WO2021117211A1

WO2021117211A1 - Cell culture device

Info

Publication number: WO2021117211A1
Application number: PCT/JP2019/048875
Authority: WO
Inventors: 達哉南; 佐々木　浩; 祐輔松本
Original assignee: オリンパス株式会社
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2021-06-17
Also published as: JPWO2021117211A1

Abstract

A cell culture device (1) comprising an observation unit (7) for observing cells (S) accommodated in a culture tank (3) together with a medium (W), a stirring mechanism (5) for stirring the medium (W) in the culture tank (3), a collecting unit (9) arranged adjacent to the observation unit (7) and collecting the cells (S) in the culture tank (3), and a controller (11) for detecting the cells (S) having desired characteristics through the observation unit (7) and collecting the detected cells (S) having desired characteristics with the collecting unit (9).

Description

Cell culture device

The present invention relates to a cell culture device.

In recent years, with the progress of stem cell research and regenerative medicine, and the development of biopharmacy such as antibody medicine, it is required to culture a large amount of cells. When culturing a large amount of cells, instead of using a flask or a petri dish, a culture tank such as a bioreactor is often used (see, for example, Patent Document 1). When cells in culture come into contact with other cells, they can be adversely affected by contact irritation. Therefore, it is desirable to collect and store the cells in the desired state at the optimum timing.

Japanese Unexamined Patent Publication No. 2011-188777

Normally, in a suspension culture system using a culture tank such as a bioreactor, the culture solution and the cells are separated by centrifuging the culture solution and the cells are collected. However, the conventional recovery method has the disadvantage that all the cultured cells are recovered, and it is not possible to recover only the desired cells.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell culture apparatus capable of recovering only desired cells at an optimum timing while continuing culturing in a suspension culture system. There is.

In order to achieve the above object, the present invention provides the following means.
One aspect of the present invention is an observation unit for observing cells housed together with a medium in a culture tank, a stirring unit for stirring the medium in the culture tank, and an observation unit adjacent to the observation unit. It includes a recovery unit for collecting the cells in the culture tank, and a control unit for detecting the cells having desired characteristics by the observation unit and collecting the detected cells having the desired characteristics by the collection unit. It is a cell culture device.

According to this aspect, the medium in the culture tank is agitated by the stirring unit, so that the cells in the culture tank are cultured in a state of being suspended in the medium. In this state, cells floating in the medium are observed by the observation unit, and cells having desired characteristics are detected by the control unit. Then, when a cell having a desired characteristic is detected, the recovery unit arranged adjacent to the observation unit is controlled by the control unit, and the cell having the desired characteristic is collected by the collection unit. Therefore, in the suspension culture system, only desired cells can be recovered at the optimum timing while continuing the culture.

The cell culture apparatus according to the above aspect includes a transparent portion capable of transmitting light, a housing having a tubular shape for accommodating the observation portion, and a tubular body in which the recovery portion can be inserted into the medium. The suction port of the collection unit is adjacent to the observation area by the observation unit in a state where the housing and the collection unit are inserted into the medium. It may be arranged as follows.

With this configuration, by inserting the housing and the recovery part into the culture tank, the cells in the medium can be observed at a desired position in the culture tank where the observation area by the observation part and the suction port of the recovery part are arranged. However, cells having the desired characteristics detected can be aspirated.
In the cell culture device according to the above aspect, the housing and the recovery unit may be integrally configured.

In the cell culture apparatus according to the above aspect, the culture tank may have an annular space capable of accommodating the medium and the cells between the cylindrical inner wall and the outer wall.
If the stirring unit forms a flow path through which the medium flows in the circumferential direction in the annular space between the inner wall and the outer wall of the culture tank, the movement direction of the cells can be specified, and the desired cells can be easily detected and recovered. be able to.

In the cell culture apparatus according to the above aspect, the recovery unit may be arranged on the downstream side in the flow direction of the medium with respect to the observation unit.
With this configuration, the cells can be recovered by the recovery unit immediately after the observation unit detects the cells having the desired characteristics, and the recovery efficiency of the desired cells can be improved.

According to the present invention, in a suspension culture system, it is possible to recover only desired cells at an optimum timing while continuing the culture.

It is a schematic block diagram of the cell culture apparatus which concerns on 1st Embodiment of this invention. It is a figure which looked at the cell culture apparatus of FIG. 1 from above. It is a schematic block diagram explaining the structure of the observation part of FIG. It is a schematic block diagram explaining the structure of the collection part of FIG. It is a schematic block diagram of the cell culture apparatus which concerns on 1st modification of 1st Embodiment of this invention. It is a figure which looked at the cell culture apparatus of FIG. 5 from above. It is a schematic block diagram of the cell culture apparatus which concerns on 2nd modification of 1st Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 3rd modification of 1st Embodiment of this invention. It is a figure which looked at the cell culture apparatus of FIG. 8 from above. It is a schematic block diagram explaining the structure of the observation part of FIG. It is a schematic block diagram of the cell culture apparatus which concerns on 2nd Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 3rd Embodiment of this invention. It is a figure explaining the shape of the housing of FIG. It is a vertical cross-sectional view explaining the structure of the observation part and the housing of FIG. It is a schematic block diagram of the cell culture apparatus which concerns on 1st modification of 3rd Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 2nd modification of 3rd Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 4th Embodiment of this invention. It is a schematic block diagram explaining the structure of the culture medium supply part of the cell culture apparatus of FIG. It is a schematic block diagram of the cell culture apparatus which concerns on 1st modification of 4th Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 2nd modification of 4th Embodiment of this invention. It is a schematic block diagram of the cell culture apparatus which concerns on 3rd modification of 4th Embodiment of this invention.

[First Embodiment]
The cell culture apparatus according to the first embodiment of the present invention will be described below with reference to the drawings.
The cell culture apparatus 1 according to the present embodiment observes the stirring mechanism (stirring unit) 5 for stirring the medium W contained in the culture tank 3 and the cells S housed together with the medium W in the culture tank 3. It includes an observation unit 7, a collection unit 9 that collects cells S in the culture tank 3, and a control unit 11 that controls the stirring mechanism 5, the observation unit 7, and the collection unit 9.

The culture tank 3 is, for example, a bottomed cylindrical container in which the upper surface 3a is closed. The culture tank 3 is formed of, for example, an optically transparent material. A recovery unit 9 is connected to the culture tank 3.

The stirring mechanism 5 rotates around the stirring shaft 5a inserted into the culture tank 3 via the upper surface 3a of the culture tank 3, a plurality of stirring blades 5b provided on the stirring shaft 5a, and the stirring shaft 5a. It is equipped with a motor 5c that rotates the shaft. In this stirring mechanism 5, when the stirring shaft 5a is rotated about the axis by the motor 5c, the stirring blade 5b rotates together with the stirring shaft 5a in the medium W, so that the stirring shaft 5 is transferred to the medium W and the cells S in the culture tank 3. A flow around 5a can be generated.

The observation unit 7 is arranged on the side of the culture tank 3. As shown in FIG. 3, the observation unit 7 irradiates an arbitrary observation surface (observation region) R in the culture tank 3 in which the medium W and the cells S are present with illumination light from the outside of the culture tank 3 (Light). By photographing the light source 13 such as Emitting Diode), the lens 15 that collects the observation light from the observation surface R irradiated with the illumination light outside the culture tank 3, and the observation light collected by the lens 15. It is provided with an imaging unit 17 such as a CCD image sensor or a CMOS image sensor that acquires an image of the observation surface R.

The collection unit 9 is arranged adjacent to the observation unit 7. As shown in FIG. 4, the recovery unit 9 includes, for example, a cell recovery container 19 for collecting medium W and cells S, a tubular member 21 such as a tube connecting the cell recovery container 19 and the culture tank 3, and cell recovery. It is provided with a negative pressure supply unit 23 such as a suction pump that applies negative pressure to the container 19.

The cell collection container 19 is, for example, a bottomed tubular container whose upper surface is closed, and the inside is sealed. The cell collection container 19 has an inflow port 19a on the upper surface for allowing the sucked medium W and cells S to flow into the inside. Further, a negative pressure supply unit 23 is connected to the cell collection container 19.

As shown in FIG. 3, the tubular member 21 has a suction port 21a at one end in the longitudinal direction for sucking the medium W and cells S in the culture tank 3 by being inserted into the culture tank 3. The suction port 21a of the tubular member 21 is arranged at a position adjacent to the observation surface R of the observation unit 7 in the culture tank 3. In the present embodiment, the suction port 21a of the tubular member 21 is arranged at a position adjacent to the observation surface R and on the downstream side of the flow of cells S generated by stirring by the stirring mechanism 5. When the observation surface R is near the inner surface of the culture tank 3, the suction port 21a may be opened to the inner surface of the culture tank 3 instead of being inserted into the culture tank 3.

The tubular member 21 is provided from the culture tank 3 by inserting one end in the longitudinal direction, that is, the suction port 21a into the culture tank 3, and connecting the other end in the longitudinal direction to the inflow port 19a of the cell collection container 19. A flow path for sucking the cells S together with the medium W is formed in the cell collection container 19.

The negative pressure supply unit 23 includes a tubular member 25 connected to the cell collection container 19 and a pump body 27.
The pump main body 27 has a receiving unit (not shown), and ON / OFF is switched when the receiving unit receives a signal from the control unit 11. When the pump body 27 is switched to the ON state, negative pressure is applied to the cell collection container 19 via the tubular member 25, so that the medium W and the cells S in the culture tank 3 pass through the tubular member 21 to the cell collection container. It is sucked into 19. When the pump body 27 is switched to the OFF state, the suction of the medium W and the cells S in the culture tank 3 is stopped.

The negative pressure supply unit 23 may include a temperature control means for controlling the temperatures of the medium W and the cells S in the cell collection container 19. In this case, the medium W and the cells S may be kept at a temperature suitable for storage, for example, 4 ° C. in the cell recovery container 19 by the temperature control means. By keeping the medium W at 4 ° C. in the cell recovery container 19, deterioration of the medium W can be prevented. Further, by keeping the cells S at 4 ° C. in the cell recovery container 19, the growth of the cells S can be suppressed.

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

For example, the control unit 11 stirs the medium W in the culture tank 3 by driving the motor 5c of the stirring mechanism 5. Further, the control unit 11 operates the observation unit 7 to periodically acquire an image of the observation surface R in the culture tank 3.

Further, the control unit 11 stores a desired feature amount of the cell S, for example, an index of the size and morphology of each cell S or each colony. The control unit 11 detects the characteristics of the cells S included in the image acquired by the observation unit 7. Then, the control unit 11 determines whether or not the cell S in the image acquired by the observation unit 7 is a desired cell S by collating the detected feature of the cell S with the stored feature amount. ..

Further, when the control unit 11 detects the cell S having a desired characteristic, the control unit 11 sends a drive signal to the negative pressure supply unit 23 of the recovery unit 9. As a result, the detected cells S having the desired characteristics are sucked into the cell collection container 19 by the negative pressure supply unit 23. The control unit 11 may transmit a signal to the negative pressure supply unit 23 by wire, or may wirelessly transmit a signal by a transmission unit (not shown). Processing such as detection, collation, determination, and transmission of a drive signal by the control unit 11 of the characteristics of the cell S is performed instantaneously.

Next, the operation of the cell culture device 1 according to the present embodiment will be described.
When the cells S are cultured by the cell culture apparatus 1 having the above configuration, the stirring mechanism 5 is driven by the control unit 11 in a state where the medium W and the cells S are contained in the culture tank 3, so that the cells S are contained in the culture tank 3. Medium W is stirred. As a result, the cells S are cultured while floating in the medium W.

Next, the observation unit 7 is controlled by the control unit 11, so that an image of the observation surface R in the culture tank 3 is periodically acquired. Then, the control unit 11 detects the characteristics of the cell S contained in the image acquired by the observation unit 7, and the detected characteristics of the cell S are collated with the desired feature amount stored in the control unit 11. To.

When the control unit 11 determines that the cell S in the image acquired by the observation unit 7 is a cell S having a desired characteristic, the control unit 11 transmits a drive signal to the negative pressure supply unit 23. Then, the pump body 27 of the negative pressure supply unit 23 is driven by the drive signal from the control unit 11 as a trigger, and the suction port 21a arranged adjacent to the observation surface R of the observation unit 7 desires the operation. The cells S having the characteristics of are aspirated.

In this case, the cells S in the culture tank 3 are moving along with the flow of the medium W, but the control by the control unit 11 is fast, and the suction port 21a of the collection unit 9 is the observation surface R of the observation unit 7. By being arranged at a position adjacent to the cell S, the cells S having the desired characteristics detected by the control unit 11 can be collected by the collection unit 9.

Therefore, according to the cell culture apparatus 1 according to the present embodiment, in the suspension culture system, only the desired cells S can be recovered at the optimum timing while continuing the culture. Further, by arranging the suction port 21a of the collection unit 9 on the downstream side of the observation surface R of the observation unit 7 in the flow direction of the cells S, the cells S having the desired characteristics detected by the control unit 11 can be collected. Efficiency can be improved.

This embodiment can be transformed into the following configuration.
As a first modification, for example, as shown in FIGS. 5 and 6, a narrow flow path 29 extending from the side wall portion 3b of the culture tank 3 to the outside of the culture tank 3 and connecting to the inside of the culture tank 3 again is provided. The observation unit 7 and the collection unit 9 may be arranged in the flow path 29.

For example, a flow path inlet 29a and a flow path outlet 29b are provided on the side wall portion 3b of the culture tank 3 at intervals in the circumferential direction of the culture tank 3, and a tubular member 29c connecting the flow path inlet 29a and the flow path outlet 29b is used. The flow path 29 may be configured. Then, the observation surface R of the observation unit 7 may be arranged in the flow path 29, and the suction port 21a of the collection unit 9 may be arranged at a position adjacent to the observation surface R. Also in this modification, it is preferable to arrange the suction port 21a of the collection unit 9 on the downstream side of the flow of the cells S with respect to the observation surface R of the observation unit 7.

In this case, the medium W and the cells S that have flowed into the flow path 29 by stirring by the stirring mechanism 5 are observed by the observation unit 7 when passing through the observation surface R in the flow path 29. Then, the cell S having a desired characteristic contained in the image acquired by the observation unit 7 is detected by the control unit 11, and the cell S having the detected desired characteristic is sucked by the collecting unit 9.

According to this modification, the cell S having the desired characteristics detected by the observation unit 7 is obtained by detecting the cell S by the observation unit 7 and sucking the cell S by the recovery unit 9 in the narrow flow path 29. It can be efficiently collected by the collection unit 9.

In this modification, for example, as shown in FIG. 6, a protrusion 31 protruding inward in the radial direction may be provided on the inner surface near the flow path inlet 29a of the culture tank 3.
The flow of the medium W is disturbed by the protrusions 31, and the flow velocity in the vicinity of the flow path inlet 29a and the flow path outlet 29b is different, so that the medium W can easily flow into the flow path inlet 29a.
Alternatively, the medium W and the cells S may be allowed to flow into the flow path 29 by arranging the liquid feed pump in the flow path 29.

As a second modification, instead of the flow path 29, for example, as shown in FIG. 7, a wall 33 for guiding cells S may be provided in the culture tank 3 on the observation surface R of the observation unit 7. Good.
In this case, a wall 33 having a curved shape is arranged along the flow direction of the cells S by the stirring mechanism 5, and a flow path is formed between the inner wall surface of the culture tank 3 and the wall 33 so as to pass on the observation surface R. You may do it.

Further, the distance between the wall 33 on the upstream side in the flow direction of the cells S and the inner wall surface of the culture tank 3 by the stirring mechanism 5 is widened, and the wall 33 on the downstream side in the flow direction of the cells S and the inner wall surface of the culture tank 3 are widened. The distance between the two and the vehicle may be narrowed. With this configuration, the medium W in the culture tank 3 can be easily guided on the observation surface R between the wall 33 and the inner wall surface of the culture tank 3.

As a third modification, for example, as shown in FIGS. 8 and 9, as a culture tank, a container having a hollow structure, that is, a medium W and cells S are housed between the cylindrical inner wall 35a and the outer wall 35b. A culture tank 35 having a possible annular space H may be adopted.

In this case, the stirring unit may be any one that can generate a flow in the circumferential direction of the culture tank 35 in the medium W in the space H. For example, instead of the stirring mechanism 5, a magnetic stirrer (not shown) may be adopted, and the magnetic stirrer may be rotated in the medium W to generate a flow in the medium W. Further, a liquid feeding pump (not shown) may be adopted as the stirring unit, and the medium W in the culture tank 3 may be circulated by the liquid feeding pump to generate a flow in the medium W.

For example, as shown in FIG. 10, the observation unit 7 is preferably arranged at an angle at which the observation surface R diagonally intersects the direction of the flow of the cells S in the space H by the stirring unit. With this configuration, the cells S do not move along the observation surface R, but the cells S pass in the direction intersecting the observation surface R, so that the cells S disappear instantly from the observation surface R. This makes it easier to measure the timing of collecting the cells S. Further, also in this modification, it is preferable to arrange the suction port 21a of the collection unit 9 on the downstream side of the flow of the cells S with respect to the observation surface R of the observation unit 7.

According to this modification, in the annular space H between the inner wall 35a and the outer wall 35b of the culture tank 3, the cells S flow in the circumferential direction of the culture tank 3 together with the medium W by stirring by the stirring mechanism 5, which is desired. It becomes easy to detect and aspirate cells S having the characteristics of.

[Second Embodiment]
Next, the cell culture apparatus according to the second embodiment of the present invention will be described.
The cell culture device 41 according to the present embodiment is different from the first embodiment in that, for example, as shown in FIG. 11, the observation unit 7 includes a retroreflective member 47 and the like. Although the cell culture device 41 includes a control unit 11, the control unit 11 is not shown in FIG.
Hereinafter, the parts having the same configuration as the cell culture apparatus 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The observation unit 7 according to the present embodiment cultures the light source 13, the objective lens 43 arranged on the side of the culture tank 3, and the illumination light from the light source 13 from the outside of the culture tank 3 via the objective lens 43. The illumination optical system 45 that irradiates the inside of the tank 3, the retroreflective member 47 that reflects the illumination light transmitted through the culture tank 3 toward the culture tank 3, and the detection that detects the illumination light collected by the objective lens 43. It is equipped with an optical system 49.

As the light source 13, in addition to the LED, a light source generally used for acquiring a phase difference image, for example, a lamp light source such as mercury, halogen, or xenon may be adopted.
The optical axis of the objective lens 43 is arranged in a substantially horizontal direction. Further, the objective lens 43 faces the culture tank 3. The focal plane F of the objective lens 43, that is, the observation plane R is arranged inside the culture tank 3.

The illumination optical system 45 includes a lens 51 that converts illumination light emitted from a light source 13 into parallel light, a diaphragm 53 having a ring slit 53a that is an annular opening, a relay optical system 55, and a half mirror 57. I have.
The ring slit 53a of the aperture 53 is arranged at a position optically conjugate with the pupil position of the objective lens 43. The illumination light from the lens 51 passes only through the ring slit 53a in the aperture 53.

The relay optical system 55 relays the illumination light from the ring slit 53a. Such a relay optical system 55 is composed of, for example, a pair of convex lenses.
The half mirror 57 reflects a part of the illumination light from the relay optical system 55, for example, 50% of the illumination light incident on the half mirror 57 from the relay optical system 55 toward the objective lens 43. Further, the half mirror 57 transmits a part of the illumination light from the objective lens 43, for example, 50% of the illumination light incident on the half mirror 57 from the objective lens 43.

The illumination light reflected by the half mirror 57 enters the objective lens 43 along the optical axis of the objective lens 43, and is emitted from the objective lens 43 toward the culture tank 3. That is, the objective lens 43 also functions as a part of the illumination optical system 45. The illumination light from the objective lens 43 passes through the side wall of the culture tank 3 and crosses the inside of the culture tank 3 in a substantially horizontal direction, and then passes through the side wall of the culture tank 3 again and is emitted to the outside of the culture tank 3. To. The position of the diaphragm 53 can be adjusted in a direction orthogonal to the optical axis of the illumination light incident on the diaphragm 53. By adjusting the position of the aperture 53, the position of the illumination light incident on the culture tank 3 from the objective lens 43 can be changed in a direction intersecting the optical axis of the illumination light.

The retroreflective member 47 is arranged so as to sandwich the culture tank 3 with the objective lens 43 in a substantially horizontal direction. The retroreflective member 47 has an array in which a large number of minute reflective elements 47a are arranged along the surface P. The surface P is a surface that intersects the optical axis of the illumination light that has passed through the culture tank 3. The reflective element 47a is, for example, a prism or spherical glass beads.

The illumination light incident on the reflective element 47a is emitted from the reflective element 47a in the opposite direction to that at the time of incident. Since the reflective element 47a 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 47 returns along the same path as the path of the illumination light incident on the retroreflective member 47. That is, the illumination light reciprocates in the same path between the inside of the culture tank 3 and the retroreflective member 47.

The surface P on which the reflective elements 47a are arranged may be either a flat surface or a curved surface. For example, the surface P may be a curved surface having a constant curvature and curved in one direction as shown in FIG. 11, or may be a curved surface curved in a plurality of directions.
The objective lens 43 and the retroreflective member 47 are arranged at positions where the stirring shaft 5a and the stirring blade 5b of the stirring mechanism 5 do not interfere with the optical path of the illumination light between the objective lens 43 and the retroreflective member 47.

The detection optical system 49 includes a phase film 59 arranged at the pupil position of the objective lens 43, a lens 15, and an imaging unit 17.
The phase film 59 has a shape corresponding to the shape of the ring slit 53a, that is, an annular shape. The phase film 59 shifts the phase of the illumination light transmitted through the phase film 59. The phase film 59 may be arranged at a position optically conjugate with the pupil position of the objective lens 43.

The suction port 21a of the collection unit 9 is arranged at a position adjacent to the observation surface R of the observation unit 7. Also in this embodiment, the suction port 21a of the collection unit 9 is arranged on the downstream side of the flow of the cells S with respect to the observation surface R of the observation unit 7.

Next, the operation of the cell culture device 41 having the above configuration will be described.
As shown in FIG. 11, the illumination light emitted from the light source 13 is emitted from the illumination optical system 45 to the culture tank 3 via the objective lens 43. After being incident on the culture tank 3, the illumination light passes through the medium W in the culture tank 3 and is emitted from the culture tank 3.

Subsequently, the illumination light is reflected by the retroreflective member 47 and re-enters the culture tank 3, and then passes through the medium W in the culture tank 3 in the opposite direction and is emitted from the culture tank 3. Therefore, the cells S floating in the medium W in the culture tank 3 are illuminated by two types of illumination methods: epi-illumination by the objective lens 43 and transmission illumination by the retroreflective member 47.

While passing through the culture tank 3 twice, a part of the illumination light is refracted by passing through the transparent cells S floating in the medium W. After passing through the culture tank 3 twice, the illumination light passes through the objective lens 43 and the half mirror 57, and is imaged on the image pickup unit 17 by the lens 15.

In the objective lens 43, the phase film 59 is arranged at a position optically conjugate with the ring slit 53a. The illumination light (refracted light) transmitted through the cells S in the culture tank 3 passes through a position different from that of the phase film 59 in the objective lens 43 and is emitted from the objective lens 43. On the other hand, the illumination light (straight light) that did not pass through the cells S in the culture tank 3 is given a phase shift by passing through the phase film 59 in the objective lens 43, and is emitted from the objective lens 43. Therefore, an optical image of the cell S with light and darkness due to the interference between the refracted light and the straight light is formed on the imaging unit 17. As a result, the imaging unit 17 acquires a phase-difference image of the cell S.

Next, the control unit 11 detects the cells S having the desired characteristics from the phase difference image acquired by the observation unit 7, and the detected cells S having the desired characteristics are sucked by the recovery unit 9.

In this case, as described above, the retroreflective member 47 reflects the illumination light along the same path as at the time of incident by a large number of minute reflecting elements 47a. Therefore, the illumination light incident on the culture tank 3 from the retroreflective member 47 is the culture tank 3 regardless of the shape of the side wall of the culture tank 3 existing between the retroreflective member 47 and the inside of the culture tank 3. The cells S inside are illuminated from the same direction at the same angle.

For example, when the side wall of the culture tank 3 has a curvature or unevenness, the side wall of the culture tank 3 exerts a lens effect with respect to illumination light. However, the lens effect is canceled by the illumination light reciprocating along the same path on the side wall of the culture tank 3. That is, the direction and angle of the illumination light incident on the culture tank 3 from the retroreflective member 47 are not affected by the side wall between the retroreflective member 47 and the inside of the culture tank 3. Therefore, even if the culture tank 3 is made of a flexible material and the side wall of the culture tank 3 is deformed over time, or even if the culture tank 3 is replaced with another culture tank 3 having a different shape and size, it is reflexive. The cells S in the culture tank 3 can be stably illuminated by the illumination light from the reflective member 47.

When the side wall of the culture tank 3 between the objective lens 43 and the inside of the culture tank 3 is flat, the illumination light incident on the culture tank 3 from the objective lens 43 travels along the optical axis of the objective lens 43. That is, coaxial epi-illumination is realized.

On the other hand, when the side wall of the culture tank 3 between the objective lens 43 and the inside of the culture tank 3 has a curvature or unevenness, the optical axis of the illumination light incident on the culture tank 3 from the objective lens 43 is the culture tank 3. It tilts with respect to the optical axis of the objective lens 43 due to the lens effect of the side wall. As a result, the position of the illumination light (straight light) returned from the retroreflective member 47 to the objective lens 43 may shift from the position of the phase film 59 in the direction intersecting the optical axis. In such a case, the illumination optical system 6 to the culture tank 3 are adjusted by adjusting the position of the aperture 53 so that the illumination light (straight light) returned from the retroreflective member 47 to the objective lens 43 passes through the phase film 59. The position of the illumination light applied to the light is adjusted.

In the present embodiment, the illumination optical system 45 irradiates the observation surface R in the culture tank 3 with the illumination light via the objective lens 43, but instead of this, the illumination optical system 45 passes through the objective lens 43, for example. The observation surface R in the culture tank 3 may be irradiated with illumination light without causing the observation. In this case, the illumination optical system 45 may include a light source arranged on the side of the objective lens 43, and the observation surface R in the culture tank 3 may be irradiated with illumination light from this light source. In order to bring the optical axis of the illumination light as close as possible to the optical axis of the objective lens 43, it is preferable that this light source is arranged in the vicinity of the objective lens 43.

Further, in the present embodiment, the phase difference image of the cell S is acquired by using the ring slit 53a and the phase film 59, but instead, a bright field image of the cell S may be acquired. That is, the illumination optical system 45 does not have to include the aperture 53, and the detection optical system 49 does not have to include the phase film 59. In this case, the epi-illuminated visual field image and the transmitted bright-field image of the cells S are acquired.

Further, in the present embodiment, a medium having a refractive index different from that of air may be filled between the objective lens 43 and the culture tank 3. The medium is, for example, water, oil, gel or water-absorbing polymer. The refractive index of the medium is preferably the same as or close to the refractive index of the medium W. The refractive index of the medium may be the same as or close to the refractive index of the material of the culture tank 3.

The medium between the objective lens 43 and the culture tank 3 reduces the lens effect of the side wall of the culture tank 3 with respect to the illumination light incident on the culture tank 3 from the objective lens 43. Thereby, when the side wall of the culture tank 3 has a curvature or unevenness, the direction and angle of the illumination light emitted from the objective lens 43 to the cells S can be stabilized. The medium may be held between the objective lens 43 and the culture tank 3 by the surface tension of the medium.

[Third Embodiment]
Next, the cell culture apparatus according to the third embodiment of the present invention will be described.
The cell culture device 61 according to the present embodiment is different from the first embodiment in that, for example, as shown in FIGS. 12 to 14, a housing 63 having a tubular shape for accommodating the observation unit 7 is adopted. Although the cell culture device 61 includes a control unit 11, the control unit 11 is not shown in FIGS. 12 to 14.
Hereinafter, the parts having the same configuration as the cell culture apparatus 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The housing 63 has an elongated tubular shape that can be inserted into the medium W via a port (not shown) on the upper surface 3a of the culture tank 3. Further, the housing 63 is made of, for example, polyvinyl chloride or the like and has flexibility. As shown in FIG. 13, the housing 63 is bent in a direction in which the tip portions 63a in the longitudinal direction intersect in the longitudinal direction.

Further, as shown in FIG. 14, the housing 63 has a transparent portion 63b that transmits illumination light and observation light at the cutting edge of the tip portion 63a. A stirring mechanism when the housing 63 is inserted into the culture tank 3 along the stirring shaft 5a by bending the tip 63a of the housing 63 and illuminating and photographing through the cutting-edge transparent portion 63b of the tip 63a. The observation surface R of the observation unit 7 can be arranged at an angle that diagonally intersects the flow direction of the medium W according to 5.

As shown in FIG. 14, the light source 13 is arranged at the tip end portion 63a of the housing 63 so as to face the transparent portion 63b.
The lens 15 is arranged side by side with the light source 13 at the tip end portion 63a of the housing 63 so as to face the transparent portion 63b.
The image pickup unit 17 is arranged at the tip end portion a of the housing 63 on the proximal end side of the lens 15.

The collection unit 9 is arranged at a position adjacent to the observation surface R of the observation unit 7. Also in this embodiment, the suction port 21a of the collection unit 9 is arranged on the downstream side of the flow of the cells S with respect to the observation surface R of the observation unit 7.

Next, the operation of the cell culture device 61 according to the present embodiment will be described.
When observing the cells S while culturing them with the cell culture device 61 having the above configuration, first, as shown in FIGS. 12 and 13, the housing 63 is inserted into the medium W via the port of the culture tank 3. To do. The housing 63 is sterilized in advance.

Next, while stirring the medium W by the stirring mechanism 5, illumination light is generated from the light source 13 in the housing 63. The illumination light emitted from the light source 13 is applied to the observation surface R in the culture tank 3 via the transparent portion 63b of the housing 63. Then, the observation light incident on the housing 63 from the observation surface R irradiated with the illumination light via the transparent portion 63b is collected by the lens 15, and the optical image of the observation light is photographed by the imaging unit 17.

The image of the observation surface R acquired by the imaging unit 17 is sent to the control unit 11. The control unit 11 detects the cells S having the desired characteristics from the image acquired by the observation unit 7, and the collection unit 9 sucks the detected cells having the desired characteristics.

According to the cell culture apparatus 61 according to the present embodiment, by inserting the housing 63 and the recovery unit 9 into the culture tank 3, the transparent portion 63b of the housing 63 and the suction port 21a of the recovery unit 9 are arranged. At a desired position in the culture tank 3, cells S having the desired characteristics detected while observing the cells S in the medium W can be aspirated.

The collection unit 9 may be inserted into the medium W in parallel with the housing 63 via the port on the upper surface 3a of the culture tank 3, and the suction port 21a may be arranged in the vicinity of the observation surface R. In this case, the housing 63 and the collection unit 9 may be integrally configured.

This embodiment can be transformed into the following configuration.
In the present embodiment, the light source 13 and the image pickup unit 17 are arranged at the tip end portion 63a of the housing 63, but as a first modification, for example, the light source 13 and the image pickup unit 17 are arranged instead. May be arranged at the base end portion of the housing 63.

In this case, the illumination light emitted from the light source 13 may be guided by a fiber (not shown) and emitted from the tip of the fiber at the tip 63a of the housing 63. Further, the observation light from the observation surface R may be received by the bundle fiber (not shown) at the tip end portion 63a of the housing 63, and may be incident on the imaging unit 17 from the bundle fiber at the base end portion of the housing 63.

In the present embodiment, the suction port 21a of the collection unit 9 is arranged on the downstream side of the observation surface R in the flow direction of the cell S, but as a second modification, for example, as shown in FIG. In addition, the suction port 21a of the collection unit 9 may be arranged at an arbitrary position adjacent to the observation surface R, such as the upstream side of the observation surface R.

It is effective when the speed of image acquisition by the observation unit 7 and the detection of the cell S by the control unit 11 is sufficiently faster than the speed of the flow of the cells S passing over the observation surface R. With this configuration, it is possible to improve the degree of freedom in the position where the suction port 21a of the collection unit 9 is arranged.

Further, as a third modification, for example, as shown in FIG. 16, a tubular protective tube 67 that covers the periphery of the housing 63 may be provided.

The protective tube 67 has an elongated shape that can be inserted into the medium W via the port of the culture tank 3. Further, the protective tube 67 is formed so that the housing 63 can be inserted and removed inside. The protective tube 67 is made of a transparent resin material such as, for example, acrylic resin (PMMA) or polyvinyl chloride. Therefore, the entire protective tube 67 constitutes an optically transparent transparent portion that transmits illumination light and observation light. In the present embodiment, the tip of the protective tube 67 in the longitudinal direction is a transparent portion 67a.

The protective tube 67 has a protrusion 69 protruding in the longitudinal direction of the protective tube 67 on the outside of the transparent portion 67a. As shown in FIG. 16, the protrusion 69 has a columnar portion 69a extending from the tip of the protective tube 67 along the longitudinal direction of the protective tube 67 and a direction intersecting the longitudinal direction of the protective tube 67 from the tip of the columnar portion 69a. It is provided with a bent portion 69b arranged at a position of blocking the front of the transparent portion 67a by bending the transparent portion 67a.

The columnar portion 69a is arranged at a position deviated from each optical axis of the light source 13 and the lens 15 with the housing 63 inserted in the protective tube 67.
The bent portion 69b is arranged on the optical axis of the light source 13 and the lens 15 with the housing 63 inserted in the protective tube 67. The bent portion 69b reflects the illumination light emitted from the light source 13 to the outside of the protective tube 67 via the transparent portion 63b of the housing 63 and the transparent portion 67a of the protective tube 67 toward the lens 15. It functions as a reflective member that obliquely illuminates the observation surface R in the culture tank 3.

According to the above configuration, the illumination light is emitted from the light source 13 in the housing 63 via the transparent portion 63b of the housing 63 and the transparent portion 67a of the protective tube 67. The illumination light emitted from the transparent portion 67a of the protective tube 67 is reflected toward the transparent portion 67a of the protective tube 67 by the bent portion 69b of the protrusion 31 in front of the transparent portion 67a of the protective tube 67. As a result, the observation surface R in the culture tank 3 between the transparent portion 67a and the bent portion 69b of the protective tube 67 is irradiated with the illumination light.

The observation light returned from the observation surface R by being irradiated with the illumination light is collected by the lens 15 via the transparent portion 67a of the protective tube 67 and the transparent portion 63b of the housing 63, and an optical image of the observation light is captured. Photographed by part 17.
Next, the control unit 11 detects the cells S having the desired characteristics from the image acquired by the observation unit 7, and the recovery unit 9 sucks the detected cells having the desired characteristics.

According to this modification, the protective tube 67 has a shape that can be inserted into the medium W via the port of the culture tank 3, so that the protective tube 67 causes the housing 63, the light source 13 in the housing 63, and the lens. With the 15 and the imaging unit 17 safely protected, the housing 63, the light source 13, the lens 15 and the imaging unit 17 can be inserted into the culture tank 3 and operated in the culture tank 3.

Further, by forming the protective tube 67 with a transparent resin material such as acrylic resin or polyvinyl chloride, the protective tube 67 is used in a UV sterilized state, and after use, only the protective tube 67 is made disposable and replaced. Can be done. As a result, it is possible to avoid contamination of the medium W as compared with the case where the housing 63 to be used repeatedly is directly inserted into the medium W.

Further, the observation surface R irradiated with the illumination light in the culture tank 3 is limited to the space between the transparent portion 63b of the protective tube 67 and the bent portion 69b of the protrusion 31, so that the transparent portion 63b of the protective tube 67 is restricted. The cells S that have invaded the space between the protrusion 31 and the bent portion 69b of the protrusion 31 can be photographed.

[Fourth Embodiment]
Next, the cell culture apparatus according to the fourth embodiment of the present invention will be described.
As shown in FIGS. 17 and 18, for example, the cell culture apparatus 71 according to the present embodiment includes a culture medium supply unit 73 that supplies the culture medium W to the culture tank 3, and the control unit 11 controls the culture medium supply unit 73. It differs from the first embodiment in that it is different from the first embodiment.
Hereinafter, the parts having the same configuration as the cell culture apparatus 1 according to the first embodiment are designated by the same reference numerals and the description thereof will be omitted.

The medium supply unit 73 includes a medium holding container 75 that holds a new medium W, and a tubular member 77 such as a tube that connects the medium holding container 75 and the culture tank 3.
The medium holding container 75 is, for example, a bottomed tubular container. As shown in FIG. 18, the medium holding container 75 has a discharge port 75a for discharging the medium W near the bottom surface.

As shown in FIG. 18, the tubular member 77 has one end in the longitudinal direction connected to the discharge port 75a of the culture medium holding container 75 and the other end in the longitudinal direction connected to the supply port 3c of the culture tank 3. As a result, the tubular member 77 forms a flow path for supplying the medium W from the medium holding container 75 to the culture tank 3. The tubular member 77 is provided with a liquid feeding pump 79 such as a perista pump that switches between supply and non-supply of the medium W from the medium holding container 75 to the culture tank 3.

The liquid feed pump 79 has a receiving unit (not shown) that receives a signal from the control unit 11. The liquid feed pump 79 is switched on / off when the receiving unit receives the signal from the control unit 11. When the liquid feed pump 79 is switched to the ON state, the medium W in the medium holding container 75 is supplied to the culture tank 3 via the tubular member 77. Further, when the liquid feed pump 79 is switched to the OFF state, the supply of the medium W from the medium holding container 75 to the culture tank 3 is stopped.

The control unit 11 switches ON / OFF of the supply of the medium W from the medium holding container 75 to the culture tank 3 by controlling the drive of the liquid feeding pump 79. Further, when the amount of the medium W sucked by the collection unit 9 exceeds a predetermined threshold value, the control unit 11 sends a drive signal to the medium supply unit 73 by a transmission unit (not shown). As a result, the medium supply unit 73 supplies the culture medium W with the new medium W.

According to the cell culture device 71 according to the present embodiment, the control unit 11 determines whether or not the amount of the medium W sucked by the recovery unit 9 exceeds a predetermined threshold value. Then, when it is determined that the amount of the medium W sucked by the collection unit 9 exceeds a predetermined threshold value, the medium supply unit 73 is controlled by the control unit 11, and the culture medium supply unit 73 controls the culture medium supply unit 73 to add new medium to the culture tank 3. W is supplied.

As described above, according to the cell culture apparatus 71 according to the present embodiment, the medium W reduced by the recovery of the cells S by the recovery unit 9 can be replenished by the medium supply unit 73. Further, since a part of the medium W in the culture tank 3 is replaced, deterioration of the medium W in the culture tank 3 can be suppressed.

This embodiment can be transformed into the following configuration.
As a first modification, for example, as shown in FIG. 19, the medium holding container 75 may be arranged above the culture tank 3 in the direction of gravity. Then, the medium W may be supplied from the medium holding container 75 to the culture tank 3 via the tubular member 77 by gravity.

In this case, instead of the liquid feed pump 79, the tubular member 77 may be provided with a gate opening / closing portion 81 such as a valve for opening / closing the flow path. Further, the gate opening / closing unit 81 may have a receiving unit (not shown) for receiving a signal from the control unit 11, and the opening / closing may be switched when the receiving unit receives the signal from the control unit 11.

According to this modification, when the gate opening / closing portion 81 is switched to the open state, the flow path of the tubular member 77 is opened, and the medium W in the medium holding container 75 passes through the tubular member 77 by gravity to the culture tank 3 Is supplied to. On the other hand, when the gate opening / closing portion 81 is switched to the closed state, the flow path of the tubular member 77 is blocked, and the supply of the medium W from the medium holding container 75 to the culture tank 3 is stopped.
Therefore, the supply and non-supply of the medium W to the culture tank 3 can be switched by a simple configuration in which the flow path of the tubular member 77 is opened and closed by the gate opening / closing portion 81.

As a second modification, for example, as shown in FIG. 20, the medium holding container 75 may further include a medium holding container 83 and a tubular member 85 that supply the medium W to the medium holding container 75.
Hereinafter, the medium holding container 75 and the tubular member 77 are also referred to as the first medium holding container 75 and the first tubular member 77, and the medium holding container 83 and the tubular member 85 are also referred to as the second medium holding container 83 and the second tubular member 85.

The second medium holding container 83 and the second tubular member 85 may have the same configurations as the first medium holding container 75 and the first tubular member 77. For example, the second medium holding container 83 may have a discharge port 83a near the bottom surface. Further, the second tubular member 85 may have a liquid feeding pump 87 such as a perista pump that switches between supply and non-supply of the medium W from the second medium holding container 83 to the first medium holding container 75.

In this case, the medium supply unit 73 is provided with temperature control means (not shown) for controlling the temperature of the medium W in the first medium holding container 75 and the temperature of the medium W in the second medium holding container 83, respectively. May be good. For example, in the first medium holding container 75, the medium W may be held at a temperature suitable for cell S culture, for example, 37 ° C., by a temperature control means. Further, in the second medium holding container 83, the medium W may be held at a temperature suitable for storage, for example, 4 ° C., by the temperature control means.

By holding the medium W at, for example, 4 ° C. in the second medium holding container 83, deterioration of the medium W can be prevented. Further, by warming the medium W supplied from the second medium holding container 83 to the first medium holding container 75 to, for example, 37 ° C. in the first medium holding container 75, the medium W having a temperature suitable for culturing is provided in the culture tank 3. It is possible to reduce the stress on the cell S due to the temperature change.

As a third modification, for example, as shown in FIG. 21, the first medium holding container 75 is arranged above the culture tank 3 in the gravity direction, and further above the first medium holding container 75 in the gravity direction. The second medium holding container 83 may be arranged in the container 83. Then, by gravity, the medium W is supplied from the second medium holding container 83 via the second tubular member 85 to the first medium holding container 75, and the medium W is supplied from the first medium holding container 75 via the first tubular member 77. The medium W may be supplied to the culture tank 3.

In this case, instead of the liquid feeding pump 79, the gate opening / closing portion 81 may be provided on the first tubular member 77. Further, instead of the liquid feeding pump 87, the second tubular member 85 may be provided with a gate opening / closing portion 89 such as a valve for opening / closing the flow path thereof. Then, the control unit 11 may control the opening / closing of the flow path of the first tubular member 77 by the gate opening / closing unit 81 and the opening / closing of the flow path of the second tubular member 85 by the gate opening / closing unit 89.

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

Further, in each of the above embodiments, for example, a configuration including a stereo optical system may be adopted as the observation unit. In this case, for example, a stereo optical system and a stereo optical system in which the observation unit 7 forms an image of two images of the same cell S floating in the medium W, which are different from each other when viewed from different viewpoints. It may be provided with a stereo imaging optical system including an imaging unit for capturing each of the two images formed by the above, and an optically transparent housing for accommodating the stereo imaging optical system.

In this case, the housing is inserted into the medium W in the culture tank 3, and the same cells S floating in the medium W have parallax from different viewpoints depending on the stereo optical system in the housing. Two images are imaged, and the two imaged images are imaged by the imaging unit. Then, the control unit 11 may detect the characteristics of the cells S included in each image of the two images acquired by the stereo imaging optical system.

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

Further, in the first embodiment, the second embodiment and the fourth embodiment, the configuration in which one observation unit 7 and one collection unit 9 are provided has been described as an example, but instead of this, for example, in the culture tank 3. A plurality of observation units 7 and recovery units 9 may be arranged by shifting their positions in the depth direction. With this configuration, the recovery efficiency of cells S having desired characteristics can be improved. In this case, the medium W and the cells S sucked by each collection unit 9 may be collected in a common cell collection container 19.

In each of the above embodiments, even in the configuration in which the stirring mechanism 5 is adopted as the stirring unit, a magnetic stirrer, a liquid feeding pump, or the like may be adopted instead.
In each of the above embodiments, the stirring mechanism 5 is inserted into the culture tank 3 via the upper surface 3a of the culture tank 3, but is inserted into the culture tank 3 via the lower surface of the culture tank 3. It may be the mode that is done.

In each of the above embodiments, the control unit 11 controls the stirring of the medium W in the culture tank 3, but the control unit 11 may not control the stirring of the medium W in the culture tank 3.
In each of the above embodiments, the characteristics of the cell S detected by the control unit 11 include, for example, the morphological characteristics of the cell S (cell S size, cell mass size, presence / absence of protrusions, number of protrusions, protrusion length, perfect circle). Degree, etc.), optical characteristics of cell S (color, brightness, wavelength characteristics, etc.), characteristics related to cell activity (cell life and death, etc.) can be mentioned. Two or more features may be combined and detected.

In each of the above embodiments, one observation unit 7 and one recovery unit 9 are provided for one culture tank 3, but two or more observation units 7 and collection units are provided for one culture tank 3. 9 may be provided. As a result, the cells S can be recovered more efficiently.

In each of the above embodiments, a main culture tank having a large capacity is separately provided, and the cells S cultured in the main culture tank are transferred together with the medium W to the culture tank 3 (hereinafter, referred to as a sub-culture tank) of each of the above embodiments. Cell recovery may be performed. In this case, the main culture tank and the sub-culture tank are connected by a flow path, and the medium W containing the cells S may be moved automatically or manually.

1,41,61,71 Cell culture device 3 Culture tank 5 Stirring mechanism 7 Observation unit 9 Recovery unit 11 Control unit 63 Housing S cell W medium

Claims

An observation unit for observing the cells contained in the culture tank together with the medium,
A stirring unit that stirs the medium in the culture tank, and a stirring unit.
A collection unit, which is arranged adjacent to the observation unit and collects the cells in the culture tank,
A cell culture apparatus including a control unit that detects cells having desired characteristics by the observation unit and collects the detected cells having the desired characteristics by the collection unit.
A housing having a transparent portion capable of transmitting light and having a tubular shape for accommodating the observation portion is provided.
The collection unit has a tubular shape that can be inserted into the medium, and has a suction port at the tip in the longitudinal direction.
The cell culture apparatus according to claim 1, wherein the suction port of the collection unit is arranged adjacent to an observation area by the observation unit in a state where the housing and the collection unit are inserted into the medium.
The cell culture apparatus according to claim 2, wherein the housing and the collection unit are integrally configured.
The cell culture apparatus according to claim 1, wherein the culture tank has an annular space capable of accommodating the medium and the cells between the cylindrical inner wall and the outer wall.
The cell culture apparatus according to any one of claims 1 to 4, wherein the recovery unit is arranged on the downstream side in the flow direction of the medium with respect to the observation unit.