JP2020174574A - Culture medium exchanging system, and culture medium exchanging method - Google Patents

Abstract

To eliminate the need for operation such as centrifugal separation to suppress damage to cells, and reduce the number of target cells included in waste liquid to improve culture efficiency.SOLUTION: A culture medium exchange system 1 comprises: an observation part 7 which measures the number of cells S included in culture medium W in a specific region R in a culture vessel 3; a discharge part 9 which discharges the culture medium W in the specific region R; and a control part 11 which causes the discharge part 9 to discharge the culture medium W in the specific region R in the case where the number of cells S included in the culture medium W in the specific region R, which is measured by observation part 7, is smaller than a predetermined threshold value, and does not cause the discharge part 9 to discharge the culture medium W in the specific region R in the case where the number of cells S included in the culture medium W in the specific region R, which is measured by the observation part 7, is a predetermined threshold value or more.SELECTED DRAWING: Figure 1

Description

The present invention relates to a medium exchange system and a medium exchange method.

In recent years, in the field of regenerative medicine using cultured cells such as iPS cells (induced pluripotent stem cells), scale-up of cell culture has been desired. Unlike adhesion culture using well plates and containers called dishes, suspension culture using a suspension culture tank called a bioreactor is mainstream for mass production of cells because the culture efficiency does not depend on the cell adhesion area. It is becoming.

In the suspension culture, as a method of exchanging the medium in the culture tank, a method of precipitating the cells in the medium by centrifugation or the like and then scooping the supernatant which is the supernatant of the medium (see, for example, Patent Document 1). Further, a method called perfusion culture (see, for example, Patent Document 2) is known in which the waste liquid and the deteriorated medium in the culture tank are discharged and a new medium is supplied to the culture tank.

Japanese Unexamined Patent Publication No. 2006-218835 JP-A-2015-136330

However, in the method described in Patent Document 1, frequent centrifugation may damage cells. In addition, centrifugation requires a lot of time and effort to culture a large amount of cells, and also requires an operation to take out the cells in the culture tank to a centrifuge, so that there is a risk of contamination. If the method of Patent Document 1 does not perform centrifugation, there is a risk that the target cells will be discarded together with the medium.

In the method described in Patent Document 2, cells remain in the liquid discharged as waste liquid from the culture tank. Therefore, there is a problem that the target cells are discarded together with the waste liquid, and the culture efficiency is lowered.

The present invention has been made in view of the above circumstances, and eliminates the need for operations such as centrifugation, suppresses damage to cells, reduces the number of target cells contained in the waste liquid, and improves culture efficiency. It is an object of the present invention to provide a medium exchange system and a medium exchange method that can be used.

In order to achieve the above object, the present invention provides the following means.
The first aspect of the present invention is an observation unit that measures the number of cells contained in a medium in a specific region in a culture vessel, a discharge unit that discharges the medium in the specific region, and the observation unit. When the number of the cells contained in the culture medium of the specific region is less than a predetermined threshold value, the medium of the specific region is discharged by the discharge unit, and the specific region is measured by the observation unit. It is a medium exchange system including a control unit that does not discharge the medium of the specific region by the discharge unit when the number of the cells contained in the medium of the region is equal to or more than a predetermined threshold value.

According to this aspect, when the number of cells contained in the medium of a specific region in the culture vessel measured by the observation unit is less than a predetermined threshold value, the control unit discharges the medium of the specific region. When the number of cells is equal to or higher than a predetermined threshold value, the medium in the specific region is not discharged by the control unit.

Therefore, only the medium having a small number of cells can be discharged from the culture vessel, the number of target cells contained in the waste liquid can be reduced, and the culture efficiency can be improved. Moreover, since it is not necessary to perform centrifugation, damage to cells can be suppressed.

The medium exchange system according to the above aspect includes a discharge channel member through which the medium is circulated, and a discharge pump for transferring the medium to the outside of the culture container via the discharge channel member. It may be that.

The medium exchange system according to the above aspect includes a suction unit that sucks the medium of the specific region from the culture container and a return unit that returns the medium sucked by the suction unit to the culture container, and the observation When the unit measures the number of the cells contained in the medium sucked by the suction unit and the control unit measures the number of the cells measured by the observation unit less than a predetermined threshold value. When the number of cells measured by the observation unit is equal to or greater than a predetermined threshold, the medium sucked by the suction unit is discharged by the discharge unit, and the medium sucked by the suction unit is returned. It may be returned to the culture medium depending on the part.

With this configuration, the number of cells is measured by the observation unit and whether or not the medium is discharged by the control unit is determined for the medium in a specific region sucked from the culture vessel by the suction unit. Therefore, it is possible to prevent the culture medium containing a cell number equal to or higher than a predetermined threshold value from being discharged from the culture vessel, and to reliably discharge only the medium having a cell number smaller than the predetermined threshold value. In addition, since the medium containing a large amount of cells is returned to the culture vessel, it is possible to suppress the disposal of target cells and prevent a decrease in culture efficiency.

In the medium exchange system according to the above aspect, the return unit includes a return flow path member for circulating the medium and a return pump for transferring the medium to the culture container via the return flow path member. May be good.

The medium exchange system according to the above aspect includes a switching mechanism for switching between a flow path discharged by the discharging unit and a flow path returned by the returning unit with respect to the medium whose number of cells has been measured by the observation unit. The control unit may control the switching of the flow path by the switching mechanism.
With this configuration, it is possible to switch between discharging the medium in which the number of cells has been counted by the observation unit as it is or returning it to the culture vessel, with a simple configuration in which the switching mechanism is only controlled by the control unit.

The medium exchange system according to the above aspect may include a recording unit that records the volume of the medium sucked by the suction unit and / or the number of cells contained in the medium sucked by the suction unit. ..
With this configuration, the quality of medium exchange can be evaluated using the volume of the medium recorded in the recording unit and the number of cells contained in the medium.

The medium exchange system according to the above aspect may also include a stirring unit that agitates the medium by rotating around a predetermined rotation axis in the culture vessel, and a recording unit that records the number of rotations of the stirring unit. Good.
The medium exchange system according to the above aspect may include a recording unit for recording the pH of the medium in the culture vessel.

In the culture medium exchange system according to the above aspect, the discharge unit has a suction port for sucking the medium of the specific region in the culture vessel, and an adjusting mechanism capable of adjusting the position of the suction port in the culture vessel. May be provided.
In the medium exchange system according to the above aspect, the suction unit has a suction port for sucking the medium in the specific region in the culture vessel, and an adjusting mechanism capable of adjusting the position of the suction port in the culture vessel. May be provided.
The medium exchange system according to the above aspect may include a recording unit that records the height from the bottom surface of the culture vessel to the suction port.

The culture medium exchange system according to the above aspect may include a culture medium supply unit that supplies the culture medium to the culture vessel.
With this configuration, the culture medium can be replenished with new medium by the amount of the medium discharged from the culture medium by the discharge unit. In addition, since a part of the medium in the culture container is replaced, deterioration of the medium in the culture container can be suppressed.

A second aspect of the present invention is a cell number acquisition step for acquiring the number of cells contained in a medium in a specific region in a culture vessel, and the cell number acquired by the cell number acquisition step is a predetermined threshold value. A determination step for determining whether or not the number of cells is relatively small, and a medium treatment step for discharging the medium in the specific region only when the determination step determines that the number of cells is less than the predetermined threshold value. It is a medium exchange method including.

According to this aspect, when the number of cells contained in the medium of a specific region in the culture vessel acquired by the cell number acquisition step is determined by the determination step to be less than a predetermined threshold value, the medium When the medium in the specific region is discharged by the treatment step and the determination step determines that the threshold value is equal to or higher than the predetermined threshold value, the medium is not discharged by the medium treatment step. Therefore, only the medium having a small number of cells can be discharged from the culture vessel, the number of target cells contained in the waste liquid can be reduced, and the culture efficiency can be improved. Moreover, since it is not necessary to perform centrifugation, damage to cells can be suppressed.

The medium exchange method according to the above aspect includes a suction step of sucking the medium of the specific region from the culture vessel, and the cell number acquisition step includes the cells contained in the medium sucked by the suction step. When the number is acquired and the determination step determines that the number of cells is less than the predetermined threshold value, the medium sucked by the suction step is discharged, and the determination step determines the number of cells. When the number of cells is determined to be equal to or higher than the predetermined threshold value, the medium sucked by the suction step may be returned to the culture vessel.

With this configuration, the number of cells is measured by the cell number acquisition step for the medium in a specific region sucked from the culture vessel by the suction step, and whether or not to discharge the medium is determined by the determination step. Therefore, it is possible to prevent the culture medium containing a cell number equal to or higher than a predetermined threshold value from being discharged from the culture vessel, and to reliably discharge only the medium having a cell number smaller than the predetermined threshold value. Further, in the medium treatment step, the medium containing a large amount of cells is returned to the culture vessel, so that the target cells can be suppressed from being discarded and the decrease in cell efficiency can be prevented.

According to the present invention, it is possible to eliminate the need for an operation such as centrifugation, suppress damage to cells, reduce the number of target cells contained in the waste liquid, and improve the culture efficiency.

It is a schematic block diagram of the culture medium exchange system which concerns on 1st Embodiment of this invention. It is a flowchart explaining the culture medium exchange method which concerns on 1st Embodiment of this invention. It is a schematic block diagram of the discharge part which concerns on the modification of 1st Embodiment. It is a schematic block diagram of the culture medium exchange system which concerns on 2nd Embodiment of this invention. It is a flowchart explaining the culture medium exchange method which concerns on 2nd Embodiment of this invention. It is a schematic block diagram of the culture medium exchange system which concerns on 3rd Embodiment of this invention. It is a flowchart explaining the culture medium exchange method which concerns on 3rd Embodiment of this invention. It is a figure explaining the height from the bottom surface of the culture vessel at the position where the culture medium is sucked in the culture vessel.

[First Embodiment]
The medium exchange system and the medium exchange method according to the first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the medium exchange system 1 according to the present embodiment has a stirring mechanism 5 for stirring the medium W contained in the culture vessel 3 and a specific region (specific region) R in the culture vessel 3. An observation unit 7 that measures the number of cells S contained in the medium W, a discharge unit 9 that discharges the medium W in the specific region R, and a control that controls the stirring mechanism 5, the observation unit 7, and the discharge unit 9. It is provided with a part 11.

The culture container 3 is, for example, a bottomed cylindrical container in which the upper surface 3a is closed. The culture vessel 3 is made of an optically transparent material.
The stirring mechanism 5 includes a stirring shaft 5a inserted into the culture vessel 3 via the upper surface 3a of the culture vessel 3, a plurality of stirring blades (stirring portions) 5b provided on the stirring shaft 5a, and a stirring shaft 5a. It is equipped with a motor 5c that rotates around the axis.

As the observing unit 7, an existing technique for observing cells S without staining and without labeling can be adopted. The observation unit 7 is, for example, an LED (Light Emitting Diode), a laser beam, or the like that irradiates a specific region R, which is an arbitrary region in which the medium W and cells S exist in the culture vessel 3, with illumination light from the outside of the culture vessel 3. The image of the specific area R is acquired by photographing the light source of the above, a lens that collects the observation light from the specific area R irradiated with the illumination light outside the culture vessel 3, and the observation light collected by the lens. It includes an imaging element such as a CCD image sensor or a CMOS image sensor, and an image analysis unit (both not shown) that processes an image acquired by the imaging element.

The specific region R of the culture vessel 3 is preferably set above the medium W in the culture vessel 3, for example, near the liquid level. This is because it is considered that the number of cells in the culture vessel 3 is the smallest in the vicinity of the liquid level of the medium W due to gravity, and unnecessary suction of the cells S is prevented.

Based on the image acquired by the image sensor, the image processing unit calculates the number of cells per observation area of the medium W in the specific region R as the number of cells S contained in the medium W in the specific region R. .. As a method of calculating the number of cells, for example, a method of determining a black part as one cell S in a state where an image is binarized, a method of extracting a contour, and a method of extracting a contour having continuity are judged as a cell S. There is a method of using a trained model in which data that the operator has determined to be a cell part in the image is accumulated.

The discharge unit 9 is a tubular member (discharge flow path member) 9A into which a suction port is inserted into the culture container 3, and a collection container (not shown) through which the medium W in the culture container 3 is passed through the tubular member 9A. It is equipped with a liquid feed pump (discharge pump) 9B for transfer.

The tubular member 9A is, for example, a soft tube such as a silicon tube. In the tubular member 9A, a suction port is inserted into the culture vessel 3 via a port 3b provided on the upper surface 3a of the culture vessel 3. Also. In the tubular member 9A, the suction port is arranged in the specific area R of the culture container 3, and the discharge port is connected to the collection container.

The liquid feed pump 9B is, for example, a peristaltic pump arranged on the tubular member 9A. When the liquid feed pump 9B is switched to the ON state by a signal from the control unit 11, the tubular member 9A is squeezed by a roller (not shown) to squeeze the medium W in the specific region R of the culture vessel 3 into the tubular member 9A. Transfer to the collection container via the route. Further, when the liquid feed pump 9B is switched to the OFF state by the signal from the control unit 11, the transfer of the medium W from the culture container 3 to the collection container is stopped.

The control unit 11 includes, for example, a storage unit such as a hard disk drive, a CPU (Central Processing Unit), and a RAM (Random Access Memory) (all not shown). The control unit 11 may be, for example, a PC (Personal Computer). The control unit 11 realizes the following functions by executing the control program stored in the memory by the CPU.

For example, the control unit 11 rotates the stirring blade 5b around the stirring shaft 5a by driving the motor 5c of the stirring mechanism 5, thereby stirring the medium W in the culture vessel 3. In addition, the control unit 11 operates the observation unit 7 to periodically acquire an image of the specific region R of the culture vessel 3, and based on the acquired image, the cells per observation area of the medium W in the specific region R. Have them measure the number.

In addition, the control unit 11 stores a predetermined threshold value indicating a desired number of cells per observation area. The control unit 11 compares the number of cells per observation area of the medium W in the specific region R measured by the observation unit 7 with a predetermined threshold value stored.

When the number of cells per observation area of the medium W in the specific region R is less than a predetermined threshold value, the control unit 11 drives the liquid feeding pump 9B of the discharge unit 9 to drive the medium W in the specific region R. To the collection container. On the other hand, when the number of cells per observation area of the medium W in the specific region R is equal to or greater than a predetermined threshold value, the control unit 11 does not drive the liquid feeding pump 9B of the discharge unit 9, and the specific region R by the discharge unit 9 does not drive. The medium W of the above is not discharged.

Next, the medium exchange method according to the present embodiment includes the cell number acquisition step SA1 for acquiring the number of cells contained in the medium W in the specific region R of the culture vessel 3, as shown in the flowchart of FIG. The determination step SA2 for determining whether or not the number of cells acquired in the cell number acquisition step SA1 is smaller than the predetermined threshold, and the determination step SA2 determine the number of cells contained in the medium W of the specific region R. It includes a medium treatment step SA3 in which the medium W in the specific region R is discharged only when it is determined that the value is less than a predetermined threshold.

Next, the operation of the medium exchange system 1 according to the present embodiment will be described.
According to the medium exchange system 1 having the above configuration, when the cells S are cultured, the stirring mechanism 5 is driven by the control unit 11 in a state where the medium W and the cells S are contained in the culture vessel 3. The medium W in the culture vessel 3 is stirred. As a result, the cells S are cultured in a floating state in the medium W.

Subsequently, when the medium W in the culture vessel 3 is exchanged, as shown in the flowchart of FIG. 2, first, the observation unit 7 is controlled by the control unit 11, so that the specific region R in the culture vessel 3 is replaced. The image of the above is periodically acquired, and the number of cells per observation area of the medium W in the specific region R is calculated based on the acquired image (step SA1).

Next, the control unit 11 compares the calculated number of cells per observation area with the stored predetermined threshold value (step SA2). When the control unit 11 determines that the number of cells per observation area of the medium W is less than a predetermined threshold value, the control unit 11 transmits a drive signal to the liquid feed pump 9B. When the liquid feed pump 9B is driven by the drive signal from the control unit 11, the medium W in the specific region R of the culture vessel 3 is discharged to the collection vessel via the tubular member 9A (step SA3). ..

On the other hand, when the control unit 11 determines that the number of cells per observation area of the medium W in the specific region R is equal to or greater than a predetermined threshold value, the control unit 11 does not transmit a drive signal to the liquid feed pump 9B, and the specific region does not transmit a drive signal. The medium W in R is not discharged (step SA4).

In this case, if the medium W in the specific region R of the culture vessel 3 is simply discharged, if a large number of cells S are present in the medium W of the specific region R, those cells S are discharged together with the medium W. It will be. In the present embodiment, when the number of cells S contained in the medium W in the specific region R of the culture vessel 3 is less than a predetermined threshold value, the medium W in the specific region R is discharged from the culture vessel 3. When the number of cells S contained in the medium W in the specific region R is equal to or greater than a predetermined threshold value, the medium W in the specific region R is not discharged.

Therefore, according to the medium exchange system 1 and the medium exchange method according to the present embodiment, only the medium W having a small number of cells can be discharged from the culture vessel 3. As a result, the number of target cells S contained in the waste liquid can be reduced, and the culture efficiency can be improved. In addition, the medium W having a small number of cells can be discharged without centrifuging when the medium is exchanged, and damage to the cells S can be suppressed.

In the present embodiment, the discharge unit 9 is provided with the liquid feed pump 9B. Instead of this, for example, as shown in FIG. 3, the discharge unit 9 may include a negative pressure supply means 9C such as a suction pump. In FIG. 3, reference numeral 9D indicates a recovery container for collecting the medium W discharged from the culture container 3.

The collection container 9D is, for example, a bottomed tubular container whose upper surface is closed, and the inside is sealed. The discharge port of the tubular member 9A of the discharge portion 9 is inserted into the upper surface of the collection container 9D. A negative pressure supply means 9C is connected to the upper surface of the collection container 9D.

When the negative pressure supply means 9C is switched to the ON state by the signal from the control unit 11, the medium W in the specific region R of the culture container 3 is passed through the tubular member 9A by applying negative pressure to the collection container 9D. When the medium W is sucked into the collection container 9D and switched to the OFF state, the suction of the medium W in the specific region R of the culture container 3 is stopped.
Even with this configuration, the same effect as when the liquid feed pump 9B is adopted can be obtained.

[Second Embodiment]
Next, the medium exchange system and the medium exchange method according to the second embodiment of the present invention will be described.
As shown in FIG. 4, the medium exchange system 21 according to the present embodiment has a suction unit 23 that sucks the medium W in the specific region R from the culture container 3 and a culture medium W that sucks the medium W sucked by the suction unit 23. It is different from the first embodiment in that it includes a return unit 25 for returning to.
Hereinafter, the parts having the same configuration as the medium exchange system 1 and the medium exchange method according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The suction unit 23 is, for example, a liquid feeding unit that transfers the tubular member 23A into which the suction port is inserted into the culture container 3 and the medium W in the culture container 3 to the return unit 25 or the discharge unit 9 via the tubular member 23A. It is equipped with a pump 23B.

The tubular member 23A is, for example, a soft tube such as a silicon tube. The tubular member 23A has a suction port arranged in a specific region R of the culture vessel 3.
The liquid feed pump 23B is, for example, a diaphragm pump arranged on the tubular member 23A, and is switched ON / OFF by a drive signal from the control unit 11. It is desirable that the liquid feed pump 23B has no risk of crushing or damaging the cells S transferred by the tubular member 23A.

The return unit 25 includes a tubular member (return flow path member) 25A into which a discharge port is inserted into the culture container 3.
The tubular member 25A has a discharge port penetrating the side wall portion of the culture container 3 and is inserted into the lower part of the culture container 3. The through hole of the culture vessel 3 through which the discharge port of the tubular member 25A penetrates is closed with an O-ring (not shown) or the like in a gap with the tubular member 25A. When the discharge port of the tubular member 25A is inserted into the lower part of the culture container 3, the cells S contained in the medium W returned to the culture container 3 are set in the upper part of the culture container 3 in the specific region R. It is possible to suppress the invasion of the culture medium.

In the present embodiment, the discharge unit 9 is not provided with the liquid feed pump 9B, and the discharge port of the tubular member 9A is connected to the collection container.
Further, the observation unit 7 further includes a flow path member 27 arranged between the tubular member 23A of the suction unit 23, the tubular member 9A of the discharge unit 9, and the tubular member 25A of the return unit 25.

The flow path member 27 has, for example, a flow path through which the medium W can pass, and allows the medium W transferred via the tubular member 23A of the suction section 23 to pass through the return section 25 or the discharge section 9. Inflow to. Further, the flow path member 27 is formed of, for example, an optically transparent material. The flow path member 27 may have a shape such as a cylinder, a square cylinder, a plate with a well, or the like that can temporarily store the medium W to such an extent that the number of cells S passing through the inside can be measured. When the flow path of the flow path member 27 is wide, the medium W may be agitated in the flow path of the flow path member 27.

Further, the observation unit 7 irradiates the flow path member 27 with the illumination light by the light source, and collects the observation light from the medium W and the cells S in the flow path member 27 irradiated with the illumination light by the lens. By taking an image of the observation light focused by the image sensor, images of the medium W and the cells S passing through the flow path member 27 are acquired. Then, the observation unit 7 observes the medium W flowing in the flow path member 27 as the number of cells contained in the medium W in the specific region R sucked by the suction unit 23 based on the image acquired by the image sensor. Calculate the number of cells per area.

The flow path member 27 is provided with a partition (switching mechanism, not shown) such as a solenoid valve for switching the inflow destination of the medium W that has passed through the inside. The partition selectively switches the inflow destination of the medium W that has passed through the flow path member 27 to one of the tubular member 9A of the discharge unit 9 and the tubular member 25A of the return unit 25, based on the switching signal from the control unit 11.

Next, in the medium exchange method according to the present embodiment, as shown in the flowchart of FIG. 5, in addition to the cell number acquisition step SA1, the determination step SA2, and the medium treatment step SA3, the medium W in the specific region R from the culture vessel 3 Includes suction step SB1 to suck. Further, in the medium exchange method, the cell number acquisition step SA1 acquires the number of cells contained in the medium W sucked by the suction step SB1. When the medium treatment step SA3 determines that the number of cells is less than a predetermined threshold by the determination step SA2, the medium W sucked by the suction step SB1 is discharged (step SA3), and the cells are discharged by the determination step SA2. When the number is determined to be equal to or higher than a predetermined threshold value, the medium W sucked by the suction step SB1 is returned to the culture vessel 3 (step SB4).

Subsequently, the operation of the medium exchange system 21 and the medium exchange method according to the present embodiment will be described.
According to the medium exchange system 21 having the above configuration, when exchanging the medium W in the culture vessel 3, the control unit 11 first drives the liquid feed pump 23B of the suction unit 23, as shown in the flowchart of FIG. By doing so, the medium W in the specific region R of the culture vessel 3 is transferred to the flow path member 27 via the tubular member 23A of the suction portion 23 (step SB1).

Next, by controlling the observation unit 7 by the control unit 11, images of the medium W and the cells S passing through the flow path member 27 are periodically acquired, and the flow path member 27 is based on the acquired images. The number of cells per observation area of the medium W flowing through the inside is calculated (step SA1). Then, the control unit 11 compares the calculated number of cells per observation area with the stored predetermined threshold value (step SA2).

When the control unit 11 determines that the number of cells per observation area of the medium W flowing in the flow path member 27 is less than a predetermined threshold value, the control unit 11 drives the partition to drive the flow path member 27. The medium W that has passed through the above water flows into the tubular member 9A of the discharge portion 9. As a result, the medium W that has passed through the flow path member 27 is transferred to the collection container via the discharge unit 9 (step SA3).

On the other hand, when the control unit 11 determines that the number of cells per observation area of the medium W flowing in the flow path member 27 is equal to or greater than a predetermined threshold value, the control unit 11 drives the partition to drive the flow path member 27. The inflow destination of the medium W that has passed through the above is switched to the tubular member 25A of the return portion 25. As a result, the medium W that has passed through the flow path member 27 is returned to the culture vessel 3 via the return unit 25 (step SB4).

That is, when the medium W in the specific region R sucked from the culture vessel 3 passes through the flow path member 27, the number of cells S contained in the medium W is measured. Then, when the number of cells is less than the predetermined threshold value, the medium W in the sucked specific region R is discharged as it is, and when the number of cells is equal to or more than the predetermined threshold value, the suctioned medium W in the specific region R is discharged. The medium W is returned to the culture vessel 3.

Therefore, according to the medium exchange system 21 and the medium exchange method according to the present embodiment, whether the number of cells is observed and discharged with respect to the medium W in the specific region R sucked from the culture vessel 3 by the suction unit 23. Since it is determined whether or not the cells are discharged, the culture medium W containing the number of cells equal to or higher than the predetermined threshold is prevented from being discharged from the culture vessel 3, and only the medium W having the number of cells less than the predetermined threshold is reliably discharged. be able to. Further, since the medium W containing a large amount of cells S is returned to the culture vessel 3, it is possible to suppress the disposal of the target cells S and prevent a decrease in culture efficiency.

In the present embodiment, the control unit 11 may issue a suction start instruction to the suction unit 23 at predetermined intervals. The suction unit 23 may continue sucking the medium W until an instruction to stop suction is sent from the control unit 11. Further, for example, when the number of cells S contained in the medium W in the specific region R of the culture vessel 3 is larger than a predetermined threshold value, or because the suction amount of the medium W in the specific region R is large, the flow path member. When the medium W is likely to overflow from 27, the suction of the medium W by the suction unit 23 may be stopped according to the instruction from the control unit 11.

[Third Embodiment]
Next, the medium exchange system and the medium exchange method according to the third embodiment of the present invention will be described.
As shown in FIG. 6, the medium exchange system 31 according to the present embodiment is different from the first embodiment in that it includes an exchange determination unit 33 that determines the medium exchange.
Hereinafter, the parts having the same configuration as the culture medium exchange systems 1 and 21 and the culture medium exchange method according to the first and second embodiments are designated by the same reference numerals and the description thereof will be omitted.

The exchange determination unit 33 is, for example, a pH sensor that detects the pH value of the medium W and a sensor control unit that determines the timing of medium exchange based on the pH value of the medium W detected by the pH sensor (both are not shown). And have.

The exchange determination unit 33 is immersed in the medium W in the culture vessel 3. The sensor control unit compares the pH value of the medium W detected by the pH sensor with a predetermined pH threshold value to determine whether the medium W has deteriorated, that is, whether it is the timing of medium replacement. (Step SC1). Then, when the sensor control unit determines that it is the timing of the culture medium exchange, the sensor control unit transmits a signal to that effect to the control unit 11. Further, in addition to the pH of the medium W, the timing of the medium exchange may be determined by setting a threshold value for the absorbance of the medium W at a specific wavelength.

When the control unit 11 receives a signal from the exchange determination unit 33 that it is time to exchange the medium, the control unit 11 controls the suction unit 23, the observation unit 7, and the partition, as shown in the flowchart of FIG. 7, in step SB1. , Step SA1, step SA2, step SA3 and step SB4.
According to this embodiment, the medium can be automatically exchanged at an appropriate timing according to the state of the medium W.

In the present embodiment, the exchange determination unit 33 determines whether or not it is the timing of medium exchange based on the pH value of the medium W. Instead of this, the exchange determination unit 33 may determine whether or not it is the timing of medium exchange based on the state of the cells S. In this case, for example, apart from the step of exchanging the medium, the observation unit 7 may acquire an image of the medium W in the specific region R, and determine the growth status of the cells S from the acquired image.

The second and third embodiments may be modified into the following configurations.
In the second and third embodiments, the partition of the solenoid valve or the like is adopted, but the partition of the solenoid valve or the like may not be adopted. In this case, for example, the suction unit 23 does not include the liquid feed pump 23B, the return unit 25 includes a liquid feed pump (return pump, not shown) such as a diaphragm pump on the tubular member 25A, and the discharge unit 9 feeds liquid. A pump 9B or a negative pressure supply means (suction pump) 9C may be provided.

In this case, by driving the liquid feed pump arranged on the tubular member 25A of the return section 25, the medium W in the specific region R of the culture vessel 3 is passed through the tubular member 23A of the suction section 23 to pass through the flow path member. Suction to 27. Then, in the flow path member 27, when the number of cells per observation area of the medium W measured by the observation unit 7 is less than a predetermined threshold value, the drive of the liquid supply pump of the return unit 25 is stopped, while the discharge unit is discharged. By driving the liquid feed pump 9B or the negative pressure supply means (suction pump) 9C of 9, the medium W that has passed through the flow path member 27 may be discharged via the discharge unit 9. On the other hand, in the flow path member 27, when the number of cells per observation area of the medium W measured by the observation unit 7 is equal to or greater than a predetermined threshold value, the flow path member 27 is driven as it is by driving the liquid feed pump of the return unit 25. The medium W that has passed through 27 may be returned to the culture vessel 3 via the return unit 25.

Further, in the second and third embodiments, the observation unit 7 is provided with the flow path member 27. Instead of this, the observation unit 7 did not have a flow path member, and the suctioned medium W acquired an image of the medium W while passing through the tubular member 23A of the suction unit 23, and sucked based on the acquired image. The number of cells per observation area of the medium W may be calculated. In this case, a partition or the like may be provided between the tubular member 23A of the suction portion 23, the tubular member 9A of the discharge portion 9, and the 25A of the return portion 25.

In the first to third embodiments, the operator may freely set a predetermined threshold value stored in the control unit 11. For example, the observation unit 7 may acquire an image of the specific region R in advance, the operator determines a preferable number of cells per observation area from the acquired image, and the determined number of cells may be set as a predetermined threshold value. ..

Until the operator sets a predetermined threshold value, in the configurations of the second and third embodiments, the threshold value is set to 0 or a value close to 0, and during that time, the medium W that has passed through the flow path member 27 is set. May be returned to the culture vessel 3 via the return unit 25. This makes it possible to prevent the cells S from being wasted until the operator determines a preferable number of cells per observation area.

Further, the control unit 11 may have a predetermined reference value, and the control unit 11 may automatically determine a predetermined threshold value based on the reference value. Further, the control unit 11 may optimize the threshold value setting by the control unit 11 by learning the standard of the predetermined threshold value set by the operator. As teacher data when learning the threshold reference, not only the threshold value set by the operator, but also the pH of the medium W at the time of measurement, the temperature in the culture vessel 3, and the rotation speed (rpm) of the stirring blade 5b. Such parameters may also be used together. This makes it possible to determine a more optimal threshold value according to changes in culture parameters.

Further, when setting the threshold value, only the target cells S are counted by image processing or visual inspection, for example, excluding dead cells or undifferentiated cells from the target for counting the number of cells. May be good. As a criterion for judging dead cells or undifferentiated cells from an image, a trained model using the judgment criteria of a worker or a third party as teacher data may be used. In addition, image processing performed based on the operator's standard or visual counting may be used as teacher data and modeled on the spot.

The first to third embodiments may be modified into the following configurations.
In each of the above embodiments, the medium W is stirred by the stirring mechanism 5 even when the medium is exchanged. As a first modification, for example, when performing medium exchange, first, the control unit 11 controls the driving of the stirring mechanism 5, and the stirring of the medium W in the culture vessel 3 is stopped, or the medium is stopped. The stirring rate of W may be reduced.

When the stirring of the medium W by the stirring mechanism 5 is stopped or the stirring speed is slowed down, most of the cells S in the medium W move to the lower side of the culture vessel 3 by gravity. Since the specific region R in the culture vessel 3 is set in the upper part of the medium W, the cells existing in the specific region R are formed by stopping the stirring of the medium W or slowing down the stirring speed when the medium exchange is performed. The number of S can be reduced. As a result, the medium can be exchanged efficiently.

Further, in each of the above embodiments, the observation unit 7 has been described as an example, but the observation unit may have a configuration in which the number of cells S contained in the medium W of the specific region R can be measured by non-staining observation. Just do it. For example, the observation unit may be configured to perform phase difference observation, oblique illumination observation, modulated contrast observation, differential interference observation, photoacoustic observation, Raman spectroscopic observation, infrared spectroscopic observation, endoscopic observation, and the like. , A retroreflective member having an array in which a plurality of minute reflective elements are arranged may be used.

As a configuration using the retroreflective member, for example, an objective lens arranged on the side of the culture container 3 or the flow path member 27 and culture from the outside of the culture container 3 or the flow path member 27 via the objective lens. The illumination optical system that irradiates the illumination light into the specific region R or the flow path member 27 of the container 3 and the illumination light that has passed through the specific region R or the flow path member 27 are specified by being reflected through the same path as the incident path. Acquired by a retroreflective member returning to the region R or the flow path member 27, an imaging element for photographing the illumination light focused by the objective lens after passing through the specific region R or the flow path member 27 again, and an imaging element. An image processing unit (both not shown) may be provided to calculate the number of cells per observation area of the medium W in the specific region R or the flow path member 27 based on the image.

In the configuration including the endoscopic observation optical system, for example, the observation unit 7 houses the light source, the lens, and the image pickup element in an elongated housing (not shown), and the housing is housed in a specific region R or a flow path member. It may be inserted in 27. Then, an image in the specific area R or the flow path member 27 is acquired with the housing inserted in the specific area R or the flow path member 27, and the inside of the specific area R or the flow path member 27 is based on the acquired image. The number of cells per observation area of the medium W may be calculated.

The configuration including a stereo measurement optical system includes, for example, a stereo optical system in which the observation unit 7 forms an image of two images having different perspectives from different viewpoints on a specific region R or a flow path member 27. It was acquired by a stereo imaging optical system including an imaging element that captures two images imaged by the stereo optical system, an optically transparent housing that houses the stereo imaging optical system, and a stereo imaging optical system. An image processing unit that calculates the number of cells per observation area of the medium W in the specific region R or the flow path member 27 based on a plurality of images may be provided.

In this case, the image processing unit identifies the position of the cells S included in each image of the two images acquired by the image sensor, and the medium W is based on the number of cells S existing in a predetermined area. The number of cells per observation area may be calculated. According to this configuration, the specific region R or the inside of the flow path member 27 can be accurately defined as a three-dimensional region. Therefore, the cell density existing in the specific region R or the three-dimensional region defined by the flow path member 27 can be accurately measured.

Further, in each of the above embodiments, the number of cells per observation area is calculated by the image processing unit based on the image acquired by the image sensor. Instead of this, for example, the observation unit 7 may include a laser light source and a detector arranged so as to face each other with the culture container 3 or the flow path member 27 interposed therebetween.

When the laser light emitted from the light source passes through the specific region R or the flow path member 27 of the culture vessel 3, it is scattered by the cells S contained in the medium W in the specific region R or the flow path member 27. The intensity of the scattered light generated at this time is proportional to the number of cells S. Therefore, the detector may detect the scattered light of the laser light and calculate the number of cells in the medium W based on the intensity of the detected scattered light.

Further, in each of the above embodiments, the culture medium exchange systems 1, 21 and 31 may include a recording unit (not shown) for recording various parameters related to the culture medium exchange. Further, if necessary, the medium exchange system 1 may include a sensor for measuring various parameters and a display unit for displaying the parameters. For example, the volume of the medium W sucked by the suction unit 23 and the number of cells S contained in the volume may be recorded by the recording unit.

Further, the rotation speed (rpm) of the stirring blade 5b in the culture vessel 3 may be recorded, or the temperature in the culture vessel 3 may be recorded. Further, the pH of the medium W in the culture vessel 3 may be recorded. For example, the quality of medium exchange can be evaluated by using numerical values such as the volume of the aspirated medium W and the number of cells contained in the volume.

Further, for example, as shown in FIG. 8, the height of the position where the suction port of the discharge unit 9 or the suction port of the suction unit 23 is arranged in the culture container 3 is measured from the bottom surface of the culture container, and the measured height is recorded. You may do it. In this case, when the medium W is sucked at a position higher than the bottom surface of the culture vessel, how many cells S are contained in the sucked medium W may be recorded as an index. FIG. 8 illustrates the discharge unit 9, but the same applies to the suction unit 23.

Further, for example, an adjusting mechanism may be provided in which the position of the suction port of the discharge unit 9 or the suction port of the suction unit 23 can be adjusted in the culture vessel 3. In this case, if the medium W in the specific region R continues to have a larger number of cells S than a predetermined threshold value, the control unit 11 drives the adjustment mechanism to drive the suction port of the discharge unit 9 or the suction unit 23. The position of the suction port may be raised by a predetermined amount.

Further, in each of the above embodiments, a medium supply unit (not shown) for supplying a new medium W to the culture vessel 3 may be provided, and perfusion culture may be used in combination. In this case, when the amount of the medium W discharged by the discharge unit 9 exceeds a predetermined threshold value, the control unit 11 drives the medium supply unit to transfer a new medium W from the medium supply unit to the culture vessel 3. It may be supplied.

Although the embodiments of the present invention have been described in detail with reference to the drawings, the specific configuration is not limited to these embodiments, and includes design changes and the like within a range that does not deviate from the gist of the present invention. .. For example, the present invention is not limited to the one applied to each of the above embodiments and modifications, and may be applied to an embodiment in which these embodiments and modifications are appropriately combined, and is not particularly limited. ..

Further, in the above embodiment, the bottomed cylindrical culture container 3 formed of an optically transparent material has been illustrated and described, but the culture tank has an arbitrary shape such as a bag shape, a spherical shape, or a box shape. Can be adopted. For example, a disposable bag-shaped culture tank may be adopted. Further, as the culture tank, any material such as hard or soft such as vinyl can be adopted. Further, the culture vessel 3 does not have to be entirely transparent, and the culture vessel 3 may partially have a transparent portion through which light such as illumination light and observation light is transmitted.

1,21,31 Medium exchange system 5b Stirring blade (stirring part)
7 Observation part 9 Discharge part 9A Tubular member (Discharge flow path member)
9B liquid feed pump (discharge pump)
11 Control unit 23 Suction unit 25 Return unit 25A Tubular member (return flow path member)
S cell W medium SA1 cell number acquisition step SA2 determination step SA3 medium treatment step SB1 suction step

Claims (14)

An observation unit that measures the number of cells contained in the medium of a specific area in the culture vessel,
A discharge section that discharges the medium in the specific region,
When the number of the cells contained in the medium of the specific region measured by the observation unit is less than a predetermined threshold value, the discharge unit discharges the medium of the specific region and the measurement is performed by the observation unit. A medium exchange system including a control unit that does not discharge the medium of the specific region by the discharge unit when the number of the cells contained in the medium of the specific region is equal to or greater than a predetermined threshold value. The medium exchange system according to claim 1, wherein the discharge unit includes a discharge flow path member for circulating the medium and a discharge pump for transferring the medium to the outside of the culture vessel via the discharge flow path member. .. A suction unit that sucks the medium of the specific region from the culture vessel,
It is provided with a return unit for returning the medium sucked by the suction unit to the culture vessel.
The observation unit measures the number of the cells contained in the medium sucked by the suction unit.
When the control unit discharges the medium sucked by the suction unit from the discharge unit when the number of cells measured by the observation unit is less than a predetermined threshold value, the medium is discharged by the discharge unit and measured by the observation unit. The medium exchange system according to claim 1 or 2, wherein when the number of cells is equal to or greater than a predetermined threshold value, the medium sucked by the suction unit is returned to the culture vessel by the return unit. The medium exchange system according to claim 3, wherein the return unit includes a return flow path member for circulating the medium and a return pump for transferring the medium to the culture vessel via the return flow path member. A switching mechanism for switching between a flow path discharged by the discharging unit and a flow path returned by the returning unit with respect to the medium whose number of cells has been measured by the observation unit is provided.
The culture medium exchange system according to claim 3 or 4, wherein the control unit controls switching of the flow path by the switching mechanism. One of claims 3 to 5, which includes a recording unit for recording the volume of the medium sucked by the suction unit and / or the number of cells contained in the medium sucked by the suction unit. The medium exchange system described. A stirring unit that stirs the medium by rotating around a predetermined rotation axis in the culture vessel,
The culture medium exchange system according to any one of claims 1 to 5, further comprising a recording unit that records the number of rotations of the stirring unit. The medium exchange system according to any one of claims 1 to 5, further comprising a recording unit for recording the pH of the medium in the culture vessel. The discharge unit has a suction port for sucking the medium of the specific region in the culture vessel.
The culture medium exchange system according to claim 1 or 2, further comprising an adjusting mechanism capable of adjusting the position of the suction port in the culture vessel. The suction unit has a suction port for sucking the medium of the specific region in the culture vessel.
The culture medium exchange system according to any one of claims 3 to 5, further comprising an adjusting mechanism capable of adjusting the position of the suction port in the culture vessel. The culture medium exchange system according to claim 9 or 10, further comprising a recording unit that records the height from the bottom surface of the culture vessel to the suction port. The medium exchange system according to any one of claims 1 to 11, further comprising a medium supply unit for supplying the medium to the culture container. The cell number acquisition step of acquiring the number of cells contained in the medium of a specific region in the culture vessel, and
A determination step for determining whether or not the number of cells acquired by the cell number acquisition step is smaller than a predetermined threshold value, and a determination step.
A medium exchange method comprising a medium treatment step of discharging a medium in the specific region only when the number of cells is determined to be less than the predetermined threshold value by the determination step. Including a suction step of sucking the medium of the particular region from the culture vessel.
The cell number acquisition step acquires the number of cells contained in the medium sucked by the suction step.
When the determination step determines that the number of cells is less than the predetermined threshold value in the medium treatment step, the medium sucked by the suction step is discharged, and the number of cells is increased by the determination step. The medium-replacement method according to claim 13, wherein when it is determined that the threshold value is equal to or higher than a predetermined threshold value, the medium sucked by the suction step is returned to the culture vessel.

Images