JP2020167959A - Medium monitoring device - Google Patents

Abstract

To correctly monitor a state of a medium on the basis of an image of a medium including cells, without complicating a device.SOLUTION: A medium monitoring device 1 comprises: an illumination light source 15 for radiating illumination light to a specific region where a culture broth and cells in a culture vessel 3 exist; a two-dimensional imaging element 27 for acquiring an image of the specific region by imaging observation light from the specific region to which the illumination light is radiated; an image analysis part 10 for dividing the image of the specific region acquired by the two-dimensional imaging element 27 into pixels of cells and pixels of a background, and calculating a representative pixel value which is a representation of the pixels of the background; and a control part 9 for repeatedly acquiring the image of the specific region by the two-dimensional imaging element 27, at a prescribed timing, then calculating the representative pixel value of the pixels of the background by the image analysis part for every image of the specific region acquired, then determining a state of the culture broth on the basis of secular change of the calculated representative pixel value.SELECTED DRAWING: Figure 1

Description

The present invention relates to a culture medium monitoring device.

In recent years, scale-up of culture has been desired in the field of regenerative medicine using cultured cells such as iPS cells (induced pluripotent stem cells). The cell culture method includes adhesive culture in which cells are cultured in a small container such as a flask and a petri dish, and a state in which the cells are suspended in the medium by stirring the medium in a large container such as a bioreactor. There is a suspension culture to culture.

In order to culture a large amount of cells, suspension culture is being adopted in place of adhesion culture because suspension culture, in which the culture efficiency is not affected by the adhesion area of cells, is superior to adhesion culture (for example, patent). See Reference 1.). The technique described in Patent Document 1 acquires an image of cells in a container in order to grasp the culture state of floating cells in the container.

In cell culture, the medium deteriorates when cells are cultured for a long period of time. Therefore, it is necessary to periodically check the degree of deterioration of the medium and replace the medium when the deterioration of the medium progresses (see, for example, Patent Document 2). The technique described in Patent Document 2 determines the state of the medium by periodically measuring the optical transmittance of the medium in which the cells are cultured, and specifies the timing of medium replacement.

JP-A-2017-140006 International Publication No. 2017/104696

When the absorbance measurement system described in Patent Document 2 is applied to the bioreactor described in Patent Document 1, both an optical system for acquiring an image of cells in a medium and an optical system for measuring a change in the transmittance of the medium are used. There is a problem that the device needs to be incorporated separately, which complicates the device.

It is also conceivable to substitute the optical system for measuring the transmittance of the medium with an optical system for acquiring an image of the cells, but when the cells are present in the medium in the region for acquiring the image, that is, the imaging region, the cells are illuminated. It will be greatly affected by light scattering and absorption. Since the cells are randomly suspended in the medium by stirring the medium, it is difficult to identify the region where the cells do not exist in the medium. There is a problem that it is not possible to distinguish whether the change in the brightness of the acquired image is due to the deterioration of the medium or the scattering of the illumination light by the cells, and the state of the medium cannot be accurately monitored.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a medium monitoring device capable of accurately monitoring the state of a medium without complicating the device.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is the specific region by photographing the illumination unit that irradiates the specific region in which the medium and the cells exist in the container with the illumination light and the observation light from the specific region irradiated with the illumination light. The image pickup unit that acquires the image of the above, and the image analysis unit that divides the image of the specific region acquired by the image pickup unit into the pixel of the cell and the pixel of the background and calculates the representative pixel value representing the pixel of the background. Then, the image pickup unit repeatedly acquires the image of the specific region at a predetermined timing, and the image analysis unit calculates the representative pixel value for each acquired image of the specific region, and the calculated representative pixel value is calculated. It is a medium monitoring apparatus including a control unit that determines the state of the medium based on the change with time.

According to this aspect, when the illumination unit irradiates a specific area in which the medium and cells in the container exist, the image pickup unit displays an image of the specific area in the container based on the observation light from the specific area. The image analysis unit calculates the representative pixel values of the background pixels that do not include the cell pixels from the acquired image of the specific region. Then, based on the time course of the representative pixel value of the background pixel calculated by the image analysis unit from each image of the specific region in the container repeatedly acquired by the control unit at a predetermined timing, the inside of the container is used. The state of the medium is determined.

In this case, although the region in which the cells are present in the medium changes with time, the cells are monitored over time only for the background pixels that do not contain the pixels of the cells extracted from the image of the specific region in the container. The state of the medium can be determined without being affected by the scattering of the illumination light. Further, by monitoring the state of the medium by using the imaging unit for observing the cells, it is not necessary to separately provide an optical system for monitoring the state of the medium, and the apparatus can be simplified. Therefore, the state of the medium can be accurately monitored based on the image of a specific area in the container without complicating the device.

The medium monitoring device according to the above aspect has an array in which a plurality of minute reflective elements are arranged, and is arranged with the container sandwiched between the illumination unit and the illumination light transmitted through the specific region in the container. The image pickup unit may acquire an image of the specific region that is re-irradiated with the illumination light reflected by the retroreflective member.

Since the retroreflective member is composed of an array in which a plurality of minute reflecting elements are arranged, the incident illumination light is reflected in the same direction as the incident direction. That is, regardless of the material, shape, size, etc. of the container, the illumination light transmitted through the specific area in the container is re-irradiated to the specific area in the container through the same optical path as the incident optical path by the retroreflective member. The image pickup unit can reliably acquire an image of the specific region. Therefore, the medium can be stably monitored even in a wide variety of culture vessels based on the state of the medium determined by the control unit.

The medium monitoring device according to the above aspect may include an oblique illumination optical system in which the illumination unit obliquely illuminates the specific region from a direction inclined with respect to the optical axis of the imaging unit.
The oblique illumination optical system makes it possible to monitor the state of the medium by using an image having a stereoscopic effect on colorless and transparent cells.

The culture medium monitoring device according to the above aspect may include a retardation optical system in which the illumination unit and the imaging unit generate a retardation image of the specific region.
The phase-difference optical system allows the state of the medium to be monitored using high-resolution, high-contrast images of cells in the medium.

The medium monitoring device according to the above aspect has a transparent portion capable of transmitting light, includes a housing for accommodating the lighting unit and the imaging unit, and has the housing inserted into the medium in the container. The illumination unit may irradiate the specific region with the illumination light via the transparent portion, and the imaging unit may acquire an image of the specific region through the transparent portion.

With this configuration, since an image of a specific region is acquired with the housing inserted in the medium in the container, there are almost no restrictions on the shape, size, material, etc. of the container to be used. Therefore, it can be applied to a wide variety of containers.

The medium monitoring device according to the above aspect obliquely illuminates the specific area by reflecting the illumination light emitted from the illumination unit to the outside of the housing via the transparent unit toward the imaging unit. It may be provided with a reflective member.
With this configuration, the range of illumination light in the container is limited to the space between the transparent part of the housing and the reflective member, and the cells that have invaded the space between the transparent part of the housing and the reflective member are allowed to enter. You can shoot.

The culture medium monitoring device according to the above aspect may include a tubular protective tube that covers the periphery of the housing, and the reflective member may be provided at the tip of the protective tube.
The protective tube can safely protect the housing and the lighting unit and the imaging unit in the housing, and operate the housing, the lighting unit, and the imaging unit in the container.

The medium monitoring device according to the above aspect may include a notification unit for notifying the user of information, and the control unit may notify the user of the timing of medium replacement by the notification unit.
Regardless of the material, shape, size, etc. of the container, the control unit accurately determines the state of the medium, so the control unit can prompt the user to change the medium at an appropriate timing via the notification unit. it can.

The medium monitoring device according to the above aspect includes a medium supply unit for supplying the medium to the container and a medium discharge unit for discharging the medium from the container, and the control unit determines that it is time to replace the medium. In this case, a part of the medium may be discharged from the container by the medium discharge unit, and a new medium may be supplied to the container by the medium supply unit.

The medium in the container can be replaced by supplying a new medium to the container by the medium supply unit while discharging a part of the medium in the container by the medium discharge unit. Further, based on the time-dependent change of the representative pixel value of the background pixel calculated by the image analysis unit, it is possible to know whether or not the medium in the container has deteriorated by the control unit, that is, whether or not it is the timing of medium replacement. Therefore, by controlling the medium supply unit and the medium discharge unit based on the time course of the representative pixel values of the background pixels, the control unit can replace the medium at an accurate timing without the trouble of the user. it can.

In the medium monitoring device according to the above aspect, the illumination unit may include a monochromatic light source that emits the illumination light having a single wavelength.
In this case, the monochromatic light source may include a white light source and a bandpass filter that extracts only a single wavelength from the light emitted from the white light source, or the monochromatic light source is an LED light source. May be good.

In the medium monitoring device according to the above aspect, the illumination unit includes a monochromatic light source that emits a plurality of monochromatic lights having different wavelengths as the illumination light, and the control unit performs each wavelength of the monochromatic light irradiated to the specific region. The state of the medium may be determined based on the time course of the representative pixel value of the background pixel in the image of each specific region acquired by the imaging unit.
With this configuration, it is possible to improve the accuracy of determining the state of the medium based on the time-dependent changes in the representative pixel values of the background pixels in each image of the specific region obtained for each wavelength of monochromatic light.

In the medium monitoring device according to the above aspect, the monochromatic light source is provided so as to be removable on the optical path of the white light source and the light emitted from the white light source, and only a single wavelength different from the light from the white light source. It may be provided with a plurality of band path filters for extracting light sources.
With this configuration, it is possible to irradiate a specific region with light having a desired wavelength simply by exchanging the bandpass filter, and it is possible to improve the versatility of the device and the operability by the operator.

In the medium monitoring device according to the above aspect, the monochromatic light source may be a plurality of LED light sources having different wavelengths.
With this configuration, a simple operation of switching the LED to be turned on is sufficient, and it is possible to eliminate the trouble of switching the bandpass filter arranged in the optical path of the illumination light.

In the culture medium monitoring device according to the above aspect, the illumination unit includes a white light source, the imaging unit includes a color CCD, and the control unit uses the background pixels in the image of the specific region acquired by the color CCD. The state of the medium may be determined based on the required relationship between the hue and pH of the medium.
With this configuration, the state of the medium can be determined more accurately by obtaining the pH of the medium from the color information of the medium.

According to the present invention, there is an effect that the state of the medium can be accurately monitored without complicating the apparatus.

It is a schematic block diagram which looked at the culture medium monitoring apparatus which concerns on 1st Embodiment of this invention from above. It is a schematic block diagram explaining the structure of the culture medium monitoring apparatus of FIG. It is a schematic block diagram explaining the structure of the optical measurement unit of FIG. It is a schematic block diagram which looked at the culture medium monitoring apparatus which concerns on the modification of 1st Embodiment of this invention from above. It is a schematic block diagram which looked at the culture medium monitoring apparatus which concerns on 2nd Embodiment of this invention from above. It is a schematic block diagram explaining the structure of the culture medium monitoring apparatus of FIG. It is a schematic block diagram explaining the structure of the culture medium monitoring apparatus which concerns on the modification of 2nd Embodiment of this invention. It is a schematic block diagram which looked at the culture medium monitoring apparatus which concerns on 3rd Embodiment of this invention from above. It is a schematic block diagram of the culture medium monitoring apparatus and the culture vessel which concerns on 4th Embodiment of this invention. 9 is a vertical cross-sectional view showing a state in which the housing constituting the medium monitoring device of FIG. 9 and the protective tube are separated. 9 is a vertical cross-sectional view showing a state in which a protective tube is attached to a housing constituting the medium monitoring device of FIG. 9. It is a vertical sectional view of the culture medium monitoring apparatus which concerns on 4th Embodiment of this invention. FIG. 12 is a vertical cross-sectional view of a housing and a protective cover constituting the medium monitoring device and the culture container of FIG. It is the schematic sectional drawing explaining the structure of the culture medium monitoring apparatus of FIG. It is a figure which shows the example of the upper image and the lower image acquired by the monitoring apparatus of FIG. 14, and the state which superposed the upper image and the lower image. It is a schematic block diagram of the optical measurement unit which concerns on the 2nd modification of each embodiment. It is a schematic block diagram of the optical measurement unit which concerns on 3rd modification of each embodiment. It is a schematic block diagram of the optical measurement unit which concerns on 4th modification of each embodiment. It is a schematic block diagram of the optical measurement unit which concerns on 5th modification of each embodiment. It is a schematic block diagram of the optical measurement unit which concerns on 6th modification of each embodiment. It is a figure which shows an example of the square color space (RGB) and the cylindrical color space (HSL) which concerns on the 7th modification of each embodiment. It is a figure which shows an example of the table which shows the relationship between the hue angle and pH which concerns on 7th modification of each embodiment. It is a schematic block diagram which shows an example of the 3 plate type color CCD which concerns on 7th modification of each embodiment. It is a schematic block diagram which shows an example of the single plate type color CCD which concerns on 7th modification of each embodiment.

[First Embodiment]
The medium monitoring apparatus according to the first embodiment of the present invention will be described below with reference to the drawings.
The medium monitoring device 1 according to the present embodiment is, for example, as shown in FIGS. 1 to 3, a stirrer 5 that stirs the culture solution (medium) W contained together with the cells S in the culture container (container) 3. The optical measurement unit 7 that acquires an image of the specific region R (see FIG. 3) in which the culture medium W and the cells S exist in the culture medium 3, the stirrer 5 and the optical measurement unit 7 are controlled, and the culture medium is used. It includes a control unit 9 for determining the state of W, a display unit 11 for displaying various information, and a warning transmission unit (notification unit) 13 for a smartphone or the like that notifies the user of the information.

The culture container 3 is, for example, a container such as a bioreactor in which cells S are suspended and cultured. The culture container 3 is a bottomed cylindrical container in which the upper surface 3a is closed. Further, the culture vessel 3 is formed of an optically transparent material, and can transmit the illumination light of the optical measurement unit 7. Phenol red or the like, which is a pH indicator, is added to the culture solution W.

The stirrer 5 has a stirring rod 5a inserted into the culture vessel 3 via the upper surface 3a of the culture vessel 3, a plurality of stirring blades 5b provided on the stirring rod 5a, and a stirring rod 5a around the longitudinal axis. It is equipped with a rotating motor 5c.

As shown in FIG. 3, the optical measurement unit 7 includes an illumination light source (illumination unit) 15 that generates illumination light, a condenser lens (illumination unit) 17 that collects illumination light emitted from the illumination light source 15. The objective lens (illumination unit) 19 that irradiates the illumination light focused by the condensing lens 17 onto the specific region R in which the culture solution W and the cells S exist in the culture vessel 3, and the specific region R in the culture vessel 3. A retroreflective member 21 that returns the transmitted illumination light toward the illumination light source 15, a half mirror 23 that branches the path of the illumination light returned by the retroreflective member 21, and an illumination light branched by the half mirror 23. It includes an imaging optical system 25 for forming an image and a two-dimensional imaging element (imaging unit) 27 for capturing an image formed by the imaging optical system 25. The specific region R corresponds to the focal plane of the objective lens 19, that is, the imaging region.

The illumination light source 15 is an LED (Light Emitting Diode) that generates, for example, 560 nm monochromatic light as illumination light. The illumination light source 15 irradiates the illumination light from the outside of the culture vessel 3 toward the specific region R in the culture vessel 3.

The objective lens 19 concentrates the illumination light from the illumination light source 15 on the specific region R in the culture vessel 3, while the retroreflective member 21 retransmits the illumination light in the culture vessel 3 and returns the illumination light. Condensing.

The retroreflective member 21 is placed on the illumination light source 15, the condenser lens 17, and the objective lens 19 in a state where the culture container 3 is sandwiched in the depth direction with respect to the objective lens 19 outside the culture container 3. They are arranged substantially opposite to each other. The illumination light source 15, the condenser lens 17, the objective lens 19, and the retroreflective member 21 are arranged at positions where the illumination light traveling in the culture vessel 3 does not interfere with the stirring rod 5a and the stirring blade 5b of the stirrer 5. ..

The retroreflective member 21 has, for example, an array in which a large number of minute reflective elements 21a are arranged along the surface P, as shown in FIGS. 1 and 3. The surface P is a surface that intersects the optical axis of the illumination light that has passed through the culture vessel 3. The surface P may be either a flat surface or a curved surface. The surface P may be, for example, a curved surface having a constant curvature and curved in one direction as shown in FIG. 1, or a curved surface curved in a plurality of directions.

The reflective element 21a is, for example, a prism or spherical glass beads. The illumination light incident on the reflecting element 21a is emitted from the reflecting element 21a by being reflected in the direction opposite to that at the time of incident. Since the reflective element 21a is minute, there is almost no shift in the path of the illumination light between the time of incident and the time of emission. Therefore, the illumination light reflected by the retroreflective member 21 returns along the same path as the path of the illumination light incident on the retroreflective member 21. That is, the illumination light reciprocates in the same path between the inside of the culture vessel 3 and the retroreflective member 21.

The position and angle at which the retroreflective member 21 is arranged can be arbitrarily set. The retroreflective member 21 may be attached to, for example, a stand or a wall (not shown), or may be attached to the side surface of the culture vessel 3. The installation position and installation angle of the retroreflective member 21 may be any position, distance, and angle that can receive the illumination light transmitted from the illumination light source 15 through the culture vessel 3.

The half mirror 23 is arranged on the optical path between the objective lens 19 and the two-dimensional image sensor 27. The half mirror 23 causes the illumination light from the illumination light source 15 to be incident on the objective lens 19, while the two-dimensional image pickup element 27 emits the illumination light that returns from the retroreflective member 21 through the objective lens 19 to the objective lens 19. Make it transparent toward.

The two-dimensional image sensor 27 is a CCD image sensor or a CMOS image sensor. The two-dimensional image sensor 27 acquires an image of a specific region R in the culture vessel 3 by photographing an image formed by the imaging optical system 25.

The control unit 9 is, for example, a PC (Personal Computer). The control unit 9 includes, for example, an interface circuit, a storage unit such as a hard disk drive, a CPU (Central Processing Unit), and a RAM (Random Access Memory) (all are not shown). Further, as shown in FIG. 1, the control unit 9 includes an image analysis unit 10 that analyzes an image acquired by the two-dimensional image sensor 27.

Various programs executed by the CPU are stored in the storage unit.
The CPU reads various programs stored in the storage unit and executes the following functions. That is, the control unit 9 turns on the illumination light source 15, drives the motor 5c of the stirrer 5, analyzes the image by the image analysis unit 10, acquires the image by the two-dimensional image sensor 27, saves the acquired image, and uses the display unit 11. It controls the display of images and the notification to the user by the display unit 11 or the warning transmission unit 13.

The image analysis unit 10 analyzes the image of the specific region R in the culture vessel 3 acquired by the two-dimensional image sensor 27, and classifies each pixel into a cell S region and a background region. For example, the image analysis unit 10 extracts the contour of the cell S in which the change in brightness is remarkable, classifies the inside of the extracted contour into the cell S, and classifies the outside of the extracted contour as the background. Further, since the background pixels have a low frequency, the image analysis unit 10 may use frequency diffraction together with the pixel classification.

Further, the image analysis unit 10 calculates a representative pixel value representing the pixels classified in the background region. For example, the image analysis unit 10 obtains the average luminance value or the intermediate luminance value of the pixels classified in the background as the representative pixel value of the pixels in the background. Hereinafter, the pixels classified into the background area are also referred to as background pixels.

Further, the control unit 9 irradiates the specific region R in the culture vessel 3 with the illumination light from the illumination light source 15 at predetermined time intervals, and causes the two-dimensional image sensor 27 to capture the illumination light transmitted through the specific region R. As a result, the image of the specific area R is repeatedly acquired at a predetermined timing. Then, the control unit 9 causes the image analysis unit 10 to calculate the representative pixel value of the background pixel for each acquired image of the specific region R, and based on the time-dependent change of the calculated representative pixel value, the culture in the culture container 3 is performed. Determine the state of the liquid W.

Further, the control unit 9 stores the initial representative pixel value of the background pixel in the image of the illumination light transmitted through the specific region R before starting the culture of the cell S. The control unit 9 compares the representative pixel value of the background pixel in the image of the specific region R acquired during the culture of the cell S with the initial representative pixel value. Then, when the representative pixel value of the background pixel is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 notifies the user that it is time to replace the medium by the display unit 11 or the warning transmitting unit 13. To do.

Next, the operation of the medium monitoring device 1 of the present embodiment will be described.
When monitoring the state of the culture solution W while culturing the cells S by the medium monitoring device 1 having the above configuration, first, the culture solution W in the culture container 3 is driven by the control unit 9 to drive the stirrer 5. It is agitated. As a result, the cells S are cultured while floating in the culture medium W.

Next, the control unit 9 turns on the illumination light source 15 at predetermined time intervals, so that the illumination light of 560 nm emitted from the illumination light source 15 passes through the condenser lens 17, the half mirror 23, and the objective lens 19. The specific region R in the culture vessel 3 is irradiated. The illumination light transmitted through the specific region R is reflected by the retroreflective member 21 and returns toward the culture vessel 3 through the same path as the incident path to the retroreflective member 21.

Then, the illumination light is transmitted through the specific region R in the culture vessel 3 again, and then is photographed by the two-dimensional image sensor 27 via the objective lens 19, the half mirror 23, and the imaging optical system 25. As a result, the two-dimensional image sensor 27 repeatedly acquires a time-lapse image of the specific region R. The time-lapse image of the specific region acquired by the two-dimensional image sensor 27 is stored by the control unit 9.

Next, by controlling the image analysis unit 10 by the control unit 9, background pixels are extracted from each image of the specific region R in the culture vessel 3 acquired at predetermined time intervals, and the extracted background pixels are extracted. The representative pixel value (luminance value) is calculated. Then, the control unit 9 monitors the time-dependent change of the representative pixel value of the background pixel for each image calculated by the image analysis unit 10.

When the representative pixel value of the background pixel is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 controls the display unit 11 or the warning transmission unit 13, and the display unit 11 or the warning transmission unit indicates that it is time to replace the medium. The user is notified by the part 13.

In this case, although the region in which the cells S exist in the culture medium W changes from moment to moment, only the background pixels that do not include the pixels of the cells S extracted from the image of the specific region R in the culture vessel 3 change with time. The state of the culture solution W can be determined without being affected by the scattering of the illumination light by the cells S.

Further, the change with time of the background pixel is measured by monitoring the state of the culture solution W using an optical system for acquiring an image of the cell S, that is, an objective lens 19, an imaging optical system 25, and a two-dimensional image sensor 27. It is not necessary to separately provide an optical system for this purpose, and the device can be simplified. Therefore, the state of the culture solution W can be accurately monitored based on the image of the specific region R in the culture vessel 3 without complicating the apparatus.

Further, the illumination light transmitted through the specific region R in the culture vessel 3 is again irradiated to the specific region R in the culture vessel 3 through the same optical path as the incident optical path by the retroreflective member 21, so that the culture vessel 3 is used. Regardless of the material, shape, size, etc. of the above, the image of the specific region R can be reliably acquired by the two-dimensional image pickup element 27. Therefore, the culture solution W can be stably monitored even in a wide variety of culture containers 3 based on the state of the culture solution W determined by the control unit 9.

This embodiment can be transformed into the following configuration.
For example, as shown in FIG. 4, the oblique illumination optical system 29 including the illumination light source 15 and the condenser lens 17 may be arranged at the position where the retroreflective member 21 is arranged.

In this case, the illumination light emitted from the illumination light source 15 of the oblique illumination optical system 29 is obliquely illuminated in the specific region R in the culture vessel 3 via the condenser lens 17. Then, the illumination light transmitted through the specific region R is focused by the objective lens 19, then imaged by the imaging optical system 25 and photographed by the two-dimensional image sensor 27.

According to this modification, the illumination light transmitted through the specific region R in the culture vessel 3 does not pass through the half mirror 23 and the retroreflective member 21, and the illumination light passes through the objective lens 19 once. Therefore, the utilization efficiency of the illumination light can be improved.

[Second Embodiment]
Next, the medium monitoring apparatus according to the second embodiment of the present invention will be described.
The medium monitoring device 31 according to the present embodiment is, for example, as shown in FIGS. 5 and 6, a medium supply unit 33 for supplying the culture medium W to the culture container 3 and a medium for discharging the culture medium W from the culture medium 3. It differs from the first embodiment in that it includes a discharge unit 41, and the control unit 9 controls the medium supply unit 33 and the medium discharge unit 41.
Hereinafter, the parts having the same configuration as the medium monitoring device 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

As shown in FIG. 6, the medium supply unit 33 forms a medium supply tank 35 holding the new culture medium W and a flow path for sending the culture medium W from the medium supply tank 35 to the culture vessel 3. A supply pipe 37 and a medium supply pump 39 that sends the culture solution W in the medium supply tank 35 to the culture vessel 3 via the medium supply pipe 37 are provided.

The medium supply tube 37 is inserted in the culture vessel 3 at a position that does not interfere with the path of the illumination light and does not interfere with the stirring blade 5b. Further, in the culture medium supply tube 37, it is preferable that the supply port 37a for supplying the culture solution W is arranged near the liquid level of the culture solution W contained in the culture container 3, for example.

The medium discharge unit 41 forms a medium discharge tank 43 for collecting the culture medium W discharged from the culture container 3 and a medium discharge pipe 45 for forming a flow path for sending the culture medium W from the culture container 3 to the medium discharge tank 43. And a medium discharge pump 47 that sends the culture solution W in the culture container 3 to the medium discharge tank 43 via the medium discharge pipe 45.

The medium discharge tube 45 is inserted in the culture vessel 3 at a position that does not interfere with the path of the illumination light and does not interfere with the stirring blade 5b. Further, in the culture medium discharge pipe 45, it is preferable that the suction port 45a for sucking out the culture solution W is arranged near the middle of the depth of the culture solution W contained in the culture container 3, for example.

The medium discharge pump 47 causes the cells S in the culture medium W to move in a state where the floating cells S are moved below the culture vessel 3 by gravity, for example, by stopping the stirring of the culture solution W or reducing the stirring speed. The culture solution W in the culture vessel 3 can be discharged at a speed that does not suck out.

The control unit 9 switches ON / OFF of the supply of the culture solution W from the culture medium supply tank 35 to the culture vessel 3 by controlling the drive of the culture medium supply pump 39. Further, the control unit 9 switches ON / OFF of the discharge of the culture solution W from the culture container 3 to the medium discharge tank 43 by controlling the drive of the medium discharge pump 47.

Further, when the representative pixel value of the background pixel calculated by the image analysis unit 10 is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 first stops driving the motor 5c of the stirrer 5. By doing so, the stirring of the culture solution W is stopped. Then, the control unit 9 drives a medium discharge pump 47 to discharge a part of the culture solution W from the culture container 3 to the medium discharge tank 43 via the medium discharge pipe 45, and further, the medium supply pump 39 is discharged. By driving, a new culture solution W is supplied from the medium supply tank 35 to the culture medium 3 via the medium supply pipe 37.

Next, the operation of the medium monitoring device 31 of the present embodiment will be described.
When the state of the culture solution W is monitored while culturing the cells S by the medium monitoring device 31 having the above configuration, the optical measurement unit 7 is controlled by the control unit 9 as in the first embodiment. Images of the specific region R in the culture vessel 3 are repeatedly acquired at time intervals, and the state of the culture solution W is determined based on the time course of the representative pixel values of the background pixels in each image of the specific region R.

When the representative pixel value of the background pixel in the image of the specific region R in the culture vessel 3 calculated by the image analysis unit 10 is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 causes the motor 5c of the stirrer 5 to move. By being controlled, the stirring of the culture solution W is stopped. As a result, the cells S suspended in the culture solution W sink to the lower part in the culture vessel 3.

Next, by driving the medium discharge pump 47 by the control unit 9, about half the amount of the culture solution W in the culture container 3 is discharged, and the discharged culture solution W is collected by the medium discharge tank 43. Since the suction port 45a of the medium discharge pipe 45 is arranged near the middle of the depth of the culture solution W, and the cells S in the culture solution W are sunk in the lower part of the culture container 3, the inside of the culture container 3 Only the culture solution W of the above can be efficiently recovered.

Next, the culture medium supply pump 39 is driven by the control unit 9, so that the culture medium supply tank 35 replenishes the culture vessel 3 with the new culture solution W. As a result, the culture solution W in the culture vessel 3 is exchanged.

After exchanging the culture solution W, the control unit 9 again irradiates the specific region R in the culture vessel 3 with illumination light and acquires an image of the specific region R at predetermined time intervals, and the background pixels of each image are acquired. The state of the culture solution W is monitored based on the time course of the representative pixel value of.

Therefore, according to the medium monitoring device 31 according to the present embodiment, the control unit 9 in the culture vessel 3 is based on the time-dependent change of the representative pixel value of the background pixel in the image of the specific region R calculated by the image analysis unit 10. It is known whether or not the culture solution W has deteriorated, that is, whether or not it is the timing of medium replacement. Therefore, the control unit 9 controls the medium supply unit 33 and the medium discharge unit 41 based on the time course of the representative pixel value of the background pixel, so that the medium can be exchanged at an accurate timing without the trouble of the user. be able to.

Further, by stopping the stirring of the culture solution W at the time of medium exchange, the cells S in the culture solution W move to the lower part in the culture container 3 by gravity. Since the suction port 45a of the medium discharge tube 45 is arranged near the middle of the depth of the culture solution W in the culture container 3, the culture medium W is contained together with the culture solution W discharged by the medium discharge unit 41. It is possible to suppress the excretion of the cell S.

This embodiment can be transformed into the following configuration.
In the present embodiment, the supply port 37a of the medium supply tube 37 is arranged near the liquid level of the culture solution W in the culture vessel 3, but instead, for example, as shown in FIG. 7, the medium is used. The supply pipe 37 may be extended to the lower part in the culture vessel 3, and the supply port 37a may be arranged near the bottom surface 3b of the culture vessel 3.

In this case, it is desirable that the supply port 37a of the culture medium supply pipe 37 is formed in an upward U-shape that folds back toward the upper surface 3a in the vicinity of the bottom surface 3b of the culture vessel 3. Further, it is preferable that the suction port 45a of the medium discharge pipe 45 is arranged at a position slightly lower than the liquid level of the culture solution W.

When exchanging the medium, the control unit 9 stops driving the motor 5c of the stirrer 5 and stops stirring the culture solution W. Further, the control unit 9 discharges the culture medium W from the culture vessel 3 and the culture medium to the culture vessel 3 by controlling the culture medium discharge pump 47 of the culture medium discharge unit 41 and the culture medium supply pump 39 of the culture medium supply unit 33. Supply of W is performed at the same time.

Since the deteriorated culture solution W that needs to be replaced has a lighter specific gravity than the new culture solution W, the upper part in the culture container 3 is the deteriorated culture solution W, and the lower part in the culture container 3 is divided into the new culture solution W. Further, when the medium is exchanged, the stirring of the culture solution W is stopped, so that the cells S in the culture solution W move to the lower part in the culture container 3 by gravity.

Therefore, according to the medium monitoring device 31 according to the present modification, by arranging the suction port 45a of the medium discharge tube 45 near the liquid surface of the culture medium W, the cells S are discharged together with the culture medium W. The risk can be reduced. Further, while the newly supplied new culture solution W stays below the culture container 3, the deteriorated culture solution W that needs to be replaced moves above the culture container 3, so that the culture solution W can be discharged and supplied. Even if they are performed at the same time, only the deteriorated culture solution W can be selectively discharged. Therefore, the time required for medium exchange can be shortened by simultaneously discharging and supplying the culture solution W.

[Third Embodiment]
Next, the medium monitoring apparatus according to the third embodiment of the present invention will be described.
In the medium monitoring device 51 according to the present embodiment, for example, as shown in FIG. 8, the optical measurement unit 7 illuminates the cells S floating in the culture medium W in order to generate a phase difference image of the cells S. A phase difference optical system 63 including an optical system (illumination unit) 53 and a detection optical system 59 that forms a phase difference image of cells S floating in the culture medium W irradiated with illumination light on the light detection unit. It differs from the first embodiment and the second embodiment in that it is provided. The medium monitoring device 51 includes a control unit 9, a display unit 11, and a warning transmission unit 13, but in FIG. 8, the control unit 9, the display unit 11, and the warning transmission unit 13 are not shown.
Hereinafter, the parts having the same configuration as the medium monitoring devices 1 and 31 according to the first embodiment and the second embodiment are designated by the same reference numerals and the description thereof will be omitted.

The illumination optical system 53 includes an illumination light source 15, a condenser lens 17, a diaphragm 55 having a ring slit 55a which is an annular opening, a relay optical system 57, a half mirror 23, and an objective lens 19. There is.

The ring slit 55a of the aperture 55 is arranged at a position optically conjugate with the pupil position of the objective lens 19. The illumination light collected by the condenser lens 17 passes only through the ring slit 55a in the aperture 55. The position of the diaphragm 55 can be adjusted in a direction orthogonal to the optical axis of the illumination light incident on the diaphragm 55.
The relay optical system 57 relays the illumination light that has passed through the ring slit 55a. The relay optical system 57 is composed of, for example, a pair of convex lenses.

The half mirror 23 reflects a part of the illumination light relayed from the illumination light source 15 by the relay optical system 57, for example, about 50% of the illumination light incident on the half mirror 23 toward the objective lens 19, while the objective A part of the illumination light incident from the lens 19 side, for example, about 50% of the illumination light incident on the half mirror 23 is transmitted.

The objective lens 19 has an optical axis arranged in a substantially horizontal direction and is arranged toward the culture vessel 3. In the objective lens 19, the focal plane F is arranged inside the culture vessel 3. The illumination light reflected by the half mirror 23 enters the objective lens 19 along the optical axis of the objective lens 19 and is emitted from the objective lens 19 toward the culture vessel 3. The illumination light emitted from the objective lens 19 passes through the side wall of the culture container 3, crosses the inside of the culture container 3 in a substantially horizontal direction, passes through the side wall of the culture container 3 again, and then passes through the side wall of the culture container 3, and then the outside of the culture container 3. Is ejected to. By adjusting the position of the diaphragm 55, the position of the illumination light incident on the culture vessel 3 from the objective lens 19 can be changed in a direction intersecting the optical axis of the illumination light.

The retroreflective member 21 is arranged so as to sandwich the culture vessel 3 with the objective lens 19 in a substantially horizontal direction.
The objective lens 19 and the retroreflective member 21 are arranged at positions where the stirring rod 5a and the stirring blade 5b of the stirrer 5 do not interfere with the path of the illumination light between the objective lens 19 and the retroreflective member 21.

The detection optical system 59 includes an objective lens 19, a phase film 61 arranged at the pupil position of the objective lens 19, an imaging optical system 25, and a two-dimensional image pickup element 27. That is, the objective lens 19 functions as an illumination optical system 53 and a detection optical system 59.

The phase film 61 has a shape corresponding to the shape of the ring slit 55a of the illumination optical system 53, that is, an annular shape. Further, the phase film 61 shifts the phase of the illumination light transmitted through the phase film 61 by λ / 4. The phase film 61 is arranged at a position conjugate with the ring slit 55a of the illumination optical system 53. The phase film 61 may be arranged at a position optically conjugate with the pupil position of the objective lens 19.

Next, the operation of the medium monitoring device 51 according to the present embodiment will be described.
First, when the state of the culture solution W is monitored while observing the cells S in phase difference by the medium monitoring device 51 having the above configuration, the illumination light source 15 is turned on by the control unit 9, so that the illumination light source 15 emits light. Illumination light is applied to the specific region R in the culture vessel 3 via the condenser lens 17, the aperture 55, the relay optical system 57, the half mirror 23, and the objective lens 19.

The illumination light applied to the specific region R is transmitted by the specific region R and then reflected by the retroreflective member 21. Then, the illumination light passes through the specific region R in the culture vessel 3 in the opposite direction, and then is focused by the objective lens 19. Therefore, the cells S floating in the culture solution W in the specific region R in the culture vessel 3 are illuminated by two types of illumination methods: epi-illumination by the objective lens 19 and transmission illumination by the retroreflective member 21.

While passing through the culture vessel 3 twice, a part of the illumination light (signal light) passes through the transparent cells S floating in the culture solution W and is refracted. After passing through the culture solution W in the culture vessel 3 twice, the illumination light passes through the objective lens 19 and the half mirror 23, and is imaged on the two-dimensional image pickup element 27 by the imaging optical system 25.

Here, in the objective lens 19, the phase film 61 is arranged at a position optically conjugate with the ring slit 55a. The illumination light (refracted light) transmitted through the cells S in the culture vessel 3 passes through a position different from that of the phase film 61 in the objective lens 19 and is emitted from the objective lens 19. On the other hand, the illumination light (straight light) that did not pass through the cells S in the culture vessel 3 is given a phase shift by passing through the phase film 61 in the objective lens 19, and is emitted from the objective lens 19. Therefore, an optical image of cells S with light and darkness due to interference between the refracted light and the straight light is formed on the two-dimensional image sensor 27. As a result, the phase difference image of the cell S is acquired by the two-dimensional image sensor 27.

Next, by controlling the optical measurement unit 7 by the control unit 9, the image of the specific region R in the culture vessel 3 is repeatedly acquired at predetermined time intervals, and the representative pixel value of the background pixel in the image of the specific region R is obtained. The state of the culture solution W is determined based on the change over time.

Then, when the representative pixel value of the background pixel is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 controls the display unit 11 or the warning transmission unit 13, and the display unit indicates that it is the timing of medium replacement. The user is notified by 11 or the warning transmitting unit 13.

In this case, as described above, the retroreflective member 21 reflects the illumination light along the same path as at the time of incident by a large number of minute reflective elements 21a. Therefore, the illumination light incident on the culture vessel 3 from the retroreflective member 21 is the culture vessel 3 regardless of the shape of the side wall of the culture vessel 3 existing between the retroreflective member 21 and the inside of the culture vessel 3. The specific area R inside is illuminated from the same direction at the same angle.

For example, when the side wall of the culture vessel 3 has a curvature or unevenness, the side wall of the culture vessel 3 exerts a lens effect on the illumination light. However, the lens effect is canceled by the illumination light reciprocating along the same path on the side wall of the culture vessel 3. That is, the direction and angle of the illumination light incident on the culture vessel 3 from the retroreflective member 21 are not affected by the side wall between the retroreflective member 21 and the inside of the culture vessel 3.

Therefore, even if the culture vessel 3 is made of a flexible material and the side wall of the culture vessel 3 is deformed over time, or even if the culture vessel 3 is replaced with another culture vessel 3 having a different shape and size, it is retrograde. The cells S in the culture vessel 3 can be stably illuminated by the illumination light from the reflective member 21.

When the side wall of the culture vessel 3 between the objective lens 19 and the inside of the culture vessel 3 is flat, the illumination light incident on the culture vessel 3 from the objective lens 19 travels along the optical axis of the objective lens 19. .. That is, coaxial epi-illumination is realized.

On the other hand, when the side wall of the culture vessel 3 between the objective lens 19 and the inside of the culture vessel 3 has a curvature or unevenness, the optical axis of the illumination light incident on the culture vessel 3 from the objective lens 19 is the culture vessel 3. Due to the lens effect of the side wall of the objective lens 19, it is tilted with respect to the optical axis of the objective lens 19. As a result, the position of the illumination light (straight light) returned from the retroreflective member 21 to the objective lens 19 may shift from the position of the phase film 61 in the direction intersecting the optical axis. In this case, the illumination light (straight light) returned from the retroreflective member 21 to the objective lens 19 by adjusting the position of the illumination light emitted from the illumination optical system 53 to the culture vessel 3 by adjusting the position of the aperture 55. ) Passes through the phase film 61.

As described above, according to the medium monitoring device 51 according to the present embodiment, the phase difference optical system 63 including the illumination optical system 53 and the detection optical system 59 provides high resolution of the cells S in the culture medium W. The state of the culture medium W can be monitored using an image having high contrast.

[Fourth Embodiment]
Next, the medium monitoring apparatus according to the fourth embodiment of the present invention will be described.
The medium monitoring device 71 according to the present embodiment is, for example, as shown in FIGS. 9 to 11, a housing that houses an illumination light source 15, an imaging optical system 25, and a two-dimensional imaging element 27 that constitute the optical measurement unit 7. The culture vessel 3 is provided with a 73 and a tubular protective tube 77 that covers the periphery of the housing 73, and the housing 73 whose circumference is covered by the protective tube 77 is inserted into the culture medium W in the culture vessel 3. It differs from the first embodiment in that an image of a specific area R in the inside is acquired. In the present embodiment, the image analysis unit 10, the display unit 11, and the warning transmission unit 13 are not shown.
Hereinafter, the parts having the same configuration as the culture medium monitoring devices 1, 31 and 51 according to the first to third embodiments are designated by the same reference numerals and the description thereof will be omitted.

In the present embodiment, as shown in FIG. 9, the culture vessel 3 is provided with a plurality of ports 3c on the upper surface 3a for inserting various tubes 75. In the example shown in FIG. 9, three ports 3c are provided on the upper surface 3a of the culture vessel 3, and cells S in the culture solution W are collected or a drug solution is administered to the culture solution W in the two ports 3c. A tube 75 is inserted. Each port 3c is provided with an O-ring (not shown) for closing the gap with the tube 75. As a result, the inside of the culture vessel 3 is maintained in a sealed state.

The housing 73 has an elongated tubular shape that can be inserted into and removed from the protective tube 77. The housing 73 is made of, for example, polyvinyl chloride or the like and has flexibility. As shown in FIG. 10, the housing 73 has a transparent portion 73a that transmits illumination light and observation light at the tip in the longitudinal direction.

The protective tube 77 has an elongated shape that can be inserted into the culture solution W via the port 3c of the culture container 3. Further, the protective tube 77 is formed so as to accommodate the housing 73 inside. When the protective tube 77 is inserted into the port 3c, the gap between the protective tube 77 and the port 3c is closed by an O-ring (not shown). The protective tube 77 is made of a transparent resin material such as acrylic resin (PMMA) or polyvinyl chloride. Therefore, the entire protective tube 77 constitutes an optically transparent transparent portion that transmits illumination light and observation light. In the present embodiment, the tip of the protective tube 77 in the longitudinal direction is the transparent portion 77a.

The protective tube 77 has a protrusion 79 protruding in the longitudinal direction of the protective tube 77 on the outside of the transparent portion 77a. As shown in FIGS. 10 and 11, for example, the protrusion 79 has a columnar portion 79a extending from the tip of the protective tube 77 along the longitudinal direction of the protective tube 77, and the protrusion 79 from the tip of the columnar portion 79a in the longitudinal direction of the protective tube 77. It is provided with a bent portion (reflecting member) 79b arranged at a position that blocks the front of the transparent portion 53a by bending in the intersecting direction.

The columnar portion 79a is arranged at a position deviated from each optical axis of the illumination light source 15 and the imaging optical system 25 with the housing 73 inserted in the protective tube 77. The bent portion 79b is housed in the protective tube 77. With the body 73 inserted, it is arranged on the optical axis of the illumination light source 15 and the imaging optical system 25. The bent portion 79b reflects the illumination light emitted from the illumination light source 15 to the outside of the protective tube 77 via the transparent portion 73a of the housing 73 and the transparent portion 77a of the protective tube 77 toward the imaging optical system 25. By doing so, it functions as a reflective member that obliquely illuminates the specific area R in the culture vessel 3.

As shown in FIGS. 10 and 11, the illumination light source 15 is arranged at the tip of the housing 73 so as to face the transparent portion 73a.
The imaging optical system 25 is arranged side by side with the illumination light source 15 at the tip of the housing 73 so as to face the transparent portion 73a. The imaging optical system 25 forms an image of the observation light incident on the housing 73 via the transparent portion 73a on the light receiving surface of the two-dimensional image sensor 27.
The two-dimensional image sensor 27 is arranged at the tip of the housing 73 on the proximal end side of the imaging optical system 25.

Next, the operation of the medium monitoring device 71 according to the present embodiment will be described.
When monitoring the state of the culture solution W while observing the cells S with the medium monitoring device 71 having the above configuration, as shown in FIGS. 9 to 11, the housing 73 whose circumference is covered with the protective tube 77 is used as the culture container. It is inserted into the culture medium W via the port 3c of 3. The housing 73 is sterilized in advance.

Next, the illumination light is emitted from the illumination light source 15 in the housing 73 via the transparent portion 73a of the housing 73 and the transparent portion 77a of the protective tube 77. The illumination light emitted from the transparent portion 77a of the protective tube 77 is reflected toward the transparent portion 77a by the bent portion 79b of the protrusion 79 in front of the transparent portion 77a. As a result, the specific region R in the culture vessel 3 between the transparent portion 77a and the bent portion 79b of the protective tube 77 is irradiated with the illumination light.

The observation light returned from the specific region R by being irradiated with the illumination light is imaged by the imaging optical system 25 via the transparent portion 77a of the protective tube 77 and the transparent portion 73a of the housing 73, and the optics of the observation light. The image is taken by the two-dimensional image sensor 27.

Next, by controlling the optical measurement unit 7 by the control unit 9, the image of the specific region R in the culture vessel 3 is repeatedly acquired at predetermined time intervals, and the representative pixel value of the background pixel in the image of the specific region R is obtained. The state of the culture solution W is determined based on the change over time.

Then, when the representative pixel value of the background pixel is lower than the initial representative pixel value by a predetermined amount or more, the control unit 9 controls the display unit 11 or the warning transmission unit 13, and the display unit indicates that it is the timing of medium replacement. The user is notified by 11 or the warning transmitting unit 13.

As described above, according to the medium monitoring device 71 according to the present embodiment, the housing is covered with the protective tube 77 by using the port 3c for inserting the tube 75 into the culture container 3. The culture container 73 can be inserted into the culture vessel 3, and further, the culture vessel to be used is used by irradiating the specific region R with illumination light and receiving the observation light from the specific region R from the housing 73 inserted in the culture solution W. A good observation image of the specific region R can be obtained with almost no restrictions on the shape, size, material, etc. of 3. Therefore, it can be applied to a wide variety of culture vessels 3, and the state of the culture solution W can be stably monitored in various culture vessels 3.

Further, since the protective tube 77 has a shape that can be inserted into the culture solution W via the port 3c of the culture container 3, the protective tube 77 allows the housing 73, the illumination light source 15 in the housing 73, and the imaging optics. With the system 25 and the two-dimensional imaging element 27 safely protected, the housing 73, the illumination light source 15, the imaging optical system 25 and the two-dimensional imaging element 27 are inserted into the culture vessel 3 and inside the culture vessel 3. Can be operated with. Further, by forming the protective tube 77 with a transparent resin material such as acrylic resin or polyvinyl chloride, the protective tube 77 is used in a UV sterilized state, and after use, only the protective tube 77 is made disposable and replaced. Can be done. As a result, it is possible to avoid contamination of the culture solution W as compared with the case where the housing 73 to be used repeatedly is directly inserted into the culture solution W.

Further, the specific region R irradiated with the illumination light in the culture vessel 3 is limited to the space between the transparent portion 77a of the protective tube 77 and the bent portion 79b of the protrusion 79, so that the transparent portion of the protective tube 77 The cells S that have invaded the space between the 77a and the bent portion 79b of the protrusion 79 can be photographed.

[Fifth Embodiment]
Next, the medium monitoring apparatus according to the fifth embodiment of the present invention will be described.
The medium monitoring device 81 according to the present embodiment has, for example, as shown in FIGS. 12 and 13, a protective cover 83 having an oblique illumination mirror 89 inside and capable of accommodating the housing 73 instead of the protective tube 77. It is different from the fourth embodiment in that the optical measurement unit 7 performs stereo measurement.
Hereinafter, the parts having the same configuration as the medium monitoring device 71 according to the fourth embodiment are designated by the same reference numerals and the description thereof will be omitted.

As shown in FIG. 14, the optical measurement unit 7 according to the present embodiment has the same cells S as the illumination light source 15 and the light guide fiber 85 that guides the illumination light emitted from the illumination light source 15. It includes a stereo optical system 87 that forms images of two images that are different from each other when viewed from different viewpoints, and a two-dimensional image pickup element 27 that captures two images formed by the stereo optical system 87, respectively.

The illumination light source 15, the stereo optical system 87, and the two-dimensional image sensor 27 are housed in the housing 73. In the present embodiment, the housing 73 has a flange portion 73b that extends in the width direction that intersects the longitudinal direction at the base end portion in the longitudinal direction.

The illumination light source 15 is arranged on the base end side of the housing 73.
The light guide fiber 85 guides the illumination light from the illumination light source 15 to the tip of the housing 73.
The stereo optical system 87 includes an objective optical system 87a that collects the observation light from the specific region R, an aperture opening 87b that divides the observation light collected by the objective optical system 87a, and an aperture opening in this order from the tip side. It includes a deflection prism 87c that deflects the observation light divided by the unit 87b, and an imaging optical system 87d that forms an image of the observation light deflected by the deflection prism 87c.

In the stereo optical system 87, one of the directions orthogonal to the optical axis of the stereo optical system 87 and orthogonal to each other is stereo. Hereinafter, the arrangement direction of the viewpoints of the stereo optical system 87 is referred to as a stereo direction.
The aperture opening 87b is arranged at the pupil position of the objective optical system 87a. In the aperture opening 87b, for example, two holes (not shown) are formed at intervals in the stereo direction.
The two-dimensional image sensor 27 is arranged on the most proximal side of the housing 73.

The protective cover 83 has a tubular shape that can be inserted into the culture solution W via the port 3c of the culture container 3 while accommodating the housing 73. Further, when the protective cover 83 is inserted into the port 3c, the gap between the protective cover 83 and the port 3c is closed by an O-ring (not shown). In addition, the protective cover 83 is sterilized and can be replaced as a disposable part that is disposable after each use. The protective cover 83 has an inner diameter dimension at which the flange portion 73b of the housing 73 abuts against the insertion port of the protective cover 83 in a state where the housing 73 is housed, and between the tip of the protective cover 83 and the tip of the housing 73. It has a length dimension in which a space is formed.

Further, the protective cover 83 has openings 83a that open on both side surfaces facing each other in the width direction at the tip portion in the longitudinal direction. These openings 83a are arranged between the tip of the protective cover 83 and the objective optical system 87a in a state where the housing 73 is inserted into the protective cover 83. Further, the opening 83a has a size that allows the cells S and the culture solution W to pass through the inside of the protective cover 83 in the width direction. These openings 83a are formed, for example, by dividing the protective cover 83 into a distal end side and a proximal end side in the longitudinal direction and connecting the distal end side and the proximal end side at two locations in the circumferential direction of the protective cover 83. It may be an opening to be closed.

At the tip of the protective cover 83, an oblique illumination mirror 89 arranged so as to face the base end side of the protective cover 83 is provided.
The reflective surface of the oblique illumination mirror 89 has an inclination of a predetermined angle with respect to the longitudinal direction of the protective cover 83. The oblique illumination mirror 89 has an angle of illumination light emitted from the light guide fiber 85 in a direction orthogonal to the stereo direction toward the stereo optical system 87 with the housing 73 housed in the protective cover 83. To reflect. As a result, in the culture vessel 3, the specific region R in which the cells S in the culture solution W that has entered the opening 83a of the protective cover 83 can be obliquely illuminated.

Since the refractive indexes in the culture solution W and the cells S are different from each other, the light is bent at the boundary between the culture solution W and the cells S. For example, the portion where the light is bent in the direction passing outside the pupil of the objective optical system 87a becomes dark on the image plane, and the portion where the light is bent in the direction passing inside the pupil of the objective optical system 87a becomes bright on the image plane. Therefore, by obliquely illuminating the cells S with the oblique illumination mirror 89, it is possible to acquire an image in which the contrast of the cells S is improved.

Next, the operation of the medium monitoring device 81 according to the present embodiment will be described.
When monitoring the state of the culture solution W while culturing the cells S by the medium monitoring device 81 having the above configuration, first, the housing 73 equipped with the protective cover 83 is passed through the port 3c of the culture container 3 and the culture solution W is used. The housing 73 is inserted inside, and the illumination light is generated from the illumination light source 15.

The illumination light emitted from the illumination light source 15 is guided by the light guide fiber 85, and is emitted from the tip of the light guide fiber 85 toward the oblique illumination mirror 89 in the protective cover 83. As a result, the illumination light reflected by the oblique illumination mirror 89 is irradiated to the specific region R in which the cells S in the culture solution W that have entered the protective cover 83 through the opening 83a are present.

Then, when the illumination light transmitted through the specific region R is incident on the stereo optical system 87 in the housing 73, the stereo optical system 87 causes two images having parallax with each other as viewed from different viewpoints with respect to the specific region R. Is tied. As a result, the two-dimensional image sensor 27 allows the plurality of cells S to have two two-dimensional images having parallax from different viewpoints for each individual cell S, for example, the upper image shown in FIG. 15 and the upper image shown in FIG. The lower image is acquired.

Next, the image analysis unit 10 analyzes one or both of the upper image and the lower image shown in FIG. 15 to extract the background pixels of those images, and then the representative pixel values of the extracted background pixels. Is calculated.

In this case, the stereo optical system 87 forms two-dimensional images of the plurality of cells S in the culture solution W, which are different from each other when viewed from different viewpoints for each individual cell S. Between the images of these two images acquired by the image pickup element 27, the positions of the same cells S in the stereo direction are shifted in opposite directions depending on the distance from the stereo optical system 87.

Therefore, since the three-dimensional position of each cell S can be known based on the amount of misalignment of each cell S, the image analysis unit 10 accurately distinguishes between the cell S contained in the specific region R and the cell S not contained in the specific region R. Can be distinguished. That is, the specific region R can be accurately defined as a three-dimensional region, and for example, the cell density existing in the three-dimensional region defined by the specific region R can be accurately measured. As a result, according to the medium monitoring device 81 according to the present embodiment, as in the fourth embodiment, the image of the specific region R in the culture vessel 3 is imaged regardless of the shape and size of the culture vessel 3 used. The representative pixel value of the background pixel can be calculated accurately.

The third to fifth embodiments can be transformed into the following configurations.
For example, each of the third to fifth embodiments is provided with the configuration of the second embodiment, that is, the medium supply unit 33 and the medium discharge unit 41, and the control unit 9 controls the medium supply unit 33 and the medium discharge unit 41. The configuration may be applied.

Further, for example, in each of the third to fifth embodiments, the configuration according to the modified example of the second embodiment, that is, the supply port 37a of the medium supply pipe 37 is arranged near the bottom surface 3b of the culture vessel 3, and the medium is used. The suction port 45a of the discharge pipe 45 is arranged near the liquid surface of the culture solution W, and the control unit 9 simultaneously discharges the culture solution W by the medium discharge unit 41 and supplies the culture solution W by the medium supply unit 33. May be applied.

In addition, each of the above embodiments can be transformed into the following configuration.
As a first modification, in the first to fifth embodiments and the modification thereof, for example, the control unit 9 and the optical measurement unit 7 are not separated, and a part or all of the control unit 9 is the optical measurement unit 7. It may be included in. For example, the control unit 9 may be housed in a housing that houses the illumination light source 15, the two-dimensional image sensor 27, and the like.

As a second modification, in the first to fifth embodiments and the modification thereof, for example, as shown in FIG. 16, the illumination light source is a white light source such as a halogen light source instead of the illumination light source 15 such as an LED. A condenser lens 17 that adopts 91 and further converts the light emitted from the white light source 91 into parallel light, and a bandpass filter 93 that cuts out a specific wavelength from the light converted into parallel light by the condenser lens 17. May be adopted.

In this case, when measuring the light intensity of the illumination light, the illumination light emitted from the white light source 91 is sent to the specific region R in the culture vessel 3 by turning on the white light source 91 or opening and closing a shutter (not shown). It may be irradiated.

According to this modification, since the halogen light source and the bandpass filter are inexpensive, the cost can be reduced. Further, since the configurations of the white light source 91 and the bandpass filter 93 have a high degree of freedom in wavelength selection, they can be applied to various culture solutions W.

As a third modification, in the first to third embodiments and the modification thereof, for example, as shown in FIG. 17, the direction of the half mirror 23 may be rotatable by 90 °. Then, by rotating the half mirror 23 by 90 °, the illumination light from the illumination light source 15 is transmitted toward the culture vessel 3 by the half mirror 23, and the illumination light from the illumination light source 15 is two-dimensionally transmitted by the half mirror 23. The case of reflecting the light toward the image pickup element 27 may be switched.

In this case, first, by arranging the half mirror 23 at the angle shown by the solid line in FIG. 17, the illumination light from the illumination light source 15 is incident on the two-dimensional image sensor 27 by the half mirror 23, and the illumination does not pass through the culture solution W. The light intensity of the light is measured by the two-dimensional image sensor 27.

Next, by switching the half mirror 23 to the angle shown by the broken line in FIG. 17, the illumination light from the illumination light source 15 is transmitted toward the culture solution W in the culture vessel 3. Then, the illumination light that is folded back by the retroreflective member 21 and transmitted back through the culture solution W is incident on the two-dimensional image sensor 27 by the half mirror 23, and the observation light that returns from the specific region R in the culture container 3 is 2 The image is taken by the two-dimensional image sensor 27.

Then, when determining the state of the culture solution W based on the time-dependent change of the representative pixel value of the background pixel in the image of the specific region R, the output of the illumination light source 15 fluctuates depending on the intensity of the illumination light that does not pass through the culture solution W. It may be possible to correct the influence of. As a result, the state of the culture solution W can be correctly evaluated even when the output of the illumination light source 15 fluctuates.

As the fourth modification, in the first to fifth embodiments and the modification thereof, the deterioration of the culture solution W may be measured by the color change.
In this modification, for example, as shown in FIG. 18, a white light source 91 such as a halogen light source is adopted as the illumination light source. Further, three bandpass filters 93A, 93B, 93C having different transmission wavelengths and a switching mechanism 95 such as a slider that selectively arranges these three bandpass filters 93A, 93B, 93C on the path of illumination light are provided. It is arranged between the condenser lens 17 and the half mirror 23. Further, as the culture solution W, Dulbecco MEM containing 0.001% phenol red and 10% fetal bovine serum is adopted.

The bandpass filter 93A is, for example, a filter (BP441) having a center wavelength of 441 nm and a bandwidth, that is, a transmission wavelength width of 10 nm. The bandpass filter 93B is, for example, a filter (BP578) having a center wavelength of 578 nm and a transmission wavelength width of 10 nm. The bandpass filter 93C is, for example, a filter (BP634) having a center wavelength of 634 nm and a transmission wavelength width of 10 nm.

First, as a setup, the relationship between the pH and the absorbance of the culture solution W is determined experimentally.
First, the observation light from the specific region R in the culture vessel 3 that does not contain both the cells S and the culture solution W is photographed by the two-dimensional image sensor 27 for each bandpass filter 93A, 93B, 93C. In this case, the representative pixel value of the background pixel in the image of the specific area R when the bandpass filter 57A is used is I _{0_441} , and the representative pixel value of the background pixel in the image of the specific area R when the bandpass filter 93B is used. I _{0_578} , and I _{0_634} is a representative pixel value of the background pixel in the image of the specific region R when the bandpass filter 93C is used. Here, since the image of the specific region R in the culture vessel 3 that does not contain both the cell S and the culture solution W is taken, the pixels of the cell S are not extracted and all the pixels are used as background pixels. Be extracted.

Next, the observation light from the specific region R in the culture vessel 3 containing the culture solution W having a known pH is photographed by the two-dimensional image sensor 27 for each bandpass filter 93A, 93B, 93C. The pH of the culture solution W may be measured, for example, by directly inserting a pH sensor (not shown) into the culture solution W. In this case, the representative pixel value of the background pixel in the image of the specific area R when the bandpass filter 93A is used is I ₄₄₁ , and the representative pixel value of the background pixel in the image of the specific area R when the bandpass filter 93B is used. The representative pixel value of the background pixel in the image of the specific region R when I ₅₇₈ and the bandpass filter 93C are used is set to I ₆₃₄ . Here, since the image of the specific region R in the culture vessel 3 containing only the culture solution W is taken, the pixels of the cell S are not extracted and all the pixels are extracted as the background pixels.

In this case, the absorbance (A ₄₄₁ , A ₅₇₈ , A ₆₃₄ ) for each wavelength in the specific region R is expressed by the following equation.
A ₄₄₁ = _-log (I ₄₄₁ / I _{0_441} )
A ₅₇₈ = _-log (I ₅₇₈ / I _{0_578} )
A ₆₃₄ = _-log (I ₆₃₄ / I _{0_634} )

The above measurement is performed on a plurality of pHs of the culture solution W, and the relational expression between the absorbance and the pH of each wavelength in the culture solution W is obtained. For example, the following equation can be obtained.
pH = log {(A _441- A ₆₃₄ ) / (A _578- A ₆₃₄ )} * 1.19 + 7.86
1.19 shows the slope of a straight line when plotting log {(A _441- A ₆₃₄ ) / (A _578- A ₆₃₄ )} vs. pH, and 7.86 shows the intercept of that straight line.

Next, as the main measurement, the change over time of the culture solution W is measured.
The bandpass filters 93A, 93B, and 93C are switched to acquire an image of the specific region R, background pixels are extracted from the acquired image, and a representative pixel value of the background pixel is calculated. Next, using the representative pixel values I _{0_441} , I _{0_578} , and I _{0_634} of the background pixels for each wavelength in the state where the cells S and the culture solution W were not contained, which were obtained in the experiment as a setup, for each wavelength in this measurement. Determine the absorbance. From the absorbance for each wavelength obtained here and the relational expression between the absorbance and the pH obtained by the setup, the change over time in the pH of the culture solution W is obtained.

According to this modification, the deterioration of the culture solution W is determined by obtaining the pH value of the culture solution W from the time-dependent change of the representative pixel value of the background pixels in the image of the specific region R of a plurality of wavelengths. The measurement accuracy of deterioration can be improved.

As a fifth modification, instead of the white light source 91 of the fourth modification, for example, as shown in FIG. 19, a plurality of LED light sources (monochromatic light sources) 15A, which emit illumination light of different wavelengths, are used as the illumination light source. 15B and 15C may be adopted. In the example shown in FIG. 19, it is assumed that the LED light source 15A emits 441 nm monochromatic light, the LED light source 15B emits 578 nm monochromatic light, and the LED light source 15C emits 634 nm monochromatic light.

In this case, instead of the condensing lens 17, the bandpass filters 93A, 93B, 93C and the switching mechanism 95, the condensing lenses 17A, 17B, 17C that condense the illumination light from the LED light sources 15A, 15B, 15C, and A mirror 97 and a dichroic mirror 99A, 99B that synthesize these light paths by reflecting or transmitting the illumination light collected by the condenser lenses 17A, 17B, and 17C may be adopted.

According to this modification, instead of switching the bandpass filters 93A, 93B, 93C as in the fourth modification, the measurement wavelength can be measured simply by switching ON / OFF of each LED light source 15A, 15B, 15C. Switching can be done.

As a sixth modification, as the first to third embodiments and as a modification thereof, for example, as shown in FIG. 20, the space between the objective lens 19 and the culture vessel 3 is refracted differently from air. A medium having a refractive index, for example, the immersion medium M of the objective lens 19 may be filled. The immersion medium M is, for example, water, oil, gel or a water-absorbing polymer. The refractive index of the immersion medium M is preferably the same as or close to the refractive index of the culture vessel 3 and the culture solution W.

According to this modification, the illumination light incident on the culture vessel 3 from the objective lens 19 and the observation light incident on the objective lens 19 from the culture vessel 3 by the immersion medium M between the objective lens 19 and the culture vessel 3 The influence of refraction at the boundary between the culture vessel 3 and the culture liquid W can be reduced. Further, the numerical aperture of the objective lens 19 can be increased to improve the resolution.

As a seventh modification, a white light source that emits wavelengths in the entire visible light range is adopted as the illumination light source as the first to third embodiments and their modifications, and a color CCD (color CCD) is used as the two-dimensional image sensor 27. (Not shown) is adopted, and the control unit 9 determines the relationship between the hue and pH of the culture solution W obtained from the background pixels of the image of the specific region R in the culture vessel 3 acquired by one imaging with the color CCD. Based on this, the state of the culture solution W may be determined.

In this case, the image analysis unit 10 extracts background pixels from the color image of the specific region R acquired by the color CCD, and uses the average value or intermediate value of each intensity of RGB in the background pixels as the representative pixel value. The color information of the culture solution W, that is, the hue angle of the culture solution W is calculated.

Specifically, as shown in FIG. 21, for example, the image analysis unit 10 assigns RGB intensities to the three axes of X (red), Y (green), and Z (blue) in a square color space ( The hue angle of the culture solution W is calculated by converting RGB) into a cylindrical color space (HSL) composed of three components of hue, saturation, and lightness. Hue indicates color information independent of saturation and brightness.

The control unit 9 stores, for example, a table as shown in FIG. 22 showing the relationship between the hue angle and the pH. Based on this table, the control unit 9 obtains the pH of the culture solution W from the hue angle calculated by the image analysis unit 10, and determines the state of the culture solution W from the change over time of the pH.
According to this modification, the state of the culture solution W can be determined more accurately by obtaining the pH of the culture solution W from the color information of the culture solution W.

In this modification, a three-plate color CCD may be adopted as the two-dimensional image sensor 27. In this case, for example, as shown in FIG. 23, the observation light from the specific region R is wavelength-separated from the dichroic prism 101 that separates the wavelengths into the wavelength regions of R (red), G (green), and B (blue). A three-sheet color CCD may be configured by a red image sensor 103A, a green image sensor 103B, and a blue image sensor 103C that capture light in each wavelength range.

Further, in the present modification, a single plate type color CCD may be adopted as the two-dimensional image sensor 27. In this case, for example, as shown in FIG. 24, a plurality of color filters 105a that transmit light in the red wavelength region, a plurality of color filters 105b that transmit light in the green wavelength region, and light in the blue wavelength region. A single plate type color CCD may be configured by one image sensor 105 in which a plurality of color filters 105c that transmit light are assigned to a plurality of pixels.

This modification can be transformed into the following configuration.
In this modification, a white light source 91 is adopted as the illumination light source, and a color CCD is adopted as the two-dimensional image sensor 27. Instead of this, a plurality of LED light sources (not shown) that emit each RGB color wavelength are adopted as the illumination light source, or a switchable bandpass filter (not shown) that extracts the white light source 91 and each RGB wavelength is used. It may be adopted. Further, a monochrome CCD (not shown) may be adopted as the two-dimensional image sensor 27.

In this case, the color of the specific region R is obtained by synchronizing the switching of the RGB illumination wavelength by switching the plurality of LED light sources or the plurality of bandpass filters and the photographing of the observation light from the specific region R by the monochrome CCD. The images may be acquired sequentially.
According to this modification, the detection sensitivity of the observation light can be improved by adopting a monochrome CCD as the two-dimensional image sensor.

As an eighth modification, in the first to fifth embodiments and their modifications, for example, the entire medium monitoring device 1, 31, 51, 71, 81 including the optical measurement unit 7 and the culture vessel 3 is darkened. The state of the culture medium W may be monitored while the culture medium W is placed in the place.
With this configuration, the intensity of the illumination light transmitted through the culture solution W can be accurately measured without being affected by the light of the lighting equipment, the light of the monitor and the outside light.

Further, in each of the above embodiments, the bottomed cylindrical culture container 3 formed of an optically transparent material has been illustrated as a container, but the culture container may be bag-shaped, spherical, box-shaped, or the like. , Any shape can be adopted. For example, a disposable bag-shaped culture container may be adopted. Further, as the culture container, 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 illumination light is transmitted.

As described above, according to each of the above embodiments, the state of the culture solution W can be measured in a non-contact manner without directly inserting the pH sensor into the culture solution W, and the risk of contamination of the culture system can be reduced.
Although the embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and design changes and the like within a range not deviating from the gist of the present invention are also included. 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, even when the medium supply unit 33, the medium discharge unit 41, and the warning transmission unit 13 are used in combination and the medium is automatically exchanged under the control of the control unit 9, the user is informed that it is the timing of the medium exchange. It may be notified.

1,31,51,71,81 Medium monitoring device 9 Control unit 10 Image analysis unit 13 Warning transmission unit (notification unit)
15 Illumination light source (illumination unit, monochromatic light source)
15A, 15B, 15C LED light source (monochromatic light source)
17 Condensing lens (illumination unit)
19 Objective lens (illumination unit)
20 Oblique illumination optical system 21 Retroreflective member 27 Two-dimensional image sensor (imaging unit)
33 Medium supply unit 41 Medium discharge unit 53 Illumination optical system (illumination unit)
63 Phase difference optical system 73 Housing 77 Protective tube 79b Bent part (reflective member)
91 White light source 93, 93A, 93B, 93C Bandpass filter S cell W culture medium (medium)

Claims (16)

An illumination unit that irradiates a specific area where the medium and cells in the container are present,
An imaging unit that acquires an image of the specific region by photographing the observation light from the specific region irradiated with the illumination light.
An image analysis unit that divides the image of the specific region acquired by the imaging unit into the pixel of the cell and the pixel of the background and calculates the representative pixel value representing the pixel of the background.
The image pickup unit repeatedly acquires an image of the specific region at a predetermined timing, and the image analysis unit calculates the representative pixel value for each acquired image of the specific region, and the calculated representative pixel value with time. A medium monitoring device including a control unit that determines the state of the medium based on changes. A retroreflective member having an array in which a plurality of minute reflective elements are arranged, arranged with the container sandwiched between the illumination unit, and reflecting the illumination light transmitted through the specific area in the container. With
The medium monitoring device according to claim 1, wherein the imaging unit acquires an image of the specific region that has been re-irradiated with the illumination light reflected by the retroreflective member. The medium monitoring device according to claim 2, wherein the illumination unit includes an oblique illumination optical system that obliquely illuminates the specific area from a direction inclined with respect to the optical axis of the imaging unit. The medium monitoring apparatus according to claim 2, wherein the illumination unit and the imaging unit include a retardation optical system that generates a retardation image of the specific region. It has a transparent portion capable of transmitting light, and includes a housing for accommodating the illumination unit and the imaging unit.
With the housing inserted into the medium in the container, the illumination unit irradiates the specific region with the illumination light via the transparent portion, and the imaging unit passes the transparent portion through the specific region. The medium monitoring apparatus according to claim 1, wherein the image of the culture medium is acquired. 5. The fifth aspect of the present invention includes a reflecting member that obliquely illuminates the specific area by reflecting the illumination light emitted from the illumination unit to the outside of the housing via the transparent unit toward the image pickup unit. The medium monitoring device described. A tubular protective tube that covers the periphery of the housing is provided.
The culture medium monitoring device according to claim 6, wherein the reflective member is provided at the tip of the protective tube. Equipped with a notification unit that notifies the user of information
The medium monitoring device according to any one of claims 1 to 7, wherein the control unit notifies the user of the timing of medium replacement by the notification unit. A medium supply unit that supplies the medium to the container,
A medium discharge unit for discharging the medium from the container is provided.
When the control unit determines that it is time to replace the medium, the medium discharge unit discharges a part of the medium from the container, and the medium supply unit supplies a new medium to the container. The culture medium monitoring apparatus according to any one of claims 1 to 8. The culture medium monitoring apparatus according to any one of claims 1 to 9, wherein the illumination unit includes a monochromatic light source that emits the illumination light having a single wavelength. The medium monitoring apparatus according to claim 10, wherein the monochromatic light source includes a white light source and a bandpass filter that extracts only a single wavelength from the light emitted from the white light source. The medium monitoring device according to claim 10, wherein the monochromatic light source is an LED light source. The illumination unit includes a monochromatic light source that emits a plurality of monochromatic lights having different wavelengths as the illumination light.
The medium is based on the time course of the representative pixel value of the background pixel in the image of each specific region acquired by the imaging unit for each wavelength of the monochromatic light irradiated to the specific region. The culture medium monitoring apparatus according to any one of claims 1 to 9, wherein the state of the medium is determined. The monochromatic light source is provided with a white light source and a plurality of bandpass filters that are detachably provided on the optical path of the light emitted from the white light source and extract only a single wavelength different from each other from the light from the white light source. 13. The medium monitoring device according to claim 13. The medium monitoring device according to claim 13, wherein the monochromatic light source is a plurality of LED light sources having different wavelengths. The lighting unit is provided with a white light source.
The imaging unit includes a color CCD
According to claim 1, the control unit determines the state of the medium based on the relationship between the hue and pH of the medium obtained from the background pixels in the image of the specific region acquired by the color CCD. Item 9. The medium monitoring device according to any one of Items 9.