JP4778746B2 - Cell culture equipment - Google Patents

Description

  The present invention relates to a cell culture device, and more specifically, to a cell culture device suitable for appropriately performing cell culture monitoring.

  An automatic cell culture device that automatically performs cell culture is a thermostat that maintains culture conditions such as temperature and concentration of a specific gas at set conditions, an incubator that is provided inside the thermostat and cultures cells, A chemical supply means for supplying chemicals from a chemical container provided outside to the incubator is provided. Such an automatic cell culture apparatus has an advantage that contamination such as contamination can be prevented in addition to the advantage of performing complicated culture operations automatically. Therefore, chemical containers for storing chemicals, chemical supply means, thermostatic chambers, incubators, containers for storing waste liquid discharged from the incubators, etc. are stored in a closed casing, and further operations such as chemical replacement are performed. It is automated so that cells do not come into direct contact with the outside through liquids or gases.

  In addition, in order to confirm the quality of the culture sample at the time of culture, component analysis of the culture medium is performed during culture, cell culture monitoring such as feedback to culture conditions such as medium exchange, recording of culture history, and warning to equipment operators, etc. It is desirable to do. When performing this cell culture monitoring, avoid sampling directly from the incubator from the viewpoint of preventing contamination. For example, install an analytical sensor in the waste liquid path of the incubator and analyze the data from the analytical sensor as needed. Is desirable.

  By the way, when cell culture monitoring is performed based on the components of the culture medium in the incubator and feedback to the culture conditions, recording of the culture history, warning to the operator, etc., the culture of the components of the culture medium in the incubator is performed. If conditions change drastically, it will hinder accurate monitoring of the culture state. In particular, at the time of medium replacement, chemicals with significantly different conditions such as the temperature of the culture solution in the previous incubator and the concentration of specific gases (for example, carbon dioxide, nitrogen gas, oxygen gas, etc.) are supplied to the incubator. Then, the metabolic rate of the cells changes due to changes in the culture conditions. As a result, in cell culture monitoring, it may be difficult to check the quality of cultured cells by monitoring changes in cell properties.

  However, the conditions such as the temperature of the replacement chemical stored in the chemical container and the conditions such as the temperature of the culture solution in the incubator are generally greatly different. For example, in the case of general mammalian cell culture, in order to suppress changes in the components of the replacement chemical, the replacement chemical is stored under the environmental conditions in the refrigerator (4 ° C., approximately 0.03% carbon dioxide (atmospheric conditions)). Is done. On the other hand, the culture solution in the incubator is an environmental condition (37 ° C., 5% carbon dioxide gas) of a carbon dioxide incubator (constant temperature bath). Therefore, when the replacement chemical is directly supplied to the incubator, the culture conditions change rapidly. For example, a general mammalian culture solution such as DMEM uses a sodium hydrogen carbonate solution as a buffer, so the environment in the medium (for example, pH, etc.) is greatly affected by the temperature and carbon dioxide concentration. To change. The environment in the medium once changed gradually changes through temperature, gas exchange, etc., but it usually takes several hours to return to the original environment. Due to such temporary changes in culture conditions, the cells themselves are also affected, and the metabolic rate of the cells changes temporarily. Make detection difficult.

An object of the present invention is to enable appropriate cell culture monitoring to confirm the quality of cultured cells even immediately after the supply of chemicals .

In order to solve the above-mentioned problems, the present invention is provided in a thermostatic chamber in which the temperature and the concentration of a specific gas are maintained at set conditions, a cell incubator provided in the thermostatic chamber, and the outside of the thermostatic layer. Assuming a cell culture device equipped with a chemical supply means for supplying chemicals stored in a chemical container to an incubator, either in the thermostatic chamber or in a closed space of the same environment as the thermostatic chamber connected to the thermostatic chamber On the other hand, there is provided a chemical holding means for holding the chemical supplied from the chemical container to the incubator for a set time, and the chemical holding means is formed by a film having thermal conductivity and transmitting the specific gas. It is characterized by holding chemicals in a container.

  Thus, before the chemical | medical agent preserve | saved in the low temperature atmospheric environment is supplied to an incubator, it is once hold | maintained at the holding | maintenance container provided in the same environment as the inside of a thermostat. And since the chemical | medical agent in a holding | maintenance container absorbs the specific gas in a thermostat through a gas permeation | transmission film | membrane and is heated, it approaches the same environmental conditions as the culture solution in an incubator. Therefore, by changing the medium when the chemical in the holding container approaches the culture condition of the culture solution or when the same culture condition is reached, changes in the culture condition can be suppressed. As a result, it is possible to accurately perform cell culture monitoring that continuously monitors changes in cell properties and confirms the quality of cultured cells.

  In this case, a control means is provided to supply and hold the chemical in the chemical container to the holding container, and the temperature of the chemical in the holding container and the concentration of the specific gas are within the allowable range for the temperature of the thermostat and the concentration of the specific gas. After matching, the culture solution in the incubator can be discharged and the medicine in the holding container can be supplied to the incubator. According to this, the change of culture conditions can be suppressed reliably. Here, the determination as to whether or not the temperature in the holding container and the constant temperature bath and the concentration of the specific gas are within the allowable range can be made, for example, based on the holding time in the holding container. That is, the heating in the holding container and the absorption amount of the specific gas correlate with the gas permeability and heat conductivity of the holding container and time. Instead, in the case of a general mammalian culture solution and the specific gas is carbon dioxide, it can also be determined by the pH of the chemical in the holding container and the incubator. That is, the pH of the chemical in the holding container is determined according to the temperature and the concentration of carbon dioxide gas.

  Further, the present invention is provided with a culture medium analysis means for analyzing the components of the culture medium in the incubator, and a cell culture monitoring control means for controlling the medium exchange of the incubator based on the analysis value of the culture medium analysis means, By this cell culture monitoring control means, it is possible to supply the medicine from the medicine container to the medicine holding means based on the analysis value of the culture solution and hold the medicine to replace the medium.

  In this case, a holding chemical analysis means for analyzing the components of the holding chemical in the holding container is further provided, and the cell culture monitoring control means has a difference between the analysis value of the culture solution and the analysis value of the holding drug at the time of medium replacement. Based on this, the culture conditions can be controlled. Thereby, the cell culture monitoring which monitors the change of the property of a cell and confirms the quality of a cultured cell can be performed more exactly.

  In the present invention, instead of a holding container having thermal conductivity and gas permeability, a medicine holding container having an open top surface is provided in the upper part of the incubator, and the medicine holding container is supplied from a medicine supply means. The medicine can be temporarily retained. In this case, when the temperature of the chemical in the chemical container and the condition of the concentration of the specific gas match within the allowable range, after discarding the culture solution in the incubator, tilt the incubator with a tilting device, The medium in the medicine holding container can be overflowed and injected into the incubator to change the medium.

According to the present invention, it is possible to appropriately perform cell culture monitoring for confirming the quality of cultured cells even immediately after supplying chemicals .

  Hereinafter, the present invention will be described based on examples.

  FIG. 1 is a diagram showing the configuration of the cell culture device of Example 1 of the present invention. In this example, the case where cell culture is performed using a mammalian culture solution will be described as an example. The cell culture apparatus of the present embodiment includes a culture vessel 1 for culturing cells, a culture vessel 2 that contains the culture vessel 1 and is a constant temperature bath in which the temperature and the concentration of a specific gas are maintained under set conditions, A storage 4 having a plurality of chemical containers 3 (only one is shown in FIG. 1 to avoid complication) is provided.

  The chemical supply means for supplying the chemical to the incubator 1 includes an injection channel 5 provided from the chemical container 3 to the incubator 1, a valve 7 provided in the injection channel 5 in order from the upstream side, a pump 8, It has a heating bag 9, a valve 10, and a pump 11. Among these chemical supply means, the valve 7 and the pump 8 are installed in the storage 4, and the heating bag 9, the valve 10, and the pump 11 are in the culture tank 2 or in the closed space 6 communicated with the culture tank 2. Is installed. The heating bag 9 is formed of a film having thermal conductivity and transmitting the specific gas.

  The culture solution in the incubator 1 is provided in the waste solution bottle 16 via the waste solution channel 12 so that the waste solution can be used. The waste liquid flow path 12 is provided with a valve 13 and a pump 14 in order from the upstream side. In addition, a pH sensor 15 is provided in the waste liquid flow path 12 on the discharge side of the pump 14.

  The culture tank 2 is provided with a temperature sensor 17, a carbon dioxide concentration sensor 18, a heater 19, and a valve 21 that adjusts the supply amount of the carbon dioxide gas 20, and the control device 22 is provided with the temperature sensor 17 and the carbon dioxide concentration sensor 18. Based on this detection signal, the heater 19 and the valve 21 are controlled so that the temperature and the carbon dioxide concentration in the culture tank 2 are maintained at the set values. Note that the culture conditions differ depending on the cells, and the nitrogen concentration and oxygen concentration may be controlled.

Next, the operation of this embodiment configured as described above will be described. For example, when a culture experiment is performed with a chemical container 3 having a capacity of 500 ml and an incubator 1 having a capacity of 500 cm ³ and a medium amount of 150 ml, and the medium is changed once in about 3 to 4 days, The culture can be performed for about 10 days to 2 weeks. The amount of culture medium and the number of culture days vary depending on the cell type and culture schedule.

  When culturing cells, the temperature sensor 17 and the carbon dioxide gas concentration sensor 18 detect the temperature and carbon dioxide gas concentration in the culture tank 2, and based on this, the controller 22 adjusts the heater 19 and the valve 21 to cultivate the cells. The temperature in the vessel 1 and the carbon dioxide gas concentration are maintained at preset conditions. For example, the conditions of temperature and carbon dioxide concentration are maintained at the same culture conditions (temperature: 37 ° C., carbon dioxide concentration: 5%) as in the case of general mammalian cells.

  During culturing, components such as pH and glucose of the culture solution in the incubator 1 change due to cell metabolism and proliferation. In order to monitor this change, the valve 13 is temporarily opened and the pump 14 is operated periodically at the time of medium exchange or between the intervals, and the culture medium in the incubator 1 is transferred to the waste liquid bottle 16 little by little. . Although the pH detection is performed in the monitoring of this embodiment, the metabolic rate of the cells changes due to the change in carbon dioxide concentration and the change in pH. Therefore, in addition to pH detection, components such as glutamine, glutamate, glucose, lactate, oxygen, ammonia, sodium, potassium, calcium, etc. are used as measurement items for recording detection results and feeding back to cell culture. May be measured.

  In this embodiment, the pH of the culture solution is detected by the pH sensor 15 as culture monitoring. This pH is detected based on the difference between the absorption peak obtained by transmitting visible light through a known cell culture solution and the known culture solution, as described in, for example, Japanese Patent Publication No. 06-034754. Is going. In the process of culturing, the medium is regularly changed.

  In FIG. 2, the flowchart of the control action at the time of the culture medium exchange of the control apparatus 22 is shown. The pH is detected during regular monitoring (S51), and it is determined whether or not the detected value has reached the threshold value for medium replacement (S52). If it is determined that the threshold value has not been reached, medium exchange is not performed. On the other hand, when it is determined that the threshold value has been reached, the valve 7 of the injection channel 5 is opened, the pump 8 is driven, and the necessary amount of the culture solution in the medicine container 3 is added to the warming bag 9 (150 ml in this example). When the required amount is injected, the valve 7 is closed and the pump 8 is stopped (S53). The injection amount at this time is detected by a liquid amount sensor (not shown).

  The culture solution injected into the heating bag 9 is held for a set time (for example, about 2 hours) (S54). The heating bag 9 is made of a resin such as silicon or plastic having thermal conductivity and gas exchange. Therefore, by providing in the closed space 6 which is the same condition as the condition in the culture tank 2, parameters such as temperature, carbon dioxide concentration, pH and the like are almost the same as the parameters of the culture solution in the incubator 1 when the set time is maintained. Become. Since the parameters of the culture solution in the incubator 1 change due to cell metabolism and proliferation, the components cannot be made the same, but this retention minimizes changes in the medium environment that occur during conventional medium exchange. Can be suppressed. The holding time in the warming bag 9 is not limited to 2 hours, and the set time is generally determined between 5 minutes and 8 hours. In addition, a heating device, a vibration device, or the like can be used to bring the parameters closer to each other more quickly.

  After maintaining the set time, in order to replace the culture solution, the valve 13 of the waste solution flow path 12 is opened, and the pump 14 is driven to discharge the culture solution in the incubator 1 to the waste solution bottle 16. When the discharge of the culture solution is completed, the valve 13 is closed, the pump 14 is stopped, the valve 10 is opened, the pump 11 is driven, and the culture solution held in the heating bag 9 is injected into the incubator 1 ( S54).

  As described above, since the medicine in the medicine container 3 is temporarily held in the heating bag 9 provided in the same environment as that in the culture tank 2 before the medicine is supplied to the incubator 1, the medicine in the holding container is While absorbing carbon dioxide gas through the gas permeable membrane, it is heated and approaches the same environmental conditions as the culture solution in the incubator 1. Therefore, changes in culture conditions can be suppressed even when the medium is changed. As a result, it is possible to accurately perform cell culture monitoring that continuously monitors changes in cell properties and confirms the quality of cultured cells. That is, since an excessive change in culture conditions can be suppressed at the time of medium exchange, changes in the medium caused by cell metabolism and proliferation can be accurately monitored even immediately after medium exchange.

  In FIG. 3, the structure of the cell culture apparatus of the other Example of this invention is shown. The present embodiment is different from the embodiment of FIG. 1 in that a temporary holding container 31 is provided in the upper part of the incubator 1 instead of the heating bag 9 holding the replacement chemical, and the temporary holding container 31 is held. It is that the incubator 1 is tilted by a tilting device and injected. Therefore, the same components as those in the embodiment of FIG.

  As shown in FIG. 3, the temporary holding container 31 is a dish-shaped shallow container provided in the upper space in the incubator 1, and the entire upper surface is opened. The temporary holding container 31 is provided with one side edge fixed to the inner wall of the incubator 1. Further, the incubator 1 is placed and held on a stage 32, and the stage 32 is cultured through a rotating shaft 33 provided at the bottom of the incubator 1 on the side where the temporary holding container 31 is fixed. It is attached to the tip of a fixing material 34 fixed to the tank 2. In addition, a tip of a bar 36 that is lifted and lowered by a lifting device 35 is provided on the lower side of the stage 32 at a position away from the rotation shaft 33. Thereby, the stage 32 can be tilted around the rotation axis 33.

  The operation of the present embodiment configured as described above will be described with reference to the flowchart of the control operation at the time of medium replacement of the control device 22 shown in FIG. As in the embodiment of FIG. 1, the pH sensor 15 detects the pH of the medium in the incubator 1 (S61), and it is determined whether or not to change the medium (S62). When it is determined that the medium is to be exchanged, the valve 7 of the injection channel 5 is opened and the pump 8 is driven, and the necessary amount of the culture solution stored in the chemical container 3 is stored in the temporary holding container 31 (150 ml in this example). When the required amount is injected, the valve 7 is closed and the pump 8 is stopped (S63). The injection amount at this time is detected by a liquid amount sensor (not shown).

  The culture solution injected into the temporary holding container 31 is held for a set time (for example, about 2 hours) (S64). Here, since the temporary holding container 31 is provided in the incubator 1 and the upper surface is opened, parameters such as temperature, carbon dioxide concentration, pH, and the like are maintained by holding the culture solution for a set time. The parameters of the culture solution in 1 are almost the same. By this holding, it is possible to minimize changes in the medium environment that occur during conventional medium exchange. The holding time is not limited to 2 hours, and the set time is generally determined between 5 minutes and 8 hours.

  After holding for the set time, the culture solution in the incubator 1 is discharged as in Example 1 (S65), and the culture solution held in the temporary holding container 31 is injected into the incubator 1 (S66). In this injection, when the bar 36 is lowered by driving the lifting / lowering device 35 from the control device 22, the stage 32 rotates and tilts around the rotation shaft 33. The temporary holding container 31 is tilted in accordance with this inclination, and the culture solution overflows from the edge of the dish-shaped container and is injected into the incubator 1. When the whole amount is injected, the lifting / lowering device 35 is operated again, the bar 36 is raised, and the incubator 1 and the stage 32 are returned to their original positions. Thereby, since the excessive change of culture conditions can be suppressed at the time of medium exchange, the change of the medium caused by cell metabolism and proliferation can be accurately monitored even immediately after the medium exchange.

  In FIG. 3, the structure of the cell culture apparatus of the other Example of this invention is shown. FIG. 1 is different from the embodiment in that a pH sensor 41 for detecting the pH of the medicine held in the heating bag 9 is provided in the heating bag 9. Since other configurations are the same as those in the embodiment of FIG. 1, the same reference numerals are given and description thereof is omitted.

  The operation of the cell culture apparatus of the present embodiment will be described using the flowchart of the medium exchange process shown in FIG.

  The pH of the medium in the incubator 1 is detected by the pH sensor 15 (S71), and it is determined from the pH detection result whether the medium is to be replaced (S72). When the medium needs to be replaced, the valve 7 of the injection channel 5 is opened, the pump 8 is driven, and the necessary amount of the culture solution stored in the chemical container 3 is added to the heating bag 9 (150 ml in this example). Inject. When the required amount is injected, the valve 7 is closed and the pump 8 is stopped (S73). The injection amount is detected by a liquid amount sensor (not shown).

  After holding the culture solution in the heating bag 9 for a set time (S74), the pH of the culture solution in the heating bag 9 is detected by the pH meter 41 (S75), and the pH of the culture solution in the incubator 1 by the pH meter 15 is detected. Detection is performed (S76). Next, the pH detection values of the pH meter 41 and the pH meter 15 are compared, and it is determined whether or not the pH of the culture solution in the warming bag 9 has converged to the allowable range of the pH of the culture solution in the incubator 1. (S77).

  Steps 74, 75 and 76 are repeated until the pH converges to an acceptable range. When the pH detection values of the pH meter 41 and the pH meter 15 converge to within an allowable range, the valve 13 is opened and the pump 14 is driven to discharge the culture solution in the incubator 1 to the waste liquid bottle 16 (S78). Next, the valve 10 is opened, the pump is driven, the culture solution held in the heating bag 9 is injected into the incubator 1 (S79), and the medium exchange is completed.

  In this example, the pH of the culture solution held in the heating bag 9 is monitored at the time of injecting the medium, and the medium is exchanged when the pH of the medium in the incubator 1 converges to an allowable range. Compared to Examples 1 and 2, the components of the culture solution in the incubator 1 and the heating bag 9 can be reliably supplied with the culture solution converged to the allowable range in accordance with the change in the pH of the medium due to cell culture. After minimizing the difference, the medium can be changed. As a result, since changes in culture conditions can be further suppressed, changes in the medium caused by cell metabolism and proliferation can be monitored more accurately even immediately after medium replacement.

  Further, in this example, the determination at the time of injecting the retained chemical into the incubator 1 is performed by comparing the detected pH value. However, the retained chemical is retained in the incubator 1 after being held for a set time without comparison. Can be injected. However, it is necessary to detect and record the pH of the retained chemical in order to perform accurate culture monitoring.

  Further, in this embodiment, a temperature sensor is further provided in the heating bag 9 to determine whether or not to change the medium so that the temperature can be compared with the comparison of the pH so that more accurate monitoring can be performed. it can. Or culture medium exchange can also be performed only by determination of temperature.

  In addition, the cell culture apparatus of each embodiment shown in FIGS. 1, 3 and 5 only shows the configuration necessary for the description of the present invention, and can be freely customized according to the user's request. . For example, as many chemical containers 3 as necessary are provided inside and outside the refrigerator, and a buffer solution such as PBS (−), a release agent such as trypsin EDTA solution, a differentiation medium different from a normal culture solution, etc. are prepared, The supply can be controlled by the control device 22.

  In this embodiment, the pH sensor is attached to the flow path to detect the pH, but the change is detected using a commercially available pH electrode or a pH indicator immobilized and surrounded by a polymer membrane. You can also. In the present embodiment, an example in which the pH sensor 15 is provided on the waste liquid flow path 12 side is shown. However, in the case of a sensor having a safe form with little biotoxicity, the sensor can be used in the incubator 1. . In addition, sampling can be automatically performed from the incubator 1 using a needle.

  In this embodiment, a reagent container, an incubator, and a waste liquid container are directly connected by a flow path system, but the solution can be delivered by a dispensing robot or the like.

  Moreover, the positional relationship of each part of a present Example is a conceptual diagram which shows the flow of solutions, such as a culture solution, Comprising: It does not show actual up-and-down relationship. In particular, when the flow of the liquid is controlled by a valve and a pump, the flow destination of the solution may be provided on the upper side.

  In addition, although each Example is the description using an adhesion type cell, it cannot be overemphasized that this invention is applicable also to a suspension cell.

  In the embodiment of the present invention, whether or not to change the medium is determined based on the detection result of the pH of the medium in the incubator 1, but the timing for changing the medium may be set according to time.

It is a figure which shows the structure of the cell culture apparatus of one Example of this invention. It is a flowchart which shows the operation | movement at the time of performing culture medium exchange of the Example of FIG. It is a figure which shows the structure of the cell culture apparatus of the other Example of this invention. It is a flowchart which shows the operation | movement at the time of performing culture medium exchange of the Example of FIG. It is a figure which shows the structure of the cell culture apparatus of the further another Example of this invention. It is a flowchart which shows the operation | movement at the time of performing culture medium exchange of the Example of FIG.

Explanation of symbols

1: Incubator 2: Culture tank 3: Chemical container 4: Storage 5: Injection flow path 6: Closed space 7, 10, 13, 21: Valve 8, 11, 14: Pump 9: Heating bag 12: Waste liquid flow Path 15, 41: pH sensor 16: Waste liquid bottle 17: Temperature sensor 18: Carbon dioxide gas concentration sensor 19: Heater 20: Carbon dioxide gas 22: Control device

Claims (6)

A thermostat in which the temperature and the concentration of the specific gas are maintained under set conditions, a cell incubator provided inside the thermostat, and a drug stored in a drug container provided outside the thermostat In a cell culture device comprising a chemical supply means for supplying to an incubator,
A chemical holding means for holding a chemical supplied from the chemical container to the incubator for a set time is provided in either one of the thermostatic chamber or a closed space of the same environment as the thermostatic chamber communicated with the thermostatic bath. The cell culture device is characterized in that the drug holding means holds the drug in a holding container formed of a film having thermal conductivity and transmitting the specific gas. After the temperature and the concentration of the specific gas chemicals before Symbol holding container are matched in concentration to the allowable range of temperature and specific gas of the thermostatic bath, and discharging the culture solution of the incubator, the holding container 2. The cell culture apparatus according to claim 1, further comprising a control means for supplying the chemical to the incubator. A thermostat in which the temperature and the concentration of the specific gas are maintained under set conditions, a cell incubator provided inside the thermostat, and a drug stored in a drug container provided outside the thermostat A medicine supply means for supplying to the incubator; a culture medium analyzing means for analyzing the components of the culture medium in the incubator; Monitoring control means, and the chemical supplied from the chemical container to the incubator is held for a set time in either the thermostatic chamber or a closed space of the same environment as the thermostatic chamber connected to the thermostatic bath The medicine holding means is provided, and the medicine holding means is formed to hold the medicine in a holding container formed by a film having thermal conductivity and transmitting the specific gas, and the cell culture monitoring control hand Supplies the chemical from the chemical container to the chemical holding means based on the analysis value of the culture solution and holds the chemical, and after discharging the culture solution in the incubator, removes the chemical in the holding vessel from the incubator A cell culturing apparatus, characterized in that the medium is exchanged by supplying the medium.   A holding chemical analysis means for analyzing a component of the holding chemical in the holding container is provided, and the cell culture monitoring control means is based on the difference between the analysis value of the culture solution and the analysis value of the holding medicine when the medium is replaced. The cell culture apparatus according to claim 3, wherein culture conditions are controlled. A thermostat in which the temperature and the concentration of the specific gas are maintained under set conditions, a cell incubator provided inside the thermostat, and a drug stored in a drug container provided outside the thermostat In a cell culture device comprising a chemical supply means for supplying to an incubator,
A medicine holding container provided at an upper part in the incubator and holding an upper surface for holding a medicine supplied from the medicine container for a set time; and inclining the incubator to overflow the medicine in the medicine holding container. A cell culture device comprising a tilting device for injecting into the incubator.   A culture medium analyzing means for analyzing a component of the culture medium in the incubator; and a cell culture monitoring control means for controlling a medium exchange of the incubator based on an analysis value of the culture medium analyzing means. The monitoring control means supplies and holds the chemical from the chemical container to the chemical holding container based on the analysis value of the culture liquid, and discharges the culture liquid in the incubator and then stores the chemical in the chemical holding container. The cell culture apparatus according to claim 5, wherein the medium is exchanged by supplying the culture medium to the incubator.

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