JP7242364B2 - Culturing method and culture unit for biological object - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method of culturing a biological object such as a cell or cell mass for a required period of time, and a culture unit applied to this method.

For long-term culturing of biological objects such as cells or cell masses, generally, a method of arranging a container such as a petri dish containing a culture medium and the biological object under a predetermined environment is adopted. In addition, Patent Literature 1 discloses a cell culture device in which a culture chamber containing cells is provided with a medium introduction/exhaust passage so that the medium can be replaced. Further, Patent Document 2 discloses a cell culture module in which cells are adhered to a fiber assembly arranged in a culture chamber and cultured for a long period of time while flowing a culture medium into and out of the culture chamber.

JP 2011-244713 A WO2014/034146

Long-term culturing of a biological subject requires that oxygen and nutrients be evenly supplied to the biological subject, and that the biological subject, which is about to grow and become large, should not be physically disturbed. The former requirement can be achieved by the flow of medium into and out of the culture chamber as shown in Patent Documents 1 and 2. On the other hand, the methods of Patent Documents 1 and 2 are insufficient for the latter request. When cells are brought into contact with a scaffolding material as in the device of Patent Document 1, or adhered to a fiber aggregate as in the module of Patent Document 2, three-dimensional cell growth occurs at the contact/attachment site. There is a problem of being blocked.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method and a culture unit for culturing a biological subject that do not inhibit the three-dimensional growth of the biological subject as much as possible.

A method for culturing a biological subject according to one aspect of the present invention comprises a recess capable of accommodating a biological subject and its culture medium, and having a bottom surface and side wall surfaces, and the bottom surface being provided with an opening. By arranging the biological object in the culture area and supplying the culture solution from the opening into the recess, a liquid flow of the culture solution blowing up from the opening into the recess is generated, and the biological object is grown. and holding the biological object for a predetermined period of about one day to several weeks while floating and rotating it in the concave portion by the liquid flow, and maintaining the biological object for the predetermined period while the biological object is in a floating state and a rotating state. is monitored, and the conditions for generating the liquid flow are changed when a predetermined change occurs in the levitation state and the rotation state of the biological object.

According to this culturing method, it is possible to supply oxygen and nutrients to the biological object by generating a liquid flow of the culture solution in the culturing region. In addition, by causing the biological object to flow within the culture area by the liquid flow, it is possible to prevent a part of the biological object from continuously facing the wall or the like that partitions the culture area in the same posture. . In other words, it is possible to culture the living body for a long period of time while moving the living body in the culture solution. As a result, the influence of the surface contacting the wall surface or the like can be suppressed, and the living body subject can freely grow three-dimensionally.

In the above method for culturing a living subject, the culture region comprises a recess having a bottom surface and a side wall surface, an opening is provided in the bottom surface or the side wall surface, and a culture solution is supplied into the recess from the opening. It is desirable to generate said liquid flow by:

According to this culturing method, a predetermined unit of the biological subject can be housed in the recess and cultured for a long period of time while being distinguished from other biological subjects. The culture medium supplied from the opening provided in the recess allows the biological object to move and supplies oxygen and nutrients, so the biological object can be cultured for a long period of time in good condition.

In the above method for culturing a living subject, the culturing region comprises a recess having a bottom surface and side walls, and the liquid flow floats the living subject in contact with the bottom surface in the culture solution. It is desirable to have a liquid flow that allows

According to this culturing method, the biological object can be cultured for a long period of time in a state where the biological object is floated in the culture medium by the liquid flow. As a result, the contact between the bottom surface and side wall surfaces of the recess and the biological object is suppressed, and the three-dimensional growth of the biological object can be further promoted.

In the method for culturing a biological subject, while the biological subject is held in a fluid state for a predetermined period of time, a change in the state of the biological subject is monitored, and when a predetermined state change occurs in the biological subject, , it is desirable to change the conditions for generating the liquid flow.

According to this culturing method, an appropriate liquid flow can be given to the biological object according to the growth of the biological object. For example, by applying a high-intensity liquid flow to a living body subject that has increased in weight due to growth, the living body body can be maintained in a fluid state.

A biological subject culture unit according to another aspect of the present invention includes a chamber including a well capable of containing a biological subject and a culture solution, an opening provided at the bottom of the well, and a pump device that sends a culture solution to a well and generates a liquid flow of the culture solution that blows up into the well from the opening ; and a pump control unit that controls the operation of the pump device, wherein the pump control unit , in a biological subject culture unit, wherein the pump device generates a liquid flow capable of causing the biological subject in contact with the bottom surface of the well to flow in the culture medium, wherein the chamber includes: a first chamber for accommodating a first biological subject in the well, and a second chamber for accommodating a plurality of second biological subjects different from the first biological subject in the well, a connection channel that connects the first chamber and the plurality of second chambers, further comprising a circulation system that circulates the culture medium through the plurality of second chambers with the first chamber as the center of circulation; The apparatus circulates the culture solution through the circulation system through the wells of the first chamber and the wells of the second chamber, thereby circulating the first biological object and the second biological object in the respective wells. It is characterized by flowing with

According to this culturing unit, it is possible to supply oxygen and nutrients to the biological subject accommodated in the well by supplying the culture medium through the opening in the well. Further, by causing the biological object to flow within the well by the liquid flow, it is possible to prevent a part of the biological object from continuously contacting the bottom surface of the well in the same posture. In other words, it is possible to culture the living body for a long period of time while moving the living body in the culture solution in the well. As a result, the influence of the contact surface with respect to the bottom surface can be suppressed, and the living body subject can freely grow three-dimensionally.

In the biological subject culture unit described above, the chambers include a first chamber for accommodating a first biological subject in the well, and a second biological subject different from the first biological subject in the well. and a second chamber containing a second chamber, and further comprising a connection flow path that connects the first chamber and the second chamber in parallel or in series, wherein the pump device is configured to pass through the connection flow path to the first Desirably, the first and second biological objects are caused to flow within the wells of the chambers and the wells of the second chamber by feeding media.

According to this culturing unit, it is possible to supply the same culture solution to a plurality of biological subjects and to culture these biological subjects for a long period of time.

A biological subject culture unit according to still another aspect of the present invention includes a chamber comprising a well capable of containing a biological subject and a culture solution, an opening provided at the bottom of the well, and a pump device that sends a culture solution to a well and generates a liquid flow of the culture solution that blows up into the well from the opening; and a pump control unit that controls the operation of the pump device, wherein the pump control unit , in the biological subject culture unit, wherein the pump device generates a liquid flow capable of causing the biological subject contacting the bottom surface of the well to flow in the culture medium, wherein the chamber comprises: It is characterized by comprising a first plate having first wells and a second plate disposed above the first plate and having second wells.

According to this culture unit, plates with wells can be stacked and arranged in a chamber. Therefore, it becomes possible to culture many biological objects at once for a long period of time.

ADVANTAGE OF THE INVENTION According to this invention, the culture|cultivation method and culture|cultivation unit of a living subject which do not inhibit three-dimensional growth of a living subject as much as possible can be provided.

FIG. 1 is a block diagram showing the configuration of a cell culture unit for realizing a method for culturing a living subject according to the first embodiment of the present invention. FIG. 2(A) is a detailed cross-sectional view of a well serving as a culture region, and FIGS. 2(B) to 2(E) are diagrams showing the relationship between the floating state of cells in the well and the observed image. 3(A) and (B) are graphs showing the results of long-term culture with or without liquid flow, FIG. 3(A) showing the projected area of cells, and FIG. 3(B) showing the state of ATP generation in cells. are shown respectively. FIG. 4(A) shows cells cultured for a long period of time with liquid flow, and FIG. 4(B) shows cells cultured for a long period of time without liquid flow. FIG. 5 is a flow chart showing an example of the operation at the start of long-term cell culture by the cell culture unit of the first embodiment. FIGS. 6A and 6B are diagrams for explaining the relationship between cell levitation and the Z height of the camera. FIG. 7 is a flow chart showing an example of the operation during long-term cell culture by the cell culture unit of the first embodiment. FIG. 8 is a block diagram showing the configuration of a cell culture unit for realizing the method for culturing a living subject according to the second embodiment of the present invention. FIG. 9 is a flow chart showing an example of the operation at the start of long-term cell culture by the cell culture unit of the second embodiment. FIG. 10 is a flow chart showing an example of the operation during long-term cell culture by the cell culture unit of the second embodiment. FIG. 11 is a flow chart showing an example of the operation during long-term cell culture by the cell culture unit of the second embodiment. FIG. 12 is a cross-sectional view showing a chamber according to a modification. FIG. 13 is a cross-sectional view showing a chamber according to a modification. FIGS. 14A to 14D are diagrams showing modified examples of wells. FIGS. 15A to 15C are diagrams showing modified examples of through-holes in wells. FIGS. 16A and 16B are diagrams showing modifications of the form of liquid flow supply to the wells.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail based on the drawings. The method and culture unit for culturing a biological subject according to the present invention can target a wide variety of biological subjects. Biological objects to which the present invention can be applied are typically exemplified by cells derived from living organisms. Here, the cells derived from the body include, for example, single cells (cells) such as blood cells and single cells, tissue fragments such as Histoculture and CTOS, cell aggregates such as spheroids and organoids, zebrafish, nematodes, and fertilized eggs. individuals, 2D or 3D colonies, and the like. In addition, examples of biological objects include tissues, microorganisms, small-sized seeds, and the like. In the embodiments described below, an example in which the biological object is cells or cell aggregates formed by aggregating several to several hundred thousand cells (hereinafter collectively referred to simply as "cells C") is shown. .

[Overall Configuration of Cell Culture Unit of First Embodiment]
FIG. 1 is a block diagram showing the configuration of a cell culturing unit U (living object culturing unit) according to the first embodiment of the present invention. The cell culture unit U includes a chamber CH having a plurality of wells 2 (recesses) capable of accommodating cells C and medium L (culture solution), a camera unit 3 for imaging the cells C accommodated in the wells 2, A pump unit 4 (pump device) for generating a liquid flow LF of the culture medium L in the well 2, and a controller 5 for controlling the operations of the camera unit 3 and the pump unit 4 and performing various processes are provided. The camera unit 3 is arranged below the chamber CH.

The chamber CH includes a container 11 with an open upper surface and a well plate 12 arranged inside the container 11 . Well plate 12 comprises a plurality of wells 2 . The wells 2 are minute depressions that serve as culture areas for the cells C, and the well plate 12 has a large number of wells 2 arranged in a matrix. The container 11 and well plate 12 are made of translucent resin or glass. This is because the cells C housed in the well 2 can be fluoroscopically imaged by the camera unit 3 arranged below the chamber CH.

A medium L is poured into the container 11, and the well plate 12 is immersed in the medium L. As shown in FIG. Container 11 includes bottom wall 13 , side walls 14 , top wall 15 and opening wall 16 . The bottom wall 13 constitutes the bottom surface of the chamber CH. A side wall 14 is erected from the peripheral edge of the bottom wall 13 and constitutes an outer frame portion of the chamber CH. The ceiling wall 15 extends horizontally from the upper end of the side wall 14 to the inside of the chamber CH, and covers the upper part of the bottom wall 13 in the vicinity of its peripheral edge. The opening wall 16 is an inclined wall extending obliquely downward from the inner end of the ceiling wall 15 toward the inside of the chamber CH. The lower end of the opening wall 16 is positioned higher than the bottom wall 13, and the peripheral edge of the well plate 12 is held at the lower end. That is, the well plate 12 is held in a state spaced upward from the bottom wall 13 . The opening wall 16 forms an opening of the chamber CH that tapers upward, and the user can carry the cells C on the well plate 12 or pick up the cells C through this opening. .

Referring to FIG. 2A, the well 2 has a cylindrical side surface 21, a bottom surface 22, a tapered opening surface 23 and a through hole 24 (an opening communicating with the well). The cylindrical side surface 21 is a portion forming the side wall surface of the well 2, and defines a cavity having a cross-sectional area capable of accommodating required cells C. As shown in FIG. The cylindrical side surface 21 is a wall surface extending in the vertical direction. The bottom surface 22 is continuous with the lower end of the cylindrical side surface 21 and consists of an inclined surface gently downwardly inclined toward the cylindrical center of the well 2 . The tapered opening surface 23 is a tapered surface that is continuous with the upper end of the cylindrical side surface 21 and widens upward. One well 2 and the adjacent well 2 are connected to each other at the upper ends of the tapered opening surfaces 23 , and the connecting portion forms a sharp ridge line portion 25 . The cylindrical side surface 21 , bottom surface 22 and tapered opening surface 23 define a cavity 20 recessed downward from the top surface of the well plate 12 . Cavity 20 is a culture area in which cells C and medium L are accommodated.

The through-hole 24 is an opening formed in the center of the bottom surface 22 , which is the lowest point of the bottom surface 22 , and is an opening that allows the cavity 20 of the well 2 and the space inside the container 11 to communicate with each other. The through hole 24 is provided to allow the culture medium poured into the container 11 to flow into the cavity 20 and to generate a liquid flow LF in the culture medium L in the cavity 20 . As shown in FIG. 1, when the medium L is poured into the container 11 to a height exceeding the well plate 12, the inner space A of the container 11 surrounded by the side walls 14, the top wall 15 and the opening wall 16 is a closed space. Become. Therefore, by pressurizing the culture medium L in the internal space A, it is possible to generate a liquid flow LF that flows into the well 2 from the through-hole 24 .

The camera unit 3 is an imaging unit that includes a lens section 31 , a camera body 32 and a lens drive section 33 and that includes a confocal optical system. The lens unit 31 is an objective lens used in an optical microscope, and includes a lens group that forms an optical image with a predetermined magnification and a lens barrel that accommodates this lens group. The camera body 32 includes an imaging device 35 (FIG. 6) such as a CCD image sensor. The lens unit 31 forms an optical image of an object to be imaged on the image plane of the imaging element 35 . The lens driving section 33 is composed of a servomotor or the like, and vertically moves the lens section 31 for focusing operation. The focal position of the lens portion 31 is determined by the parfocal distance from the height position h of the body surface 34 . The focusing operation may be performed by vertically moving the chamber CH instead of moving the lens unit 31 .

The pump unit 4 is composed of, for example, a circulation pump such as a tube pump, and functions to send fluid to each well 2 through the through holes 24 of the well plate 12 to generate liquid flow in the liquid in the wells 2 . In this embodiment, the fluid that the pump unit 4 sends to the wells 2 is the culture medium L, and the liquid in the wells 2 is also the culture medium L. The pump unit 4 includes a pump body 41 , a liquid-sending tube 42 and a liquid-sucking tube 43 .

The pump main body 41 performs a pump operation to send the liquid to the liquid-sending tube 42 and to generate a suction force of the liquid to the liquid-sucking tube 43 . When a tube pump is used, the pump main body 41 includes a rotor that is rotationally driven by a drive motor and a curved tube portion that is squeezed by the rotor. One end 421 of the liquid-sending tube 42 is connected to the pump main body 41 , while the other end 422 penetrates the ceiling wall 15 of the well plate 12 and opens into the internal space A of the container 11 . One end 431 of the liquid suction tube 43 opens into the internal space A at a position opposite to the other end 422 , and the other end 432 is connected to the pump main body 41 .

When the pump main body 41 operates, the culture medium L circulates through the through holes 24 . That is, when the pump main body 41 operates and the medium L is sent to the liquid-sending tube 42 , the sent medium L enters the internal space A of the container 11 . The internal space A is in a semi-sealed state by the bottom wall 13, the side wall 14, the top wall 15, the opening wall 16, and the well plate 12 of the container 11, and the only part through which the pressure can be released is the through hole 24. Therefore, as shown in FIGS. 2B and 2D, a liquid flow LF is generated that flows from the internal space A through the through-hole 24 into the cavity 20 of the well 2 . Although the amount of liquid in the container 11 increases due to the supply of the culture medium L from the liquid-sending tube 42 , the increased amount is sucked by the liquid-absorbing tube 43 and goes to the pump main body 41 .

The controller 5 performs control necessary for long-term culture of the cells C. The controller 5 controls the operation of the pump unit 4 to generate a liquid flow LF that blows up from the through hole 24 into the cavity 20 of the well 2. , the cell C is caused to flow within the cavity 20 by this liquid flow LF. In addition, the controller 5 performs control to maintain the state for a predetermined period while causing the cells C to flow within the cavity 20 . Furthermore, the controller 5 controls the operation of the camera unit 3 to capture an image of the cells C in the well 2 and monitor changes in the state of the cells C. FIG. Then, when a predetermined state change occurs in the cell C, control is performed to change the conditions for generating the liquid flow LF. Details of the functional configuration of the controller 5 will be described later.

[Method for long-term culture of cells]
In the method for culturing cells C according to the present embodiment, a liquid flow of the medium L is generated in the cavity 20 of the well 2, and the cells C contained in the cavity 20 are floated and rotated by the liquid flow LF. and hold it for a predetermined period. Although the holding period is arbitrary, it is a long period, for example, from one day to several weeks. An example of carrying out this culture method using the cell culture unit U described above will be described.

FIG. 2A shows a state in which cells C are put into wells 2 of well plate 12 immersed in culture medium L. FIG. When the liquid flow LF is not generated from the through-hole 24 , the cells C settle under their own weight and come into contact with the bottom surface 22 . FIG. 2(A) shows a state in which cells C having a roughly elliptical shape lie sideways and are in contact with the bottom surface 22 . In this way, when the cells C are cultured for a long period of time while some of the cells C are in contact with some member, the three-dimensional growth is inhibited due to the restraint of the cells C at the contact points, the non-distribution of nutrients, and the like. becomes easier.

Therefore, in this embodiment, the cells C are allowed to flow within the cavity 20 so that part of the cells C do not continue to contact the cylindrical side surface 21 and the bottom surface 22 of the well 2 . More desirably, it is floated within the cell C cavity 20 so as not to contact the cylindrical side surface 21 and the bottom surface 22 as much as possible. A liquid flow LF generated by the pump unit 4 is used as means for realizing such flow or floating of the cells C, as shown in FIGS. 2(B) and 2(D). The liquid flow LF is generated by supplying the medium L in the container 11 to the well 2 through the through-hole 24 .

Assume that the liquid flow LF is generated in a state where the cell C is in contact with the bottom surface 22 as shown in FIG. 2(A). Since the liquid flow LF is a liquid flow that blows upward through the through holes 24, the cells C are pushed by the liquid flow LF and start floating. Then, as shown in FIGS. 2B and 2D, the cells C rise to a predetermined height within the well 2 according to the strength of the liquid flow LF. In general, cells C, such as cell aggregates, do not have a true spherical shape, are asymmetrical in shape and mass, and have unevenness on the surface. Therefore, when the liquid flow LF hits the cell C, the cell C rotates as indicated by the arrow R in the figure.

The flow state of the cells C in the well 2 can be confirmed by the captured image of the camera unit 3 arranged below the chamber CH. FIG. 2(C) shows a two-dimensional image captured by the camera unit 3 when the cell C is in the posture of FIG. 2(B), and FIG. 2(E) shows the cell C in the posture of FIG. 2 shows a two-dimensional image captured by the camera unit 3 when . When the cell C is rotating, if the camera unit 3 is made to continuously perform the imaging operation, the cell C will be imaged in different shapes. Therefore, by analyzing the continuously captured images, it is possible to determine whether the cell C is rotating. Further, by focusing on the characteristic portion of the shape or color of the cell C and observing the movement of the characteristic portion with continuous images, it is possible to grasp whether or not the cell C has made one turn. Furthermore, the rotation of the cell C allows the camera unit 3 fixedly arranged below to acquire a multifaceted surface image of the cell C. FIG. Accordingly, it is also possible to comprehend the three-dimensional shape of the cell C by synthesizing the acquired images.

Desirable liquid flow LF conditions generated during long-term culture are as follows. First, the flow velocity of the liquid flow LF must be at least as high as to float the cells C in contact with the bottom surface 22 in the culture medium L. On the other hand, the liquid flow LF must not have such a speed that the cells C jump out of the well 2 . In addition, it is desirable to select a flow rate of the liquid flow LF that allows the cells C to float to a stable height position and rotate the cells C at that height position without causing the cells C to rise and fall in the well 2 .

If the culture of the cells C is continued, the cells C grow and generally undergo a state change in which their volume and mass increase. In this case, the required floating state of the cells C cannot be obtained with the conventional strength of the liquid flow LF. Therefore, it is desirable to monitor the floating state of the cells C with the camera unit 3 and change the conditions for generating the liquid flow LF when a significant change in the floating state or the rotating state of the cells C is observed. For example, by applying a high-intensity liquid flow to the cells C, which have increased in weight due to growth, the cells C can be maintained in a floating state.

[Evaluation of long-term culture]
3A and 3B are graphs showing the results of long-term culture tests with and without liquid flow LF, FIG. 3A shows the projected area of cell C, and FIG. 1 is a graph showing the production status of ATP (adenosine triphosphate) of each. In this test, an example chamber in which liquid flow LF was generated and a comparative example chamber in which liquid flow LF was not generated were prepared. A well plate 12 having a large number of wells 2 arranged in a matrix was set in each chamber. The well plate 12 was immersed in a culture medium composed of a predetermined medium inside the container 11 . In each well plate 12, 180 spheroids (an example of a biological object) were individually accommodated in well 2, and long-term culture was initiated.

In the example chamber, immediately after the start of culture, a liquid flow LF was generated so that the spheroids floated in the well 2, and this was continued. On the other hand, in the comparative example chamber, culture was performed in a state in which the spheroids were in contact with the bottom surface 22 by their own weight without generating the liquid flow LF. Then, 3 days after the start of culture, part of the medium was replaced. Thereafter, culture was continued in both chambers, and the projected area of Cell C and the production of ATP were evaluated 13 days later.

The projected area in FIG. 3A is the area obtained from the two-dimensional image of the cell C imaged by the camera unit 3 . The bar graph in FIG. 3(A) shows the values obtained by averaging the projected areas of 180 spheroids. It can be seen that the projected area of the spheroids cultured in the example chamber with liquid flow is clearly larger than that of the spheroids cultured in the comparative example chamber without liquid flow.

The state of ATP production in FIG. 3(B) is an index for evaluating bioactivity of cultured spheroids. The projected area shown in FIG. 3(A) is for evaluating the two-dimensional size of the spheroids, and the three-dimensional shape cannot be determined. In some cases, the dead spheroids have collapsed and the projected area has apparently increased. Organisms degrade ATP to create activity energy, so when spheroids grow and are healthy, ATP production will increase. Therefore, by measuring the state of ATP production, it is possible to grasp the growth state of spheroids.

The graph in FIG. 3(B) shows that the ATP measurement reagent was administered to the spheroids cultured in the chambers of Examples and Comparative Examples, the ATP amount of each spheroid was measured, and the ATP amount of 180 spheroids was averaged. is shown. It can be seen that the amount of ATP in the spheroids cultured in the example chamber with liquid flow is clearly greater than the amount of ATP cultured in the comparative example chamber without liquid flow.

FIG. 4(A) shows cells C in an example chamber cultured for 13 days with a liquid flow LF, and FIG. 4(B) shows cells C in a comparative example chamber cultured for 13 days without generating a liquid flow LF. FIG. 4 shows cell C; It can be seen that the cells C in the example chamber are generally large in size and grow into various three-dimensional shapes. On the other hand, it can be seen that the cell C in the comparative chamber is generally small in size and does not proliferate well. As described above, it was confirmed that the long-term culture with the generation of the liquid flow LF enables the cells C to grow larger and into various three-dimensional shapes as compared to the case where the liquid flow LF is not generated.

[Controller functional configuration]
Next, the functional configuration of the controller 5 of FIG. 1 that realizes long-term culture with the liquid flow LF as described above will be described. The controller 5 is composed of a microcomputer or the like, and operates to include a pump control section 51, a camera control section 52, an image processing section 53, a data processing section 54, and a storage section 55 by executing a predetermined program. .

The pump control section 51 controls the operation of the pump unit 4 . The pump control unit 51 generates the liquid flow LF and increases/decreases the flow velocity of the liquid flow LF by controlling the discharge force from the liquid feeding tube 42 of the pump unit 4 . Specifically, the pump control unit 51 causes the pump unit 4 to generate a liquid flow LF capable of causing the cells C in contact with the bottom surface 22 of the well 2 to float in the culture medium L and to flow or rotate. . When the pump unit 4 is a tube pump, the pump controller 51 controls the rotational speed of the rotor provided in the pump main body 41 so as to generate the liquid flow LF that can float and rotate the cells C as required.

The camera control section 52 controls operations of the camera unit 3 . The camera control section 52 causes the camera unit 3 to continuously image the cells C in the well 2 while the pump unit 4 is generating the liquid flow LF. That is, the camera control unit 52 causes the camera unit 3 to capture continuous images of the cells C floating, flowing or rotating in the well 2 . Specifically, the camera control unit 52 sends a control pulse to the lens driving unit 33 to move the lens unit 31 vertically at a pitch of several tens of μm, for example, in order to focus on the cell C in the well 2. give. Also, the camera control unit 52 controls the camera body 32 to perform an imaging operation at a predetermined timing.

The image processing unit 53 performs processing for identifying the three-dimensional shape of the cell C based on the image captured by the camera unit 3 . The image processing unit 53 performs image processing such as edge detection processing and pattern recognition processing accompanied by feature amount extraction on the image data acquired by the camera body 32, and in the image acquired at each imaging timing, the cell C Identify the shape of The image processing unit 53 also performs processing for extracting characteristic portions of the cell C for use in grasping the rotation state of the cell C. FIG.

The data processing unit 54 executes various types of processing based on the shape data of the cell C specified by the image processing unit 53, the position data of the characteristic portion, and the like. For example, the data processing unit 54 determines whether or not the cell C is floating from the bottom surface 22 of the well 2 based on the focus position information set by the lens driving unit 33 . Further, the data processing unit 54 synthesizes the shape data of the cell C specified for each image by the image processing unit 53, and performs processing for creating the three-dimensional shape of the cell C. FIG. Furthermore, based on the shape data of the cell C specified by the image processing unit 53, the data processing unit 54 determines whether the cell C is rotating or stationary, how many times the cell is rotating, and so on. A process for determining the rotation state of C is performed. Specifically, based on continuous captured images of one cell C, whether or not the cell C has rotated once and the rotation speed of the cell C are derived from the expression state or movement state of the characteristic portion. do.

The storage unit 55 stores various setting data related to the cell culture unit U and table data. In addition, the storage unit 55 associates and stores the state of floating and rotation of the cell C with the performance of the driving data of the pump main body 41 corresponding to the conditions for generating the liquid flow LF. It is desirable to accumulate a large amount of this kind of data and optimize the drive of the pump main body 41 by AI learning.

[Operation flow of the cell culture unit of the first embodiment]
Next, an example of the operation flow of the cell culture unit U of the first embodiment will be described mainly with reference to FIGS. 1 and 5. FIG. FIG. 5 is a flow chart showing an example of the operation at the start of long-term cell culture by the cell culture unit U of the first embodiment. Assume that cells C are put into wells 2 of the well plate 12 and that the cells C are in contact with the bottom surface 22 before the start of the flow. The flow shown in FIG. 5 is a flow for floating the cells C to a predetermined height in the well 2 when the long-term culture of the cells C is started.

First, the camera unit 3 is controlled by the camera control unit 52 of the controller 5, and the position of the camera unit 3 is adjusted (step S1). is executed (step S2). At this point, the liquid flow LF is not generated. In this focusing operation, the lens drive unit 33 moves the lens unit 31 up and down, and the image processing unit 53 performs edge detection processing and contrast evaluation on the obtained image. Then, the height position (Z-direction height) of the lens portion 31 with the best contrast is detected, and the Z-direction height is set as the in-focus position. Image information such as image data at the in-focus position, Z-direction height data, and cell C area data are stored in the storage unit 55 .

FIG. 6A is a diagram showing a state in which the position adjustment in step S1 and the focusing operation in step S2 are being performed. A focused optical image of the cell C is formed on the image plane of the imaging element 35 provided in the camera body 32 . The focal position of the lens unit 31, which is an objective lens for imaging a minute object such as a cell C, is determined by the parfocal distance Zh from the surface 34 with the body. Therefore, when the cell C is in focus, the distance between the cell C and the barrel surface 34 becomes the parfocal distance Zh. That is, by moving the camera unit 3 to the height in the Z direction shown in FIG. 6A (step S1), it is possible to focus on the cell C in contact with the bottom surface 22 (step S2). Therefore, the Z-direction height of the cell C can be grasped by obtaining the Z-direction height of the waist surface 34 . Since the depth of field a actually exists, the focal position has a certain width in the Z direction. A certain reference height Z0 is set, and the Z-direction height Z1 of the body surface 34 with respect to the reference height Z0 when the cell C in contact with the bottom surface 22 is focused is stored in the storage unit 55. be done. This Z1 corresponds to the Z-direction height of the cell C when it is not floating above the bottom surface 22 .

After that, the camera control unit 52 moves the camera unit 3 in the Z direction so that the height of the camera unit 3 in the Z direction is at a predetermined height position (step S3). This movement is, for example, a movement to raise the camera unit 3 in the Z direction so that the body mounting surface 34 is positioned at the position Z2 shown in FIG. This movement is for setting the focal position of the camera unit 3 .

Subsequently, the pump control unit 51 controls the pump unit 4 to gradually increase the liquid flow LF from zero, and detects the flow velocity F of the liquid flow LF that floats the cell C to a floating height within a predetermined range. is executed. In addition, it is desirable to form a table in advance of the relationship between the operation rank of the pump body 41, for example, the amount of liquid discharged per unit time, and the flow velocity F of the liquid flow LF that can be generated. Moreover, the floating height may be the floating height in a state where the well is completely separated from the bottom surface of the well, or the floating height is partially in contact with the bottom surface of the well.

The pump control unit 51 operates the pump main body 41 so as to generate the liquid flow LF with the flow velocity F of the lowest rank (step S4). Next, the camera control unit 52 controls the lens driving unit 33 to cause the lens unit 31 to perform a focusing operation on the cell C while the liquid flow LF having the flow velocity F is generated (step S5). Actually, since the height in the Z direction of the camera unit 3 has already been set in step S3 so that the in-focus position is at the target height position Z2, the period of waiting for the cell C to float up to the in-focus position. Become. Then, it is determined whether or not the cell C has reached a predetermined height in the Z direction (step S6). That is, it is determined whether or not the cell C is being imaged in focus by the camera unit 3 in which the mounting surface 34 is set at a predetermined Z-direction height.

FIG. 6B shows a state in which the cell C is floated by the generation of the liquid flow LF through the through-hole 24 and the lens part 31 is focused on the floated cell C. FIG. The parfocal distance Zh is a fixed distance, and the Z-direction height of the body attachment surface 34 is increased from Z1 to Z2 in step S3. Focusing on the floated cell C means that the cell C has also floated by the height from Z1 to Z2. That is, the distance Z2-Z1 is the cell C floating height. If the floating height is too small, the cell C interferes with the bottom surface 22 and becomes unrotatable or impeded in rotation. On the other hand, if the floating height is too large, cells C may jump out of well 2 .

In view of these, the "predetermined height" used as the criterion in step S6 is set to a height corresponding to the lower layer position of the well 2, that is, a position closer to the bottom surface 22, where the cells C can freely rotate. It is desirable to Further, when the flow velocity F of the liquid flow LF increases, the floating height of the cell C naturally tends to increase, while the vertical swing of the cell C tends to increase during the floating. From this point of view as well, it is desirable to suppress the vertical swing without increasing the flying height too much. In this case, the cell C can be easily maintained in a state of being floated within a predetermined Z-direction height range, so that a state in which the camera unit 3 can easily capture an image can be formed.

In step S6, if the Z-direction height of the cell C has not reached the predetermined height (NO in step S6), it means that the cell C is not sufficiently floated. In this case, the pump control unit 51 increments the flow velocity F of the liquid flow LF to F=F+1 rank (step S7). It should be noted that "one rank" is a predetermined increase unit of the pump discharge amount, and is interlocked with the increase of the flow velocity F. Then, the pump control unit 51 controls the operation of the pump main body 41 so as to achieve the newly given flow velocity F (step S4).

In step S6, when the Z-direction height of the cell C has reached a predetermined height (YES in step S6), the cell C has risen to a predetermined height. In this case, the pump control unit 51 maintains the operating state of the pump main body 41 and fixes the flow rate F thereof. At the same time, proper temperature control of the well 2 and atmospheric gas setting are performed. The preparatory processing for long-term culture is thus completed, and long-term culture of cells C is started (step S8). Cell C often exhibits the behavior of rolling after surfacing. Therefore, the cell C has started to rotate at or before this step S8.

FIG. 7 is a flow chart showing an example of the operation during long-term culture of cells C by the cell culture unit U after finishing the processes up to step S8. At this point, the pump controller 51 continues to cause the pump unit 4 to generate the liquid flow LF at the flow rate F set in step S4. The controller 5 determines whether or not the timing for monitoring the cell C has come (step S11). This monitor timing can be set, for example, in a span of about one hour to one day. When the monitor timing arrives (YES in step S11), the camera control unit 52 detects the state information of the cell C by setting the Z-direction height of the base surface 34 to Z2 in step S3. 3, an image of the cell C is captured (step S12).

Subsequently, based on the obtained image of the cell C, the state of the cell C is specified, and the state is recorded and analyzed (step S13). Specifically, the image processing unit 53 performs predetermined image processing on the acquired image of the cell C, so that data such as the outer shape and color of the cell C can be grasped. This data is stored in the storage unit 55 in association with the acquisition timing. Further, based on the data, the data processing unit 54 performs a process of determining the number of turns of the cells C, a process of extracting various sizes of the cells C, a process of identifying colors and textures, a process of evaluating the fluorescence of the cells C, and the like. conduct. Based on these processing results, for example, analysis processing such as cell growth amount analysis based on changes in the size of cells C, quality analysis such as life and death based on cell texture, life and death of cells C based on fluorescence conditions, and expression analysis of specific proteins. , are executed by the data processing unit 54 . The above processing results and analysis results are also stored in the storage unit 55 . Then, the relationship between the past flow velocity F and the rotation data of the cells C and the cell quality data is referred to, and a process of comparing it with the data acquired this time is performed (step S14).

Subsequently, a process of analyzing the floating state of the cell C is executed (step S15). Specifically, the data processing unit 54 determines the degree of focus on the cell C based on image processing data from the image processing unit 53 . If the degree of focusing is good, the cell C will continue to float at the height position of the parfocal distance Zh shown in FIG. 6(B). On the other hand, when the degree of focus is low, the cell C does not exist at the set height position. For example, when the cell C grows and increases in mass, it tends to sink, and when it dies or deteriorates and its mass decreases, it tends to float.

Then, the data processing unit 54 determines whether or not the floating state and the rotating state of the cell C are within a predetermined range that can be recognized as "good" based on the processing results of steps S14 and S15 (step S16). . If it is not within the “good” range (NO in step S16), the pump control unit 51 changes the flow rate F of the liquid flow LF to F=F+1 rank or F =F-1 (step S17). Then, the process returns to step S12 and similar analysis processing is repeated. On the other hand, if the floating state and rotation state of the cell C are "good" (YES in step S16), the pump control unit 51 maintains the current flow velocity F of the liquid flow LF (step S18).

[Second embodiment]
FIG. 8 is a block diagram showing the configuration of a cell culture unit U1 for realizing the method for culturing a living subject according to the second embodiment of the present invention. In the first embodiment, an example in which one chamber is used is shown, but in the second embodiment, an example in which a plurality of chambers are connected in series and in parallel through connection channels is shown. The cell culture unit U1 contains five chambers 0,1,2,3,4. These chambers 0, 1, 2, 3, 4 are intended to each contain a different cell.

As cell resources, for example, patient-derived normal cells, diseased cells such as cancer cells, and iPS/ES cell-derived differentiated cells can be used. Here, heart cells (first biological object) in the wells of chamber 0 (first chamber), brain cells (second biological object) in the wells of chamber 1 (second chamber), and An example is shown in which lung cells are accommodated in wells, liver cells are accommodated in chamber 3 wells, and intestinal cells are accommodated in chamber 4 wells. In addition, these chambers 0, 1, 2, 3, and 4 are provided with liquid level sensors 0, 1, 2, 3, and 4 that detect the liquid level of the medium (culture solution) in each chamber. there is

The cell culture unit U1 further includes a group of first flow control valves FCV0, 1, 2, 3, 4 installed corresponding to each of the chambers 0, 1, 2, 3, 4; A group of FCVs 10, 11, 12, 13, and 14, which is a group of two flow control valves, a pump group consisting of a first pump P1, a second pump P2, a replenishment pump P3, and a recovery pump P4, a reservoir medium tank T1, and a waste liquid tank. T2 and a controller 500.

The cell culture unit U1 has a circulatory system that circulates the medium to the other chambers 1, 2, 3, and 4 with the chamber 0 containing heart cells as the center of circulation, and replenishes the medium or collects the medium as necessary. It has a replenishment system and a recovery system. The circulatory system includes a venous tube 61, an arterial tube 62, arterial side tubes 631 to 634, and venous side tubes 641 to 634 as connecting channels that connect the chambers 0, 1, 2, 3, and 4 in series and parallel. 644 included.

The downstream end of the venous tube 61 is connected to the inlet side of the chamber 0, and the upstream end of the arterial tube 62 is connected to the outlet side thereof. Connected to the downstream end of the arterial tube 62 is an arterial parallel tube 63 in the form of a manifold tube. Connected to the upstream end of the venous tube 61 is a parallel venous tube 64 also in the form of a manifold tube. From the artery parallel tube 63, the downstream ends thereof are respectively connected to the first tube 631 connected to the chamber 1, the second tube 632 connected to the chamber 2, the third tube 633 connected to the chamber 3, and the chamber 4. Each upstream end of the fourth tube 634 is branched. In addition, the vein parallel tube 64 includes a fifth tube 641 whose upstream end is connected to the chamber 1, a sixth tube 642 connected to the chamber 2, a seventh tube 643 connected to the chamber 3, and a chamber 4. Each downstream end of the connected eighth tube 644 is connected. That is, the heart cell chamber 0 and other chambers 1, 2, 3 and 4 are connected in series, and the chambers 1, 2, 3 and 4 are connected in parallel.

The arterial tube 62 has the FCV 10, the first tube 631 has the FCV 11, the second tube 632 has the FCV 12, the third tube 633 has the FCV 13, and the fourth tube 634 has the FCV 14, respectively. In addition, FCV0 is arranged in the intravenous tube 61, FCV1 is arranged in the fifth tube 641, FCV2 is arranged in the sixth tube 642, FCV3 is arranged in the seventh tube 643, and FCV4 is arranged in the eighth tube 644. there is A first pump P1 is arranged in the venous tube 61 and a second pump P2 is arranged in the arterial tube 62 .

By driving the second pump P2, the culture medium is pumped out from the chamber 0 through the arterial tube 62. FIG. The amount of medium delivered is adjusted by the degree of FCV10 opening. The culture medium sent to the second pump P2 is supplied in parallel to each of the chambers 1, 2, 3 and 4 via the artery parallel tube 63 and tubes 631-634. The amount of medium supplied to each chamber 1, 2, 3, 4 is determined by the opening of the second flow control valve group FCV11, 12, 13, 14 and/or the first flow control valve group FCV0, Adjustable by 1, 2, 3, 4 degrees of opening.

A suction force is generated by driving the first pump P1, and the medium is sucked from the chambers 1, 2, 3, and 4 via the tubes 641 to 644 and the vein parallel tube 64. FIG. The aspirated medium is returned to chamber 0 through venous tubing 61 . That is, by driving the first pump P1 and the second pump P2, the culture medium can be circulated starting from the chamber 0 and returning to the chamber 0 via the chambers 1, 2, 3 and 4. By closing the FCV0 arranged in the intravenous tube 61, the circulation of the culture medium can be stopped. Also, by closing the FCVs 1, 2, 3, 4 respectively arranged in the tubes 641-644, the chambers 1, 2, 3, 4 can be separated from the circulatory system.

Here, two pumps, the first pump P1 and the second pump P2, are used to circulate the medium in chambers 0, 1, 2, 3 and 4, which are open type chambers as shown in FIG. This is because it is assumed to be used. The container 11 shown in FIG. 1 has an opening wall 16 on the upper surface, and the internal space A of the container 11 communicates with the outside air through the through holes 24 of the well plate 12 . This is because two pumps are required to control the outflow of medium from chamber 0 and to control the inflow of medium into chamber 0 because the circulation circuit is not closed. If the chambers 0, 1, 2, 3, and 4 are of a closed type with the upper surface of the opening wall 16 sealed, one of the first pump P1 and the second pump P2 is omitted. Also good.

The supply system consists of a supply pump P3 and a reservoir medium tank T1. A fresh medium is stored in the reservoir medium tank T1. Note that the reservoir medium tank T1 may contain components other than the medium, such as reagents. In this case, the medium containing the reagent can be circulated in the circulatory system at an appropriate timing. The supply pump P3 pumps medium from the reservoir medium tank T1 to the arterial parallel tube 63. As shown in FIG. When the amount of culture medium circulating in the circulatory system runs short, the replenishment pump P3 is driven so that the required amount of culture medium is supplied to the arterial parallel tube 63 . The recovery system comprises a recovery pump P4 and a waste liquid tank T2. Recovery pump P4 aspirates and recovers excess medium from the circulation system through arterial parallel tubing 63 . The recovered culture medium is stored in the waste liquid tank T2. This waste liquid tank T2 may be treated as an analysis recovery tank T2. In this case, it is possible to realize an embodiment in which part of the medium circulating in the circulation system is recovered in the analysis recovery tank T2, and the components of the recovered medium are analyzed.

The controller 500 comprehensively controls the operation of the cell culture unit U1, and functionally controls a pump control unit 501, a first FCV control unit 502, a second FCV control unit 503, a data processing unit 504, and a storage unit 505. have. Although not shown in FIG. 8, chambers 0, 1, 2, 3, and 4 are provided with camera units for capturing images of the cells housed in the wells of each chamber.

The pump control unit 501 controls the first pump P1, the second pump P2, the replenishment pump P3, and the recovery pump P4, and generates required medium discharge force or medium suction force at a predetermined timing. The first FCV control unit 502 performs control to adjust the opening degrees of FCVs 0, 1, 2, 3, and 4. The second FCV control unit 503 controls the opening degrees of the FCVs 10, 11, 12, 13, and 14. FIG. The data processing unit 504 performs the same data processing and analysis processing as the data processing unit 54 of the first embodiment based on the image data of the cells in each chamber. The storage unit 505 stores various setting data and table data regarding the cell culture unit U1.

[Operation Flow of Cell Culture Unit of Second Embodiment]
Next, the operation flow of the cell culture unit U1 of the second embodiment will be described. FIG. 9 is a flowchart showing an example of the operation at the start of long-term cell culture by the cell culture unit U1. The controller 500 confirms that chambers 0, 1, 2, 3, and 4 have been set in a predetermined state (step S21). This confirmation operation is, for example, acceptance of an operation start instruction from the user. Up to this point, chambers 0, 1, 2, 3, 4 are pre-populated with the specified cells and media. Circulatory system tubes such as the venous tube 61 and the arterial tube 62 are also pre-filled with culture medium.

Next, the second FCV control unit 503 sets the opening degrees of the FCVs 10, 11, 12, 13, and 14 to prescribed values predetermined for each cell (step S22). Note that the first FCV control unit 502 also sets the opening degrees of FCVs 0, 1, 2, 3, and 4 to specified values. Next, the pump controller 501 drives the first pump P1 and the second pump P2 (step S23). As a result, starting from chamber 0 containing heart cells, the culture medium flows out from artery tube 62, passes through parallel-connected chambers 1, 2, 3, and 4, and flows from vein tube 61 to chamber 0. Circulation of the returning medium is initiated. As a result, the cells in each chamber 0, 1, 2, 3, 4 flow or float in the wells.

After that, the controller 500 acquires detected values of medium liquid levels in the chambers from the liquid level sensors 0, 1, 2, 3, and 4 attached to the respective chambers 0, 1, 2, 3, and 4. (step S24). Using this detection value data, the data processing unit 504 determines whether or not the liquid level in each chamber 0, 1, 2, 3, 4 is the default value, that is, whether each chamber 0, 1, 2 , 3 and 4 is judged to be insufficient or excessive (step S25). As one embodiment, for chambers 1, 2, 3, and 4 other than cardiac cells, among FCVs 1, 2, 3, and 4, only the FCV of the chamber to be adjusted is sequentially "opened" and the others are "closed." , the shortage/excess of the culture medium may be determined individually.

If the culture medium is determined to be "insufficient" in step S25, the pump control unit 501 drives the supply pump P3 to supply the culture medium from the reservoir culture medium tank T1 to the artery parallel tube 63 (step S26). On the other hand, if the culture medium is determined to be "excess" in step S25, the pump control unit 501 drives the recovery pump P4 to suck the culture medium from the parallel artery tube 63 and flow it into the waste liquid tank T2 (step S27). Then, the process returns to step S24, and the operation of reconfirming the liquid level in the chamber is repeated.

On the other hand, if the culture medium is determined to be the "predetermined value" in step S25, and if either the supply pump P3 or recovery pump P4 is being driven, the pump control unit 501 stops this (step S28). Then, when it is confirmed that the cells in the chambers 0, 1, 2, 3, and 4 have risen to a predetermined height by the same method as in the first embodiment, the camera unit (not shown) confirms that the cells at that time Maintain the flow rate of the liquid stream. Thereby, long-term culture of cells is started (step S29). During long-term cell culture, the camera unit observes, for example, the state of cell growth.

FIG. 10 is a flow chart showing the process of taking out the chamber during long-term cell culture by the cell culture unit U1. During long-term cell culture, controller 500 determines whether there is a request to remove a particular chamber (step S31). The removal request is a request for processing to disconnect one or more of the chambers 1, 2, 3, and 4 from the circulation system while continuing the circulation of the medium so that they can be removed from the cell culture unit U1. If there is no takeout request, it waits.

If there is the takeout request (YES in step S31), the controller 500 performs the following process. Here, it is assumed that there is a request to take out the chamber 1 for brain cells. In this case, the second FCV control unit 503 throttles the FCV 11 to "close" (step S32). Further, the first FCV control unit 502 performs control to "close" the FCV1 (step S33). As a result, the chamber 1 is disconnected from the culture medium circulation circuit through the arterial parallel tube 63 and the venous parallel tube 64 .

Since the chamber 1 is cut off from the circulation circuit, the hydraulic pressures of the other chambers 2, 3, 4 will rise. In this case, the flow velocity of the liquid flow in these chambers 2, 3 and 4 increases, which may cause cells in each well to float excessively or jump out of the well. Therefore, the second FCV control unit 503 proportionally divides the amount of "closing" the FCV 11 to decrease the opening degrees of the FCVs 12, 13 and 14 (step S34). Further, the pump control unit 501 suppresses the output of the first pump P1 by the amount that the chamber 1 is disconnected. This allows the chambers 2, 3, and 4 to maintain the liquid flow velocity before the chamber 1 is cut off. After that, the user's work includes returning the removed chamber 1 to its original state after observation, treatment with a chemical solution, or removal of some cells, and assembling a new chamber containing cells in place of the chamber 1. is assumed.

FIG. 11 is a flow chart showing the liquid level management operation in the chamber during long-term cell culture by the cell culture unit U1. The controller 500 acquires detected values of medium liquid levels in the respective chambers 0, 1, 2, 3, and 4 from the liquid level sensors 0, 1, 2, 3, and 4 at predetermined sampling timings ( step S41). During long-term culture, changes may occur in the height of the medium liquid surface and the state of the liquid flow LF due to factors such as evaporation of the medium and proliferation or reduction of cells. A camera unit that constantly monitors the decrease in the medium with the output values of the liquid level sensors 0, 1, 2, 3, and 4, and the fluctuations in the cell flow or floating state caused by changes in the state of the liquid flow LF. can be found in the images.

Next, the data processing unit 504 determines whether there is an abnormality in the height of the medium surface in each chamber, that is, whether the medium in each chamber 0, 1, 2, 3, 4 is insufficient or excessive. Also, it is determined whether or not there is an abnormality in the floating state of each cell (step S42). If there is no abnormality in the height of the medium liquid level or in the floating state of the cells (NO in step S42), the current strength of the liquid flow LF is maintained.

On the other hand, if an abnormality is found in the height of the medium liquid level or in the floating state of the cells (YES in step S42), a predetermined action is taken (step S43). For example, when it is detected that the floating height of the cells in the chamber 1 is lowered, the second FCV control unit 503 increases the opening of the FCV 11 to increase the liquid flow LF, and conversely, the cells float. If the height is too high, the degree of opening of the FCV 11 is decreased to reduce the liquid flow LF. Further, when a decrease in the height of the culture medium liquid level is detected, the pump control unit 501 drives the replenishment pump P3 to replenish the circulation system with the culture medium.

According to the cell culture unit U1 of the second embodiment described above, it is possible to supply the same culture medium to a plurality of types of cells while culturing these cells for a long period of time. As a similar cell culture unit, there is an existing unit in which cells are placed in a microchannel through which a culture solution is circulated and reacted with the culture solution. However, it is difficult for existing units to handle a large amount of cells due to the use of microchannels. On the other hand, in the cell culture unit U1, a well plate 12 having a large number of wells 2 can be arranged in each chamber 0, 1, 2, 3, 4, so that a large amount of cells can be cultured or reacted at the same time. Also, in the existing unit, the cells being cultured cannot be taken out from the microchannel, but in the cell culture unit U1, the cells can be easily taken out as shown in the flow of FIG. Furthermore, since the cell culture unit U1 has a configuration in which the chambers are connected by tubes, there is an advantage that the number of chambers can be easily increased or decreased, and the arrangement of the chambers can be easily changed. In addition, there is also the advantage that intercellular transmitters (signaling substances) such as hormones, cytokines, and neurotransmitters can be circulated to cells in chambers 0, 1, 2, 3, and 4 and their reactions can be observed.

[Other embodiments]
Although the main embodiments of the present invention have been described above, the present invention is not limited to these, and various modified embodiments are possible. For example, the present invention can employ modified embodiments as shown below.

(1) FIG. 1 illustrates a chamber CH having only one well plate 12 . FIG. 12 is a cross-sectional view showing a chamber CH1 according to a modification. In the chamber CH1, a lower well plate 12A (first plate) and an upper well plate 12B (second plate) are arranged in upper and lower layers. The lower well plate 12A has wells 2 (first wells) with through holes 24, like the well plate 12 shown in FIG. The upper well plate 12B is spaced above the lower well plate 12A and similarly has wells 2 (second wells) with through holes 24 therein.

By using this chamber CH1, the liquid flow passing through the through-holes 24 of the lower well plate 12A further passes through the through-holes 24 of the upper well plate 12B, thereby not only the wells 2 of the lower well plate 12A but also the upper wells. Liquid flow can also be generated in well 2 of plate 12B. Therefore, the lower well plate 12A cultures the first cells Ca, and the upper well plate 12B cultures the second cells Cb different from the first cells Ca. Can be cultured.

(2) FIG. 13 is a cross-sectional view showing a chamber CH2 according to another modification. The chamber CH2 has a lower well plate 12A, a middle well plate 12C and a lid 46. As shown in FIG. The lower well plate 12A is the same as the well plate 12 shown in FIG. The middle well plate 12C is arranged above the lower well plate 12A at intervals and has wells 2 capable of containing cells. The lid 46 seals the top of the middle well plate 12C. The lower well plate 12A is immersed in the first culture medium LA. On the other hand, a second culture medium LB different from the first culture medium LA is filled between the middle well plate 12C and the lid body 46 .

The lower well plate 12A holds the first cells Ca, and the middle well plate 12C holds the second cells Cb. The middle well plate 12C has a filter function that allows the first medium LA and the second medium LA to permeate each other, but does not allow free mixing of the two mediums and entry/exit of both cells. The first culture medium LA is supplied to the lower well plate 12A by a liquid-sending tube 42 and a liquid-absorbing tube 43 arranged with respect to the container 11, and a liquid flow is generated. On the other hand, the middle well plate 12C is supplied with the second culture medium LB by the liquid-feeding tube 44 and the liquid-absorbing tube 45 arranged with respect to the lid 46, and a liquid flow is generated. In this way, a configuration may be adopted in which different cells are held in two layers, upper and lower, in one chamber, and a liquid flow is independently generated for each cell.

(3) In the above-described second embodiment, the example in which the liquid level sensors 0, 1, 2, 3, and 4 are attached to the respective chambers 0, 1, 2, 3, and 4 is shown. Instead of the liquid level sensors 0, 1, 2, 3 and 4, other means may be used to monitor the liquid level of the culture medium. For example, a float is placed on the liquid surface of the medium, and the float is also observed with the camera unit 3 arranged for observing cells, and the height of the float is grasped to monitor the liquid surface of the medium. You can do it.

(4) In the above embodiment, as illustrated in FIG. 2A, the well 2 including the cylindrical side surface 21, the bottom surface 22, the tapered opening surface 23, and the through hole 24 is illustrated. The shape of the well 2 can be changed variously. FIGS. 14A to 14D are diagrams showing wells 2A to 2D according to modifications. A well 2A in FIG. 14A exemplifies a well having a cannonball-shaped cross section. FIG. 14B illustrates a well 2B with a simple rectangular cross section, and FIG. 14C illustrates a well 2C with a triangular cross section. Further, FIG. 14D illustrates a well 2D in which cavities with a rectangular cross section and a trapezoidal cross section are connected in the vertical direction. All of the wells 2A to 2D are provided with through-holes 24 for generating the liquid flow LF at the lowest positions.

(5) Also, the number and formation positions of the through-holes 24 can be variously changed as long as the liquid flow LF capable of floating and rotating the cells C can be formed. FIGS. 15A to 15C are diagrams showing wells 2E to 2G provided with through holes 24A to 24E according to modifications. A well 2E in FIG. 15A shows an example in which two through holes 24A and 24B are formed near the bottom. FIG. 15B illustrates a well 2F having one through hole 24C on its side surface. FIG. 15C illustrates a well 2G in which through holes 24D and 24E are formed in the bottom and side surfaces, respectively.

(6) Furthermore, the shape of the opening communicating with the well can also be changed in various ways. FIG. 16(A) illustrates a well 2H having a through hole 24F that penetrates the wall surface of the well in an oblique direction rather than in an orthogonal direction. This through-hole 24F makes it easier to generate a vortex in the well 2H. FIG. 16(B) illustrates a well 2I in which the opening is not a through hole but a nozzle 26 is used. The tip of the nozzle 26 faces the cavity of the well 2I and is capable of ejecting liquid.

According to the present invention described above, oxygen and nutrients can be supplied to the cells C by generating the liquid flow LF of the culture medium L in the wells 2 serving as the cell C culture regions. In addition, by causing the cells C to flow, float, and rotate within the well 2 by the liquid flow LF, some of the cells C are prevented from continuing to contact the cylindrical side surface 21 and the bottom surface 22 of the well 2 in the same posture. can be That is, it is possible to culture the cells C for a long period of time while migrating in the medium L. As a result, the influence of the surface contacting the cylindrical side surface 21 and the bottom surface 22 can be suppressed, and the cells C can freely grow three-dimensionally.

U cell culture unit (culturing unit for biological object)
C cell (biological object)
L medium (culture solution)
LF liquid flow CH chamber 12 well plate 2 well recesses)
20 cavity (culture area)
24 through hole (opening)
3 camera unit 4 pump unit (pump device)
5 controller 51 pump control unit

Claims (3)

arranging the biological object in a culture area that is capable of accommodating the biological object and its culture medium and that is composed of a recess having a bottom surface and a side wall surface, the bottom surface being provided with an opening;
generating a liquid flow of the culture solution blowing up into the recess from the opening by supplying the culture solution into the recess from the opening ;
holding the biological object for a predetermined period of about one day to several weeks while floating and rotating it in the concave portion by the liquid flow ;
monitoring the floating state and the rotating state of the biological object while holding the biological object for the predetermined period;
A method of culturing a biological object, wherein when a predetermined change occurs in the floating state and the rotational state of the biological object, the condition for generating the liquid flow is changed. a chamber comprising a well capable of containing a biological object and a culture medium, and an opening provided at the bottom of the well;
a pump device that sends a culture solution to the well through the opening and generates a liquid flow of the culture solution that blows up into the well from the opening ;
a pump control unit that controls the operation of the pump device,
In a biological subject culture unit, the pump control unit causes the pump device to generate a liquid flow that allows the biological subject in contact with the bottom surface of the well to flow in the culture solution. ,
As the chambers, a first chamber for accommodating a first biological subject in the well, a second chamber for accommodating a plurality of second biological subjects different from the first biological subject in the well, has
a circulation system that connects the first chamber and the plurality of second chambers, the circulation system circulating the culture medium through the plurality of second chambers with the first chamber as the center of circulation;
The pump device circulates the culture solution through the circulation system to the wells of the first chamber and the wells of the second chamber, thereby pumping the first biological object and the second biological object respectively. A living subject culture unit that is flowed within a well . a chamber comprising a well capable of containing a biological object and a culture medium, and an opening provided at the bottom of the well;
a pump device that sends a culture solution to the well through the opening and generates a liquid flow of the culture solution that blows up into the well from the opening;
a pump control unit that controls the operation of the pump device,
In a biological subject culture unit, the pump control unit causes the pump device to generate a liquid flow that allows the biological subject in contact with the bottom surface of the well to flow in the culture solution. ,
A culture unit for a living subject, wherein the chamber comprises a first plate having first wells and a second plate disposed above the first plate and having second wells.

Images