JP6795380B2 - Culture solution regulator - Google Patents

Description

The present invention relates to a culture solution adjusting device for adjusting the properties of the culture solution.

Conventionally, some devices of this type include a dissolved oxygen concentration measuring device, a dissolved carbon dioxide concentration measuring device, and a pH measuring device (see Patent Document 1). The ones described in Patent Document 1 are based on the values measured by these measuring instruments, the amount of air mixed gas supplied to the gas exchange means, the amount of oxygen-containing bubbles supplied from the aeration means, and the amount of oxygen-containing bubbles supplied from the gas supply device. It is said that it will control the amount of gas supplied.

Japanese Unexamined Patent Publication No. 2014-18174

However, in the case described in Patent Document 1, it is unclear how the gas exchange means, the aeration means, and the gas supply device are coordinated and controlled to adjust the properties of the culture solution, and there is still room for improvement. It is supposed to leave.

The present invention has been made to solve these problems, and a main object of the present invention is to provide a culture solution adjusting device capable of easily adjusting the properties of the culture solution.

The first means for solving the above problems is
A culture solution adjusting device that adjusts the properties of a culture solution that is a pH equilibrium solution.
The first flow rate adjusting unit that adjusts the flow rate of oxygen supplied at a predetermined pressure,
A second flow rate adjusting unit that adjusts the flow rate of carbon dioxide supplied at the predetermined pressure, and
A third flow rate adjusting unit that adjusts the flow rate of the inert gas supplied at the predetermined pressure, and
A mixing unit that mixes the oxygen, the carbon dioxide, and the inert gas whose flow rates have been adjusted by the first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate adjusting unit, respectively.
A storage unit to which the mixed gas flowing out from the mixing unit is supplied and stores the culture solution,
An oxygen concentration sensor that detects the dissolved oxygen concentration of the culture solution and
A pH sensor that detects the pH of the culture solution and
The flow rate of the oxygen is set based on the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, and based on the pH detected by the pH sensor and the target value of the pH. A flow rate setting unit that sets the flow rate of the carbon dioxide and sets the flow rate of the inert gas by subtracting the set oxygen flow rate and the set carbon dioxide flow rate from the predetermined flow rate.
The first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate so as to be the flow rate of the oxygen, the flow rate of carbon dioxide, and the flow rate of the inert gas set by the flow rate setting unit. The flow control unit that controls the adjustment unit and
To be equipped.

According to the above configuration, the flow rate of oxygen supplied at a predetermined pressure is adjusted by the first flow rate adjusting unit, and the flow rate of carbon dioxide supplied at a predetermined pressure is adjusted by the second flow rate adjusting unit and supplied at a predetermined pressure. The flow rate of the inert gas is adjusted by the third flow rate adjusting unit. Oxygen, carbon dioxide, and an inert gas whose flow rates have been adjusted by the first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate adjusting unit are mixed in the mixing unit to form a mixed gas. Then, the mixed gas flowing out from the mixing section is supplied to the culture solution stored in the storage section.

Here, the dissolved oxygen concentration of the culture solution is detected by the oxygen concentration sensor, and the pH of the culture solution is detected by the pH sensor. The dissolved oxygen concentration of the culture solution changes according to the oxygen partial pressure of the mixed gas, and the pH of the culture solution changes according to the carbon dioxide partial pressure of the mixed gas. If the pressure of each gas is equal at a predetermined pressure and the total flow rate of the mixed gas is constant, the partial pressure of each gas is proportional to the flow rate of each gas.

In this regard, the flow rate setting unit sets the flow rate of oxygen based on the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, and sets the pH and the target value of pH detected by the pH sensor. Based on this, the flow rate of carbon dioxide is set, and the flow rate of the inert gas is set by subtracting the set flow rate of oxygen and the set flow rate of carbon dioxide from the predetermined flow rate. Therefore, the flow rate of oxygen for setting the dissolved oxygen concentration to the target value, the flow rate of carbon dioxide for setting the pH to the target value, and the flow rate of the inert gas can be easily set. Then, the first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate adjusting unit are controlled so as to have the oxygen flow rate, the carbon dioxide flow rate, and the inert gas flow rate set by the flow rate setting unit. To. Therefore, according to the above-mentioned culture solution adjusting device, the properties of the culture solution can be easily adjusted. The target value of the dissolved oxygen concentration and the target value of pH may be preset values or may be input by the user each time.

In the second means, the flow rate setting unit has a first deviation, which is a deviation between the dissolved oxygen concentration detected by the oxygen concentration sensor and a target value of the dissolved oxygen concentration, and a flow rate of the oxygen to be set. The first deviation is applied to the first data in which the relationship is defined in advance to set the flow rate of the oxygen, and the second deviation, which is the deviation between the pH detected by the pH sensor and the target value of the pH. , The flow rate of the carbon dioxide is set by applying the second deviation to the second data in which the relationship with the flow rate of the carbon dioxide to be set is defined in advance.

According to the above configuration, the relationship between the first deviation, which is the deviation between the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, and the set oxygen flow rate is defined in advance by the first data. ing. Then, the flow rate setting unit applies the first deviation to the first data to set the oxygen flow rate. Therefore, the flow rate of oxygen for setting the dissolved oxygen concentration to the target value can be set more easily. Further, the relationship between the second deviation, which is the deviation between the pH detected by the pH sensor and the target value of pH, and the set flow rate of carbon dioxide is defined in advance by the second data. Then, the flow rate setting unit sets the flow rate of carbon dioxide by applying the second deviation to the second data. Therefore, the flow rate of carbon dioxide for setting the pH to the target value can be set more easily. The first data and the second data can be defined in advance based on an experiment or the like.

In the third means, the first data includes an oxygen flow rate table in which the relationship between each dissolved oxygen concentration when the dissolved oxygen concentration of the culture solution is balanced and each flow rate of oxygen in the mixed gas is predetermined. The oxygen flow rate table includes a first coefficient table in which the relationship between the coefficient multiplied by the oxygen flow rate corresponding to the target value of the dissolved oxygen concentration and the first deviation is defined in advance, and the second data is the culture solution. A carbon dioxide flow rate table in which the relationship between each pH when the pH is equilibrium and each flow rate of carbon dioxide in the mixed gas is defined in advance, and a flow rate of carbon dioxide corresponding to the target value of the pH in the carbon dioxide flow rate table. It includes a second coefficient table in which the relationship between the multiplication coefficient and the second deviation is defined in advance.

According to the above configuration, the relationship between each dissolved oxygen concentration and each flow rate of oxygen in the mixed gas when the dissolved oxygen concentration of the culture solution is in equilibrium is defined in advance by the oxygen flow rate table. Then, the relationship between the coefficient multiplied by the flow rate of oxygen corresponding to the target value of the dissolved oxygen concentration in the oxygen flow rate table and the first deviation is defined in advance by the first coefficient table. Therefore, the oxygen flow rate is set by multiplying the oxygen flow rate when the dissolved oxygen concentration of the culture solution is balanced by a coefficient corresponding to the first deviation. Therefore, the dissolved oxygen concentration can be quickly adjusted to the target value while referring to the flow rate of oxygen when the dissolved oxygen concentration is in equilibrium.

Further, the relationship between each pH when the pH of the culture solution is in equilibrium and each flow rate of carbon dioxide in the mixed gas is defined in advance by the carbon dioxide flow rate table. Then, in the carbon dioxide flow rate table, the relationship between the coefficient multiplied by the flow rate of carbon dioxide corresponding to the target value of pH and the second deviation is defined in advance by the second coefficient table. Therefore, the flow rate of carbon dioxide is set by multiplying the flow rate of carbon dioxide when the pH of the culture solution is in equilibrium by a coefficient corresponding to the second deviation. Therefore, the pH can be quickly adjusted to the target value while referring to the flow rate of carbon dioxide when the pH is in equilibrium.

The amount of oxygen dissolved and the amount of carbon dioxide dissolved in the culture solution vary depending on the temperature. In this regard, in the fourth means, the oxygen flow rate table changes the relationship between each dissolved oxygen concentration when the dissolved oxygen concentration of the culture solution is balanced and each flow rate of oxygen in the mixed gas according to each temperature. The carbon dioxide flow rate table is defined in advance, and the relationship between each pH when the pH of the culture solution is balanced and each flow rate of carbon dioxide in the mixed gas is predetermined according to each temperature. The configuration is adopted. Therefore, even if the dissolved amount of oxygen and the dissolved amount of carbon dioxide in the culture solution change depending on the temperature, the dissolved oxygen concentration and pH can be appropriately adjusted to the target values.

In a configuration in which the flow rate of oxygen and the flow rate of carbon dioxide are set, and the flow rate of the inert gas is set by subtracting the set flow rate of oxygen and the set flow rate of carbon dioxide from the predetermined flow rate, the mixed gas having a predetermined flow rate is always used. Will flow. Therefore, even after the dissolved oxygen concentration and pH are adjusted to the target values, the mixed gas may be wasted.

In this respect, in the fifth means, in the flow rate setting unit, the first deviation, which is the deviation between the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, is larger than the first predetermined deviation. When the second deviation, which is small and is the deviation between the pH and the target value of the pH detected by the pH sensor, is smaller than the second predetermined deviation, the oxygen flow rate, the carbon dioxide flow rate, and the carbon dioxide flow rate are described. A configuration is adopted in which the flow rate of the inert gas is set to 0. Therefore, when the first deviation is smaller than the first predetermined deviation and the second deviation is smaller than the second predetermined deviation, the flow of the mixed gas can be stopped. Therefore, it is possible to prevent the mixed gas from being wasted.

In the sixth means, a culture section for culturing cells with the culture solution, a first delivery section for delivering the culture solution from the storage section to the culture section, and the culture solution from the culture section to the storage section are delivered. A second sending unit for sending is provided.

According to the above configuration, the culture solution is delivered from the storage section to the culture section by the first delivery section. Then, the cells are cultured in the culture medium in the culture section, and the dissolved oxygen concentration and pH of the culture solution change depending on the activity of the cells. In this regard, the second delivery unit sends the culture solution from the culture unit to the storage unit. Then, in the storage section, the dissolved oxygen concentration and pH of the culture solution are adjusted to the target values again. Therefore, the culture solution can be circulated between the storage section and the culture section for continuous use, and the properties of the culture solution supplied to the culture section can be maintained at the target value.

The seventh means includes an operation unit that is operated when inputting the target value of the dissolved oxygen concentration and the target value of the pH.

According to the above configuration, the user can input the target value of the dissolved oxygen concentration and the target value of pH by operating the operation unit. Then, the flow rate setting unit sets the flow rate of oxygen for setting the dissolved oxygen concentration to the target value, the flow rate of carbon dioxide for setting the pH to the target value, and the flow rate of the inert gas. Therefore, the user can automatically adjust the properties of the culture solution to the target values simply by operating the operation unit and inputting the target value of the dissolved oxygen concentration and the target value of pH.

The schematic diagram which shows the culture solution adjusting apparatus. A graph showing the relationship between the oxygen volume ratio, the dissolved oxygen concentration, and the temperature in an equilibrium state. The graph which shows the relationship between the natural logarithmic value of the carbon dioxide volume ratio in an equilibrium state, pH and temperature. The figure which shows the oxygen flow rate table. The figure which shows the carbon dioxide flow rate table. The figure which shows the 1st coefficient table. The figure which shows the 2nd coefficient table. A time chart showing changes in dissolved oxygen concentration.

Hereinafter, an embodiment embodied in a culture medium adjusting device for adjusting the properties of the culture medium for culturing cells will be described with reference to the drawings.

As shown in FIG. 1, the culture solution adjusting device 10 includes a control unit 11, a culture solution bottle 20, an oxygen concentration sensor 21, a pH sensor 22, a temperature sensor 23, a culture container 30, pumps 31, 32 and the like.

The control unit 11 includes a mass flow controller (MFC) 12, 13, 14, a joint 15, an operation unit 16, a display unit 17, a microcomputer (MC) 18, and the like.

Oxygen from the oxygen cylinder 41 is adjusted to a predetermined pressure by the regulator (pressure regulating valve) 45 and supplied to the MFC 12 (corresponding to the first flow rate adjusting unit). Carbon dioxide from the carbon dioxide cylinder 42 is adjusted to a predetermined pressure by the regulator 46 and supplied to the MFC 13 (corresponding to the second flow rate adjusting unit). Nitrogen (corresponding to an inert gas) from the nitrogen cylinder 43 is adjusted to a predetermined pressure by the regulator 47 and supplied to the MFC 14 (corresponding to the third flow rate adjusting unit). That is, oxygen, carbon dioxide, and nitrogen having predetermined pressures equal to each other are supplied to the MFCs 12, 13, and 14, respectively. MFCs 12, 13 and 14 adjust the flow rates of oxygen, carbon dioxide and nitrogen, respectively. The adjustment state of the gas flow rate by the MFCs 12, 13 and 14 is controlled by the MC18.

Oxygen, carbon dioxide, and nitrogen whose flow rates have been adjusted by MFCs 12, 13, and 14, respectively, merge at the joint 15. The joint 15 connects the pipes 12a, 13a, 14a connected to the MFCs 12, 13 and 14, respectively, to the pipe 15a. That is, the joint 15 (corresponding to the mixing portion) mixes oxygen, carbon dioxide, and nitrogen supplied from the MFCs 12, 13, and 14, respectively, and the mixed gas thereof is supplied to the pipe 15a.

The pipe 15a is connected to a culture solution bottle 20 (corresponding to a storage portion) for storing the culture solution L1. Specifically, the pipe 15a is connected to the culture solution bottle 20 so as to supply the mixed gas into the culture solution L1. The culture solution L1 which is a pH equilibrium solution is stored in the culture solution bottle 20. As the reagent to be added to the culture broth, a reagent that is free in the pH range of 6 to 8 and has a pH buffering function, such as bicarbonate, can be adopted. As the bicarbonate, sodium bicarbonate (sodium hydrogen carbonate), potassium hydrogen carbonate, calcium hydrogen carbonate and the like can be adopted. In addition, serum, amino acids, vitamins, sugars and the like are added to the culture solution L1 as nutrients necessary for cell activity. In addition, an indicator such as phenol red is added to the culture solution L1 in order to visually confirm the pH.

The culture solution bottle 20 is provided with an oxygen concentration sensor 21 that detects the dissolved oxygen concentration DO of the culture solution L1, a pH sensor 22 that detects the pH of the culture solution L1, and a temperature sensor 23 that detects the temperature of the culture solution L1. ing.

A pump 31 is connected to the culture solution bottle 20 via a pipe 25. A culture vessel 30 (corresponding to the culture unit) is connected to the pump 31 (corresponding to the first delivery unit) via a pipe 26. The pump 31 sucks the culture solution L1 in the culture solution bottle 20 through the pipe 25, and discharges the culture solution L1 to the culture container 30 through the pipe 26. That is, the pump 31 delivers the culture solution L1 from the culture solution bottle 20 to the culture container 30.

The culture solution L2 is stored in the culture container 30. The culture medium L2 (medium) contains cells to be cultured. As the cells, animal cells, plant cells, insect cells, bacteria, yeasts, fungi and the like can be adopted. Further, the cell may be a tissue composed of a plurality of cells or a tissue composed of a plurality of types of cells. Then, the cells are cultured in the culture medium L2 in the culture vessel 30, and the dissolved oxygen concentration and pH of the culture solution L2 change depending on the activity of the cells.

A pump 32 is connected to the culture vessel 30 via a pipe 27. A culture solution bottle 20 is connected to the pump 32 (corresponding to the second delivery unit) via a pipe 28. The pump 32 sucks the culture solution L2 in the culture container 30 through the pipe 27, and discharges the culture solution L2 to the culture solution bottle 20 through the pipe 28. That is, the pump 32 delivers the culture solution L2 from the culture container 30 to the culture solution bottle 20.

The operation unit 16 of the control unit 11 is composed of a dial, a switch, or the like. The operation unit 16 is operated when the user inputs the target value of the dissolved oxygen concentration DO and the target value of pH of the culture solution L1. The target value of the dissolved oxygen concentration DO and the target value of pH input by the operation of the operation unit 16 are output to the MC18.

The display unit 17 is composed of a liquid crystal display or the like, and has a target value (ppm) of dissolved oxygen concentration DO, a detected value (ppm) of dissolved oxygen concentration DO, a target value of pH, a detected value of pH, and a detected value of temperature. (° C.), oxygen flow rate (ml / min), carbon dioxide flow rate (ml / min), total flow rate of mixed gas (ml / min), etc. are displayed. The display state of the display unit 17 is controlled by the MC18.

The control unit 11 includes an insertion portion for inserting the SD card 19 (storage medium). The MC 18 writes data to the SD card 19 inserted in the insertion unit, and reads the data written to the SD card 19. Specifically, the MC 18 is sequentially detected by the target value of the dissolved oxygen concentration DO, the dissolved oxygen concentration DO sequentially detected by the oxygen concentration sensor 21, the target value of pH, the pH sequentially detected by the pH sensor 22, and the temperature sensor 23. The temperature T and the like are written to the SD card 19 at a predetermined cycle.

The MC18 (corresponding to a flow rate setting unit and a flow rate control unit) is a microcomputer provided with a CPU, ROM, RAM, an input / output interface, and the like. The MC18 controls the MFCs 12, 13 and 14 so that the dissolved oxygen concentration DO and the pH of the culture solution L1 become target values based on the values detected by the sensors 21, 22, 23.

FIG. 2 is a graph showing the relationship between the oxygen volume ratio in the mixed gas, the dissolved oxygen concentration DO in the culture solution L1, and the temperature T in the culture solution L1 in a state where the mixed gas is supplied to the culture solution L1 and is in equilibrium. is there. The dissolved oxygen concentration DO of the culture solution L1 changes according to the oxygen partial pressure of the mixed gas. If the pressure of each gas in the mixed gas is equal at a predetermined pressure and the total volume (total flow rate) of the mixed gas is constant, the partial pressure of each gas is proportional to the volume (flow rate) of each gas. Therefore, the larger the oxygen volume ratio in the mixed gas, the higher the dissolved oxygen concentration DO. Specifically, the dissolved oxygen concentration DO is proportional to the oxygen volume ratio in the mixed gas. Further, the amount of oxygen dissolved in the culture solution L1 changes according to the temperature T. Specifically, as the temperature T increases, the amount of oxygen dissolved in the culture solution L1 decreases, and the dissolved oxygen concentration DO decreases.

FIG. 3 shows the relationship between the natural logarithmic value of the carbon dioxide volume ratio in the mixed gas, the pH of the culture solution L1, and the temperature T of the culture solution L1 in a state where the mixed gas is supplied to the culture solution L1 and is in equilibrium. It is a graph. Since hydrogen ions are generated by dissolving carbon dioxide in the culture solution L1, the pH of the culture solution L1 changes according to the partial pressure of carbon dioxide in the mixed gas. If the pressure of each gas in the mixed gas is equal at a predetermined pressure and the total volume (total flow rate) of the mixed gas is constant, the partial pressure of each gas is proportional to the volume (flow rate) of each gas. Therefore, the larger the carbon dioxide volume ratio in the mixed gas, the smaller the pH. Specifically, the amount of decrease in pH is proportional to the amount of increase in the natural logarithmic value of the carbon dioxide volume ratio in the mixed gas. Further, the amount of carbon dioxide dissolved in the culture solution L1 changes according to the temperature T. Specifically, the higher the temperature T, the smaller the amount of carbon dioxide dissolved in the culture solution L1 and the higher the pH.

FIG. 4 shows an oxygen flow rate that defines the relationship between each dissolved oxygen concentration DO and each flow rate of oxygen in the mixed gas and each temperature T when the dissolved oxygen concentration DO of the culture solution L1 is balanced based on the graph of FIG. It is a figure which shows the table. For example, the oxygen flow rate when equilibrating at the dissolved oxygen concentration DO1 (for example, 1 ppm) at the temperature T1 is the flow rate RO11, and the oxygen flow rate when equilibrating at the dissolved oxygen concentration DOn (for example, 20 ppm) at the temperature Tm is the flow rate ROnm. The flow rate RO11 increases in order from the flow rate ROn1, and the flow rate RO11 increases in order from the flow rate RO1m. And the flow rate ROnm is the largest. The flow rate RO under the conditions not included in the three graphs of FIG. 2 is calculated by proportionally interpolating between the graphs.

FIG. 5 is a diagram showing a carbon dioxide flow rate table that defines the relationship between each pH when the pH of the culture solution L1 is in equilibrium, each flow rate of carbon dioxide in the mixed gas, and each temperature T based on the graph of FIG. Is. For example, the carbon dioxide flow rate when equilibrated at pH 1 (predetermined pH value, for example 6.9) at temperature T1 is the flow rate RC11, and when equilibrated at pH n (predetermined pH value, eg 7.9) at temperature Tm. The carbon dioxide flow rate is RC nm. The flow rate RC11 decreases in order from the flow rate RCn1, and the flow rate RC11 increases in order from the flow rate RC1m. And the flow rate RC1m is the largest. The flow rate RC under the conditions not included in the three graphs of FIG. 3 is calculated by proportionally interpolating between the graphs.

Then, the MC 18 sets the flow rate of oxygen based on the dissolved oxygen concentration DO detected by the oxygen concentration sensor 21 and the target value of the dissolved oxygen concentration DO. For example, the reference flow rate is the oxygen flow rate ROnm when the dissolved oxygen concentration DOn is balanced at the temperature Tm, and the reference flow rate is a coefficient corresponding to the first deviation dOM, which is the deviation between the detected value of the dissolved oxygen concentration DO and the target value DOn. Set the oxygen flow rate by multiplying by ROnm.

FIG. 6 is a first coefficient table that defines the relationship between the coefficients POnm and MOnm multiplied by the reference flow rate ROnm of oxygen corresponding to the target value DOn of the dissolved oxygen concentration DO in the oxygen flow rate table of FIG. 4 and the first deviation dOM. It is a figure which shows. The first coefficient table can be defined in advance based on experiments and the like.

For example, when the detected value is larger by the deviation dO1 on the positive side with respect to the target value DO1 (for example, 1 ppm) of the dissolved oxygen concentration DO, the first coefficient is the first coefficient PO11. Further, when the detected value is larger by dOM (dO1 <dOM) on the positive side with respect to the target value DOn (for example, 20 ppm) of the dissolved oxygen concentration DO, the first coefficient is the first coefficient POnm. It decreases in order from the first coefficient PO11 to the first coefficient PO1m, and decreases in order from the first coefficient POn1 to the first coefficient POnm. On the other hand, when the detected value is smaller by the deviation dO1 (0 <dO1) on the negative side with respect to the target value DO1 of the dissolved oxygen concentration DO, the first coefficient is the first coefficient MO11. Further, when the detected value is smaller by dOM (dO1 <dOM) on the negative side with respect to the target value DOn of the dissolved oxygen concentration DO, the first coefficient is the first coefficient MOnm. The first coefficient MO11 increases in order from the first coefficient MO1m, and the first coefficient MOn1 increases in order from the first coefficient MOnm.

Similarly, the MC 18 sets the flow rate of carbon dioxide based on the pH detected by the pH sensor 22 and the target value of pH. For example, by using the carbon dioxide flow rate RCnm when pH n is balanced at the temperature Tm as the reference flow rate, and multiplying the reference flow rate RCnm by a coefficient corresponding to the second deviation dPm, which is the deviation between the detected value of pH and the target value DOn. Set the flow rate of carbon dioxide.

FIG. 7 shows a second coefficient table that defines the relationship between the coefficients PCnm and MCnm multiplied by the reference flow rate RCnm of carbon dioxide corresponding to the target value pHn of pH in the carbon dioxide flow rate table of FIG. 5 and the second deviation dPm. It is a figure which shows. The second coefficient table can be defined in advance based on an experiment or the like.

For example, the second coefficient is the second coefficient PC11 when the detected value is larger by the deviation dP1 on the positive side with respect to the target value pH1 (predetermined pH value, for example, 6.9) of pH. Further, when the detected value is larger by dPm (dP1 <dPm) on the positive side with respect to the target pH value pHn (predetermined pH value, for example, 7.9), the second coefficient is the second coefficient PCnm. The second coefficient PC11 increases in order from the second coefficient PC1m, and the second coefficient PCn1 increases in order from the second coefficient PCnm. On the other hand, the second coefficient is the second coefficient MC11 when the detected value is smaller by the deviation dP1 (0 <dP1) on the negative side with respect to the target value pH1 of pH. Further, when the detected value is smaller by dPm (dP1 <dPm) on the negative side with respect to the target value pHn of pH, the second coefficient is the second coefficient MCnm. The second coefficient MC11 becomes smaller in order from the second coefficient MC1m, and the second coefficient MCn1 becomes smaller in order from the second coefficient MCnm.

The first data is composed of the oxygen flow rate table and the first coefficient table, and the second data is composed of the carbon dioxide flow rate table and the second coefficient table. That is, the first data defines in advance the relationship between the first deviation, which is the deviation between the dissolved oxygen concentration DO detected by the oxygen concentration sensor 21 and the target value of the dissolved oxygen concentration DO, and the set oxygen flow rate. There is. In addition, the second data prescribes the relationship between the second deviation, which is the deviation between the pH detected by the pH sensor 22 and the target value of pH, and the set flow rate of carbon dioxide.

The MC18 sets the oxygen flow rate and the carbon dioxide flow rate based on the above, and sets the nitrogen flow rate by subtracting the set oxygen flow rate and the set carbon dioxide flow rate from the predetermined flow rate (for example, 750 ml). To do. Then, the MC18 controls the MFCs 12, 13 and 14, respectively, so as to have the set oxygen flow rate, carbon dioxide flow rate, and inert gas flow rate.

FIG. 8 is a time chart showing changes in the dissolved oxygen concentration DO when the properties of the culture solution L1 are adjusted by the culture solution adjusting device 10. Here, it is assumed that the temperature of the culture solution L1 detected by the temperature sensor 23 is the temperature Tm, and the target value of the dissolved oxygen concentration DO is set to the target value DOn.

Before the time t1, for example, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn is between + dO3 and + dO4, the oxygen flow rate ROnm in the oxygen flow rate table of FIG. 4 is the first coefficient of FIG. It is set to the oxygen flow rate (ROnm × POn4) multiplied by the first coefficient POn4 in the table. Similarly, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn is between + dO1 to + dO2, the oxygen flow rate ROnm in the oxygen flow rate table of FIG. 4 is the first in the first coefficient table of FIG. It is set to the oxygen flow rate (ROnm × POn2) multiplied by the coefficient POn2.

At time t1, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn becomes + dO1 or less, the flow rate (ROnm × POn2) is maintained until the predetermined time TA elapses. Then, at the time t2 when the predetermined time TA has elapsed, the flow rate of oxygen is the flow rate (ROnm × POn1), provided that the deviation of the detected value with respect to the target value DOn is maintained within the target range (+ dO2-−dO2). Is set to. While the deviation of the detected value with respect to the target value DOn is maintained within the target range, the oxygen flow rate is maintained at the flow rate (ROnm × POn1).

At time t3, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn becomes −dO2 or less, the oxygen flow rate ROnm in the oxygen flow rate table of FIG. 4 is multiplied by the first coefficient MOn3 in the first coefficient table of FIG. The flow rate of oxygen (ROnm × MOn3) is set.

At time t4, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn becomes −dO1 or more, the flow rate (ROnm × MOn2) is maintained until the predetermined time TA elapses. Then, at the time t5 when the predetermined time TA has elapsed, the flow rate of oxygen is the flow rate (ROnm × MOn1), provided that the deviation of the detected value with respect to the target value DOn is maintained within the target range (+ dO2-−dO2). Is set to. While the deviation of the detected value with respect to the target value DOn is maintained within the target range, the oxygen flow rate is maintained at the flow rate (ROnm × MOn1).

At time t6, when the deviation of the value detected by the oxygen concentration sensor 21 with respect to the target value DOn becomes + dO2 or more, the oxygen flow rate ROnm in the oxygen flow rate table of FIG. 4 is multiplied by the first coefficient POn3 in the first coefficient table of FIG. It is set to the oxygen flow rate (ROnm × POn3). Further, the pH of the culture solution L1 is also adjusted in the same manner by the culture solution adjusting device 10.

The present embodiment described in detail above has the following advantages.

The MC18 sets the flow rate of oxygen based on the dissolved oxygen concentration DO detected by the oxygen concentration sensor 21 and the target value of the dissolved oxygen concentration DO, and sets the pH and the target value of pH detected by the pH sensor 22. Based on this, the flow rate of carbon dioxide is set, and the flow rate of nitrogen is set by subtracting the set flow rate of oxygen and the set flow rate of carbon dioxide from the predetermined flow rate. Therefore, the flow rate of oxygen for setting the dissolved oxygen concentration DO to the target value, the flow rate of carbon dioxide for setting the pH to the target value, and the flow rate of nitrogen can be easily set. Then, the MFC 12, MFC 13, and MFC 14 are controlled so as to have the set oxygen flow rate, carbon dioxide flow rate, and nitrogen flow rate. Therefore, according to the culture solution adjusting device 10, the properties of the culture solution L1 can be easily adjusted.

The relationship between the first deviation dOm, which is the deviation between the dissolved oxygen concentration DO detected by the oxygen concentration sensor 21 and the target value of the dissolved oxygen concentration DO, and the set oxygen flow rate is defined in advance by the first data. There is. Then, the MC 18 applies the first deviation dOm to the first data to set the oxygen flow rate. Therefore, the flow rate of oxygen for setting the dissolved oxygen concentration DO to the target value can be set more easily.

The relationship between the second deviation dPm, which is the deviation between the pH detected by the pH sensor 22 and the target value of pH, and the set flow rate of carbon dioxide is defined in advance by the second data. Then, the MC18 applies the second deviation dPm to the second data to set the flow rate of carbon dioxide. Therefore, the flow rate of carbon dioxide for setting the pH to the target value can be set more easily.

The relationship between each dissolved oxygen concentration DO and each flow rate of oxygen in the mixed gas when the dissolved oxygen concentration DO of the culture solution L1 is in equilibrium is defined in advance by the oxygen flow rate table of FIG. Then, the relationship between the first coefficient multiplied by the flow rate of oxygen corresponding to the target value of the dissolved oxygen concentration DO in the oxygen flow rate table and the first deviation dOM is defined in advance by the first coefficient table of FIG. Therefore, the oxygen flow rate is set by multiplying the oxygen flow rate when the dissolved oxygen concentration DO of the culture solution L1 is balanced by a first coefficient according to the first deviation dOm. Therefore, the dissolved oxygen concentration DO can be quickly adjusted to the target value while referring to the flow rate of oxygen when the dissolved oxygen concentration DO is in equilibrium.

The relationship between each pH when the pH of the culture solution L1 is in equilibrium and each flow rate of carbon dioxide in the mixed gas is defined in advance by the carbon dioxide flow rate table of FIG. Then, in the carbon dioxide flow rate table, the relationship between the second coefficient multiplied by the flow rate of carbon dioxide corresponding to the target value of pH and the second deviation dPm is defined in advance by the second coefficient table. Therefore, the flow rate of carbon dioxide is set by multiplying the flow rate of carbon dioxide when the pH of the culture solution L1 is in equilibrium by a second coefficient corresponding to the second deviation dPm. Therefore, the pH can be quickly adjusted to the target value while referring to the flow rate of carbon dioxide when the pH is in equilibrium.

-The oxygen flow rate table prescribes the relationship between each dissolved oxygen concentration DO and each flow rate of oxygen in the mixed gas when the dissolved oxygen concentration DO of the culture solution L1 is in equilibrium, according to each temperature Tm, and carbon dioxide In the carbon flow rate table, the relationship between each pH when the pH of the culture solution L1 is balanced and each flow rate of carbon dioxide in the mixed gas is defined in advance according to each temperature Tm. Therefore, even if the dissolved amount of oxygen and the dissolved amount of carbon dioxide in the culture solution L1 change according to the temperature Tm, the dissolved oxygen concentration DO and the pH can be appropriately adjusted to the target values.

-The pump 31 sends the culture solution L1 from the culture solution bottle 20 to the culture container 30. Then, the cells are cultured in the culture medium L2 in the culture vessel 30, and the dissolved oxygen concentration DO and pH of the culture solution L2 change depending on the activity of the cells. At this point, the pump 32 sends the culture solution L2 from the culture container 30 to the culture solution bottle 20. Then, in the culture solution bottle 20, the dissolved oxygen concentration DO and pH of the culture solution L1 are adjusted to the target values again. Therefore, the culture solutions L1 and L2 can be circulated between the culture solution bottle 20 and the culture container 30 for continuous use, and the properties of the culture solution L1 supplied to the culture container 30 can be maintained at the target value. Can be done.

-The user can input the target value of the dissolved oxygen concentration DO and the target value of pH by operating the operation unit 16. Then, the MC18 sets the flow rate of oxygen for setting the dissolved oxygen concentration DO to the target value, the flow rate of carbon dioxide for setting the pH to the target value, and the flow rate of nitrogen. Therefore, the user can automatically adjust the properties of the culture solution L1 to the target value only by operating the operation unit 16 and inputting the target value of the dissolved oxygen concentration DO and the target value of the pH.

It should be noted that the above embodiment can be modified and implemented as follows.

-The MC18 may read the target value of the dissolved oxygen concentration DO and the target value of pH from the SD card 19. Further, the control unit 11 may be provided with an input / output interface for inputting / outputting data to / from the outside.

A buffer tank (corresponding to a mixing portion) for temporarily storing and mixing oxygen, carbon dioxide, and nitrogen supplied from MFCs 12, 13, and 14, respectively, may be provided in place of the joint 15. Further, as the inert gas, argon or helium can be used instead of nitrogen.

The predetermined flow rate, which is the total flow rate of the mixed gas, is not limited to 750 ml and can be set arbitrarily. In that case, the pressure (corresponding to the predetermined pressure) of each gas adjusted by the regulators (pressure regulating valves) 45, 46, 47 may be changed according to the predetermined flow rate.

-If the amount of oxygen dissolved in the culture solution L1 does not change much with temperature, the relationship between each dissolved oxygen concentration DO in the oxygen flow rate table and each flow rate of oxygen in the mixed gas is uniformly defined with respect to the temperature. You may. Similarly, when the amount of carbon dioxide dissolved in the culture solution L1 does not change much with temperature, the relationship between each pH in the carbon dioxide flow rate table and each flow rate of carbon dioxide in the mixed gas is uniformly set with respect to the temperature. It may be specified. In that case, the temperature sensor 23 can be omitted.

-Instead of the oxygen flow rate table of FIG. 4, the volume ratio of oxygen to the dissolved oxygen concentration DON and the temperature Tm at equilibrium (that is, the flow rate of oxygen) may be calculated based on the relational expression representing the graph of FIG. .. Similarly, instead of the carbon dioxide flow rate table of FIG. 5, the natural logarithmic value (that is, the flow rate of carbon dioxide) of the carbon dioxide volume ratio with respect to pHn and temperature Tm at equilibrium is calculated based on the relational expression representing the graph of FIG. It may be calculated.

-The oxygen flow rate table and the first coefficient table can be combined into one table (corresponding to the first data). In that case, each flow rate of oxygen to be set may be specified in advance for each target value of the dissolved oxygen concentration DO and each first deviation dOM. Similarly, the carbon dioxide flow rate table and the second coefficient table can be combined into one table (corresponding to the second data). In that case, each flow rate of carbon dioxide to be set may be specified in advance for each target value of pH and each second deviation dPm.

-It is also possible to set the oxygen flow rate by PID control, PI control, or the like based on the target value and the detected value of the dissolved oxygen concentration DO. Similarly, the flow rate of carbon dioxide can be set by PID control, PI control, or the like based on the target value and the detected value of pH. In that case as well, the nitrogen flow rate may be set by subtracting the set oxygen flow rate and the set carbon dioxide flow rate from the predetermined flow rate, which is the total flow rate of the mixed gas.

-In a configuration in which the flow rate of oxygen and the flow rate of carbon dioxide are set, and the flow rate of nitrogen (inert gas) is set by subtracting the set flow rate of oxygen and the set flow rate of carbon dioxide from the predetermined flow rate, the flow rate is always predetermined. A mixed gas with a flow rate will flow. Therefore, even after the dissolved oxygen concentration DO and pH are adjusted within the target range, the mixed gas may be wasted.

In this respect, the MC18 (flow rate setting unit) has a first deviation dOM, which is a deviation between the dissolved oxygen concentration DO and the target value of the dissolved oxygen concentration DO detected by the oxygen concentration sensor 21, and is smaller than the first predetermined deviation. When the second deviation dPm, which is the deviation between the pH detected by the pH sensor 22 and the target value of pH, is smaller than the second predetermined deviation, the oxygen flow rate, the carbon dioxide flow rate, and the nitrogen flow rate are set to 0. You may. As the first predetermined deviation, for example, the first deviation dO1 shown in FIG. 6 can be adopted. As the second predetermined deviation, for example, the second deviation dP1 shown in FIG. 7 can be adopted. According to such a configuration, when the first deviation dOm is smaller than the first predetermined deviation and the second deviation dPm is smaller than the second predetermined deviation, the flow of the mixed gas can be stopped. Therefore, it is possible to prevent the mixed gas from being wasted.

-The culture solution L2 in the culture container 30 can be used for a certain period of time and then discarded without being returned to the culture solution bottle 20. In that case, the culture solution bottle 20 may be appropriately replenished with the culture solution L1 according to the decrease in the culture solution L1 in the culture solution bottle 20.

-It is also possible to adopt a configuration in which the culture solution bottle 20 and the culture container 30 are integrated into a container. That is, cells may be contained in the culture solution in the container, and the properties of the culture solution may be adjusted. In that case, the pumps 31 and 32 and the culture vessel 30 are unnecessary.

10 ... Culture solution adjusting device, 11 ... Control unit, 12 ... MFC (first flow rate adjusting unit), 13 ... MFC (second flow rate adjusting unit), 14 ... MFC (third flow rate adjusting unit), 15 ... Joint (mixing) Unit), 16 ... Operation unit, 18 ... Microcomputer (flow rate setting unit, flow rate control unit), 20 ... Culture solution bottle (storage unit), 21 ... Oxygen concentration sensor, 22 ... pH sensor, 23 ... Temperature sensor, 30 ... Culture container (culture section), 31 ... Pump (first delivery section), 32 ... Pump (second delivery section).

Claims (7)

A culture solution adjusting device that adjusts the properties of a culture solution that is a pH equilibrium solution.
The first flow rate adjusting unit that adjusts the flow rate of oxygen supplied at a predetermined pressure,
A second flow rate adjusting unit that adjusts the flow rate of carbon dioxide supplied at the predetermined pressure, and
A third flow rate adjusting unit that adjusts the flow rate of the inert gas supplied at the predetermined pressure, and
A mixing unit that mixes the oxygen, the carbon dioxide, and the inert gas whose flow rates have been adjusted by the first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate adjusting unit, respectively.
A storage unit to which the mixed gas flowing out from the mixing unit is supplied and stores the culture solution,
An oxygen concentration sensor that detects the dissolved oxygen concentration of the culture solution and
A pH sensor that detects the pH of the culture solution and
The flow rate of the oxygen is set based on the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, and based on the pH detected by the pH sensor and the target value of the pH. A flow rate setting unit that sets the flow rate of the carbon dioxide and sets the flow rate of the inert gas by subtracting the set oxygen flow rate and the set carbon dioxide flow rate from the predetermined flow rate.
The first flow rate adjusting unit, the second flow rate adjusting unit, and the third flow rate so as to be the flow rate of the oxygen, the flow rate of carbon dioxide, and the flow rate of the inert gas set by the flow rate setting unit. The flow control unit that controls the adjustment unit and
A culture solution adjusting device comprising. The flow rate setting unit defines in advance the relationship between the first deviation, which is the deviation between the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, and the set flow rate of the oxygen. The first deviation is applied to one data to set the flow rate of the oxygen, the second deviation which is the deviation between the pH detected by the pH sensor and the target value of the pH, and the carbon dioxide to be set. The culture solution adjusting device according to claim 1, wherein the second deviation is applied to the second data in which the relationship with the flow rate of carbon dioxide is set in advance. The first data includes an oxygen flow rate table in which the relationship between each dissolved oxygen concentration when the dissolved oxygen concentration of the culture solution is in equilibrium and each flow rate of oxygen in the mixed gas is defined in advance, and the dissolved oxygen in the oxygen flow rate table. Includes a first coefficient table that prescribes the relationship between the coefficient multiplied by the oxygen flow rate corresponding to the target value of oxygen concentration and the first deviation.
The second data includes a carbon dioxide flow rate table in which the relationship between each pH when the pH of the culture solution is balanced and each flow rate of carbon dioxide in the mixed gas is defined in advance, and the pH in the carbon dioxide flow rate table. The culture solution adjusting device according to claim 2, further comprising a coefficient for multiplying the flow rate of carbon dioxide corresponding to a target value and a second coefficient table in which the relationship between the second deviation is defined in advance. In the oxygen flow rate table, the relationship between each dissolved oxygen concentration when the dissolved oxygen concentration of the culture solution is in equilibrium and each flow rate of oxygen in the mixed gas is defined in advance according to each temperature.
The carbon dioxide flow rate table according to claim 3, wherein the relationship between each pH when the pH of the culture solution is balanced and each flow rate of carbon dioxide in the mixed gas is defined in advance according to each temperature. Culture solution adjusting device. In the flow rate setting unit, the first deviation, which is the deviation between the dissolved oxygen concentration detected by the oxygen concentration sensor and the target value of the dissolved oxygen concentration, is smaller than the first predetermined deviation, and is detected by the pH sensor. When the second deviation, which is the deviation between the pH and the target value of the pH, is smaller than the second predetermined deviation, the oxygen flow rate, the carbon dioxide flow rate, and the inert gas flow rate are set to 0. The culture solution adjusting device according to any one of claims 1 to 4. A culture section for culturing cells with the culture solution, and
A first delivery unit that delivers the culture solution from the storage unit to the culture unit,
A second delivery unit that delivers the culture solution from the culture unit to the storage unit,
The culture solution adjusting device according to any one of claims 1 to 5. The culture solution adjusting device according to any one of claims 1 to 6, further comprising an operation unit operated when inputting the target value of the dissolved oxygen concentration and the target value of the pH.

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