JP2018000130A - Cell culture device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cell culture devices which culture cells such as stem cells efficiently and are capable of stably obtaining culture cells with high quality.SOLUTION: A cell culture device has a culture tank for conducting three-dimensional culture of cells suspended in a culture solution; a culture unit comprising circulation means for circulating the culture solution so that the culture solution forms an upward flow in the culture tank; a cell distribution detection unit for detecting a cell distribution in the culture tank; and a control unit for controlling the circulation rate of the culture solution by the circulation means based on the cell distribution detected by the cell distribution detection unit.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a cell culture device.

  As a cell culture device, for example, Patent Literature 1 discloses a cell culture device having a cell culture unit for performing three-dimensional culture and a unit for feeding various chemical solutions, culture solutions, waste solutions, and the like. This cell culture device is a device for culturing cells on a carrier suspended in a culture solution, and it automatically performs operations such as medium exchange and passaging that have been performed manually by workers. To do so, a control unit is provided to control each unit.

JP2015-136330A

  In recent years, with the expansion of applications in the medical field, cell culture apparatuses are required to improve the efficiency of culture and to improve the quality and homogenization of the obtained cultured cells.

  The present disclosure describes a cell culture apparatus capable of efficiently performing cell culture of stem cells and the like and stably obtaining high-quality cultured cells.

  A cell culture device according to one embodiment of the present disclosure includes a culture tank that performs three-dimensional culture of cells suspended in a culture solution, and a circulation that circulates the culture solution so that the culture solution forms an upward flow in the culture vessel. A culture unit including means, a cell distribution detection unit for detecting cell distribution in the culture tank, and a control unit for controlling the circulation rate of the culture medium by the circulation means based on the cell distribution detected by the cell distribution detection unit; .

  According to the cell culture device according to one embodiment of the present disclosure, cell culture of stem cells and the like can be efficiently performed, and high-quality cultured cells can be stably obtained.

It is a figure showing a schematic structure of a cell culture device concerning one embodiment of this indication. It is a flowchart which shows the execution procedure of the circulation rate control in the cell culture apparatus of FIG.

  A cell culture device according to one embodiment of the present disclosure includes a culture tank that performs three-dimensional culture of cells suspended in a culture solution, and a circulation that circulates the culture solution so that the culture solution forms an upward flow in the culture vessel. A culture unit including means, a cell distribution detection unit for detecting cell distribution in the culture tank, and a control unit for controlling the circulation rate of the culture medium by the circulation means based on the cell distribution detected by the cell distribution detection unit; .

  In this cell culture device, the cells are suspended in the culture solution, and are cultured while floating in the culture solution due to the balance between the upward flow by the circulation means and the dead weight. The cell distribution detection unit can detect the equilibrium state between the upward flow caused by the circulation means and the dead weight of the cell by detecting the distribution of the cells floating in the culture solution in the culture tank. The control unit controls the circulation speed of the culture solution by the circulation means, thereby adjusting the equilibrium state between the upward flow caused by the circulation means and the dead weight of the cells, and appropriately maintaining the cell distribution in the culture tank.

  Here, in the conventional cell culture apparatus, the cell distribution in the culture solution is biased to the lower or upper stage from an appropriate position in the culture tank, and thus, the cultured cells are excessively agglomerated and it is difficult to perform homogeneous culture. There was a case. In addition, since the cell distribution gradually changes due to changes in the size of the cell mass during the culturing process, it was difficult to maintain the cell distribution at an appropriate position in the culture tank.

  In the cell culture device according to the above aspect, the cell distribution detection unit detects the cell distribution, and the control unit controls the circulation rate of the culture solution based on the detection result. In addition, even if the cell distribution changes due to changes in cell size, etc. during the culturing process, the equilibrium state between the upflow by the circulation means and the cell's own weight is adjusted by the detection by the cell distribution detection unit and the control by the control unit. . For this reason, in the cell culture apparatus according to the above aspect, it is possible to appropriately maintain the cell distribution and perform homogeneous culture.

  In some embodiments, the cell distribution detection unit includes turbidity measuring means for measuring the turbidity of the culture solution in the culture tank at a plurality of locations in the vertical direction. In this case, the cell distribution in the culture solution can be detected based on the level of turbidity measured at a plurality of measurement locations. In addition to the detection of cell distribution, the amount of increase in the number of cells in the culture solution can also be estimated based on the change in turbidity from the start of culture.

  In some embodiments, the culture unit includes culture environment detection means for detecting a culture environment in the culture tank and culture environment adjustment means for adjusting the culture environment in the culture tank. In this case, it is possible to easily confirm whether or not the intended culture environment is maintained by detecting the culture environment in the culture tank by the culture environment detection means, and if necessary, the culture environment adjustment means. The culture environment in the culture tank can be easily adjusted. The culture environment adjusting means may be controlled by the control unit, and the control unit may control the adjustment of the culture environment by the culture environment adjusting means based on the culture environment detected by the culture environment detecting means.

  In some embodiments, the cell culture apparatus collects a culture solution supply unit that supplies a culture solution to the culture unit, a wash solution supply unit that supplies a wash solution to the culture unit, and a waste solution containing the culture solution or the wash solution from the culture unit. And a waste liquid recovery unit. In this case, operations such as culture medium exchange and passage in cell culture can be easily performed by operating each unit.

  In some embodiments, the control unit is configured to supply the culture solution from the culture solution supply unit to the culture unit, supply the cleaning solution from the wash solution supply unit to the culture unit, and supply the waste solution from the culture unit to the waste solution recovery unit. Control the feeding of each liquid. In this case, operations such as culture medium exchange and passage in cell culture can be performed collectively by the control unit, and the operation can be automated.

  In some embodiments, the culture solution supply unit includes a culture solution storage unit that stores the culture solution at a predetermined temperature, and a culture solution heating unit that heats the culture solution to a temperature equal to or higher than a predetermined temperature. The culture solution is stored at a temperature suitable for storage in the culture solution storage means, heated to a temperature suitable for culture in the culture solution heating means, and supplied to the culture tank. In this case, when it becomes necessary to supply the culture solution to the culture tank, the culture solution stored in the culture solution storage means can be heated and used by the culture solution heating means, so that the culture solution is replenished from outside the apparatus. The work procedure is simplified as compared with the case of doing so.

  In some embodiments, the cell culture device further includes a liquid volume detection unit that detects a volume of the culture solution in the culture tank, and the control unit is based on the liquid volume detected by the liquid volume detection unit. Control of the feeding of the culture solution from the culture solution supply unit to the culture unit. In this case, the liquid quantity in the culture tank is detected by the liquid quantity detection means, and based on the detection result, the culture liquid is supplied into the culture tank under the control of the control unit. Thereby, the supply amount of the culture solution is precisely controlled, and the adverse effect on the cultured cells due to the large amount of supply amount of the culture solution is avoided.

  In some embodiments, the cell culture device further includes an enzyme supply unit that supplies an enzyme solution containing an enzyme to the culture unit, and the control unit controls the supply of the enzyme solution from the enzyme supply unit to the culture unit. To do. In this case, the required amount of enzyme solution can be easily supplied to the culture unit at the required timing under the control of the control unit.

  In some embodiments, the enzyme supply unit includes an enzyme storage unit that stores the enzyme solution at a predetermined temperature. Thereby, the enzyme solution is stored at a temperature suitable for storage.

  In some embodiments, the cell culture apparatus further includes a dissolved oxygen amount detection unit that detects a dissolved oxygen amount of a culture solution circulating in the culture unit, and the culture unit supplies oxygen to the culture solution. The control unit further includes a means for controlling the amount of oxygen supplied by the oxygen supply means based on the amount of dissolved oxygen detected by the dissolved oxygen amount detection means. In this case, since the required amount of oxygen is supplied to the culture solution under the control of the control unit, cell culture can be easily performed while maintaining a constant dissolved oxygen amount.

  In some embodiments, the cell culture device further comprises pH detection means for detecting the pH of the culture solution in the culture tank, and the control unit is configured to control the culture solution based on the pH detected by the pH detection means. The time for replacement is determined, and notification to the user or replacement of the culture medium is performed. In this case, the pH detection means detects the change in the pH of the culture solution, and the control unit automatically determines the replacement time of the culture solution based on the detection result. Etc. are prevented. The control unit may notify the user of the replacement time via the interface unit or the like. Further, the control unit may automatically replace the culture solution after determining the replacement time by controlling the culture solution supply unit, the washing solution supply unit, and the waste solution supply unit.

  In some embodiments, the cell culture device may further include an inhibitor supply unit that supplies a Rho kinase inhibitor to the culture unit, and the control unit includes the concentration of the Rho kinase inhibitor in the culture medium in the culture tank. The amount of Rho kinase inhibitor supplied by the inhibitor supply unit is controlled so that becomes a predetermined concentration. In this case, the required amount of Rho kinase inhibitor can be easily supplied to the culture unit at the required timing under the control of the control unit.

  In some embodiments, the inhibitor supply unit includes an inhibitor storage means for storing the Rho kinase inhibitor at a predetermined temperature. Thereby, the Rho kinase inhibitor is stored at a temperature suitable for storage.

  In some embodiments, the cell culture device further includes a chemical solution supply unit that supplies the chemical solution to the culture unit, and the control unit controls the feeding of the chemical solution from the chemical solution supply unit to the culture unit. In this case, the required amount of the chemical solution can be easily supplied to the culture unit at the required timing by the control of the control unit.

  In some embodiments, the chemical solution supply unit includes a chemical solution storage unit that stores the chemical solution at a predetermined temperature. Thereby, a chemical | medical solution is stored at the temperature suitable for storage.

  In some embodiments, the cell culture apparatus further includes: a photographing cell collection unit that collects part of the cells from the culture unit; and a photographing unit that obtains a micrograph of the cells collected by the photographing cell collection unit. I have. In this case, since the micrograph of the cell after completion | finish of culture | cultivation in the cell culture apparatus or in the middle of culture | cultivation can be acquired easily, evaluation of the cultured cell, evaluation of culture conditions, etc. can be performed more correctly.

  In some embodiments, the control unit controls the collection of cells by the imaging cell recovery unit and the acquisition of micrographs by the imaging unit. In this case, a microphotograph at a desired timing can be easily (in some cases automatically) acquired by control by the control unit.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  With reference to FIG. 1, the cell culture apparatus 1 which concerns on one Embodiment of this invention is demonstrated. The cell culture apparatus 1 includes a culture tank 11 that performs three-dimensional culture of cells suspended in the culture solution F1, and a circulation pump 12 that circulates the culture solution F1 so that the culture solution F1 flows upward in the culture vessel. And an oxygen supply tank 13 that is provided in the middle of the circulation of the culture solution F1 and supplies oxygen to the culture solution F1. In the cell culture device 1, a culture unit is configured by a culture tank 11, a circulation pump 12, an oxygen supply tank 13, and a line (pipe or pipe) that connects these to each other. The culture tank 11 and the oxygen supply tank 13 are connected by a first circulation line L1, and the culture solution F1 extracted from the upper stage of the culture tank 11 is sent to the oxygen supply tank 13 via the first circulation line L1. To be liquidated. The oxygen supply tank 13 and the circulation pump 12 are connected by a second circulation line L2, and the culture solution F1 supplied with oxygen as necessary in the oxygen supply tank 13 passes through the second circulation line L2. The liquid is sent to the circulation pump 12. The circulation pump 12 and the culture tank 11 are connected by a third circulation line L3, and the culture solution F1 fed from the circulation pump 12 is supplied to the lower stage of the culture tank 11 through the third circulation line L3. Is done. The culture solution F1 supplied through the third circulation line L3 moves in an upward flow from the lower part to the upper part of the culture tank 11, and is sent out of the culture tank 11 from the first circulation line L1. .

  The cell culture device 1 is provided with a turbidimeter 15 for measuring the turbidity of the culture solution F1 in the culture tank 11 at a plurality of locations in the vertical direction. In the cell culture device 1, the cell distribution in the culture solution F <b> 1 in the culture tank 11 is detected by the turbidimeter 15. The cell culture device 1 includes the turbidimeter 15 as a cell distribution detection unit. However, the cell distribution detection unit is not limited thereto, and includes, for example, a camera that acquires an image of the culture solution F1. Good.

  The cell culture apparatus 1 is provided with a microbubble pump 14 as auxiliary means for assisting the supply of oxygen to the culture solution F1 in the oxygen supply tank 13. The microbubble pump 14 is connected to the oxygen supply tank 13 via a fourth circulation line L4 branched from the second circulation line L2 and a fifth circulation line L5 connected to the first circulation line L1. A part of the culture solution F1 passing through the second circulation line L2 is sent to the microbubble pump 14 via the fourth circulation line L4, and the microbubble pump 14 passes through the fifth circulation line L5 and the first circulation line L1. And returned to the oxygen supply tank 13. The microbubble pump 14 generates microbubbles in the culture solution F1 and promotes dissolution of oxygen in the culture solution F1. In addition, the cell culture apparatus 1 includes a dissolved oxygen meter 16 that detects the amount of dissolved oxygen in the culture solution F1 in the culture tank 11. The dissolved oxygen meter 16 may detect the amount of dissolved oxygen before the start of culture, may always detect the amount of dissolved oxygen during the culture, or may detect the amount of dissolved oxygen at predetermined intervals during the culture.

  The cell culture device 1 further includes a pH meter 17 that detects the pH of the culture solution F1 in the culture tank 11. The pH meter 17 may detect the pH before the start of culture, may always detect the pH during the culture, or may detect the pH at predetermined intervals during the culture. The detection result of the pH by the pH meter can be a criterion for determining the replacement time of the culture solution F1.

  The culture tank 11 is disposed in an incubator 21 for keeping the culture environment in the culture tank 11 constant. The incubator 21 is adjusted and maintained in a predetermined culture environment (for example, a temperature of 35 to 38 ° C. and a carbon dioxide concentration of 5%), and prevents the culture environment from fluctuating due to the external environment. In the cell culture apparatus 1, an oxygen supply tank 13 is also arranged in the incubator in order to avoid a temperature change of the culture solution F1 with oxygen supply. The cell culture device 1 may be provided with a thermometer 22 for measuring the temperature in the culture tank 11 or the temperature of the culture solution F1 in the culture tank 11. In this case, the inside of the culture tank 11 is subjected to a predetermined culture by the thermometer 22. It can be confirmed that the environment is adjusted and maintained. In addition to the thermometer 22, the cell culture device 1 may further include a detection unit that detects a culture environment other than the temperature.

  The cell culture device 1 connects a culture solution storage tank 31 for storing the culture solution F1, a culture solution heating tank 32 for heating the culture solution F1, and a culture solution storage tank 31 and a culture solution heating tank 32. A culture liquid supply line L6 is further provided, and a culture liquid supply pump 33 is provided in the culture liquid supply line L6. The culture solution F1 stored in the culture solution storage tank 31 is supplied to the culture solution heating tank 32 via the culture solution supply line L6 as necessary, and is cultured in the culture solution heating tank 32. It is heated to a suitable temperature. The culture solution F1 heated in the culture solution heating tank 32 is supplied from the culture solution heating tank 32 via a heated culture solution supply line L7 that connects the culture solution heating tank 32 and the culture tank 11. The solution is sent to the culture tank 11. The culture solution storage tank 31 and the culture solution supply line L6 are connected via a sterile connector 34. Thereby, when replacing | exchanging the culture solution storage tank 31, the aseptic condition after the culture solution supply line L6 can be maintained easily. The culture solution storage tank 31 is disposed in the refrigeration unit 35. The refrigeration unit 35 holds the culture solution storage tank 31 at a temperature (for example, 2 to 6 ° C.) suitable for storing the culture solution F1. The culture solution heating tank 32 is disposed in the incubator 21, and the incubator 21 heats the culture solution F1 in the culture solution heating tank 32 to a temperature suitable for culture (for example, 35 to 38 ° C.).

  The cell culture device 1 further includes a cell recovery line L8 serving as a recovery path for recovering cells cultured from the culture tank 11, and a cell recovery pump 41 is provided in the cell recovery line L8. A sterile connector 42 is provided at the outlet of the cell collection line L8, and can be aseptically connected to the cell collection vial 43. In the cell culture device 1, after connecting the cell collection vial 43 to the sterile connector 42, the cultured cells can be collected by circulating the cells from the culture tank 11 to the cell collection line L 8 by operating the cell collection pump 41.

  The cell culture apparatus 1 further includes a cleaning liquid storage tank 51 that stores the cleaning liquid F2 and a cleaning liquid supply pump 52 that supplies the cleaning liquid F2 from the cleaning liquid storage tank 51 to the culture tank 11. The cleaning liquid storage tank 51 is connected to the culture tank 11 via the cleaning liquid supply line L9, and the cleaning liquid F2 stored in the cleaning liquid storage tank 51 is supplied to the cleaning liquid supply line L9 by the cleaning liquid supply pump 52 as necessary. Then, the solution is fed to the culture tank 11. The washing liquid feeding line L9 is connected to the third circulation line L3 that is the circulation path of the culture unit, and the washing liquid F2 is fed to the culture tank 11 via the washing liquid feeding line L9 and the third circulation line L3. In some cases, after circulating in the circulation path of the culture unit, it is discharged from the culture unit as a waste liquid. The cleaning liquid storage tank 51 and the cleaning liquid supply line L 9 are connected via a sterile connector 53. Thereby, when exchanging the cleaning liquid storage tank 51, the aseptic condition after the cleaning liquid feeding line L9 can be easily maintained.

  The washing solution F2 is not particularly limited, and may be a known washing solution used for cell culture. For example, the cleaning liquid F2 may be Phosphate Buffered Salin (PBS). The washing solution F2 may be a basal medium suitable for cells to be cultured. For example, DMEM, F-12, RPMI, etc. are typical media.

  The cell culture device 1 further includes a waste liquid storage tank 61 that stores the waste liquid F3 collected from the culture unit. The cell culture device 1 connects the first waste liquid line L10 connecting the culture tank 11 and the waste liquid storage tank 61 as the first waste liquid path, and connects the oxygen supply tank 13 and the waste liquid storage tank 61 as the second waste liquid path. The second waste liquid line L11 is provided. The first waste liquid line L10 is provided with a first waste liquid pump 62 for sending the waste liquid discharged from the culture tank 11 to the waste liquid storage tank 61. The second waste liquid line L11 is discharged from the oxygen supply tank 13. A second waste liquid pump 63 for supplying the waste liquid to be discharged to the waste liquid storage tank 61 is provided. The first waste liquid line L10 and the second waste liquid line L11 join before reaching the waste liquid storage tank 61 and are connected to the waste liquid storage tank 61 via the aseptic connector 64. The waste liquid storage tank 61 can be replaced by the aseptic connector 64 while easily maintaining the sterility of the first waste liquid line L10 and the second waste liquid line L11. The waste liquid stored in the waste liquid storage tank 61 may be a waste liquid containing a culture solution or a washing solution, for example, a used culture solution at the time of replacing the culture solution, a used culture solution discarded before cell recovery or passage, It may be a waste liquid generated after cleaning with a cleaning liquid.

  The cell culture device 1 further includes an enzyme solution storage tank 71 that stores an enzyme solution F4 containing an enzyme, and an enzyme solution supply pump 72 that supplies the enzyme solution F4 from the enzyme solution storage tank 71 to the culture tank 11. Yes. The enzyme solution storage tank 71 is connected to the culture tank 11 via an enzyme solution supply line L12, and the enzyme solution F4 stored in the enzyme solution storage tank 71 is transferred by an enzyme solution supply pump 72 as necessary. The solution is fed to the culture tank 11 through the enzyme solution feeding line L12. The enzyme solution storage tank 71 and the enzyme solution supply line L12 are connected via a sterilization connector 73, so that when the enzyme solution storage tank 71 is replaced, the aseptic condition after the enzyme solution supply line L12 is easily maintained. it can. The enzyme solution storage tank 71 is disposed in the refrigeration unit 35 and is held together with the culture solution storage tank 31 at a predetermined temperature (for example, 2 to 6 ° C.). When the temperature suitable for storing the enzyme solution F4 is different from the temperature suitable for storing the culture solution F1, the enzyme solution storage tank 71 is held in the refrigeration unit 35 at a temperature different from that of the culture solution storage tank 31. Alternatively, it may be arranged in another refrigeration unit different from the refrigeration unit 35. The enzyme may be a known chemical solution used for cell dispersion. Examples of the enzyme include Accutase, Trypsin-EDTA, Dispase, and Collagenase. In one embodiment, a drug having a function of cell dispersion may be used instead of the enzyme. Examples of such drugs include EDTA solutions.

  The cell culture apparatus 1 further includes an inhibitor storage tank 81 for storing the Rho kinase inhibitor, and an inhibitor liquid supply pump 82 for supplying the Rho kinase inhibitor from the inhibitor storage tank 81 to the culture tank 11. . The inhibitor storage tank 81 is connected to the culture tank 11 via the inhibitor liquid supply line L13, and the Rho kinase inhibitor stored in the inhibitor storage tank 81 is added to the inhibitor liquid supply pump 82 as necessary. Is fed to the culture tank 11 via the inhibitor feeding line L13. The inhibitor storage tank 81 and the inhibitor liquid supply line L13 are connected via a sterilization connector 83, so that when the inhibitor storage tank 81 is replaced, the sterile condition after the inhibitor liquid supply line L13 is easily maintained. it can. The inhibitor storage tank 81 is disposed in the refrigeration unit 35 and is held together with the culture solution storage tank 31 at a predetermined temperature (for example, 2 to 6 ° C.). When the temperature suitable for storage of the Rho kinase inhibitor is different from the temperature suitable for storage of the culture solution F1, the inhibitor storage tank 81 is held in the refrigeration unit 35 at a temperature different from that of the culture solution storage tank 31. It may be arranged in another refrigeration unit different from the refrigeration unit 35.

  The cell culture device 1 further includes a chemical solution storage tank 91 that stores the chemical solution, and a chemical solution feed pump 92 that sends the chemical solution from the chemical solution storage tank 91 to the culture tank 11. The chemical solution storage tank 91 is connected to the culture tank 11 via the chemical solution supply line L14, and the chemical solution stored in the chemical solution storage tank 91 passes through the chemical solution supply line L14 by the chemical solution supply pump 92 as necessary. To the culture tank 11. The chemical solution storage tank 91 and the chemical solution supply line L14 are connected via an aseptic connector 93, so that when the chemical solution storage tank 91 is replaced, the aseptic condition after the chemical solution supply line L14 can be easily maintained. The medicinal solution storage tank 91 is disposed in the refrigeration unit 35 and is held together with the culture solution storage tank 31 at a predetermined temperature (for example, 2 to 6 ° C.). When the temperature suitable for storing the chemical solution is different from the temperature suitable for storing the culture solution F1, the chemical solution storage tank 91 may be held in the refrigeration unit 35 at a temperature different from that of the culture solution storage tank 31. You may arrange | position in the other refrigeration unit different from the unit 35. FIG. The chemical solution may be a known chemical solution used for cell culture. For example, the drug solution may be a drug related to maintenance of undifferentiated cytokines, promotion of differentiation, activation, etc., and examples thereof include Activin, b-FGF, SCF, TGF-beta, and the like.

  The cell culture device 1 further includes a photographing unit 101 and a photographing liquid supply line L15 branched from the cell recovery line L8 and connected to the photographing unit 101. The photographing liquid supply line L15 includes a photographing unit. 101 is provided with a photographing liquid feeding pump 102 for feeding a measurement sample containing cells. The photographing unit 101 is connected to a buffer tank 103 that stores a photographing buffer F5. The photographing unit 101 photographs the measurement sample fed by the photographing liquid feeding pump 102 and acquires a micrograph of the cells included in the measurement sample. The measurement sample after obtaining the micrograph may be discarded from the photographing unit 101 as a waste liquid. Further, the measurement sample after obtaining the micrograph may be sent to the cell recovery line L8 via the imaging liquid supply line L15 and may be recovered in the cell recovery vial 43. At this time, the imaging liquid supply pump 102 may play a role of supplying the measurement sample from the imaging unit 101 to the cell recovery line L8. The imaging unit 101 and the buffer tank 103 are arranged in the incubator 21 and are kept at a predetermined temperature (for example, 35 to 38 ° C.) in order to observe the cell state in the culture environment more accurately.

  The cell culture device 1 further includes a controller 111 as a control unit. The controller 111 acquires turbidity information detected by the turbidimeter 15, controls the circulation pump 12 based on the turbidity information, and adjusts the circulation speed of the culture solution F1.

  With reference to FIG. 2, the execution procedure of the circulation speed control by the controller 111 will be described. First, the controller 111 acquires detection information (input value) related to turbidity from the turbidimeter 15 (step S01). The turbidimeter 15 measures the turbidity of the culture solution F1 in the culture tank 11 at a plurality of locations in the vertical direction, and the controller 111 receives information detected at each location. Next, the controller 111 converts the plurality of input values from the turbidimeter 15 into predetermined engineering values, respectively (step S02). In addition, when the controller 111 acquires the detection information of the turbidimeter 15 as an engineering value, step S02 may be omitted.

  Next, the controller 111 compares the turbidity in the vertical direction of the culture solution F1 in the culture tank 11 based on the acquired plurality of engineering values (step S03), and creates a turbidity distribution (step S04). A measurement position where the turbidity is maximum is extracted from a plurality of measurement positions by the profilometer 15 (step S05). By these steps, the controller 111 obtains information on cell distribution in the culture solution F1 in the culture tank 11.

  The controller 111 determines the circulation rate of the culture solution F1 and controls the circulation pump 12 based on the obtained cell distribution information. Specifically, first, based on the relationship between the position where the cells should be distributed in the culture solution F1 and the measurement position by the turbidimeter 15 in advance, the circulation rate is increased by a predetermined amount for each measurement position. Is set so that the circulation speed is lowered by a predetermined lowering amount, or the circulation speed is maintained. The controller 111 refers to this setting and determines whether or not the target measurement position (for example, the maximum turbidity measurement position extracted in step S05) is the speed increase setting (step S06). When the controller 111 determines that the speed increase setting is set (step S06: YES), the controller 111 temporarily determines the increase amount of the circulation speed with reference to the set increase amount (step S07). If it is determined that the speed increase setting is not set (step S06: NO), the controller 111 determines whether or not the target measurement position is the speed decrease setting (step S08) and determines that the target measurement position is the speed decrease setting (step S08). (Step S08: YES), the lowering amount of the circulation speed is provisionally determined with reference to the set lowering amount (Step S09). The controller 111 determines the provisional value of the circulation speed after circulation (from the increase amount provisionally determined in step S07 or the decrease amount provisionally determined in step S08) and the initial circulation speed (circulation speed before execution of the circulation speed control) ( The circulation speed before execution + the amount of increase or the circulation speed before execution−the amount of decrease is calculated (step S10). If it is determined in step S08 that the speed drop is not set (step S08: NO), the initial circulation speed is calculated as a temporary value of the circulation speed after control.

  Next, the controller 111 determines whether or not the calculated temporary value exceeds the upper limit value of the circulation rate in the cell culture device 1 (step S11). When the controller 111 determines that the temporary value exceeds the upper limit value (step S11: YES), the controller 111 replaces the temporary value with the upper limit value (step S12). If controller 111 determines that the temporary value does not exceed the upper limit value (step S11: NO), it then determines whether the temporary value is less than the lower limit value (step S13). When the controller 111 determines that the temporary value is less than the lower limit value (step S13: YES), the controller 111 replaces the temporary value with the lower limit value (step S14). If the controller 111 determines that the provisional value is not less than the lower limit (step S14: NO), the provisional value is maintained. Then, the controller 111 determines the provisional value set through steps S11 to S14 as the post-control circulation speed (step S15). The controller 111 controls the circulation pump 12 based on the determined circulation speed (step S16).

  In addition, the execution procedure of the circulation speed control by the controller 111 is not limited to the above, and the circulation pump 12 may be controlled based on the detection result by the turbidimeter 15. The controller 111 may further acquire information measured from a meter other than the turbidimeter 15 and may further control a pump other than the circulation pump 12.

  The controller 111 may acquire the detection result of the dissolved oxygen meter 16 and control the microbubble pump 14 based on the acquired detection result. When the dissolved oxygen amount detected by the dissolved oxygen meter 16 exceeds the predetermined value, the controller 111 decreases the amount of microbubbles generated by the microbubble pump 14 so that the predetermined value of the dissolved oxygen amount is maintained. When the dissolved oxygen amount detected by the total 16 is less than the specified value, the amount of microbubbles generated by the microbubble pump 14 may be increased.

  The controller 111 may acquire the detection result of the pH meter 17, determine the replacement time of the culture solution F1 based on the acquired detection result, and notify the user or replace the culture solution F1. Moreover, the controller 111 may control the culture solution feeding pump 33, the washing solution feeding pump 52, the first waste solution pump 62, and the second waste solution pump 63, respectively. In this case, the controller 111 can control each pump according to its own judgment or a user's instruction, and can perform operations such as exchanging the culture medium and passage. The cell culture device 1 may further include a liquid level detector (not shown) that detects the volume of the culture solution F1 in the culture tank 11, and the controller 111 acquires the detection result of the liquid level detector. Then, the amount of the culture solution fed by the culture solution feeding pump 33 may be controlled based on the acquired detection result.

  The controller 111 can control the cell collection pump 41. In this case, for example, the controller 111 automatically sends the cell recovery pump 41 after the culturing time set in advance (further, if necessary, the culture liquid feeding pump 33, the washing liquid feeding pump 52, the first waste liquid pump 62, And the second waste liquid pump 63) can be controlled to automatically collect cells.

  The controller 111 may control the enzyme solution feeding pump 72. In this case, the enzyme solution F4 can be sent to the culture tank 11 based on a user instruction or a preset setting. Further, the controller 111 may control the inhibitor feeding pump 82, and in this case, the Rho kinase inhibitor can be fed to the culture tank 11 based on a user instruction or a prior setting. Moreover, the controller 111 may control the chemical liquid feeding pump 92, and in this case, the chemical liquid can be fed to the culture tank 11 based on a user instruction or a prior setting. Further, the controller 111 may control the imaging liquid supply pump 102. In this case, the measurement sample can be supplied to the imaging unit 101 based on a user instruction or a pre-setting. Furthermore, the controller 111 may control the taking of the micrograph by the photographing unit 101, and in this case, the micrograph of the cell can be automatically acquired based on the user's instruction or the presetting.

  In such a cell culture device 1, the cells are suspended in the culture solution F1, and are cultured while floating in the culture solution F1 due to the balance between the upward flow of the culture solution F1 and its own weight. The controller 111 controls the circulation speed of the culture solution F1 by the circulation pump 12, thereby adjusting the equilibrium state between the upward flow of the culture solution F1 and the dead weight of the cells, and appropriately maintaining the cell distribution in the culture tank 11. it can.

  Moreover, in the cell culture apparatus 1, the turbidimeter 15 detects the turbidity of the culture solution F1 in the culture tank 11, and the controller 111 controls the circulation speed according to the above execution procedure. Thereby, even if the cell distribution changes due to a change in cell size or the like during the culturing process, the equilibrium state between the upward flow of the culture solution F1 and the dead weight of the cells can be automatically adjusted by the control of the controller 111. For this reason, in the cell culture apparatus 1, it is possible to carry out homogeneous culture while appropriately maintaining the cell distribution.

  Moreover, in the cell culture apparatus 1, the turbidimeter 15 measures the turbidity of the culture solution F1 in the culture tank 11 at a plurality of locations in the vertical direction. Here, when the number of cells cultured in the culture tank 11 increases, the turbidity of the culture solution F1 increases. Further, when the size of the cultured cell (or the size of the cell mass) increases, the cell distribution gradually moves downward in the vertical direction due to its own weight. According to the turbidimeter 15 described above, the culture state can be monitored.

  Moreover, in the cell culture apparatus 1, the dissolved oxygen meter 16 detects the amount of dissolved oxygen in the culture solution F1, and the controller 111 can control the microbubble pump 14 based on the detection result. For this reason, in the cell culture apparatus 1, the cell culture which maintained the fixed amount of dissolved oxygen can be easily implemented by control of the controller 111.

  In the cell culture device 1, the pH of the culture solution F1 is detected by the pH meter 17, and the controller 111 determines the replacement time of the culture solution F1 based on the detection result, and notifies the user or replaces the culture solution F1. Can be executed. In such a cell culture device 1, the controller 111 automatically determines the replacement time of the culture medium F1, and prevents the deterioration of the quality of the cultured cells due to the passing of the replacement time.

  In the cell culture apparatus 1, the controller 111 can control the culture liquid feeding pump 33, the cleaning liquid feeding pump 52, the first waste liquid pump 62, and the second waste liquid pump 63, respectively. For this reason, in the cell culture apparatus 1, according to the determination of the controller 111 or a user's instruction | indication, work, such as replacement | exchange of a culture solution F1, a subculture, can be implemented automatically.

  Further, in the cell culture device 1, the controller 111 can control the enzyme solution feeding pump 72, the inhibitor solution feeding pump 82, and the chemical solution feeding pump 92, respectively. Therefore, in the cell culture device 1, the enzyme solution, the Rho kinase inhibitor, and the chemical solution can be automatically supplied to the culture tank 11 at a preset amount and timing under the control of the controller 111.

  Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment. For example, the culture tank 11 may have a truncated cone shape in which the horizontal cross-sectional area increases from the bottom toward the top. Thereby, a velocity gradient is provided in the upward flow, and the correlation between the circulation velocity and the cell distribution becomes remarkable.

DESCRIPTION OF SYMBOLS 1 Cell culture apparatus 11 Culture tank 12 Circulation pump 13 Oxygen supply tank 14 Micro bubble pump 15 Turbidimeter 16 Dissolved oxygen meter 17 pH meter 21 Incubator 22 Thermometer 31 Culture solution storage tank 32 Culture solution heating tank 33 Culture solution feed Liquid pump 34 Aseptic connector 35 Refrigeration unit 41 Cell recovery pump 42 Aseptic connector 43 Cell recovery vial 51 Cleaning liquid storage tank 52 Cleaning liquid feed pump 53 Aseptic connector 61 Waste liquid storage tank 62 First waste liquid pump 63 Second waste liquid pump 64 Aseptic connector 71 Enzyme Liquid storage tank 72 Enzymatic liquid feed pump 73 Aseptic connector 81 Inhibitor storage tank 82 Inhibitor liquid feed pump 83 Aseptic connector 91 Chemical liquid storage tank 92 Chemical liquid feed pump 93 Aseptic connector 101 Imaging unit 102 Imaging liquid pump 103 Buffer tank 111 Controller F1 Culture solution F2 Wash solution F3 Waste solution F4 Enzyme solution F5 Imaging buffer L1 First circulation line L2 Second circulation line L3 Third circulation line L4 Fourth circulation line L5 Fifth circulation line L6 Culture solution feeding line L7 Heated Culture solution feed line L8 Cell recovery line L9 Wash solution feed line L10 First waste solution line L11 Second waste solution line L12 Enzyme solution feed line L13 Inhibitor solution feed line L14 Chemical solution feed line L15 Shooting solution feed line

Claims (17)

A culture tank that performs three-dimensional culture of cells suspended in the culture medium, and a culture unit that includes a circulation means for circulating the culture liquid so that the culture liquid forms an upward flow in the culture tank;
A cell distribution detection unit for detecting a cell distribution in the culture tank;
Based on the cell distribution detected by the cell distribution detection unit, a control unit for controlling the circulation rate of the culture solution by the circulation means;
A cell culture device.   The cell culture device according to claim 1, wherein the cell distribution detection unit includes turbidity measuring means for measuring the turbidity of the culture solution in the culture tank at a plurality of locations in the vertical direction.   The cell culture according to claim 1 or 2, wherein the culture unit includes culture environment detection means for detecting a culture environment in the culture tank and culture environment adjustment means for adjusting the culture environment in the culture tank. apparatus. A culture solution supply unit for supplying the culture solution to the culture unit;
A cleaning liquid supply unit for supplying a cleaning liquid to the culture unit;
A waste liquid recovery unit for recovering a waste liquid containing the culture liquid or the washing liquid from the culture unit;
The cell culture device according to any one of claims 1 to 3, further comprising:   The control unit is configured to supply the culture solution from the culture solution supply unit to the culture unit, supply the cleaning solution from the cleaning solution supply unit to the culture unit, and from the culture unit to the waste solution recovery unit. The cell culture device according to claim 4, wherein each of the liquid wastes is controlled.   The culture solution supply unit includes a culture solution storage unit that stores the culture solution at a predetermined temperature, and a culture solution heating unit that heats the culture solution to a temperature equal to or higher than the predetermined temperature. 5. The cell culture apparatus according to 5. A liquid amount detecting means for detecting the amount of the culture solution in the culture tank;
The said control unit controls the liquid feeding of the said culture solution from the said culture solution supply unit to the said culture unit based on the liquid amount detected by the said liquid amount detection means. The cell culture device according to Item. An enzyme supply unit for supplying an enzyme solution containing an enzyme to the culture unit;
The cell culture device according to any one of claims 1 to 7, wherein the control unit controls feeding of the enzyme solution from the enzyme supply unit to the culture unit.   The cell culture device according to claim 8, wherein the enzyme supply unit includes an enzyme storage unit that stores the enzyme solution at a predetermined temperature. Further comprising a dissolved oxygen content detection means for detecting the dissolved oxygen content of the culture fluid circulating in the culture unit,
The culture unit further includes oxygen supply means for supplying oxygen to the culture solution,
The cell culture according to any one of claims 1 to 9, wherein the control unit controls the amount of oxygen supplied by the oxygen supply unit based on the amount of dissolved oxygen detected by the dissolved oxygen amount detection unit. apparatus. Further comprising pH detection means for detecting the pH of the culture solution in the culture tank;
11. The control unit according to any one of claims 1 to 10, wherein the control unit determines a replacement time of the culture medium based on a pH detected by the pH detection means, and notifies a user or replaces the culture medium. The cell culture device according to Item. An inhibitor supply unit for supplying a Rho kinase inhibitor to the culture unit;
The control unit controls the supply amount of the Rho kinase inhibitor by the inhibitor supply unit so that the concentration of the Rho kinase inhibitor in the culture medium in the culture tank becomes a predetermined concentration. Item 12. The cell culture device according to any one of Items 1 to 11.   The cell culture device according to claim 12, wherein the inhibitor supply unit includes an inhibitor storage means for storing the Rho kinase inhibitor at a predetermined temperature. A chemical solution supply unit for supplying a chemical solution to the culture unit;
The cell culture device according to any one of claims 1 to 13, wherein the control unit controls the feeding of the chemical solution from the chemical solution supply unit to the culture unit.   The cell culture device according to claim 14, wherein the chemical solution supply unit includes a chemical solution storage means for storing the chemical solution at a predetermined temperature. A cell collection unit for photographing that collects a part of the cells from the culture unit;
An imaging unit for obtaining a micrograph of the cells recovered by the imaging cell recovery unit;
The cell culture device according to any one of claims 1 to 15, further comprising:   The cell culture device according to claim 16, wherein the control unit controls the collection of the cells by the cell-collecting unit for photographing and the acquisition of the micrograph by the photographing unit.

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