WO2019102593A1

WO2019102593A1 - Cell culture device

Info

Publication number: WO2019102593A1
Application number: PCT/JP2017/042251
Authority: WO
Inventors: 倫教志田; 浩介石井
Original assignee: 株式会社Ihi
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2019-05-31

Abstract

A cell culture device which comprises: a culture unit comprising a culture tank for three-dimensionally culturing cells suspended in a culture solution and a circulation means for circulating the culture solution so that an upward flow of the culture solution is generated in the culture tank; a cell distribution detection unit detecting the cell distribution in the culture tank; and a control unit controlling the circulation speed of the culture solution by the circulation means depending on the cell distribution detected by the cell distribution detection unit.

Description

Cell culture device

The present disclosure relates to cell culture devices.

As a cell culture apparatus, for example, Patent Document 1 discloses a cell culture apparatus having a cell culture unit that performs three-dimensional culture, and a unit that sends various liquid chemicals, a culture solution, waste liquid, and the like. This cell culture apparatus is an apparatus for culturing cells on a carrier suspended in a culture solution, and automatically performs operations such as medium replacement and passaging, which have conventionally been performed manually by workers. A control unit is provided to control each unit in order to do so.

JP, 2015-136330, A

In recent years, as the application to the medical field has been expanded, in the cell culture apparatus, there are demands for more efficient culture and higher quality and homogenization of the obtained cultured cells.

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

A cell culture apparatus according to one aspect of the present disclosure includes a culture vessel for performing three-dimensional culture of cells suspended in a culture solution, and a circulation for circulating the culture solution so that the culture solution forms an ascending 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 fluid by the circulation means based on the cell distribution detected by the cell distribution detection unit And.

According to the cell culture apparatus according to one aspect 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 circulation rate control in the cell culture apparatus of FIG.

In this cell culture apparatus, cells are suspended in a culture solution and cultured while being suspended in the culture solution by the balance between the upward flow by circulation means and dead weight. The cell distribution detection unit can detect the balance between the upward flow by the circulating means and the weight of the cells by detecting the distribution of cells suspended in the culture solution in the culture tank. The control unit can control the circulation rate of the culture solution by the circulation means to control the balance between the upward flow by the circulation means and the dead weight of the cells, and can appropriately maintain the cell distribution in the culture tank.

Here, in the conventional cell culture apparatus, when the cell distribution in the culture solution is biased lower or upper than the appropriate position in the culture tank, the cultured cells are excessively aggregated, and homogeneous culture is difficult. It could have been. In addition, since the cell distribution gradually changes due to a change in the size of a cell mass and the like in the culture process, it has been 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 speed of the culture fluid based on the detection result. Also, even if the cell distribution changes due to changes in cell size etc during the culture process, the detection by the cell distribution detection unit and the control by the control unit regulate the balance between the upward flow by circulating means and the cell's own weight . For this reason, in the cell culture device according to the above aspect, it is possible to carry out homogeneous culture while appropriately maintaining the cell distribution.

In some embodiments, the cell distribution detection unit includes turbidity measurement means for measuring the turbidity of the culture solution in the culture vessel at multiple points in the vertical direction. In this case, the distribution of cells in the culture solution can be detected by the degree of turbidity measured at a plurality of measurement points. In addition to the detection of cell distribution, it is also possible to estimate the amount of increase in the number of cells in the culture solution based on the change in turbidity from the start of culture.

In some embodiments, the culture unit includes culture environment detection means for detecting the culture environment in the culture vessel, and culture environment adjustment means for adjusting the culture environment in the culture vessel. In this case, by detecting the culture environment in the culture tank by the culture environment detection means, it is possible to easily confirm whether or not the desired culture environment is maintained, and the culture environment adjustment means as needed. The culture environment in the culture vessel 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 recovers a culture fluid supply unit that supplies culture fluid to the culture unit, a washing solution supply unit that supplies the washing solution to the culture unit, and a waste liquid containing the culture fluid or the washing 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 carried out by the operation of each unit.

In some embodiments, the control unit sends the culture solution from the culture solution supply unit to the culture unit, sends the washing solution from the wash solution supply unit to the culture unit, and discharges the waste solution from the culture unit to the waste solution recovery unit. Control each liquid transfer. 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 culture solution storage means for storing the culture solution at a predetermined temperature, and culture solution heating means for heating the culture solution to a temperature higher than or equal to 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 unit can be used by heating it by the culture solution heating unit, so that the culture solution can be replenished from outside the apparatus The work procedure is simplified compared to the case of

In some embodiments, the cell culture apparatus further comprises fluid volume detection means for detecting the fluid volume of the culture fluid in the culture vessel, and the control unit is based on the fluid volume detected by the fluid volume detection means. And controlling the transfer of the culture solution from the culture solution supply unit to the culture unit. In this case, the liquid volume detection means detects the liquid volume in the culture tank, and the culture solution is supplied into the culture tank under the control of the control unit based on the detection result. 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 the supply amount of the culture solution can be avoided.

In some embodiments, the cell culture apparatus further comprises an enzyme supply unit that supplies an enzyme solution containing an enzyme to the culture unit, and the control unit controls transfer of the enzyme solution from the enzyme supply unit to the culture unit. Do. In this case, the control unit can easily supply the required amount of enzyme solution to the culture unit at the required timing.

In some embodiments, the enzyme supply unit comprises an enzyme storage means for storing the enzyme solution at a predetermined temperature. The enzyme solution is thereby stored at a temperature suitable for storage.

In some embodiments, the cell culture apparatus further comprises a dissolved oxygen amount detection means for detecting the amount of dissolved oxygen in the culture fluid circulating in the culture unit, and the culture unit supplies oxygen to the culture fluid. The control unit further controls the supply amount of oxygen by the oxygen supply unit based on the dissolved oxygen amount detected by the dissolved oxygen amount detection unit. In this case, since the necessary amount of oxygen is supplied to the culture solution by the control of the control unit, it is possible to easily carry out cell culture maintaining a fixed amount of dissolved oxygen.

In some embodiments, the cell culture apparatus further comprises pH detection means for detecting the pH of the culture solution in the culture vessel, and the control unit controls the culture solution based on the pH detected by the pH detection means. Determine the replacement time and notify the user or replace the culture solution. In this case, the pH detection means detects a change in pH of the culture solution, and the control unit automatically determines the culture solution replacement time based on the detection result. Etc. are prevented. The control unit may notify the user of the replacement time via an interface unit or the like. Further, the control unit may automatically exchange the culture solution after the determination of the replacement time by controlling the culture solution supply unit, the washing solution supply unit, and the waste solution supply unit described above.

In some embodiments, the cell culture apparatus may further comprise an inhibitor supply unit that supplies Rho kinase inhibitor to the culture unit, and the control unit controls the concentration of Rho kinase inhibitor in the culture solution in the culture vessel. The amount of Rho kinase inhibitor supplied by the inhibitor supply unit is controlled so as to achieve a predetermined concentration. In this case, the control unit can easily supply the necessary amount of Rho kinase inhibitor to the culture unit at the necessary timing.

In some embodiments, the inhibitor delivery unit comprises an inhibitor storage means for storing the Rho kinase inhibitor at a predetermined temperature. This causes the Rho kinase inhibitor to be stored at a temperature suitable for storage.

In some embodiments, the cell culture apparatus further comprises a drug solution supply unit for supplying a drug solution to the culture unit, and the control unit controls the delivery of the drug solution from the drug solution supply unit to the culture unit. In this case, the control unit can easily supply the necessary amount of drug solution to the culture unit at the required timing.

In some embodiments, the chemical solution supply unit includes chemical solution storage means for storing the chemical solution at a predetermined temperature. Thereby, the drug solution is stored at a temperature suitable for storage.

In some embodiments, the cell culture apparatus further includes: a cell collection unit for photographing that collects a part of cells from the culture unit; and a photographing unit that acquires a micrograph of the cells collected by the cell collection unit for photographing. Have. In this case, since it is possible to easily obtain a microphotograph of cells after or during culture in the cell culture apparatus, it is possible to more accurately evaluate cultured cells, evaluate culture conditions, and the like.

In some embodiments, the control unit controls the collection of cells by the imaging cell collection unit and the acquisition of photomicrographs by the imaging unit. In this case, the control unit can easily (possibly automatically) acquire a micrograph at a desired timing.

Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

A cell culture device 1 according to an embodiment of the present disclosure will be described with reference to FIG. The cell culture apparatus 1 includes a culture vessel 11 for performing three-dimensional culture of cells suspended in a culture solution F1, and a circulation pump 12 for circulating the culture solution F1 so that the culture solution F1 forms an upward flow in the culture vessel. And an oxygen supply tank 13 provided in the middle of circulation of the culture solution F1 and supplying oxygen to the culture solution F1. In the cell culture apparatus 1, a culture unit is constituted by the culture tank 11, the circulation pump 12, the oxygen supply tank 13, and a line (a pipe or a pipe) connecting these to each other. The culture tank 11 and the oxygen supply tank 13 are connected by the first circulation line L1, and the culture fluid F1 extracted from the upper portion of the culture tank 11 is fed to the oxygen supply tank 13 via the first circulation line L1. Be liquid. The oxygen supply tank 13 and the circulation pump 12 are connected by the second circulation line L2, and the culture fluid F1 supplied with oxygen as necessary in the oxygen supply tank 13 is connected via the second circulation line L2. The fluid is sent to the circulation pump 12. The circulation pump 12 and the culture tank 11 are connected by the third circulation line L3, and the culture fluid F1 sent from the circulation pump 12 is supplied to the lower part of the culture tank 11 through the third circulation line L3. Be done. The culture fluid F1 supplied via the third circulation line L3 moves upward from the lower part to the upper part of the culture tank 11 and is transferred out of the culture tank 11 from the first circulation line L1. .

The cell culture apparatus 1 is provided with a turbidimeter 15 which measures 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 apparatus 1, the turbidimeter 15 detects the cell distribution in the culture solution F1 in the culture tank 11. Although the cell culture apparatus 1 includes the turbidimeter 15 as a cell distribution detection unit, the cell distribution detection unit is not limited to this, and includes, for example, a camera for acquiring an image of the culture fluid F1 and the like. Good.

The cell culture device 1 is provided with a micro bubble pump 14 as an auxiliary means for assisting the supply of oxygen to the culture fluid F1 in the oxygen supply tank 13. The micro bubble 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 portion of the culture fluid 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 feeds the fifth circulation line L5 and the first circulation line L1. It is returned to the oxygen supply tank 13 via The micro bubble pump 14 generates micro bubbles in the culture fluid F1 and promotes the dissolution of oxygen in the culture fluid F1. Moreover, the cell culture apparatus 1 is equipped with the dissolved oxygen meter 16 which detects the dissolved oxygen amount in the culture solution F1 in the culture tank 11. As shown in FIG. The dissolved oxygen meter 16 may detect the amount of dissolved oxygen before starting the culture, may constantly detect the amount of dissolved oxygen during the culture, and may detect the amount of dissolved oxygen at predetermined intervals during the culture.

The cell culture apparatus 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 the culture, may constantly detect the pH during the culture, and may detect the pH at predetermined intervals during the culture. The result of detection of pH by a pH meter can be used as a criterion of the replacement time of the culture fluid 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 adjusts and maintains the inside to a predetermined culture environment (for example, a temperature of 35 to 38 ° C., a carbon dioxide concentration of 5%), and prevents the culture environment from being changed by the external environment. In the cell culture apparatus 1, the oxygen supply tank 13 is also disposed in the incubator in order to avoid the temperature change of the culture solution F1 with oxygen supply. The cell culture apparatus 1 may be provided with a thermometer 22 for measuring the temperature in the culture tank 11 or the temperature of the culture fluid F1 in the culture tank 11. In this case, the inside of the culture tank 11 is 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 apparatus 1 may further include detection means for detecting a culture environment other than temperature.

The cell culture apparatus 1 connects the culture solution storage tank 31 for storing the culture solution F1, the culture solution heating tank 32 for heating the culture solution F1, the culture solution storage tank 31, and the culture solution heating tank 32. A culture fluid delivery line L6 is further provided, and a culture fluid delivery pump 33 is provided in the culture fluid delivery line L6. The culture solution F1 stored in the culture solution storage tank 31 is transferred to the culture solution heating tank 32 via the culture solution supply line L6 as necessary, and culture is performed in the culture solution heating tank 32. It is warmed to a suitable temperature. The culture fluid F1 heated in the culture fluid heating tank 32 is fed from the culture fluid heating tank 32 via the heated culture fluid delivery line L7 connecting the culture fluid heating tank 32 and the culture tank 11. The liquid is sent to the culture tank 11. The culture solution storage tank 31 and the culture solution delivery line L6 are connected via the aseptic connector 34. As a result, when the culture solution storage tank 31 is replaced, it is possible to easily maintain the aseptic condition after the culture solution delivery line L6. The culture solution storage tank 31 is disposed in the refrigeration unit 35. The refrigeration unit 35 maintains the culture solution storage tank 31 at a temperature (for example, 2 to 6 ° C.) suitable for storage of 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 (for example, 35 to 38 ° C.) suitable for culture.

The cell culture apparatus 1 further includes a cell recovery line L8 serving as a recovery path for recovering cells cultured in the culture tank 11. The cell recovery line L8 is provided with a cell recovery pump 41. 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 apparatus 1, after the cell recovery vial 43 is connected to the sterile connector 42, the cultured cells can be recovered by circulating the cells from the inside of the culture tank 11 to the cell recovery line L8 by the operation of the cell recovery pump 41.

The cell culture apparatus 1 further includes a washing solution storage tank 51 for storing the washing solution F 2, and a washing solution sending pump 52 for sending the washing solution F 2 from the washing solution storage tank 51 to the culture tank 11. The washing solution storage tank 51 is connected to the culture tank 11 through the washing solution feeding line L9, and the washing solution F2 stored in the washing solution storage tank 51 is fed by the washing solution feeding pump 52 as needed. Is sent to the culture tank 11 via the The washing solution feed line L9 is connected to the third circulation line L3 which is a circulation route of the culture unit, and the washing solution F2 is sent to the culture tank 11 via the washing solution feed line L9 and the third circulation line L3. After being optionally circulated in the circulation route of the culture unit, it is discharged from the culture unit as waste liquid. The cleaning solution storage tank 51 and the cleaning solution supply line L9 are connected via the aseptic connector 53. Thus, when replacing the cleaning solution storage tank 51, the aseptic condition after the cleaning solution supply 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 washing solution F2 may be Phosphate Buffered Salin (PBS). In addition, the washing solution F2 may be a basal medium or the like suitable for cells to be cultured. For example, DMEM, F-12, RPMI and the like are representative media.

The cell culture apparatus 1 further includes a waste liquid storage tank 61 for storing the waste liquid F3 collected from the culture unit. The cell culture apparatus 1 also connects a first waste liquid line L10 connecting the culture tank 11 and the waste liquid storage tank 61 as a first waste liquid path, and connects an oxygen supply tank 13 and a waste liquid storage tank 61 as a 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 feeding the waste liquid discharged from the culture tank 11 to the waste liquid storage tank 61, and the second waste liquid line L11 is discharged from the oxygen supply tank 13 A second waste liquid pump 63 is provided to feed the waste liquid to the waste liquid storage tank 61. The first waste liquid line L10 and the second waste liquid line L11 join together before reaching the waste liquid storage tank 61, and are connected to the waste liquid storage tank 61 via the aseptic connector 64. The sterile connector 64 allows the waste liquid storage tank 61 to be replaced while easily maintaining the aseptic condition of the first waste liquid line L10 and the second waste liquid line L11. The waste solution stored in the waste solution storage tank 61 may be a waste solution containing a culture solution or a washing solution, for example, a used culture solution at the time of culture medium replacement, a used culture solution discarded before cell recovery or passage. It may be a waste liquid or the like generated after the cleaning with the cleaning liquid.

The cell culture apparatus 1 further includes an enzyme solution storage tank 71 for storing an enzyme solution F4 containing an enzyme, and an enzyme solution delivery pump 72 for transferring the enzyme solution F4 from the enzyme solution storage tank 71 to the culture tank 11 There is. The enzyme solution storage tank 71 is connected to the culture tank 11 via the enzyme solution feed line L12, and the enzyme solution F4 stored in the enzyme solution storage tank 71 is optionally sent by the enzyme solution feed pump 72. The liquid is fed to the culture tank 11 via the enzyme liquid feed line L12. The enzyme solution storage tank 71 and the enzyme solution delivery line L12 are connected via the aseptic connector 73, whereby when replacing the enzyme solution storage tank 71, the sterile condition after the enzyme solution delivery line L12 can be 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 under 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 maintained at a temperature different from that of the culture solution storage tank 31 in the refrigeration unit 35. It may well be arranged in another refrigeration unit different from the refrigeration unit 35. The enzyme may be a known drug used for cell dispersion. For example, enzymes include Accutase, Trypsin-EDTA, Dispase, and Collagenase. In one aspect, instead of the enzyme, a drug having a function of cell dispersion may be used. Such agents include EDTA solution and the like.

The cell culture apparatus 1 further includes an inhibitor storage tank 81 for storing Rho kinase inhibitor, and an inhibitor delivery pump 82 for transferring Rho kinase inhibitor to the culture tank 11 from the inhibitor storage tank 81. . The inhibitor storage tank 81 is connected to the culture tank 11 via the inhibitor delivery line L13, and the Rho kinase inhibitor stored in the inhibitor storage tank 81 is an inhibitor delivery pump 82 as needed. Thus, the liquid is fed to the culture tank 11 via the inhibitor delivery line L13. The inhibitor storage tank 81 and the inhibitor delivery line L13 are connected via the aseptic connector 83, whereby when the inhibitor storage tank 81 is replaced, the sterility of the inhibitor delivery line L13 and thereafter can be 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 under 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 fluid F1, the inhibitor storage tank 81 is maintained at a temperature different from that of the culture fluid storage tank 31 in the refrigeration unit 35. It may be disposed in another refrigeration unit different from the refrigeration unit 35.

The cell culture apparatus 1 further includes a drug solution storage tank 91 for storing a drug solution, and a drug solution sending pump 92 for sending a drug solution from the drug 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 delivery line L14, and the chemical solution stored in the chemical solution storage tank 91 is transferred by the chemical solution delivery pump 92 as needed. The liquid is sent to the culture tank 11 via The drug solution storage tank 91 and the drug solution sending line L14 are connected via the aseptic connector 93, whereby when replacing the drug solution storage tank 91, it is possible to easily maintain the aseptic condition of the drug solution sending line L14 and thereafter. The chemical solution storage tank 91 is disposed in the refrigeration unit 35, and is held together with the culture solution storage tank 31 under a predetermined temperature (for example, 2 to 6 ° C.). When the temperature suitable for storing the drug solution is different from the temperature suitable for storing the culture solution F1, the drug solution storage tank 91 may be kept at a temperature different from that of the culture solution storage tank 31 in the refrigeration unit 35, It may be disposed in another refrigeration unit different from the unit 35. The drug solution may be a known drug solution used for cell culture. For example, the drug solution may be an agent related to maintenance of undifferentiation such as cytokine, promotion of differentiation, activation, etc., and includes Activin, b-FGF, SCF, TGF-beta and the like.

The cell culture apparatus 1 further includes an imaging unit 101, and a liquid transfer line L15 for imaging which is branched from the cell collection line L8 and connected to the imaging unit 101. The imaging liquid supply line L15 includes the imaging unit A photographing liquid-sending pump 102 is provided for feeding a measurement sample containing cells at 101. Further, a buffer tank 103 for storing a shooting buffer F5 is connected to the shooting unit 101. The imaging unit 101 images the measurement sample fed by the imaging liquid feed pump 102, and obtains a micrograph of the cells contained in the measurement sample. The measurement sample after obtaining the microphotograph may be discarded from the imaging unit 101 as a waste liquid. In addition, the measurement sample after obtaining the microphotograph may be sent to the cell collection line L8 via the imaging liquid supply line L15 and collected in the cell collection vial 43. At this time, the imaging liquid transfer pump 102 may play a role of transporting the measurement sample from the imaging unit 101 to the cell recovery line L8. The imaging unit 101 and the buffer tank 103 are disposed in the incubator 21 and maintained 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 apparatus 1 further includes a controller 111 as a control unit. The controller 111 acquires information on the turbidity detected by the turbidity meter 15, controls the circulation pump 12 based on the information on the turbidity, and adjusts the circulation speed of the culture fluid F1.

An execution procedure of circulation speed control by the controller 111 will be described with reference to FIG. First, the controller 111 acquires detection information (input value) relating to turbidity from the turbidity meter 15 (step S01). The turbidity meter 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 turbidity meter 15 into predetermined engineering values (step S02). In addition, when the controller 111 acquires the detection information of the turbidity meter 15 as an engineering value, step S02 may be omitted.

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

The controller 111 determines the circulation speed of the culture fluid F1 and controls the circulation pump 12 based on the information of the obtained cell distribution. Specifically, first, based on the relationship between the position where cells should be distributed in culture fluid F1 and the measurement position by turbidity meter 15, the circulating speed is increased by a predetermined amount for each measurement position. Setting to decrease the circulation speed by a predetermined decrease amount or to maintain the circulation speed. The controller 111 refers to this setting and determines whether or not the measurement position of the object (for example, the measurement position of the maximum turbidity extracted in step S05) is the speed increase setting (step S06). When the controller 111 determines that the speed increase setting is made (step S06: YES), the controller 111 refers to the set increase amount to temporarily determine the increase amount of the circulation speed (step S07). If it is determined that the speed increase setting is not set (step S06: NO), the controller 111 determines whether the target measurement position is the speed decrease setting (step S08), and if it is determined that the speed decrease setting is Step S08: YES) With reference to the set descent amount, the descent amount of the circulating speed is temporarily determined (step S09). The controller 111 uses the increase amount temporarily determined in step S07 or the decrease amount temporarily determined in step S08, and the initial circulation speed (the circulation speed before execution of the circulation speed control), a provisional value of the circulation speed after circulation ( The circulation speed before the execution + the increase amount, or the circulation speed before the execution−the decrease amount) is calculated (step S10). If it is determined in step S08 that the speed reduction setting 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 the calculated temporary value exceeds the upper limit value of the circulation rate in the cell culture device 1 (step S11). If the controller 111 determines that the provisional value exceeds the upper limit (step S11: YES), it replaces the provisional value with the upper limit (step S12). When the controller 111 determines that the temporary value does not exceed the upper limit (step S11: NO), it then determines whether the temporary value is less than the lower limit (step S13). If the controller 111 determines that the temporary value is less than the lower limit value (step S13: YES), the temporary value is replaced with the lower limit value (step S14). In addition, when the controller 111 determines that the temporary value is not less than the lower limit (step S14: NO), the temporary value is maintained. Then, the controller 111 determines the temporary value set through steps S11 to S14 as the circulation speed after control (step S15). The controller 111 controls the circulation pump 12 based on the determined circulation speed (step S16).

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 turbidity meter 15. Moreover, the controller 111 may further acquire information measured from a meter other than the turbidity meter 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 micro bubble pump 14 based on the acquired detection result. The controller 111 reduces the amount of microbubbles generated by the microbubble pump 14 when the amount of dissolved oxygen detected by the dissolved oxygen meter 16 exceeds the predetermined value so that the predetermined value of the amount of dissolved oxygen is maintained. If the amount of dissolved oxygen 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 fluid F1 based on the acquired detection result, and notify the user or exchange the culture fluid F1. In addition, the controller 111 may control the culture solution delivery pump 33, the washing solution delivery 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 culture medium replacement and passage. In addition, the cell culture apparatus 1 may further include a liquid volume detector (not shown) for detecting the liquid volume of the culture fluid F1 in the culture tank 11, and the controller 111 acquires the detection result of the liquid volume detector. Then, on the basis of the acquired detection result, the amount of culture solution delivered by the culture solution delivery pump 33 may be controlled.

The controller 111 can control the cell recovery pump 41. In this case, for example, the controller 111 automatically performs the cell recovery pump 41 (in addition, if necessary, the culture fluid delivery pump 33, the washing solution delivery pump 52, the first waste solution pump 62, And the second waste liquid pump 63) to automatically collect the cells.

The controller 111 may control the enzyme solution delivery pump 72, and in this case, the enzyme solution F4 can be delivered to the culture tank 11 based on the user's instruction or the previous setting. In addition, the controller 111 may control the inhibitor delivery pump 82, and in this case, the Rho kinase inhibitor can be delivered to the culture tank 11 based on the user's instruction or the previous setting. In addition, the controller 111 may control the chemical solution delivery pump 92, and in this case, the chemical solution can be delivered to the culture tank 11 based on the user's instruction or the previous setting. Further, the controller 111 may control the liquid supply pump 102 for imaging, and in this case, the measurement sample can be supplied to the imaging unit 101 based on a user's instruction or a prior setting. Furthermore, the controller 111 may control the photographing of the microphotograph by the photographing unit 101, and in this case, the microphotograph of the cell can be automatically acquired based on the user's instruction or the prior setting.

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

Further, in the cell culture apparatus 1, the turbidimeter 15 detects the turbidity of the culture fluid F1 in the culture tank 11, and the controller 111 controls the circulation speed in the above execution procedure. Thereby, even if the cell distribution changes due to a change in cell size or the like in the culture process, the controller 111 can automatically adjust the equilibrium state between the upward flow of the culture fluid F1 and the weight of the cell. For this reason, in the cell culture apparatus 1, homogeneous distribution can be performed while maintaining the cell distribution appropriately.

Moreover, in the cell culture apparatus 1, the turbidimeter 15 measures the turbidity of the culture solution F1 in the culture tank 11 in multiple places of the perpendicular direction. Here, when the number of cells cultured in the culture tank 11 increases, the turbidity of the culture solution F1 increases. In addition, as the size of the cells to be cultured (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 above-mentioned turbidimeter 15, it also becomes possible to monitor these culture conditions.

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

In the cell culture apparatus 1, the pH meter 17 detects the pH of the culture fluid F1, and the controller 111 determines the replacement time of the culture fluid F1 based on the detection result, and notifies the user or the culture fluid F1 is exchanged. Can do In such a cell culture apparatus 1, the controller 111 automatically determines the replacement time of the culture solution F1 and prevents the deterioration of the cultured cells due to the passing of the replacement time.

Further, in the cell culture apparatus 1, the controller 111 can control the culture solution delivery pump 33, the washing solution delivery pump 52, the first waste solution pump 62, and the second waste solution pump 63. For this reason, in the cell culture apparatus 1, operations such as replacement of the culture fluid F1 and passage can be performed automatically according to the determination of the controller 111 or the instruction of the user.

Further, in the cell culture apparatus 1, the controller 111 can control the enzyme solution delivery pump 72, the inhibitor delivery solution pump 82, and the chemical solution delivery pump 92. For this reason, in the cell culture apparatus 1, the enzyme solution, the Rho kinase inhibitor and the drug solution can be automatically supplied to the culture tank 11 at the amount and timing set in advance by the control of the controller 111.

As mentioned above, although one embodiment of this indication was described, this indication is not limited to the above-mentioned embodiment. For example, the culture vessel 11 may have a frusto-conical shape in which the cross-sectional area in the horizontal direction increases from the bottom toward the top. As a result, a velocity gradient is provided to the upflow, and the correlation between the circulation velocity and the cell distribution becomes remarkable.

According to the cell culture apparatus of the present disclosure, cell culture of stem cells and the like can be performed efficiently, and high quality cultured cells can be stably obtained.

DESCRIPTION OF SYMBOLS 1 cell culture apparatus 11 culture tank 12 pump for circulation 13 oxygen supply tank 14 micro bubble pump 15 turbidity meter 16 dissolved oxygen meter 17 pH meter 21 incubator 22 thermometer 31 culture solution storage tank 32 culture solution heating tank 33 culture solution delivery Liquid pump 34 Aseptic connector 35 Refrigeration unit 41 Cell collection pump 42 Aseptic connector 43 Cell collection vial 51 Washing liquid storage tank 52 Washing liquid feeding 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 Enzyme liquid supply pump 73 Aseptic connector 81 Inhibitor storage tank 82 Inhibitor liquid supply pump 83 Aseptic connector 91 Chemical liquid storage tank 92 Chemical liquid supply pump 93 Aseptic connector 101 Imaging unit 102 Imaging liquid delivery pump 103 Buffer tank 111 Controller F1 Culture solution F2 Washing 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 delivery line L7 Warmed Culture solution delivery line L8 Cell recovery line L9 Washing solution delivery line L10 First waste liquid line L11 Second waste solution line L12 Enzyme solution delivery line L13 Inhibitor delivery line L14 Chemical solution delivery line L15 Imaging solution delivery line

Claims

A culture vessel for performing three-dimensional culture of cells suspended in a culture solution, and a culture unit including circulation means for circulating the culture solution so that the culture solution forms an ascending flow in the culture vessel;
A cell distribution detection unit for detecting cell distribution in the culture vessel;
A control unit that controls the circulation speed of the culture fluid by the circulation unit based on the cell distribution detected by the cell distribution detection unit;
A cell culture apparatus comprising:
The cell culture device according to claim 1, wherein the cell distribution detection unit includes turbidity measurement 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 a culture environment in the culture tank. apparatus.
A culture solution supply unit for supplying the culture solution to the culture unit;
A cleaning solution supply unit for supplying a cleaning solution to the culture unit;
A waste liquid recovery unit for collecting waste liquid containing the culture fluid 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 sends the culture solution from the culture solution supply unit to the culture unit, sends the washing solution from the wash solution supply unit to the culture unit, and sends the culture solution from the culture unit to the waste solution recovery unit The cell culture apparatus according to claim 4, wherein each liquid feed of the waste liquid is controlled.
5. The culture solution storage unit according to claim 4, wherein the culture solution supply unit includes culture solution storage means for storing the culture solution at a predetermined temperature, and culture solution heating means for heating the culture solution to a temperature higher than the predetermined temperature. The cell culture apparatus as described in 5.
The apparatus further comprises a liquid amount detecting means for detecting the liquid amount of the culture solution in the culture tank,
The control unit controls transfer of the culture solution from the culture solution supply unit to the culture unit based on the liquid volume detected by the liquid volume detection means. The cell culture apparatus as described in a term.
It further comprises 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 the transfer 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.
It further comprises a dissolved oxygen amount detection means for detecting the dissolved oxygen amount of the culture fluid circulating in the culture unit,
The culture unit further includes an 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 supply amount of oxygen by the oxygen supply unit based on the dissolved oxygen amount detected by the dissolved oxygen amount detection unit. apparatus.
The apparatus further comprises pH detection means for detecting the pH of the culture solution in the culture vessel,
The control unit determines the replacement time of the culture solution based on the pH detected by the pH detection means, and notifies the user or exchanges the culture solution. The cell culture apparatus as described in a term.
It further comprises an inhibitor supply unit for supplying Rho kinase inhibitor to the culture unit,
The control unit controls the supply amount of the Rho kinase inhibitor by the inhibitor supply unit such that the concentration of the Rho kinase inhibitor in the culture solution 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 comprises an inhibitor storage means for storing the Rho kinase inhibitor at a predetermined temperature.
It further comprises 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 transfer of the drug solution from the drug solution supply unit to the culture unit.
The cell culture device according to claim 14, wherein the drug solution supply unit includes a drug solution storage unit for storing the drug solution at a predetermined temperature.
A photographing cell collection unit for collecting a part of the cells from the culture unit;
An imaging unit for acquiring 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 recovery of the cells by the imaging cell recovery unit and acquisition of the photomicrograph by the imaging unit.