CULTURE DEVICE AND CULTURE METHOD FOR DYNAMICALLY CONTROLLING pH IN CULTURE VESSEL

Abstract

[Problem] The present invention addresses the problem of providing a culture device and a culture method capable of measuring the pH in a culture vessel and controlling the pH in the culture vessel. Alternatively, the present invention addresses the problem of providing a culture device and a culture method which can control the pH in a culture vessel so as to achieve a pH in accordance with the growth stage or the proliferation stage of a culture object. [Solution] The present invention is provided with a culture chamber in which a culture vessel containing at least a culture medium and / or a culture object can be installed, a pH measurement unit capable of measuring the pH in the culture vessel, and a control unit for controlling the feed or abolition of gas into the culture chamber. The control unit controls the feed or the like of gas into the culture chamber so as to achieve a preliminarily set pH in accordance with the growth stage or the proliferation stage of the culture object.

Description

Cultivation device and method for dynamically controlling pH in a culture vessel

[0001] The present invention relates to a culture device and a culture method, and more particularly to a culture device and a culture method that are capable of controlling the pH in a culture vessel by measuring the pH in the culture vessel and controlling the supply or stop of gas into the culture chamber.

[0002] When culturing using a general culturing device, the device is controlled so that the temperature, humidity, carbon dioxide concentration, and oxygen concentration within the device are maintained at set values.

[0003] The main purpose of controlling the carbon dioxide concentration here is to prevent pH fluctuations within the culture vessel, but generally the carbon dioxide concentration provided by the culture medium manufacturer is set as a set value as a guideline for maintaining the pH within the culture vessel, and the device is simply controlled to maintain the carbon dioxide concentration at the set value.

[0004] However, the relationship between the cells or microorganisms being cultured and pH is close. For example, the pH of human blood is generally maintained at 7.4, and a pH below 7.2 can be fatal. Therefore, strict control of the pH within the culture vessel is essential.

[0005] Conventional technology merely attempts to maintain an estimated pH within the culture vessel by controlling only the carbon dioxide concentration, and is unable to precisely control the pH within the culture vessel taking into account differences in pH between culture medium lots, pH variations due to biased carbon dioxide concentrations within the device, and pH fluctuations due to the metabolism and activity of individual culture subjects, which can result in variations in pH within the culture vessel affecting the results of the culture. Furthermore, in these circumstances, many culture technicians and researchers understand the importance of pH within the culture vessel, and perform time-consuming tasks such as regularly measuring the pH within the culture vessel using various analytical devices such as blood gas analyzers to check for any abnormalities in the culture process.

[0006] In this regard, Patent Document 1 discloses a device and system that can monitor the pH and other factors inside an incubator and issue an alarm if the pH deviates from a specified range. However, this device and system only issues an alarm when an abnormality occurs, and separate measures are required to maintain the pH inside the culture vessel when an abnormality occurs, so there is still a burden on culture technicians and researchers, and further technological development is desired.

[0007] On the other hand, Non-Patent Document 1 reports that better culture results were obtained when changing the pH depending on the cell growth stage in human embryo culture. Non-Patent Document 2 also reports that, as shown in Figure 5, human embryos are cultured after in vitro fertilization from a cleavage stage embryo to a morula and then to a blastocyst, and that the optimal pH differs depending on the growth stage, and that increasing the pH at the end of the cleavage stage embryo allows it to grow into a high-quality blastocyst, improving the effectiveness of infertility treatment.

[0008] Furthermore, Non-Patent Document 3 reports that the culture results were improved by changing the medium components and varying the pH during the culture of mouse embryos, and Non-Patent Document 4 reports that CO ２ It has been reported that when the carbon dioxide concentration was adjusted and the bacteria were cultured at different pH levels, differences were observed at the genetic level.

[0009] Furthermore, it has been reported that in human cell culture or microbial culture, when the culture subject grows and becomes highly confluent, changing the pH in the culture vessel can improve subsequent culture results.

[0010] In conventional technologies, when changing the pH in a culture vessel according to the growth or proliferation stage of the culture subject, it is necessary to temporarily remove the culture subject from the culture device, and then perform time-consuming tasks such as changing the culture medium, adding additives, and transferring the culture subject to a culture device with a different environment. These tasks may result in a decrease in culture quality and efficiency. Therefore, there is a need for the development of a technology that can easily and optimally control the pH in a culture vessel without requiring complicated operations and time-consuming tasks.

[0011] Patent No. 5150837

[0012] J. E. Swain, “Is there an optimal pH for culture media used in clinical IVF?”, Human Reproduction Update, 2012, Vol. 18, Issue 3, p. 333-339E. Adolfsson et al. , “Shift in pH during transition to the embryonic genome impacts embryo development”, ESHRE, 2016A. Abdala et al. , “Different CO2 settings (6.0% vs 7.0%) do have an impact on extracellular pH of culture medium (pHe) and euploidy “rates rather than on blastocyst development: a sibling oocyte study”, Journal of Assisted Reproduction and Genetics (2021) Vol. 38, p. 2915-2923M. Hentemann et al. , “Differential pH in embryo culture”, Fertility and Sterility Vol. 95, Issue 4, March 15, 2011

[0013] Therefore, an object of the present invention is to provide a culture device and a culture method that can measure the pH in a culture vessel and control the pH in the culture vessel, or to provide a culture device and a culture method that can control the pH in the culture vessel so that the pH is appropriate for the growth or proliferation stage of the culture subject.

[0014] In order to solve the above problems, the culture device of the present invention is characterized by comprising a culture chamber in which a culture vessel containing at least a culture medium and / or a culture subject can be placed, a pH measurement unit capable of measuring the pH in the culture vessel, and a control unit that controls the supply or stop of a single gas or a mixed gas into the culture chamber.

[0015] In this way, the pH within the culture vessel is controlled based on the measurement data of the pH within the culture vessel, making it possible to precisely control the pH within the culture vessel, taking into account, for example, differences in pH between culture media and pH fluctuations due to the metabolism and activity of individual culture subjects, and providing a culture environment that eliminates uncertainties caused by the pH within the culture vessel.

[0016] The control unit can also control the supply of gas into the culture chamber so that the pH is set to a predetermined value depending on the growth stage or proliferation stage of the culture object.

[0017] This makes it possible to provide an optimal culture environment according to the growth or proliferation stage of the culture subject without requiring the efforts and complicated operations of culture technicians and researchers, thereby contributing to improving culture quality and results.

[0018] The culture device of the present invention further has a judgment unit that judges the growth stage or proliferation stage of the culture object, and the control unit can also control the supply of gas into the culture chamber, etc., based on the judgment result output from the judgment unit.

[0019] As the growth or proliferation of the culture subject progresses, lactic acid and other waste products generated by the metabolism and activity of the culture subject itself are released into the culture medium, causing the pH inside the culture vessel to change.The determination unit can also determine the growth or proliferation stage of the culture subject based on the change in pH inside the culture vessel measured by the pH measurement unit.

[0020] Alternatively, the culture device of the present invention may optionally further include an imaging device that photographs the culture object, and the determination unit may be configured to determine the growth stage or proliferation stage of the culture object based on the image of the culture object photographed by the imaging device.

[0021] The control unit can also predict transient or immediate changes in pH within the culture vessel when gas supply to the culture chamber is started or stopped, and take this into consideration when controlling the supply of gas to the culture chamber, etc.

[0022] Depending on the culture conditions, it may be difficult to immediately change the pH in the culture vessel to the pH setting value even if the carbon dioxide concentration is changed due to the influence of the buffering action of the culture medium, etc. However, the culture device of the present invention can predict transient or immediate changes in pH, and can quickly bring the pH in the culture vessel to the target pH setting value and control it to maintain it.

[0023] The culture device of the present invention may also optionally be equipped with a stirring means capable of stirring the culture medium and / or the culture object in the culture vessel, or a gas exchange means for permeating gas into the culture medium and / or the culture object in the culture vessel, thereby allowing the pH in the culture vessel to be rapidly changed.

[0024] Furthermore, the culture device of the present invention may optionally be equipped with an abnormality detection unit capable of detecting abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth state of the culture subject, thereby making it possible to quickly grasp abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth state of the culture subject.

[0025] The culture method of the present invention is characterized by comprising a step of measuring the pH in a culture vessel containing a culture medium and / or a culture subject placed in a culture chamber, and a step of controlling the supply or stop of gas into the culture chamber based on the measurement result of the pH in the culture vessel.

[0026] Furthermore, the culture method of the present invention may also include a step of controlling the supply of gas into the culture chamber so that the pH is set to a predetermined value according to the growth stage or proliferation stage of the culture subject.

[0027] The culture method of the present invention may also include a step of controlling the supply of gas into the culture chamber based on the determination result output from a determination unit that determines the growth stage or proliferation stage of the culture object.

[0028] The culture method of the present invention may also include a step of determining the growth stage or proliferation stage of the culture object based on a change in pH within the culture vessel.

[0029] The culture method of the present invention can also include a step of predicting transient or immediate changes in pH within the culture vessel when gas supply to the culture chamber is started or stopped, and taking this into consideration to control the supply of gas to the culture chamber.

[0030] The culture device and culture method of the present invention make it possible to control the pH in the culture vessel taking into consideration differences in pH between culture media and pH fluctuations due to the metabolism and activity of individual culture subjects, which was not possible with conventional technology, and provide a stable culture environment that eliminates uncertainties caused by pH in the culture vessel. Furthermore, the present invention makes it possible to easily change the culture environment to an optimal pH depending on the growth or proliferation stage of the culture subject, which contributes to improving culture quality and culture results.

[0031] 1. An open end view showing one embodiment of the culture device of the present invention. 2. An example of a correlation diagram between pH and carbon dioxide concentration in a culture vessel that can be used in the culture device and culture method of the present invention. 3. A flow diagram illustrating the pH control procedure for the culture device and culture method of the present invention. 4. A flow diagram illustrating the pH control procedure for the culture device and culture method of the present invention. 5. A diagram illustrating the growth stages of a human embryo. 6. A diagram illustrating the proliferation stages of human somatic stem cells. 7. An open end view showing one embodiment of the culture device equipped with an imaging device of the present invention. 8. A block diagram showing one embodiment of the culture device equipped with an imaging device of the present invention. 9. A flow diagram illustrating the pH control procedure for the culture device and culture method of the present invention.

[0032] An embodiment of the culture apparatus and culture method of the present invention will be described below with reference to the drawings. Note that although this embodiment describes several features, not all of these features are necessarily required for the invention, and they may be combined in any desired manner.

[0033] FIG. 1 shows a culture apparatus A according to an embodiment of the present invention.

[0034] The culture device A is equipped with a culture chamber I, an outer door D1, an inner door D2, a shelf F, a pH measurement unit M, a control unit C, an air supply unit K, and a carbon dioxide gas supply unit G, and is capable of storing a culture vessel V in the culture chamber I.

[0035] The opening of the culture chamber I is composed of an outer door D1 and an inner door D2. Because the inner door D2 is made of glass, the inside of the culture chamber I and the culture vessels V stored therein can be observed from the outside without opening the inner door D2. This allows the culture apparatus A to prevent changes in the culture chamber due to the opening and closing of the door, and to provide a stable culture environment.

[0036] There are no particular limitations on the culture vessel V as long as it can accommodate at least the culture medium and / or the culture subject and can be placed in a culture room, and it is of course acceptable to appropriately accommodate additives necessary for culture. Furthermore, the culture vessel V may be optionally divided into areas within the vessel, such as an area that accommodates the culture subject and an area that does not. The culture subject is also not particularly limited, and may be, for example, cells such as animal cells and plant cells, tissues, or microorganisms.

[0037] The pH measurement unit M is composed of a pH measurement control unit m1 and a pH detection unit m2, but there are no particular restrictions on the pH measurement unit that can be used in the culture device and culture method of the present invention as long as it is capable of measuring the pH inside the culture vessel.For example, there is no problem if the pH detection unit m2 and the culture vessel V are molded as a single unit, or if it is a form that can be attached and detached from the culture device A as appropriate.

[0038] The pH detection unit m2 can use various pH detection methods, and it is expected that a detection method using a pH electrode or a detection method using a pH indicator or fluorescent dye will be used, for example.

[0039] Specifically, as shown in Fig. 1, a pH electrode can be placed as a pH detection unit m2 in the culture vessel V to measure the pH inside the culture vessel. In this case, the pH detection unit m2 may be placed in an area of ​​the culture vessel V that contains the culture subject, or may be placed in an area that does not contain the culture subject to measure the pH. Alternatively, it is also possible to measure the pH by placing a separate vessel containing the culture medium to be used for culture near the culture vessel V and placing the pH detection unit m2 therein.

[0040] Another possible method is to add a pH indicator such as phenol red to the culture medium and measure the amount of light emitted in response to the pH using the pH detection unit m2. In this case, the pH indicator may be added to the area containing the culture subject, or it is of course optional to add the pH indicator to an area not containing the culture subject. Alternatively, a method is also possible in which a fluorescent dye is added to the culture medium and the pH detection unit m2 measures the fluorescence intensity in response to the pH, and the pH is measured. Adding the fluorescent dye to an area not containing the culture subject allows for continuous non-invasive measurement of the pH.

[0041] The control unit C receives pH measurement data from the pH measurement unit M, calculates the difference between the pH in the culture vessel and the pH set value, calculates the required carbon dioxide concentration based on this, and controls the atmosphere supply unit K or the carbon dioxide supply unit G to control the pH in the culture vessel.

[0042] 3, the pH in the culture vessel is measured by a pH measurement unit M, and the pH measurement data is transmitted to a control unit C. The connection between the control unit C and the pH measurement unit M is not particularly limited, and may be either wired or wireless (Wi-Fi, Bluetooth, etc.).

[0043] The control unit C calculates the deviation between the pH measurement data and the pH setpoint, and if the deviation is greater than ±α, calculates a target value for increasing or decreasing the carbon dioxide concentration according to the required increase or decrease in pH based on the correlation between pH and carbon dioxide concentration. ±α is a value set to eliminate the effects of measurement error, transient response, and the like. The pH in the culture vessel depends on the buffering action of bicarbonate added to the culture medium and the dissolved carbon dioxide, and the correlation between the pH and carbon dioxide concentration in the culture vessel is inversely proportional as shown in Figure 2. Because the slope and linearity of this correlation vary depending on culture conditions such as the type of culture medium and the size of the culture apparatus, it is possible to calculate a precise target value for increasing or decreasing the carbon dioxide concentration by determining the correlation in advance under the culture conditions to be implemented.

[0044] Next, the control unit C opens and closes the valves in the respective flow paths connecting the atmosphere supply unit K or the carbon dioxide gas supply unit G to the culture chamber I, and controls the supply or stop of the atmosphere or carbon dioxide gas so that the carbon dioxide gas concentration reaches the target carbon dioxide gas increase / decrease value.

[0045] Alternatively, as shown in FIG. 4, the control unit C can grasp the magnitude relationship between the pH measurement data received from the pH measurement unit M and the pH set value, and if the deviation from the pH set value is small, continue supplying carbon dioxide gas, and if the deviation from the pH set value is large, control the carbon dioxide gas supply unit G to stop supplying carbon dioxide gas.

[0046] The pH control can be performed continuously, but can be set to be performed at any interval taking into consideration the buffering action of the culture medium. Furthermore, the pH control is not limited to the supply of carbon dioxide gas or air, and any type of gas can be used as long as it can change the pH inside the culture vessel, and a single gas or a mixed gas may be used.

[0047] As described above, the culture device and culture method of the present invention control the pH based on the measurement data of the pH inside the culture vessel, making it possible to precisely control the pH inside the culture vessel taking into account differences in pH between culture media and fluctuations in pH due to the metabolism and activity of individual culture subjects, and providing a stable culture environment that eliminates uncertainties caused by the pH inside the culture vessel.

[0048] The control unit C calculates the necessary carbon dioxide concentration to achieve a predetermined pH depending on the growth or proliferation stage of the culture subject, and can also control the pH inside the culture vessel by controlling the atmosphere supply unit K or the carbon dioxide supply unit G.

[0049] Specifically, a data recording unit R1 (not shown) provided in the control unit C or accessible by the control unit C via an electrical communication network records the elapsed time from the start of culture and the optimal pH corresponding to the growth or proliferation stage of the cultured object at each elapsed time. The control unit C reads the optimal pH corresponding to the elapsed time from the start of culture from the data recording unit R1 and sets it as the pH set value. The control unit C then calculates the difference between the pH set value and the pH measurement data received from the pH measurement unit M, and controls the pH in the culture vessel as described above.

[0050] As a result, the culture device and culture method of the present invention can control the pH in the culture vessel to an optimal pH depending on the growth or proliferation stage of the culture subject, without requiring complicated operations or time-consuming procedures.

[0051] The culture device A may also include a determination unit E (not shown) that determines the growth stage or proliferation stage of the culture object. The control unit C may also control the supply of gas into the culture chamber based on the determination result output from the determination unit E.

[0052] As the culture subject grows or proliferates, waste products such as lactic acid generated by the subject's own metabolism and activity are released into the medium, causing changes in the pH within the culture vessel. The determination unit E calculates the difference between the pH measurement data received from the pH measurement unit M and the set pH value, and determines the change in pH within the culture vessel corresponding to the growth or proliferation stage of the subject. The determination unit E accesses a data recording unit R2 (not shown), which is provided in the determination unit E or accessible by the determination unit E via an electrical communication network, and determines the growth or proliferation stage of the subject based on the change in pH within the culture vessel. The data recording unit R2 records the growth or proliferation stage of the subject when cultured under specified culture conditions, as well as the change in pH within the culture vessel corresponding to each stage. The determination unit E outputs the growth or proliferation stage corresponding to the calculated change in pH within the culture vessel from the data recording unit R2 and transmits the determination result to the control unit C. Note that changes in pH within the culture vessel are affected by factors such as the type of culture medium, the type of culture medium, the amount of culture medium (thickness within the culture vessel), and the amount of carbon dioxide gas supplied. These factors are taken into account when recording the changes in pH within the culture vessel in the data recording unit R2. Next, the control unit C accesses a data recording unit R3 (not shown) that is provided in the control unit C or that the control unit C can access via an electrical communication network, and reads out the optimal pH within the culture vessel corresponding to the determination result of the growth or proliferation stage of the culture vessel received from the determination unit E. The data recording unit R3 records the growth or proliferation stage of the culture vessel and the optimal pH within the culture vessel at each stage. The control unit C sets the read-out optimal pH within the culture vessel as the pH setpoint, calculates the deviation between the pH setpoint and the pH measurement data received from the pH measurement unit M, and controls the pH within the culture vessel as described above.

[0053] In this way, the culture apparatus and culture method of the present invention can determine the growth or proliferation stage of the culture object from changes in pH within the culture vessel, and can control the pH based on the determination results.

[0054] The control unit C can also predict transient or immediate changes in pH within the culture vessel when gas supply to the culture chamber is started or stopped, and control the supply of gas to the culture chamber taking this into consideration.

[0055] Controlling the gas supply is essential for timely changes in the pH of the medium according to the growth of the embryos. Below, we will explain the change in pH of the medium when the carbon dioxide concentration is simply changed, with reference to an example shown in Table 1.

[0056]

[0057] Table 1 shows the change in pH over time in 100 μL of KSOM medium, a medium for mouse embryos, placed in a culture vessel with the surface covered with mineral oil, as the carbon dioxide concentration in the culture chamber was increased from 5% to 10%.

[0058] In embryo culture, the surface of the medium is sometimes covered with oil to prevent it from drying out, but this oil can slow the penetration of carbon dioxide into the medium, making it take time for the pH to stabilize. The results shown in Table 1 are thought to be due to the time lag mentioned above occurring before the pH stabilizes.

[0059] In this regard, in the culture of human embryos, the period from fertilization to implantation is approximately 3 to 7 days. Therefore, in order to adjust the culture medium pH to an appropriate level during this time depending on the stage of embryo development, the time lag described above may not be negligible in relation to the culture results, making it essential to dynamically manage and adjust the culture medium pH value.

[0060] Therefore, the control unit C can timely adjust the pH of the culture medium using the following method. Specifically, the control unit C accesses a data recording unit R4 (not shown) that is provided in the control unit C or that the control unit C can access via an electrical communication network, and reads the record of the pH in the culture vessel when the carbon dioxide concentration is changed under specified culture conditions. Based on the read record, the control unit C predicts the transient or immediate change in the pH in the culture vessel that will occur when the gas supply rate is changed to a specified carbon dioxide concentration, calculates the carbon dioxide concentration required to quickly change the pH in the culture vessel to the pH setpoint, and controls the supply of gas into the culture chamber, etc. Furthermore, after the pH in the culture vessel has been changed to the pH setpoint, the control unit C calculates the carbon dioxide concentration required to maintain the pH setpoint and controls the supply of gas into the culture chamber, etc. The control unit C can also read the record of the pH in the culture vessel from the data recording unit R4, taking into account the initial pH change rate after the carbon dioxide concentration is changed. Depending on the culture conditions, it may be difficult to immediately change the pH in the culture vessel even if the carbon dioxide concentration is changed due to the influence of the buffering action of the culture medium, etc. However, in this way, culture apparatus A can predict the transient or immediate change in pH in the culture vessel when gas supply to the culture chamber is started or stopped, and can quickly change the pH in the culture vessel to the pH set value.

[0061] The operation of the control unit C and the resulting changes in pH will be described below with reference to Table 2. In Table 2, (a) shows the set value of the gas supply concentration at each time, (b) shows the change in gas concentration in the culture chamber, and (c) shows the change in culture medium pH. The vertical axis of (a) shows the set value S of the gas supply concentration, the vertical axis of (b) shows the measured value G of the gas concentration in the culture chamber, and the vertical axis of (c) shows the measured value P of the culture medium pH, and each horizontal axis shows time H. This shows how the culture medium pH is adjusted in a timely manner by predicting and controlling the optimal gas supply concentration to adjust to a target pH value based on the transient changes in the culture medium pH that were measured and recorded in advance along with the changes in the gas concentration in the culture chamber.

[0062]

[0063] Here, in the example shown in Table 2, the relationship between the gas concentration G in the culture chamber and the culture medium pH value P is shown when the control unit C is used to change the culture medium pH from a state in which the gas concentration G in the culture chamber is maintained at G1 and the measured value P of the culture medium pH is maintained at P1 to P2, and the gas supply concentration S is set to S1 and controlled by reading and predicting from the data recording unit R4.

[0064] Furthermore, the control unit C can minimize the time lag and adjust the pH more timely by temporarily supplying gas at a concentration higher than the originally required gas supply concentration S. The operation of this control unit C and the resulting change in pH will be described with reference to Table 3.

[0065]

[0066] In the example shown in Table 3, the control unit C temporarily supplies gas at a gas supply concentration S2 that is higher than the gas supply concentration S1 that is normally required for adjustment, and adjusts the gas supply concentration to S1 at time H4. This allows the medium pH to reach the target pH value P2 at time H5, which is earlier than the arrival time H3 in the example shown in Table 2.

[0067] The value of this gas supply concentration S2 can be obtained as a predicted control value by recording in advance the transient changes in culture medium pH due to changes in gas supply concentration S, or conditions such as the culture target, the initial value before the change in culture medium pH and the changed value, the type and volume of culture medium, and the type and volume of mineral oil, and by incorporating these into an approximate formula while taking into account and adjusting for slight deviations contained in the actual measurement results.

[0068] The culture apparatus A may optionally be provided with a stirring means, such as a pump, capable of stirring the medium and / or the culture medium in the culture vessel, thereby enabling the pH in the culture vessel to be quickly changed to a predetermined pH.

[0069] The culture apparatus A may also be equipped with a gas exchange means for supplying air or carbon dioxide gas via the air supply unit K or carbon dioxide gas supply unit G. Specifically, air or carbon dioxide gas can be supplied to the medium and / or the culture target in the culture vessel while bubbling, or can be supplied through hollow fibers. This increases the surface area of ​​the medium, facilitating the penetration of air or carbon dioxide gas into the medium, and allowing the pH in the culture vessel to be quickly adjusted to a predetermined pH.

[0070] The culture device A also includes an anomaly detection unit S (not shown) that can detect abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth conditions of the cultured object. For example, if contaminants such as mold are introduced into the culture vessel, the pH of the culture medium will change rapidly. Alternatively, if the growth conditions of the cultured object are poor, changes in the pH of the culture medium due to the metabolism or activity of the cultured object itself will be delayed. The anomaly detection unit S detects an abnormality in the culture medium in the culture vessel and / or abnormalities in the growth conditions of the cultured object when the pH measurement data received from the pH measurement unit M shows a predetermined change within a certain period of time. If such an abnormality is detected, the anomaly detection unit S can also display an "Abnormality" warning on a terminal device (not shown) accessible via a telecommunications network. Thus, the culture device and culture method of the present invention can quickly detect abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth conditions of the cultured object, thereby providing a stable culture environment.

[0071] 7 and 8 show a culture apparatus B according to an embodiment of the present invention, which is equipped with an imaging device P.

[0072] The culture device B is provided with an imaging device P and has the same configuration as the culture device A.

[0073] The imaging device P is composed of an imaging device control unit p1, a camera unit p2, an objective lens p3, and a light p4, but the imaging device that can be used with the culture device and culture method of the present invention is not limited to this embodiment, and there are no particular restrictions as long as it is possible to photograph the culture object, and it is of course also possible to use one in which multiple culture vessels and an imaging device are integrated, such as a time-lapse culture device.

[0074] The culture device B photographs the culture object continuously or at predetermined time intervals using an imaging device P, and performs image analysis on the photographed images in a judgment unit E. When it is determined that the culture object has reached a predetermined state, the culture device B controls the pH inside the culture vessel by controlling the air supply unit K or the carbon dioxide gas supply unit G so that the pH inside the culture vessel becomes optimal for the growth or proliferation stage of the culture object.

[0075] For example, when a human embryo is to be cultured, first, the embryo is photographed continuously or at predetermined time intervals by the imaging device P according to the flow shown in FIG.

[0076] The imaging device P transmits the captured image from the imaging device control unit p1 to the control unit C provided in the culture device B. The control unit C then transmits the image to the determination unit E. Alternatively, the imaging device P can be configured to transmit the captured image from the imaging device control unit p1 to the determination unit E. There are no particular restrictions on the connection format of the imaging device control unit p1, the control unit C, and the determination unit E, and they may be wired or wireless (Wi-Fi, Bluetooth, etc.). It is also optional to configure the control unit C and the determination unit E as an integrated unit.

[0077] Next, the determination unit E performs image analysis on the image. As shown in FIG. 5 , the number of cells in a human embryo increases due to cell division and its external shape changes depending on the growth stage. A feature extraction unit e1 (not shown) included in the determination unit E extracts the number of cells, shape, size, and other characteristics of the embryo based on the image. An image data recording unit e2 (not shown) included in the determination unit E records image information of the embryo and information on the corresponding growth stage. The image data recording unit e2 recognizes the image of the embryo based on the feature information extracted by the feature extraction unit e1 and reads out the corresponding growth stage to determine the growth stage of the embryo. The image data recording unit e2 may also be configured to record information on the elapsed time since the start of culture. The determination unit E can calculate the elapsed time since the start of culture based on information on the time the image was captured and perform image analysis taking the elapsed time into account, thereby enabling accurate determination of the growth stage.

[0078] The evaluation unit E transmits the evaluation result to the control unit C. When the embryo reaches the morula stage, which requires a change in the pH setpoint, the control unit C reads the optimal pH in the culture vessel from the data recording unit R3 and changes the pH setpoint. Furthermore, the control unit C calculates the deviation between the pH measurement data and the pH setpoint, and calculates a target value for increasing or decreasing the carbon dioxide concentration according to the required increase or decrease in pH based on the correlation between pH and carbon dioxide concentration. The control unit C opens and closes valves in the respective flow paths connecting the atmosphere supply unit K or the carbon dioxide supply unit G to the culture chamber I, controlling the supply or stop of the atmosphere or carbon dioxide gas so that the carbon dioxide gas concentration reaches the target increase or decrease value. If it takes time for the medium to reach the optimal pH after the carbon dioxide gas concentration is changed, the pH setpoint may be changed to the optimal pH taking that time into account when it is determined that the embryo has reached an octa-section embryo, which is one stage before the morula stage.

[0079] 6, for example, when culturing human somatic stem cells or microorganisms, the imaging device P captures images of a predetermined area of ​​the culture vessel V continuously or at predetermined time intervals, and transmits the images from the imaging device control unit p1 to the determination unit E. The determination unit E can also perform image analysis on the images received from the imaging device control unit p1 and determine the proliferation stage by calculating the density of cells or microorganisms per unit area.

[0080] The determination unit E transmits the determination result to the control unit C. When the cell or microorganism density per unit area reaches a predetermined value, the control unit C accesses a data recording unit R5 (not shown) provided in the control unit C or accessible by the control unit C via an electrical communication network and reads out the corresponding optimal pH. The data recording unit R5 records the cell or microorganism density per unit area and the optimal pH corresponding to each density. The control unit C changes the pH set value to the optimal pH, calculates the deviation between the pH measurement data and the pH set value, and calculates a target value for increasing or decreasing the carbon dioxide concentration according to the required increase or decrease in pH based on the correlation between pH and carbon dioxide concentration. The control unit C opens and closes valves in the respective flow paths connecting the atmosphere supply unit K and the carbon dioxide supply unit G to the culture chamber I, and controls the supply or stop of the atmosphere or carbon dioxide so that the carbon dioxide concentration reaches the target increase or decrease value.

[0081] Of course, in the case of the culture device B, when it is not necessary to change the pH setting value depending on the growth or proliferation stage of the culture subject, control is performed to maintain a predetermined pH setting value, similar to the pH control described for the culture device A, and the timing of the pH control and the timing of the photographing by the imaging device P can each be performed at any timing.

[0082] In this way, the culture device and culture method of the present invention can provide an optimal culture environment according to the growth or proliferation stage of the culture subject without requiring the efforts or complicated operations of embryologists or researchers. Therefore, for example, in the culture of human embryos, it can increase the probability of development from a fertilized egg to a blastocyst, and ultimately contribute to increasing the success rate of infertility treatment, and can also contribute to improving the culture quality and results in the culture of human cells and microorganisms, for example.

[0083] The anomaly detection unit S included in the culture device B can also detect anomalies in the culture medium in the culture vessel and / or anomalies in the growth state of the culture object based on images of the culture medium and / or the culture object in the culture vessel captured by the imaging device P. Specifically, the anomaly detection unit S receives images of the culture medium and / or the culture object in the culture vessel captured by the imaging device P. The anomaly detection unit S performs image analysis on the images. For example, since the external shape, size, etc. of a human embryo and a contaminant such as mold differ, the anomaly detection unit S detects an anomaly in the culture medium when a feature extraction unit s (not shown) included in the anomaly detection unit S extracts features that differ from those of the culture object. For example, since the external shape, etc. of a human embryo changes depending on the growth stage as described above, the anomaly detection unit S detects an anomaly in the growth state of the culture object when there is no predetermined change within a certain period of time in the features of the culture object extracted by the feature extraction unit s. When such an abnormality is detected, the abnormality detection unit S displays a warning such as "abnormal culture medium" or "abnormal growth state of the culture subject" on a terminal device or the like accessible via an electrical communication network, and can also display the image received from the imaging device P. In this way, the culture device and culture method of the present invention can detect abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth state of the culture subject in more detail based on images.

[0084] The present application encompasses the following inventions: [1] A culture device comprising a culture chamber capable of accommodating a culture vessel containing at least a culture medium and / or a culture subject, a pH measurement unit capable of measuring the pH in the culture vessel, and a control unit that controls the supply or stop of a single gas or a mixed gas into the culture chamber. [2] The culture device described in [1], wherein the control unit controls the supply of gas, etc. into the culture chamber to achieve a pH that is preset according to the growth or proliferation stage of the culture subject. [3] The culture device described in [1] or [2], further comprising a determination unit that determines the growth or proliferation stage of the culture subject, and the control unit controls the supply of gas, etc. into the culture chamber based on the determination result output from the determination unit. [4] The culture device described in [3], wherein the determination unit determines the growth or proliferation stage of the culture subject based on a change in pH in the culture vessel measured by the pH measurement unit. [5] The culture device described in [3] or [4], further comprising an imaging device that photographs the culture subject, and the determination unit determines the growth or proliferation stage of the culture subject based on an image of the culture subject photographed by the imaging device. [6] The culture device according to any one of [1] to [5], wherein the control unit predicts a transient or immediate change in pH in the culture vessel when gas supply to the culture chamber is started or stopped, and controls the gas supply, etc. to the culture chamber taking this into consideration. [7] The culture device according to any one of [1] to [6], which has a stirring means capable of stirring the culture medium and / or the culture object in the culture vessel. [8] The culture device according to any one of [1] to [7], which has a gas exchange means for permeating the gas into the culture medium and / or the culture object in the culture vessel. [9] The culture device according to any one of [1] to [8], which further has an abnormality detection unit capable of detecting an abnormality in the culture medium in the culture vessel and / or an abnormality in the growth state of the culture object.

[10] The culture device according to any one of [1] to [9], wherein the pH measurement unit is detachable.

[11] A culture method comprising the steps of measuring the pH in a culture vessel containing a culture medium and / or a culture object placed in a culture chamber, and controlling the supply or stop of gas to the culture chamber based on the measurement result of the pH in the culture vessel.

[12] The culture method according to

[11] , comprising a step of controlling the gas supply, etc. into the culture chamber so that the pH is set to a predetermined value according to the growth or proliferation stage of the culture object.

[13] The culture method according to

[11] or

[12] , comprising a step of controlling the gas supply, etc. into the culture chamber based on a determination result output from a determination unit that determines the growth or proliferation stage of the culture object.

[14] The culture method according to

[13] , comprising a step of determining the growth or proliferation stage of the culture object based on a change in pH within the culture vessel.

[15] The culture method according to any of

[11] to

[14] , comprising a step of predicting a transient or immediate change in pH within the culture vessel when gas supply to the culture chamber is started or stopped, and controlling the gas supply, etc. into the culture chamber taking this into consideration.

[0085] As described above, the present invention can control the pH within a culture vessel based on measured data of the pH within the culture vessel, and enables control of the pH within the culture vessel taking into account differences in pH between culture media and pH fluctuations due to the metabolism and activity of individual culture subjects, which was not possible with conventional technology, making it possible to provide a stable culture environment that eliminates culture uncertainties caused by the pH within the culture vessel. Furthermore, it is possible to easily change the culture environment to an optimal pH according to the growth and proliferation stage of the culture subject without requiring the time and effort of culture technicians or researchers or complicated operations, making it extremely useful for application to culture devices and culture methods related to the cultivation of any culture subject.

[0086] A Cultivation device I Cultivation room D1 Outer door D2 Inner door F Shelf M pH measurement unit m1 pH measurement control unit m2 pH detection unit C Control unit E Determination unit K Air supply unit G Carbon dioxide gas supply unit V Cultivation vessel B Cultivation device P Imaging device p1 Imaging device control unit p2 Camera unit p3 Objective lens p4 Lighting T1 Cleavage stage embryo period T2 Blastocyst period T3 Low confluence state of culture subject T4 High confluence state of culture subject

Claims

1. A culture device characterized by comprising a culture chamber in which a culture vessel containing at least a culture medium and / or a culture subject can be placed, a pH measurement unit capable of measuring the pH within the culture vessel, and a control unit that controls the supply or stop of a single gas or a mixed gas into the culture chamber.

2. The culture device according to claim 1, wherein the control unit controls the supply of gas into the culture chamber so as to achieve a predetermined pH depending on the growth or proliferation stage of the culture subject.

3. A culture device as described in claim 1 or 2, further comprising a judgment unit that judges the growth stage or proliferation stage of the culture object, and the control unit controls the supply of gas into the culture chamber, etc. based on the judgment result output from the judgment unit.

4. The culture device according to claim 3, wherein the determination unit determines the growth stage or proliferation stage of the culture object based on the change in pH in the culture vessel measured by the pH measurement unit.

5. A culture device as described in claim 3 or 4, further comprising an imaging device that photographs the culture object, and the determination unit determines the growth stage or proliferation stage of the culture object based on the image of the culture object photographed by the imaging device.

6. A culture device according to any one of claims 1 to 5, wherein the control unit predicts transient or immediate changes in pH within the culture vessel when gas supply to the culture chamber is switched on or off, and controls the supply of gas to the culture chamber taking this into account.

7. The culture device according to any one of claims 1 to 6, which has stirring means capable of stirring the medium and / or the culture object in the culture vessel.

8. The culture apparatus according to any one of claims 1 to 7, further comprising a gas exchange means for allowing the gas to permeate the medium and / or the culture object in the culture vessel.

9. The culture device according to any one of claims 1 to 8, further comprising an abnormality detection unit capable of detecting abnormalities in the culture medium in the culture vessel and / or abnormalities in the growth state of the culture subject.

10. The culture device according to any one of claims 1 to 9, wherein the pH measuring unit is detachable.

11. A culture method comprising the steps of measuring the pH in a culture vessel containing a culture medium and / or a culture subject placed in a culture chamber, and controlling the supply or stop of gas into the culture chamber based on the measurement result of the pH in the culture vessel.

12. A culture method as described in claim 11, which includes a step of controlling the supply of gas, etc. into the culture chamber so that the pH is set to a predetermined value according to the growth or proliferation stage of the culture subject.

13. A culture method according to claim 11 or 12, further comprising a step of controlling the supply of gas into the culture chamber based on the determination result output from a determination unit that determines the growth stage or proliferation stage of the culture object.

14. The culture method according to claim 13, further comprising a step of determining the growth or proliferation stage of the culture object based on a change in pH within the culture vessel.

15. A culture method according to any one of claims 11 to 14, comprising a step of predicting a transient or immediate change in pH in the culture vessel when gas supply to the culture chamber is started or stopped, and taking this into account to control the supply of gas to the culture chamber.

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