless and transparent. 
     As illustrated in  FIG. 4 , the substrate  2  includes a hole  2   d  such as a circular hole, which is provided in an area of the substrate  2  where the reservoir tank  11  is disposed. This is because the state of the culture liquid in the reservoir tank  11  can be visually observed through the hole  2   d . When the state of the culture liquid in the reservoir tank  11  deteriorates, turbidity and discoloration occur. Similarly, it is preferable that the substrate  2  includes a hole provided in an area of the substrate  2  where the waste liquid tank  21  is disposed. 
     As illustrated in  FIG. 5 , the diaphragm adjustment mechanism  13  can form a diaphragm for narrowing the flow path opening by pressing the flexible tube which is the flow path L 12  so as to be held. As explained above, the diaphragm adjustment mechanism  13  discretely readjusts the flow rate of each flexible tube of the flow path L 12 . By forming the diaphragm in the flow path L 12 , pressure P 1  on an upstream side of the diaphragm becomes larger than pressure P 2  on a downstream side thereof. As a result, bubbles or the like are less likely to be generated in the culture liquid when the pump group  12   a  arranged on the upstream side of the diaphragm sucks and discharges the culture liquid, thus accurately controlling the flow rate. 
     Flow of Culture Liquid in Culture Dish 
     Here, in the present embodiment, it is configured so that the culture liquid is discharged while being supplied to the culture dish  3  so that the moving linear velocity V of the culture liquid from the supply port  4  toward the discharge port  5  in the culture dish  3  is less than the maximum velocity at which shear stress is not applied to the cultured cells  6 . 
       FIG. 6  is a diagram illustrating an incidence rate of cells affected by shear stress appearing in the cultured cells  6  with respect to the moving linear velocity V of the culture liquid from the supply port  4  toward the discharge port  5 . The incidence rate of cells affected by shear stress is defined as an area ratio of cells, in which cell death or cell mutation occurs, to normal cells. As illustrated in  FIG. 6 , the incidence rate of cells affected by shear stress is 0 when it is less than maximum velocity Vmax. Therefore, by discharging the culture liquid while supplying it to the culture dish  3  at the moving linear velocity V which is less than the maximum velocity Vmax, it is possible not to exert influence due to the shear stress on the cultured cells  6 . Specific maximum velocity Vmax is about 0.3 m/min when the cultured cells  6  are ES cells of mice. 
     Moreover, in the present embodiment, because it is configured to continuously supply and discharge the culture liquid to and from the culture dish  3 , there is no need to exchange the culture liquid over a long period of time, so that it is possible to obtain a proper cell with a large cell area. Furthermore, each opening of the supply port  4  and the discharge port  5  is large, which is not less than a size such that at least waste product is not clogged when it passes through the opening. Therefore, because clogging does not occur in the filter or the like, also from this point of view, there is no need to exchange the culture liquid over a long period of time, thus, obtaining a proper cell with a large cell area. 
     When it is ensured that the cultured cells  6  are not affected by the shear stress, the culture liquid is preferably discharged while being supplied to the culture dish  3  so that the velocity is a diffusion velocity V 1  or less due to molecular motion of the culture liquid. For the diffusion due to the molecular motion of the culture liquid, unlike artificial diffusion, the flow of the culture liquid does not apply the shear stress to the cultured cells  6 . As a result, it is possible to obtain an appropriate cell which is not damaged and has no stress. 
     Because the diffusion velocity V 1  is an extremely small value as compared with the maximum velocity Vmax and each amount of supply and discharge of the culture liquid can be reduced, it is possible to grow the cultured cells  6  at low cost. A minimum moving linear velocity is a velocity at which the cultured cells  6  can obtain necessary nutrients without cell death of the cultured cells  6 , and is different for each cultured cell  6 . Therefore, it is preferable that the moving linear velocity to be set be the same as or equal to the diffusion velocity. The minimum moving linear velocity is generally about ⅓ of the diffusion velocity. 
     Here, assuming that an average distance is x[cm] at which molecules move within time t[s], the diffusion phenomenon can be expressed as 
         x =(4 Dt )̂(1/2)
 
     Wherein D is a diffusion coefficient, which is about D=(1 to 2)×10̂(−5)[cm̂2/s] at room temperature. 
     Therefore, an average distance x at which molecules in the culture liquid move for one minute is 0.4 to 0.7 mm, and the diffusion velocity V 1  is 0.4 to 0.7 mm/min. 
     On the other hand, it is considered that the moving linear velocity V of the culture liquid from the supply port  4  toward the discharge port  5  is the diffusion velocity V 1  or less. The cross sectional area from the supply port  4  toward the discharge port  5  in the culture dish  3  is 129 mm̂2 when the depth of the culture liquid is 1.5 mm and the width thereof is 86 mm. Therefore, assuming that the flow rate of the culture liquid is set to 42 μL/min, the moving linear velocity V is a value obtained by dividing a flow rate by cross sectional area, which is 0.379 mm/min. In this case, the moving linear velocity V becomes smaller than the diffusion velocity V 1 . 
       FIG. 7  is a diagram illustrating a state image of the culture liquid flowing from the supply-port side face  3   a  toward the discharge-port side face  3   b  when the flow rate is set to the flow rate of the culture liquid. In addition,  FIG. 7  is colored by adding methylene blue to the culture liquid to be supplied. As illustrated in  FIG. 7 , because the supplied culture liquid is supplied from the six supply ports  4 , six curves are formed. It is found in each front-edge area E 1  of the six curves that the supplied culture liquid diffuses into the existing culture liquid. The front-edge area E 1  moves toward the discharge-port side face  3   b  as time passes. Moreover, the supplied culture liquid becomes dominant on the supply-port side face  3   a  as time passes. The diffusion velocity is quicker by meniscus on flow-direction side faces  3   c  and  3   d  of the culture dish  3  parallel to the direction in which the culture liquid moves toward the discharge-port side face  3   b.    
     The flow rate of the culture liquid to be supplied is controlled so as to be supplied or discharged at a flow rate not exceeding the diffusion state. In other words, the supply amount of the culture liquid is a flow rate that refills the flow rate of the culture liquid diffused from the supply port  4 . 
       FIG. 8  illustrates a change in relative concentration in association with the passage of time on a straight line C illustrated in  FIG. 7 . A range R is based on the position of the supply-port side face  3   a . As illustrated in  FIG. 8 , the front-edge area E 1 , in which a relative concentration D is inclined in a bell shape, moves toward the discharge-port side face  3   b  at the diffusion velocity V 1  in association with the passage of time. On the other hand, in the supply-port side face  3   a  from the front-edge area E 1 , the front-edge area E 1  moves at the moving linear velocity V not more than the diffusion velocity V 1 , the relative concentration becomes constant, and the supplied culture liquid is dominant. 
     Flow Control Process of Culture Liquid 
       FIG. 9  is a flowchart illustrating a procedure for controlling a flow rate of the culture liquid by the flow rate controller  30 . As illustrated in  FIG. 9 , at first, the cultured cells  6  are stuck to the bottom face of the culture dish  3 , and thereafter, the supply-side micro flow rate pump is driven while the discharge-side micro flow rate pump  22  is stopped and the culture liquid is supplied so that the culture liquid is filled in the culture dish  3  (Step S 101 ). Thereafter, the supply-side micro flow rate pump  12  is stopped, the discharge-side micro flow rate pump  22  is driven to discharge the culture liquid, and the depth of the culture liquid is set so as to be a predetermined depth (Step S 102 ). Thereafter, at the predetermined depth, the flow rate is controlled on the supply-side micro flow rate pump  12  and on the discharge-side micro flow rate pump  22  so as to supply and discharge the culture liquid at a constant flow rate so that the moving linear velocity of the culture liquid from the supply port  4  toward the discharge port  5  is less than the maximum velocity at which the shear stress is not applied to the cultured cells  6  or so that the moving linear velocity is preferably equal to or less than the diffusion velocity of the culture liquid (Step S 103 ). 
     Experimental Result 
     The cell culture apparatus  1  was used to seed A549 cells (human alveolar basal epithelial carcinoma cells) in the culture dish  3 , supply the culture liquid at a diffusion velocity or less and discharge the culture liquid for five days, so that the A549 cells were cultured. Then, it was checked whether shear stress was applied to the cells. Whether the shear stress was applied thereto is understood by checking a phosphorylation state in an NO pathway and a PKC pathway. Experimental results indicate that no eNOS phosphate peptides were identified in the NO pathway. Moreover, KRTS (S73) phosphate peptides were not identified in the PKC pathway, and there was no change in phosphorylation of KRTS (S73). From these results, it can be estimated that no shear stress is applied to the cells. 
     Arrangement of Supply Ports and Discharge Ports 
     In the embodiment, as illustrated in the upper portion of  FIG. 10 , the opening positions of the supply ports  4  are arranged at equal intervals linearly in a horizontal direction within a culture liquid level with respect to the supply-port side face  3   a , however, as illustrated in the lower portion of  FIG. 10 , the opening positions of the supply ports  4  may be shifted to the center side. In this case, the influence due to the meniscus is reduced, the front-edge areas E 1  can be moved more linearly. The same goes to the opening positions of the discharge ports  5 . 
     When the depth of the culture liquid is deep, as illustrated in  FIG. 11 , planar arrangement may be adopted so that the opening positions of the supply ports  4  are dispersed also in the depth direction. The same goes to the opening positions of the discharge ports  5 . 
     Suppression of Meniscus Generation 
     Incidentally, as illustrated in  FIG. 7 , the moving linear velocity of the culture liquid in the flow-direction side faces  3   c  and  3   d  was quick by the meniscus. Therefore, to suppress generation of meniscus, a fluorine-based water repellent agent was applied to the flow-direction side faces  3   c  and  3   d , the supply-port side face  3   a , and the discharge-port side face  3   b . As a result, even if the opening arrangement of the supply ports  4  was as illustrated in the upper portion of  FIG. 10 , the moving linear velocity of the culture liquid in the flow-direction side faces  3   c  and  3   d  could be suppressed as illustrated in  FIG. 12 . In other words, by applying the fluorine-based water repellent agent to the flow-direction side faces  3   c  and  3   d , the supply-port side face  3 a, and the discharge-port side face  3   b , generation of meniscus can be suppressed, and the moving linear velocity of the culture liquid can be made uniform. Because generation of meniscus is only to be suppressed, the fluorine-based water repellent agent only has to be applied to at least the flow-direction side faces  3   c  and  3   d . In addition, the water repellent agent is not limited to a fluorine-based material, and may be a material having water repellency. 
     Application of the water repellent agent makes smaller the surface free energy of the flow-direction side faces  3   c  and  3   d , the supply-port side face  3   a , and the discharge-port side face  3   b , than that of the bottom face of the culture dish  3 . Therefore, instead of the application of the water repellent agent, the flow-direction side faces  3   c  and  3   d , the supply-port side face  3   a , and the discharge-port side face  3   b  of the culture dish  3 , where each has a surface material having the surface free energy smaller than the surface free energy of the bottom face of the culture dish  3 , may be used. 
     Horizontal Adjustment Mechanism of Culture Dish 
     The front edge of the flow of the culture liquid illustrated in  FIG. 12  is oblique to the flow direction. This is because the culture dish  3  is not horizontally disposed, but is inclined to the width direction and the depth of the culture liquid is different in the width direction. Therefore, as illustrated in  FIG. 13 , a culture-dish substrate  2   e  to which the culture dish  3  is fixed is provided on the substrate  2 , and four horizontal adjustment mechanisms  51  ( 51   a ,  51   b ,  51   c ,  51   d ) are provided between the substrate  2  and the culture-dish substrate  2   e  at positions corresponding to four corners of the culture dish  3 . Moreover, two levels  52  for detecting the horizontality between the X direction and the Y direction are fixed to the upper side of the culture-dish substrate  2   e . However, if there is any device that can detect the horizontality of the XY plane, the level may be one unit. 
       FIG. 14  is a diagram illustrating a configuration of the specific horizontal adjustment mechanism  51   c . In the horizontal adjustment mechanism  51   c , when an adjustment dial  53  is rotated, a threaded portion  53   a  formed on the front end side of the adjustment dial  53  rotates. An inclined member  54  screwed to the threaded portion  53   a  moves in the Y direction according to the rotation of the threaded portion  53   a . An elevating member  55  is disposed on the upper portion of the inclined member  54 . An inclined portion  54   a  of the upper portion of the inclined member  54  and an inclined portion  55   a  of the lower portion of the elevating member  55  slidably abut each other. Therefore, the elevating member  55  moves in a Z direction, which is a height direction, according to the movement of the inclined member  54  in the Y direction. Then, an operator refers to the horizontal state indicated by the levels  52 , adjusts the positions of the four horizontal adjustment mechanisms  51   a ,  51   b ,  51   c , and  51   d  in the Z direction, and can horizontally adjust the culture-dish substrate  2   e . Resultantly, a bottom face  3   e  of the culture dish  3  can be adjusted horizontally. 
     When the bottom face  3   e  of the culture dish  3  is horizontally adjusted by the horizontal adjustment mechanisms  51 , the depth of the culture liquid to be supplied to and discharged from the culture dish  3  can be made constant. As a result, the moving linear velocity of the culture liquid in the flow direction (X direction) can be made uniform. 
     Although the horizontal adjustment mechanisms  51  are manual type, automatic horizontal adjustment mechanisms are preferable. For example, it may be configured so that position images of bubbles indicated by the levels  52  are acquired by an imaging device and a motor-driven adjustment dial is controlled to rotate so that the bubbles move to a horizontal position. By automating the horizontal adjustment, the horizontal state can be always maintained automatically even during culture of the cultured cells  6 . 
     Connection Between Liquid Feeding Portion and Liquid Discharging Portion 
     As illustrated in  FIG. 15 , it may be configured so that a connecting portion  61  between the reservoir tank  11  and the inlet of the supply-side micro flow rate pump  12 , a connecting portion  62  between the outlet of the supply-side micro flow rate pump  12  and the supply port  4 , a connecting portion  63  between the discharge port  5  and the inlet of the discharge-side micro flow rate pump  22 , and a connecting portion  64  between the outlet of the discharge-side micro flow rate pump  22  and the waste liquid tank  21  are directly connected by sockets and plugs with built-in automatic opening/closing valves respectively and can be detachable and one-touch connected. 
     As illustrated in  FIG. 16 , it may be configured so that a multistage configuration in which the reservoir tank  11  is provided on an upper side of a supply-side flow path  75  that connects between the supply port  4  and the outlet of the supply-side micro flow rate pump  12  is disposed horizontally in a manner that one end of the multistage configuration provided the reservoir tank  11  therein is directed to the supply port  4  of the culture dish  3 , and at the other end of the multistage configuration provided the reservoir tank  11  therein, the inlet of the supply-side micro flow rate pump  12  is detachably connected to the reservoir tank  11  and the outlet of the supply-side micro flow rate pump  12  is detachably connected to the inlet of the supply-side flow path  75 . 
     In other words, it is configured so that a connecting portion  71  between the inlet of the supply-side micro flow rate pump  12  and the reservoir tank  11  and a connecting portion  72  between the outlet of the supply-side micro flow rate pump  12  and the inlet of the supply-side flow path  75  are directly connected by sockets and plugs with built-in automatic opening/closing valves respectively and can be detachable and one-touch connected. 
     Likewise, it may be configured so that a multistage configuration in which the waste liquid tank  21  is provided on an upper side of a discharge-side flow path  76  that connects between the discharge port  5  and the inlet of the discharge-side micro flow rate pump  22  is disposed horizontally in a manner that one end of the multistage configuration provided the waste liquid tank  21  therein is directed to the discharge port  5  of the culture dish  3 , and at the other end of the multistage configuration provided the waste liquid tank  21  therein, the outlet of the discharge-side micro flow rate pump  22  is detachably connected to the waste liquid tank  21  and the inlet of the discharge-side micro flow rate pump  22  is detachably connected to the outlet of the discharge-side flow path  76 . 
     In other words, it is configured so that a connecting portion  74  between the outlet of the discharge-side micro flow rate pump  22  and the waste liquid tank  21  and a connecting portion  73  between the outlet of the discharge-side micro flow rate pump  22  and the outlet of the discharge-side flow path  76  are directly connected by sockets and plugs with built-in automatic opening/closing valves respectively and can be detachable and one-touch connected. 
     Transportation Form of Cell Culture Apparatus 
     Because the cell culture apparatus is in a flat plate shape, it can be stacked in multiple stages. Therefore, for example, as illustrated in  FIG. 17 , the cell culture apparatus  1  can be transported by using a transportation box  80 , capable of keeping the heat in, in which it is stacked in multiple stages. Power connectors  81  of the cell culture apparatuses  1  are respectively connected to power connectors  82  connected to a battery  83 . The power connectors  82  are connected to the battery  83  in such a manner as a string of beads. Therefore, for example, even if the cell culture apparatus  1  at the third stage from the top is removed, only the power connector  81  and the power connector  82  of the removed cell culture apparatus  1  are removed, and the other cell culture apparatuses  1  can continuously culture the cells. 
     When transporting the cell culture apparatuses  1  in the transportation box  80  or the like, the culture dish  3  is covered with a lid, and it is preferable to fill the space in the culture dish  3  with the culture liquid. This makes it possible to reduce the influence of shear stress on the cultured cells in the culture dish  3  and to perform continuous culture until the cultured cells reach the destination. 
     Application of Closed Perfusion System Flow Path 
     In the embodiment, the flexible tube is used for the flow path between the culture dish  3  and the discharge-side micro flow rate pump  22 , and the user connects between the flexible tube and the discharge port of the discharge-side micro flow rate pump  22 . In a modification of the embodiment, the closed perfusion system flow path in which no opening portions are provided on all the flow paths is formed. 
       FIG. 18  is a plan view illustrating a configuration of a cell culture apparatus  200  using the closed perfusion system flow path.  FIG. 19  is an A-A line cross-sectional view of the cell culture apparatus  200  illustrated in  FIG. 18 . As illustrated in  FIG. 18  and  FIG. 19 , the cell culture apparatus  200  includes culture dishes  203   a  and  203   b , a reservoir tank  211 , a waste liquid tank  223 , and motors  213   a  and  213   b , which are provided on a substrate  201 . A flow path plate  220  is disposed on the upper side of the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223 . Supply-side micro flow rate pumps  212   a  and  212   b  are disposed on the motors  213   a  and  213   b , respectively. Concave portions are formed in the substrate  201  so that the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223  are arranged in the concave portions. An opening is formed in each bottom face of the concave portions. 
     A flow path is embedded in the flow path plate  220 . The flow path plate  220  internally forms a flow path by forming a groove as a flow path on opposing surfaces between an upper plate  221  and a lower plate  222  and joining the opposing surfaces between the upper plate  221  and the lower plate  222 . The flow path includes a first flow path L 231  between the reservoir tank  211  and the supply-side micro flow rate pump  212   a , a second flow path L 232  between the supply-side micro flow rate pump  212   a  and the culture dishes  203   a  and  203   b , a third flow path L 233  between the culture dishes  203   a  and  203   b  and the discharge-side micro flow rate pump  212   b , and a fourth flow path L 234  between the discharge-side micro flow rate pump  212   b  and the waste liquid tank  223 . 
     An inlet  251  between the first flow path L 231  and an inlet pipe L 241  of the supply-side micro flow rate pump  212   a  is pin port connected thereto, and an outlet  252  between the second flow path L 232  and an outlet pipe L 242  of the supply-side micro flow rate pump  212   a  is pin port connected thereto. An inlet  255  between the third flow path L 233  and an inlet pipe L 243  of the discharge-side micro flow rate pump  212   b  is pin port connected thereto, and an outlet  256  between the fourth flow path L 234  and an outlet pipe L 244  of the discharge-side micro flow rate pump  212   b  is pin port connected thereto. Thus, the culture liquid within the reservoir tank  211  flows through the closed perfusion system flow path until it is collected in the waste liquid tank  223 . Therefore, it is possible to prevent contamination to the culture liquid by workers during assembly of the cell culture apparatus  200 . In addition, it is preferable that the pin port connection is previously in a connected state in order to prevent contamination. This eliminates works of the workers forming a flow path at the time of assembly of the cell culture apparatus  200 . 
     Lower engaging portions  251  for being inserted into respective openings of the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223  arranged on the substrate  201  to position the flow path plate  220  are formed on the lower side of the flow path plate  220 . Moreover, upper engaging portions  252  for preventing displacement of respective lids  261  closing the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223  are formed on the upper side of the flow path plate  220 . Openings  301  are formed in respective areas of the flow path plate  220  corresponding to respective openings of the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223 . The flow path plate  220  is provided with nozzles to form respective flow paths into the culture dishes  203   a  and  203   b , the reservoir tank  211 , and the waste liquid tank  223 . 
     The cell culture apparatus  200  is configured to provide one supply port  253  and one discharge port  254  in each of the culture dishes  203   a  and  203   b . This is because there may be a case where when a plurality of supply ports branched from one flow path are provided in one culture dish at the time of supplying the culture liquid at a low flow rate, it is difficult to equally branch the flow rate to the respective supply ports. 
     Moreover, in this modification, liquid level detection sensors SD for detecting a liquid level of the culture liquid in the culture dishes  203   a  and  203   b  are provided on the flow path plate  220 . A flow rate controller, not illustrated, corresponding to the flow rate controller  30  of  FIG. 2  performs flow rate control so that the liquid level detected by the liquid level detection sensor SD is constant. This makes it possible to maintain a constant moving velocity of the culture liquid. 
     Furthermore, in the modification, an inclination adjustment mechanism for diagonally adjusting the bottom face of each of the culture dishes  203   a  and  203   b  is provided at A portion of the substrate  201  illustrated in  FIG. 19 . The inclination adjustment mechanism can be implemented in the same configuration as that of the horizontal adjustment mechanism  51  illustrated in  FIG. 13  and  FIG. 14 . The inclination by the inclination adjustment mechanism is formed so that the discharge port  254  side in the bottom face of each of the culture dishes  203   a  and  203   b  is made lower. This makes it possible to effectively remove dead cells occurring during culture from the discharge port  254 . Therefore, it is preferable to match the position of the discharge port  254  with the inclination direction of the inclination adjustment mechanism. The supply port  253  is provided at the position facing the discharge port  254 . Therefore, when the culture dish is inclined by the inclination adjustment mechanism, the supply port  253  is provided at a higher position as compared with the discharge port  254 . It is also preferable to provide the discharge port of the reservoir tank  211  at a lower position when inclined, similarly to the discharge port  254 . 
     In connecting the pump  212   a  and the motor  213   a , there is a mechanism that the input shaft of the motor  213   a  can be connected to the pump  212   a  while rotating. Specifically, the input shaft of the motor  213   a  can be moved up and down by a spring, and when the position of the fitting hole of the pump  212   a  and the position of the input shaft of the motor  213   a  are matched, both of them can be connected by the input shaft being pushed thereinto by the spring. Likewise, in connecting the pump  212   b  and the motor  213   b , there is a mechanism that the input shaft of the motor  213   b  can be connected to the pump  212   b  while rotating. 
     In the embodiment, because the flow rate of the culture liquid to be supplied and discharged is quite low, the consumption of the culture liquid can be reduced even if cells having a large cell area are cultured. In addition, even if a culture period until cells required for recent regenerative medicine are obtained is a long period of time such as about one month, the cost of cell culture can be reduced without consuming a large amount of culture liquid. 
     Incidentally, the culture liquid is a liquid medium. The liquid medium includes a synthetic medium and a natural medium. The synthetic medium is, unlike the natural medium, a mixture of physiological saline solution (salt solution) with chemical substances such as saccharides and vitamins. The synthetic medium is required to have a composition of a salt solution in which osmotic pressure, pH, ionic composition, and the like are optimum for tissues and cells of specific mammals, reptiles, fish, insects, plants, and the like. In order to find a salt solution in which composition of the salt solution is optimized, the cell culture apparatus according to the present embodiment can be used. In other words, by supplying and discharging the salt solution not including nutrients to and from the cell culture apparatus and observing the survival state of the cells in the cell culture apparatus, the composition of an optimal salt solution can be found. In this case, the cell culture apparatus according to the present embodiment can continuously exclude waste products from the cells for a long time and can remove other factors such as stress and damage for survival of the cells, and can, therefore, determine the composition of the optimal salt solution. When finding an optimal synthetic medium, it is only necessary to further change the composition of various nutrients and observe the survival state of the cells. 
     In other words, the cell culture apparatus according to the present embodiment supplies a liquid, such as the culture liquid including nutrients required to grow cultured cells and the salt solution not including nutrients required for life support of cultured cells for a short period of time, to the culture dish  3  and discharges the liquid therefrom. 
     REFERENCE SIGNS LIST 
       1 ,  200  CELL CULTURE APPARATUS 
       2 ,  201  SUBSTRATE 
       2   a ,  2   b  FLANGE 
       2   c ,  2   d  HOLE 
       2   e  CULTURE-DISH SUBSTRATE 
       3 ,  203   a ,  203   b  CULTURE DISH 
       3   a  SUPPLY-PORT SIDE FACE 
       3   b  DISCHARGE-PORT SIDE FACE 
       3   c ,  3   d  FLOW-DIRECTION SIDE FACE 
       3   e  BOTTOM FACE 
       4 ,  253  SUPPLY PORT 
       5 ,  254  DISCHARGE PORT 
       6  CULTURED CELLS 
       10  LIQUID FEEDING PORTION 
       11 ,  211  RESERVOIR TANK 
       12 ,  212   a ,  212   b  SUPPLY-SIDE MICRO FLOW RATE PUMP 
       12   a ,  22   a  PUMP GROUP 
       12   b ,  22   b  SPEED REDUCER 
       12   c ,  22   c ,  213   a ,  213   b  MOTOR 
       13 ,  23  DIAPHRAGM ADJUSTMENT MECHANISM 
       20  LIQUID DISCHARGING PORTION 
       21 ,  223  WASTE LIQUID TANK 
       22  DISCHARGE-SIDE MICRO FLOW RATE PUMP 
       30  FLOW RATE CONTROLLER 
       40  TEMPERATURE CONTROLLER 
       41  TRANSPARENT CONDUCTIVE FILM HEATER 
       42 ,  44  TEMPERATURE SENSOR 
       43  PELTIER ELEMENT 
       50  POWER SUPPLY 
       51  ( 51   a ,  51   b ,  51   c ,  51   d ) HORIZONTAL ADJUSTMENT MECHANISM 
       52  LEVEL 
       53  ADJUSTMENT DIAL 
       53   a  THREADED PORTION 
       54  INCLINED MEMBER 
       54   a ,  55   a  INCLINED PORTION 
       55  ELEVATING MEMBER 
       61  to  64 ,  71  to  74  CONNECTING PORTION 
       75  SUPPLY-SIDE FLOW PATH 
       76  DISCHARGE-SIDE FLOW PATH 
       80  TRANSPORTATION BOX 
       81 ,  82  POWER CONNECTOR 
       83  BUTTERY 
       103   a ,  103   b ,  111   a ,  111   b ,  121   a ,  121   b  HOLDING MEMBER 
       220  FLOW PATH PLATE 
     C STRAIGHT LINE 
     D RELATIVE CONCENTRATION 
     E 1  FRONT-EDGE AREA 
     L 11 , L 12 , L 21 , L 22  FLOW PATH 
     L 231  FIRST FLOW PATH 
     L 232  SECOND FLOW PATH 
     L 233  THIRD FLOW PATH 
     L 234  FOURTH FLOW PATH 
     P 1 , P 2  PRESSURE 
     R RANGE 
     SD LIQUID LEVEL DETECTION SENSOR 
     V MOVING LINEAR VELOCITY 
     V 1  DIFFUSION VELOCITY 
     Vmax MAXIMUM VELOCITY