WO2023054080A1

WO2023054080A1 - Cell culturing method and cell culturing system

Info

Publication number: WO2023054080A1
Application number: PCT/JP2022/035071
Authority: WO
Inventors: Masatsugu Igarashi; Ichirou HIRAHARA
Original assignee: Terumo Kabushiki Kaisha
Priority date: 2021-09-30
Filing date: 2022-09-21
Publication date: 2023-04-06

Abstract

The present invention relates to a cell culturing method and a cell culturing system (10). The cell culturing method includes a measurement step and a culture determination step. In the measurement step, a glutamic acid concentration of a collected culture medium is measured. In the culture determination step, a determination is made as to whether or not a cell culturing state is in a final stage of a logarithmic growth phase, based on a measured glutamic acid concentration, which is the glutamic acid concentration that was measured. The cell culturing system (10) includes a sampling flow path (98), a biosensor (90), and a culture determination unit (162).

Description

CELL CULTURING METHOD AND CELL CULTURING SYSTEM

The present invention relates to a cell culturing method and a cell culturing system.

In the case of culturing cells, a cell propagation process is divided into three phases, namely, an induction phase, a logarithmic growth phase, and a growth arrest phase. The induction phase is an interval until the cells become adapted to a new culturing environment. The logarithmic growth phase is an interval during which the cells, which have completed the induction phase, increase exponentially. The growth arrest phase is an interval during which cell growth is stopped due to the cell density having reached an upper limit. In culturing of the cells in this manner, the activity of the cells decreases during the growth arrest phase, and the cells eventually die. Therefore, it is desirable to perform collection of the cells at a timing of a final stage of the logarithmic growth phase when the number of cells is large, and further, the activity of the cells is high.

For example, in JP 6824050 B2, a cell culturing method is disclosed in which a glutamine concentration of a culture medium (culture solution) obtained from a bioreactor is measured, and a cell culturing state is determined based on the measured glutamine concentration.

In the conventional technique discussed above, the glutamine concentration of the culture medium is measured. The glutamine concentration of the culture medium progresses with a relatively small amount of change from the logarithmic growth phase until the growth arrest phase. Therefore, based on the glutamine concentration, it is not an easy matter to grasp the timing at which the final stage of the logarithmic growth phase occurs.

The present invention has the object of solving the aforementioned problem.

One aspect of the present invention is characterized by a cell culturing method, comprising a measurement step of measuring a glutamic acid concentration of a culture medium used in performing culturing of the cells, and a culture determination step of determining whether or not a cell culturing state is in a final stage of a logarithmic growth phase, based on a measured glutamic acid concentration, which is the glutamic acid concentration that was measured.

Another aspect of the present invention is characterized by a cell culturing system configured to perform culturing of the cells, the cell culturing system comprising a sampling flow path configured to obtain a culture medium used in performing culturing of the cells, a biosensor installed in the sampling flow path, and configured to measure a glutamic acid concentration of the culture medium flowing through the sampling flow path, and a culture determination unit configured to determine whether or not a cell culturing state is in a final stage of a logarithmic growth phase, based on a measured glutamic acid concentration, which is the glutamic acid concentration that was measured by the biosensor.

According to the present invention, based on the measured glutamic acid concentration, it is possible to accurately grasp the timing at which the cell culturing state is in the final stage of the logarithmic growth phase. Consequently, the cells can be collected at a timing at which the final stage of the logarithmic growth phase occurs. Thus, it is possible to collect a large number of highly active cells.

FIG. 1 is a schematic circuit diagram of a cell culturing system according to an embodiment of the present invention; FIG. 2 is a configuration diagram of a controller shown in FIG. 1; FIG. 3 is a flowchart for explaining a cell culturing method in which the cell culturing system shown in FIG. 1 is used; FIG. 4 is an explanatory diagram showing a culture preparation step; FIG. 5 is an explanatory diagram showing a priming step; FIG. 6 is a flowchart for explaining a culturing step; FIG. 7 is an explanatory diagram showing a sampling step; FIG. 8 is a graph showing changes in a glutamic acid concentration; FIG. 9 is an explanatory diagram showing a stripping step; and FIG. 10 is an explanatory diagram showing a collection step.

As shown in FIG. 1, a cell culturing system 10 according to an embodiment of the present invention cultures (propagates) within a culture medium cells that have been separated from biological tissue. The cells used in the cell culturing system 10 are adherent cells. However, the cells used in the cell culturing system 10 may be planktonic cells. More specifically, as examples of the cells used in the cell culturing system 10, there may be cited ES cells, iPS cells, mesenchymal stem cells, and the like. The cells used in the cell culturing system 10 are not limited to the cell types described above.

The cell culturing system 10 is equipped with a cell culturing device 12, a support device 14, and a controller 16. Liquids containing at least one of a cell solution, a culture medium, a cleaning solution, and a stripping solution flow in the cell culturing device 12.

The cell solution is a solution containing cells. The culture medium is a culture medium for causing the cells to propagate. The culture medium is selected depending on the cells to be cultured. As the culture medium, there may be used, for example, an MEM (Minimum Essential Medium), a Balanced Salt Solution (BSS), an FBS (fetal bovine serum), and MEMalpha with L-alanine-L-glutamine dipeptide (GlutaMAX), and the like. The cleaning solution cleans the interior of the cell culturing device 12. As the cleaning solution, for example, water, a buffer solution, or a physiological saline solution or the like is used. As examples of the buffer solution, there may be cited PBS (Phosphate Buffered Salts) and TBS (Tris-Buffered Saline) or the like. The stripping solution strips the cells from a later-described bioreactor 26 of the cell culturing device 12. As the stripping solution, for example, trypsin or an EDTA solution is used. The culture medium, the cleaning solution, and the stripping solution are not limited to the liquids described above.

The cell culturing device 12 is discarded after being used one time (every time that a predetermined number of cells have been cultured). Stated otherwise, the cell culturing device 12 is a disposable product. The cell culturing device 12 comprises a supply unit 18, a collection container 20, a waste liquid accommodation unit 22, and a culturing body 24.

The supply unit 18 supplies the cell solution, the culture medium, the cleaning solution, and the stripping solution to the culturing body 24. The collection container 20 collects the cells that are cultured in the culturing body 24. The waste liquid accommodation unit 22 accommodates the waste liquid that is generated in the culturing body 24. Each of the collection container 20 and the waste liquid accommodation unit 22, for example, is a medical bag obtained by molding a soft resin material into a bag-like shape. As examples of the soft resin material, there may be cited polyvinyl chloride and polyolefin. However, each of the collection container 20 and the waste liquid accommodation unit 22 may be a tank or the like constituted by a hard resin.

The culturing body 24 includes the bioreactor 26, a culturing circuit 28, a gas exchange unit 30, a sensor unit 32, and a sampling unit 34.

The bioreactor 26 includes a plurality of hollow fiber membranes 36, and a cylindrical housing 38. The plurality of hollow fiber membranes 36 are accommodated inside the housing 38. One end of the respective hollow fiber membranes 36 is fixed to one end of the housing 38. Another end of the respective hollow fiber membranes 36 is fixed to another end of the housing 38.

The respective hollow fiber membranes 36 are made of a polymer material. More specifically, as the material constituting the respective hollow fiber membranes 36, there may be cited polypropylene, polyolefin resin, polysulfone, polyether sulfone, polyacrylonitrile, polytetrafluoroethylene, polystyrene, polymethylmethacrylate, cellulose acetate, cellulose triacetate, regenerated cellulose, and the like. However, the material constituting the respective hollow fiber membranes 36 is not limited to the aforementioned materials.

The bioreactor 26 is provided with a first region 40 and a second region 42. The first region 40 is defined by inner holes of a plurality of hollow fiber membranes 36. The second region 42 is defined by a space between an inner peripheral surface of the housing 38 and outer peripheral surfaces of the plurality of hollow fiber membranes 36. Each of the hollow fiber membranes 36 includes a plurality of non-illustrated pores therein. The first region 40 and the second region 42 communicate with each other through the plurality of pores of the respective hollow fiber membranes 36. The diameter of the pores is of a size that allows small molecules (for example, water, ions, oxygen, lactic acid, etc.) to pass therethrough, while preventing the passage of macromolecules (cells, etc.) therethrough. The diameter of the respective pores, for example, is greater than or equal to 0.005 micrometers and less than or equal to 10 micrometers.

A first inlet port 44, a first outlet port 46, a second inlet port 48, and a second outlet port 50 are installed in the housing 38. The first inlet port 44 is installed at one end of the housing 38. The first inlet port 44 communicates with the first region 40 via an inlet positioned at one end of the plurality of hollow fiber membranes 36. The first outlet port 46 is installed at another end of the housing 38. The first outlet port 46 communicates with the first region 40 via an outlet positioned at the other end of the plurality of hollow fiber membranes 36.

The second inlet port 48 and the second outlet port 50 are installed on an outer peripheral surface of the housing 38. The second inlet port 48 is positioned between a center of the housing 38 and the first inlet port 44 in the longitudinal direction of the housing 38. The second outlet port 50 is positioned between the center of the housing 38 and the first outlet port 46 in the longitudinal direction of the housing 38. Each of the second inlet port 48 and the second outlet port 50 communicates with the second region 42.

The culturing circuit 28 includes flow paths which are extended in a linear shape. More specifically, the culturing circuit 28 includes a plurality of tubes through which the liquids flow. The respective tubes are made of a soft resin material.

The culturing circuit 28 is not limited to the configuration described above. The culturing circuit 28 may include, for example, a sheet member including the flow paths therein through which the liquids flow. The sheet member is constituted by two sheets made of a soft resin material which are stacked on each other in a thickness direction. Locations within the two sheets other than portions thereof that make up the flow paths are joined (fusion bonded) mutually to each other. Within the two sheets, flow path wall parts that make up the flow paths are not joined (fusion bonded) to each other. Within the sheet members, the flow path wall parts preferably bulge outward in a natural state in which liquid is not flowing through the flow paths. Extra portions on both sides of the sheet member in directions intersecting the flow paths may be cut off.

The culturing circuit 28 comprises a first supply flow path 52, a first circulation flow path 54, a second supply flow path 56, a second circulation flow path 58, a collection flow path 60, and a waste liquid flow path 62. One end of the first supply flow path 52 is connected to the supply unit 18. The supply unit 18 supplies the cell solution, the culture medium, the cleaning solution, and the stripping solution one at a time at a predetermined timing to the first supply flow path 52. Another end of the first supply flow path 52 merges with the first circulation flow path 54.

Within the first circulation flow path 54, a first merging section 64, which is a portion to which the first supply flow path 52 is connected, is positioned at an intermediate portion in a direction in which the first circulation flow path 54 extends. One end of the first circulation flow path 54 is connected to the first inlet port 44. Another end of the first circulation flow path 54 is connected to the first outlet port 46. The first circulation flow path 54 communicates with the inner holes (the first region 40) of the plurality of hollow fiber membranes 36.

One end of the second supply flow path 56 is connected to the supply unit 18. The supply unit 18 supplies the culture medium and the cleaning solution one at a time at a predetermined timing to the second supply flow path 56. Another end of the second supply flow path 56 merges with the second circulation flow path 58.

Within the second circulation flow path 58, a second merging section 66, which is a portion to which the second supply flow path 56 is connected, is positioned at an intermediate portion in a direction in which the second circulation flow path 58 extends. One end of the second supply flow path 56 is connected to the second inlet port 48. Another end of the second supply flow path 56 is connected to the second outlet port 50. The second circulation flow path 58 communicates with the space (the second region 42) between the plurality of hollow fiber membranes 36 and the housing 38.

The collection flow path 60 extends from the first circulation flow path 54. Within the first circulation flow path 54, a collection branching section 70, which is a portion to which the collection flow path 60 is connected, is positioned between the first merging section 64 and the first outlet port 46 in the first circulation flow path 54. An extending end of the collection flow path 60 is connected to the collection container 20.

The waste liquid flow path 62 is a flow path for discarding the liquids that flow through the first circulation flow path 54 and the second circulation flow path 58. The waste liquid flow path 62 includes a first waste liquid flow path 72, a second waste liquid flow path 74, and a third waste liquid flow path 76. The first waste liquid flow path 72 extends from the first circulation flow path 54. Within the first circulation flow path 54, a first branching section 78, which is a portion to which the first waste liquid flow path 72 is connected, is positioned between the first outlet port 46 and the collection branching section 70 in the first circulation flow path 54.

The second waste liquid flow path 74 extends from the second circulation flow path 58. Within the second circulation flow path 58, a second branching section 80, which is a portion to which the second waste liquid flow path 74 is connected, is positioned between the second merging section 66 and the second outlet port 50 in the second circulation flow path 58.

An extending end of the first waste liquid flow path 72 and an extending end of the second waste liquid flow path 74 are connected to one end of the third waste liquid flow path 76. Stated otherwise, the one end of the third waste liquid flow path 76 is an intermediate merging section 82 where the extending end of the first waste liquid flow path 72 and the extending end of the second waste liquid flow path 74 merge. Another end of the third waste liquid flow path 76 is connected to the waste liquid accommodation unit 22.

The gas exchange unit 30 is installed in the second circulation flow path 58 between the second merging section 66 and the second inlet port 48. The gas exchange unit 30 allows a gas having predetermined components to pass through the liquid (culture medium) that flows through the second circulation flow path 58. The gas used in the gas exchange unit 30 includes, for example, components therein that are similar to those of natural air. Stated otherwise, the gas contains nitrogen, oxygen, and carbon dioxide. More specifically, the gas contains, for example, 75% nitrogen, 20% oxygen, and 5% carbon dioxide by volume.

The sensor unit 32 is installed in the third waste liquid flow path 76. The sensor unit 32 is an integrally molded product. The sensor unit 32 includes a gas sensor 84 and a pH sensor 86. The gas sensor 84 measures a gas concentration of the liquid flowing through the third waste liquid flow path 76. More specifically, the gas sensor 84 includes an oxygen sensor and a carbon dioxide sensor. The oxygen sensor measures an oxygen concentration of the liquid flowing through the third waste liquid flow path 76. The carbon dioxide sensor measures a carbon dioxide concentration of the liquid flowing through the third waste liquid flow path 76. The pH sensor 86 measures a pH (hydrogen ion index) of the liquid flowing through the third waste liquid flow path 76.

The sampling unit 34 is connected to a portion within the third waste liquid flow path 76 between the sensor unit 32 and the waste liquid accommodation unit 22. The sampling unit 34 is equipped with a measurement circuit 88, a biosensor 90, and a cleaning solution accommodation unit 92.

The measurement circuit 88 includes flow paths which are extended in a linear shape. The measurement circuit 88 includes a plurality of tubes through which the liquids flow. The respective tubes are made of a soft resin material. However, the measurement circuit 88 may include, for example, a sheet member including the flow paths therein through which the liquids flow. The sheet member is configured in the same manner as the sheet member constituting the aforementioned culturing circuit 28. The measurement circuit 88 includes a sampling flow path 98 and an introduction flow path 100.

The sampling flow path 98 has a first end 106 and a second end 108. The first end 106 is one end of the sampling flow path 98. The second end 108 is another end of the sampling flow path 98. Each of the first end 106 and the second end 108 is connected to the third waste liquid flow path 76. Within the third waste liquid flow path 76, a third branching section 110, which is a portion to which the first end 106 of the sampling flow path 98 is connected, is positioned between the sensor unit 32 and the waste liquid accommodation unit 22 in the third waste liquid flow path 76. Within the third waste liquid flow path 76, a third merging section 112, which is a portion to which the second end 108 of the sampling flow path 98 is connected, is positioned between the third branching section 110 and the waste liquid accommodation unit 22 in the third waste liquid flow path 76.

One end of the introduction flow path 100 is connected to the cleaning solution accommodation unit 92. Another end of the introduction flow path 100 is connected to the sampling flow path 98. In the sampling flow path 98, a fourth merging section 114, which is a portion to which the introduction flow path 100 is connected, is positioned at an intermediate portion in a direction in which the sampling flow path 98 extends.

The biosensor 90 is installed in the sampling flow path 98 in a portion thereof between the fourth merging section 114 and the second end 108. The biosensor 90 is an integrally molded enzyme sensor. The biosensor 90 includes, for example, a glucose sensor 120, a lactic acid sensor 122, and a glutamic acid sensor 123. Each of the glucose sensor 120, the lactic acid sensor 122, and the glutamic acid sensor 123 is placed in contact with the liquid flowing through the sampling flow path 98. The glucose sensor 120 measures a glucose concentration of the liquid flowing through the sampling flow path 98. The lactic acid sensor 122 measures a lactic acid concentration of the liquid flowing through the sampling flow path 98. The glutamic acid sensor 123 measures a glutamic acid concentration of the liquid flowing through the sampling flow path 98.

The biosensor 90 is not limited to being an enzyme sensor, and may also be a non-enzyme sensor.

The cleaning solution accommodation unit 92 is a medical bag, in the same manner as the waste liquid accommodation unit 22 described above. However, the cleaning solution accommodation unit 92 may be a tank or the like constituted by a hard resin. The cleaning solution is accommodated in the cleaning solution accommodation unit 92. The cleaning solution is a solution for cleaning the biosensor 90. As the cleaning solution, for example, a solution is used which is similar to the cleaning solution supplied from the aforementioned supply unit 18 to the culturing circuit 28.

The cell culturing device 12 is set on the support device 14. The support device 14 includes a cassette that supports the cell culturing device 12. The support device 14 is a reusable product that is capable of being used a plurality of times.

The support device 14 is equipped with a plurality of pumps 124 and a plurality of clamps 126. Each of the plurality of pumps 124 imparts a flowing force to the liquids inside the flow paths by squeezing the flow path wall parts of the cell culturing device 12. More specifically, each of the plurality of pumps 124 includes a non-illustrated pressing member. The pressing member includes, for example, a rotating member, and a plurality of pressing rollers. The plurality of pressing rollers are attached to an outer circumferential portion of the rotating member. The plurality of pressing rollers are arranged at intervals with spaces left therebetween in the circumferential direction of the rotating member. Each of the pressing rollers rubs against the outer surfaces of the flow path wall parts of the cell culturing device 12.

As shown in FIG. 2, the supply unit 18, the gas exchange unit 30, the sensor unit 32, the biosensor 90, the plurality of pumps 124, and the plurality of clamps 126 are connected wirelessly or over wires to the controller 16. The controller 16 is equipped with a computation unit 154 (processing unit), a storage unit 156, and a notification unit 157. The computation unit 154 is constituted, for example, by a processor (processing circuit) such as a CPU (Central Processing Unit), a GPU (Graphics Processing Unit), or the like.

The computation unit 154 includes a pump control unit 158, a clamp control unit 160, a culture determination unit 162, a sensor control unit 164, a notification control unit 166, and a gas exchange control unit 168. By executing a program stored in the storage unit 156, the computation unit 154 realizes the pump control unit 158, the clamp control unit 160, the culture determination unit 162, the sensor control unit 164, the notification control unit 166, and the gas exchange control unit 168.

The computation unit 154 may realize at least a portion of the pump control unit 158, the clamp control unit 160, the culture determination unit 162, the sensor control unit 164, the notification control unit 166, and the gas exchange control unit 168 by way of an integrated circuit. As an example of such an integrated circuit, there may be cited an ASIC (Application Specific Integrated Circuit), an FPGA (Field-Programmable Gate Array), and the like.

The storage unit 156 includes a volatile memory and a non-volatile memory. As an example of the volatile memory, there may be cited a RAM (Random Access Memory) or the like. Such a volatile memory is used as a working memory of the processor, and data and the like required for carrying out processing or calculations are temporarily stored therein. As an example of the non-volatile memory, there may be cited a ROM (Read Only Memory), a flash memory, or the like. Such a non-volatile memory is used as a storage memory. Programs, tables, maps, etc., are stored in the non-volatile memory. At least a portion of the storage unit 156 may be incorporated in the processor or the integrated circuit as were described above.

The pump control unit 158 controls the plurality of pumps 124. The clamp control unit 160 controls the plurality of clamps 126. The culture determination unit 162 carries out a predetermined determination process. The sensor control unit 164 controls the biosensor 90 and the sensor unit 32. The notification control unit 166 controls the notification unit 157 based on a determination result of the culture determination unit 162. The gas exchange control unit 168 controls the gas exchange unit 30.

The notification unit 157, for example, generates at least one of sound, light, and vibrations. The notification unit 157 may include a display unit (display) for displaying characters, figures, symbols, or the like. In the present embodiment, the notification unit 157 is incorporated in the controller 16. However, the notification unit 157 may be incorporated in a mobile device carried by a user. In this case, the controller 16 is wirelessly connected to the mobile device.

Next, a description will be given concerning a cell culturing method in which the cell culturing system 10 is used.

As shown in FIG. 3, the cell culturing method includes a mounting step, a culture preparation step, a priming step, a culturing step, a notification step, a stripping step, and a collection step.

In the mounting step (step S1), the cell culturing device 12 is set on the support device 14.

Next, the culture preparation step (step S2) is carried out. In the culture preparation step, as shown in FIG. 4, the clamp control unit 160 controls the plurality of clamps 126, and together therewith, the pump control unit 158 controls the plurality of pumps 124, thereby supplying the cleaning solution from the supply unit 18 to the first supply flow path 52 and the second supply flow path 56. The cleaning solution supplied to the first supply flow path 52 is guided to the waste liquid accommodation unit 22 via the first circulation flow path 54 and the waste liquid flow path 62. The cleaning solution supplied to the second supply flow path 56 is guided to the waste liquid accommodation unit 22 via the second circulation flow path 58 and the waste liquid flow path 62. In accordance therewith, the culturing circuit 28 and the bioreactor 26 are filled with the cleaning solution.

Thereafter, by supplying the culture medium from the supply unit 18 to the first supply flow path 52 and the second supply flow path 56, the culturing circuit 28 and the bioreactor 26 are filled with the culture medium. Stated otherwise, the cleaning solution existing in the culturing circuit 28 and the bioreactor 26 is replaced by the culture medium.

Next, the priming step (step S3 of FIG. 3) is carried out. In the priming step, as shown in FIG. 5, the cleaning solution flows from the cleaning solution accommodation unit 92 to the waste liquid accommodation unit 22 via the introduction flow path 100, the sampling flow path 98, and the third waste liquid flow path 76.

After the priming step, the culturing step (step S4 of FIG. 3) is carried out. In the culturing step, as shown in FIG. 6, a seeding step (step S5) is performed. In the seeding step, by the cell solution being supplied from the supply unit 18 to the first supply flow path 52, the cells within the cell solution are adhered to the inner surfaces of each of the hollow fiber membranes 36 of the bioreactor 26.

Next, cell culturing is initiated (step S6). Specifically, the culture medium is supplied from the supply unit 18 to the culturing circuit 28. At this time, the gas exchange control unit 168 controls the gas exchange unit 30 to thereby carry out gas exchange on the culture medium flowing through the second circulation flow path 58. In the bioreactor 26, the culture medium in the first region 40 and the culture medium in the second region 42 are exchanged through the pores of each of the hollow fiber membranes 36. A portion of the culture medium inside the first circulation flow path 54 is guided to the third waste liquid flow path 76 via the first waste liquid flow path 72. A portion of the culture medium inside the second circulation flow path 58 is guided to the third waste liquid flow path 76 via the second waste liquid flow path 74.

Next, the sampling step (step S7) is carried out. The sampling step is carried out at an appropriate timing after the start of cell culturing. In the sampling step, for example, as shown in FIG. 7, the culture medium that was guided to the third waste liquid flow path 76 flows from the third branching section 110 into the sampling flow path 98. The culture medium that has flowed into the sampling flow path 98 flows into the waste liquid accommodation unit 22 via the biosensor 90, the third merging section 112, and the third waste liquid flow path 76.

Thereafter, as shown in FIG. 6, the measurement step (step S8) is carried out. In the measurement step, the sensor control unit 164 controls the biosensor 90, and measures the concentration of a predetermined component of the culture medium. More specifically, the sensor control unit 164 controls the glucose sensor 120 to measure the glucose concentration of the culture medium. The glucose sensor 120 transmits a measured glucose concentration, which is the glucose concentration that was measured, to the controller 16.

The sensor control unit 164 controls the lactic acid sensor 122 to measure the lactic acid concentration of the culture medium. The lactic acid sensor 122 transmits a measured lactic acid concentration, which is the lactic acid concentration of the culture medium that was measured, to the controller 16.

The sensor control unit 164 controls the glutamic acid sensor 123 to measure the glutamic acid concentration of the culture medium. The glutamic acid sensor 123 transmits a measured glutamic acid concentration, which is the glutamic acid concentration of the culture medium that was measured, to the controller 16. The measured glucose concentration, the measured lactic acid concentration, and the measured glutamic acid concentration are stored in the storage unit 156.

Thereafter, the biosensor 90 is cleaned (step S9). More specifically, the cleaning solution flows from the cleaning solution accommodation unit 92 to the waste liquid accommodation unit 22 via the introduction flow path 100, the sampling flow path 98, and the third waste liquid flow path 76. In accordance therewith, the biosensor 90 is subjected to cleaning by the cleaning solution.

Incidentally, in the present embodiment, the cell propagation process is divided into three phases, namely, an induction phase, a logarithmic growth phase, and a growth arrest phase. In such a case, in order to collect as large a number of highly active cells as possible, it is essential to grasp the timing at which the final stage of the logarithmic growth phase (immediately prior to the growth arrest phase) occurs.

Therefore, as shown in FIG. 6, the culture determination unit 162 determines whether or not the cell culturing state is in the final stage of the logarithmic growth phase (step S10: culture determination step). The present inventors have discovered that the glutamic acid concentration in the culture medium changes relatively significantly in the final stage of the logarithmic growth phase. Therefore, in step S10, based on the measured glutamic acid concentration, the culture determination unit 162 determines whether or not the cell culturing state is in the final stage of the logarithmic growth phase.

According to the present embodiment, the culture determination unit 162 determines whether or not the measured glutamic acid concentration exceeds the threshold value Ma (see FIG. 8). In the case that the measured glutamic acid concentration has exceeded the threshold value Ma, the culture determination unit 162 determines that the cell culturing state is in the final stage of the logarithmic growth phase. In the case that the measured glutamic acid concentration is less than or equal to the threshold value Ma, the culture determination unit 162 determines that the cell culturing state is not in the final stage of the logarithmic growth phase.

The threshold value Ma is appropriately set according to the type of cells to be cultured, the type of culture medium, and the like. The threshold value Ma is stored in the storage unit 156. Hereinafter, a description will be given concerning a method for determining the threshold value Ma that is stored in the storage unit 156. FIG. 8 is a graph showing changes in average values of the measured glutamic acid concentration of a plurality of cell samples. The graph of FIG. 8 is created at a stage prior to carrying out the cell culturing method.

In the case of creating such a graph, first, a plurality of cell samples containing cells, which are of the same type as the cells of the present embodiment, are prepared. Subsequently, during culturing of the plurality of cell samples, the glutamic acid concentration of each of the cell samples is measured at each of predetermined time intervals. In addition, at each of respective measurement times, average sample concentrations, which are average values of a plurality of measured glutamic acid concentrations, are calculated. Thereafter, by mutually connecting the sample average concentrations at each of the measurement times, the graph shown in FIG. 8 is obtained.

In FIG. 8, the average sample concentration does not change significantly from the induction phase of cell propagation until just prior to the final stage of the logarithmic growth phase. However, the average sample concentration varies significantly in the final stage of the logarithmic growth phase of cell propagation.

More specifically, in the final stage of the logarithmic growth phase, the average sample concentration sharply increases from time Ta to time Tb, and thereafter, sharply decreases from time Tb to time Tc. The average sample concentration shows a peak value Mp at time Tb. Moreover, the period from time Ta until time Tc corresponds to the final stage of the logarithmic growth phase in the cell propagation process.

In such a case, the threshold value Ma, for example, is preferably set to be higher than a highest value Mu of the measured glutamic acid concentration of the plurality of cell samples at a stage prior to the final stage of the logarithmic growth phase, and to be lower than a lowest value Ml of the measured glutamic acid concentration of the plurality of cell samples at time Tb. In accordance therewith, based on the measured glutamic acid concentration, it is possible to accurately detect the final stage of the logarithmic growth phase of the cells.

Further, in step S10, the culture determination unit 162 may determine whether or not an absolute value of a rate of change in the measured glutamic acid concentration exceeds a reference rate of change. In the case that the absolute value of the rate of change in the measured glutamic acid concentration has exceeded the reference rate of change, the culture determination unit 162 determines that the cell culturing state is in the final stage of the logarithmic growth phase. In the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, the culture determination unit 162 determines that the cell culturing state is not in the final stage of the logarithmic growth phase.

The rate of change in the measured glutamic acid concentration is an amount of change per unit time in the measured glutamic acid concentration. The unit time, for example, is a time interval during which the glutamic acid concentration in the culture medium is measured. The unit time (the time interval during which the glutamic acid concentration is measured) may be one hour, and may also be one day (24 hours). The reference rate of change is appropriately set depending on the type of cells to be cultured, the type of culture medium, and the like. The reference rate of change is stored in the storage unit 156.

The reference rate of change is set, for example, based on the data (the graph) shown in FIG. 8. As shown in FIG. 8, the reference rate of change, for example, is preferably set to be greater than an absolute value of a maximum amount of change “Delta Ca” in the average sample concentration per unit time at a stage prior to the final stage of the logarithmic growth phase, and to be less than an absolute value of an amount of change “Delta Cb” in the average sample concentration from time Ta until time Tb. Moreover, the reference rate of change may be set to a value that is greater than the absolute value of the maximum amount of change “Delta Ca”, and less than the absolute value of the amount of change “Delta Cc” in the average sample concentration from time Tb until time Tc. In accordance therewith, based on the measured glutamic acid concentration, it is possible to accurately detect the final stage of the logarithmic growth phase of the cells.

Furthermore, in step S10, immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds the first reference rate of change, in the case that the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds a second reference rate of change, the culture determination unit 162 may determine that the cell culturing state is in the final stage of the logarithmic growth phase. The first reference rate of change and the second reference rate of change are set in the same manner as the reference rate of change discussed above. Moreover, the first reference rate of change may be the same value as the second reference rate of change, may be greater than the second reference rate of change, or may be less than the second reference rate of change. In the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, the culture determination unit 162 determines that the cell culturing state is not in the final stage of the logarithmic growth phase. In accordance therewith, based on the measured glutamic acid concentration, it is possible to more accurately detect the final stage of the logarithmic growth phase of the cells.

As shown in FIG. 6, in the case it is determined by the culture determination unit 162 that the cell culturing state is not in the final stage of the logarithmic growth phase (step S10: NO), the process returns to step S7.

In the case it is determined by the culture determination unit 162 that the cell culturing state is in the final stage of the logarithmic growth phase (step S10: YES), as shown in FIG. 3, the notification step (step S11) is carried out.

In the notification step, the notification control unit 166 controls the notification unit 157 to issue a notification to the effect that the cell culturing state is in the final stage of the logarithmic growth phase. More specifically, if the notification unit 157 includes a speaker, the notification control unit 166, for example, controls the notification unit 157 to generate a voice saying "It is time to collect the cells." from the speaker. Further, if the notification unit 157 includes a light emitting unit such as a lamp or the like, the notification control unit 166 may control the notification unit 157 to cause the light emitting unit to emit light. Furthermore, if the notification unit 157 includes a vibration generating unit, the notification control unit 166 may control the notification unit 157 to cause vibrations to be generated from the vibration generating unit. Further, if the notification unit 157 includes a display unit (display), the notification control unit 166 may control the notification unit 157 to cause characters such as "It is time to collect cells." or the like to be displayed on the display unit. In accordance therewith, the user can be notified that the cell culturing state is in the final stage of the logarithmic growth phase (that it is time to collect the cells). Therefore, the user can efficiently undertake preparations for a process (for example, subculturing) after recovery of the cells.

After the notification step, the stripping step (step S12) is carried out. In the stripping step, as shown in FIG. 9, the clamp control unit 160 controls the plurality of clamps 126, and together therewith, the pump control unit 158 controls the plurality of pumps 124, thereby supplying the stripping solution from the supply unit 18 to the first supply flow path 52. Upon doing so, the stripping solution is guided from the supply unit 18 to the bioreactor 26 via the first supply flow path 52 and the first circulation flow path 54. In the bioreactor 26, the stripping solution strips the cultured cells from the inner surfaces of each of the hollow fiber membranes 36.

Thereafter, as shown in FIG. 3, the collection step (step S13) is carried out. In the collection step, as shown in FIG. 10, the clamp control unit 160 controls the plurality of clamps 126, and together therewith, the pump control unit 158 controls the plurality of pumps 124, whereby the liquid containing the cells inside the first circulation flow path 54 is guided to the collection container 20 via the collection flow path 60. In accordance therewith, the flow of operations of the cell culturing method comes to an end.

The present embodiment exhibits the following advantageous effects.

According to the present embodiment, based on the measured glutamic acid concentration, it is possible to accurately grasp the timing at which the cell culturing state is in the final stage of the logarithmic growth phase. Consequently, the cells can be collected in the final stage of the logarithmic growth phase. Thus, it is possible to collect a large number of highly active cells.

In the culture determination step, in the case that the measured glutamic acid concentration has exceeded the threshold value Ma, the culture determination unit 162 determines that the cell culturing state is in the final stage of the logarithmic growth phase. Further, in the culture determination step, in the case that the measured glutamic acid concentration is less than or equal to the threshold value Ma, the culture determination unit 162 determines that the cell culturing state is not in the final stage of the logarithmic growth phase.

Further, in the present embodiment, in the culture determination step, in the case that the absolute value of the rate of change in the measured glutamic acid concentration has exceeded the reference rate of change, the culture determination unit 162 may determine that the cell culturing state is in the final stage of the logarithmic growth phase. In this case, in the culture determination step, in the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, the culture determination unit 162 determines that the cell culturing state is not in the final stage of the logarithmic growth phase.

Furthermore, in the present embodiment, in the culture determination step, immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds the first reference rate of change, in the case that the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds a second reference rate of change, a determination may be made that the cell culturing state is in the final stage of the logarithmic growth phase. In this case, in the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, a determination is made that the cell culturing state is not in the final stage of the logarithmic growth phase.

In this manner, since the culture determination unit 162 uses the threshold value Ma or the reference rate of change (including the first reference rate of change and the second reference rate of change), it is possible to easily determine whether or not the cell culturing state has reached the final stage of the logarithmic growth phase.

In the notification step, the notification control unit 166 controls the notification unit 157 to thereby cause a notification of predetermined information to be issued from the notification unit 157, in the case of it having been determined by the culture determination unit 162 that the cell culturing state is in the final stage of the logarithmic growth phase.

In accordance therewith, the user can be easily notified that the cell culturing state is in the final stage of the logarithmic growth phase. Therefore, the user can efficiently undertake preparations for collecting the cells and the like.

In the cell culturing method, the cells are cultured inside the bioreactor 26. In accordance therewith, the cells can be efficiently cultured.

In the collection step, the clamp control unit 160 controls the collection clamps 126, thereby causing the collection flow path 60 to open. Further, in the collection step, the pump control unit 158 controls the pumps 124, and guides the cells from the bioreactor 26 into the collection container 20 via the culturing circuit 28.

In accordance therewith, the cells that were cultured in the bioreactor 26 can be automatically accommodated in the collection container 20.

The present invention is not limited to the embodiment described above, and various alternative configurations could be adopted therein without deviating from the essence and gist of the present invention. The sampling unit 34 may be connected to either the first waste liquid flow path 72 or the second waste liquid flow path 74. Further, the sampling unit 34 may be connected to a portion between the second outlet port 50 and the second branching section 80 in the second circulation flow path 58. In the cell culturing system 10, instead of the sampling unit 34, a sampling port may be installed to which a syringe or the like is capable of being connected. In the cell culturing system 10, the notification unit 157 and the notification control unit 166 need not necessarily be provided.

In the present invention, in the cell culturing method and the cell culturing system, instead of culturing the cells inside the bioreactor 26, the cells may be cultured using a flask or a Petri dish. Further, in the cell culturing method, the glutamic acid concentration of the culture medium inside the cell culturing container, such as the bioreactor 26, the flask, the Petri dish, or the like, may be directly measured without collecting the cells. In this case, in the cell culturing method, the sampling step is not included.

According to the present embodiment, the following inventive content is disclosed.

According to the above-described embodiment, the cell culturing method is disclosed, comprising the measurement step of measuring the glutamic acid concentration of the culture medium used in performing culturing of the cells, and the culture determination step of determining whether or not the cell culturing state is in the final stage of the logarithmic growth phase, based on the measured glutamic acid concentration, which is the glutamic acid concentration that was measured.

In the above-described cell culturing method, in the culture determination step, in the case that the glutamic acid concentration has exceeded the threshold value (Ma), a determination may be made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the glutamic acid concentration is less than or equal to the threshold value, a determination may be made that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing method, in the culture determination step, in the case that the absolute value of the rate of change in the measured glutamic acid concentration has exceeded the reference rate of change, a determination may be made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, a determination may be made that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing method, in the culture determination step, in the case that immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds the first reference rate of change, the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds the second reference rate of change, a determination may be made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, a determination may be made that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing method, there may further be included the notification step of issuing a notification of predetermined information, in the case that a determination is made in the culture determination step that the cell culturing state is in the final stage of the logarithmic growth phase.

In the above-described cell culturing method, the cells may be cultured inside the bioreactor (26).

In the above-described cell culturing method, there may further be included the collection step of collecting the cells inside the bioreactor, in the case that a determination is made in the culture determination step that the cell culturing state is in the final stage of the logarithmic growth phase.

In the above-described cell culturing method, in the collection step, the cells inside the bioreactor may be guided to the collection container (20) by the pumps (124) that are installed in the flow path connected to the bioreactor.

In the above-described cell culturing method, there may further be included the sampling step of obtaining the culture medium.

According to the above-described embodiment, the cell culturing system (10) that performs culturing of the cells is disclosed, the cell culturing system comprising the sampling flow path (98) in which the culture medium used in performing culturing of the cells is obtained, the biosensor (90) installed in the sampling flow path, and which measures the glutamic acid concentration of the culture medium flowing through the sampling flow path, and the culture determination unit (162) which determines whether or not the cell culturing state is in the final stage of the logarithmic growth phase, based on the measured glutamic acid concentration, which is the glutamic acid concentration that was measured.

In the above-described cell culturing system, in the case that the glutamic acid concentration has exceeded the threshold value, the culture determination unit may determine that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the glutamic acid concentration is less than or equal to the threshold value, the culture determination unit may determine that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing system, in the case that the absolute value of the rate of change in the measured glutamic acid concentration has exceeded the reference rate of change, the culture determination unit may determine that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, the culture determination unit may determine that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing system, in the case that immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds the first reference rate of change, the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds the second reference rate of change, the culture determination unit may determine that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, the culture determination unit may determine that the cell culturing state is not in the final stage of the logarithmic growth phase.

In the above-described cell culturing system, there may further be provided the notification control unit (166) that controls the notification unit (157) to issue a predetermined notification from the notification unit, in the case that a determination is made by the culture determination unit that the cell culturing state is in the final stage of the logarithmic growth phase.

In the above-described cell culturing system, there may further be provided the bioreactor in which the cells are cultured.

In the above-described cell culturing system, there may further be provided the culturing circuit (28) connected to the bioreactor, the collection container connected to the culturing circuit, the pump which guides the cells from the bioreactor to the collection container via the culturing circuit by imparting fluidity to the liquid inside the culturing circuit, the clamps (126) that open and close the collection flow path (60) forming at least a portion of the pathway from the bioreactor to the collection container in the culturing circuit, the pump control unit (158) that controls the pumps, and the clamp control unit (160) that controls the clamps, wherein, in the case it is determined by the culture determination unit that the cell culturing state is in the final stage of the logarithmic growth phase, the clamp control unit may open the clamps to thereby cause the collection flow path to open, and together therewith, the pump control unit may control the pumps to guide the cells from the bioreactor to the collection container via the culturing circuit.

Description of Reference Numerals

10 … cell culturing system
26 … bioreactor
60 … collection flow path
90 … biosensor
98 … sampling flow path
124 … pumps
126 … clamps
157 … notification unit
158 … pump control unit
160 … clamp control unit
162 … culture determination unit
166 … notification control unit
Ma … threshold value

Claims

A cell culturing method, comprising:
a measurement step of measuring a glutamic acid concentration of a culture medium used in performing culturing of the cells; and
a culture determination step of determining whether or not a cell culturing state is in a final stage of a logarithmic growth phase, based on a measured glutamic acid concentration, which is the glutamic acid concentration that was measured.
The cell culturing method according to claim 1, wherein, in the culture determination step, in the case that the glutamic acid concentration has exceeded a threshold value, a determination is made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the glutamic acid concentration is less than or equal to the threshold value, a determination is made that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing method according to claim 1, wherein, in the culture determination step, in the case that an absolute value of a rate of change in the measured glutamic acid concentration has exceeded a reference rate of change, a determination is made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, a determination is made that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing method according to claim 1, wherein, in the culture determination step, in the case that immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds a first reference rate of change, the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds a second reference rate of change, a determination is made that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, a determination is made that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing method according to any one of claims 1 to 4, further comprising a notification step of issuing a notification of predetermined information, in the case that a determination is made in the culture determination step that the cell culturing state is in the final stage of the logarithmic growth phase.
The cell culturing method according to any one of claims 1 to 5, wherein the cells are cultured inside a bioreactor.
The cell culturing method according to claim 6, further comprising a collection step of collecting the cells inside the bioreactor, in the case that a determination is made in the culture determination step that the cell culturing state is in the final stage of the logarithmic growth phase.
The cell culturing method according to claim 7, wherein, in the collection step, the cells inside the bioreactor are guided to a collection container by a pump installed in a flow path connected to the bioreactor.
The cell culturing method according to any one of claims 1 to 8, further comprising a sampling step of obtaining the culture medium.
A cell culturing system configured to perform culturing of the cells, the cell culturing system comprising:
a sampling flow path configured to obtain a culture medium used in performing culturing of the cells;
a biosensor installed in the sampling flow path, and configured to measure a glutamic acid concentration of the culture medium flowing through the sampling flow path; and
a culture determination unit configured to determine whether or not a cell culturing state is in a final stage of a logarithmic growth phase, based on a measured glutamic acid concentration, which is the glutamic acid concentration that was measured.
The cell culturing system according to claim 10, wherein, in the case that the glutamic acid concentration has exceeded a threshold value, the culture determination unit determines that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the glutamic acid concentration is less than or equal to the threshold value, the culture determination unit determines that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing system according to claim 10, wherein, in the case that an absolute value of a rate of change in the measured glutamic acid concentration has exceeded a reference rate of change, the culture determination unit determines that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the absolute value of the rate of change in the measured glutamic acid concentration is less than or equal to the reference rate of change, the culture determination unit determines that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing system according to claim 10, wherein in the case that immediately after the measured glutamic acid concentration has risen so that the rate of change in the measured glutamic acid concentration exceeds a first reference rate of change, the measured glutamic acid concentration has descended so that the absolute value of the rate of change in the measured glutamic acid concentration exceeds a second reference rate of change, the culture determination unit determines that the cell culturing state is in the final stage of the logarithmic growth phase, whereas, in the case that the rate of change is less than or equal to the first reference rate of change when the measured glutamic acid concentration rises, or is less than or equal to the absolute value of the second rate of change when the measured glutamic acid concentration descends, the culture determination unit determines that the cell culturing state is not in the final stage of the logarithmic growth phase.
The cell culturing system according to any one of claims 10 to 13, further comprising a notification control unit configured to control a notification unit to issue a predetermined notification from the notification unit, in the case that a determination is made by the culture determination unit that the cell culturing state is in the final stage of the logarithmic growth phase.
The cell culturing system according to any one of claims 10 to 14, further comprising a bioreactor configured to culture the cells.
The cell culturing system according to claim 15, further comprising:
a culturing circuit connected to the bioreactor;
a collection container connected to the culturing circuit;
a pump configured to guide the cells from the bioreactor to the collection container via the culturing circuit by imparting fluidity to a liquid inside the culturing circuit;
a clamp configured to open and close a collection flow path forming at least a portion of a pathway from the bioreactor to the collection container in the culturing circuit;
a pump control unit configured to control the pump; and
a clamp control unit configured to control the clamp;
wherein, in the case it is determined by the culture determination unit that the cell culturing state is in the final stage of the logarithmic growth phase, the clamp control unit opens the clamp to thereby cause the collection flow path to open, together with the pump control unit controlling the pump to guide the cells from the bioreactor to the collection container via the culturing circuit.