JP7565033B2 - Culture medium circulation device - Google Patents

Description

The present invention relates to a culture medium circulation device that circulates culture medium between wells in a multi-well plate having multiple wells.

In recent years, drug discovery research based on cultured cells and tissues has become more active. In particular, since the creation of iPS cells, it has become relatively easy to produce tissue and organ cells, and these tissues and organs are used to verify the effects and side effects of drugs and other substances. Traditionally, such verification has been carried out on a single tissue or organ, but tissues and organs do not function independently within the body, but rather function while communicating with other tissues and organs. For this reason, it is not possible to fully understand the effects on the entire organism by only targeting a single tissue or organ.

As a result, a technology has recently been developed that uses microfluidic devices to place multiple tissues or organs on a chip called "organ on a chip," enabling communication between these tissues and organs and then verifying the effects of drugs, etc. (see, for example, Patent Document 1).

Special table 2019-505214 publication

However, with chips like those described above, whenever the combination of tissues or organs is changed or the flow path is changed, the microchip design and production must be changed. This makes the chip less versatile and can lead to high costs.

In addition, culture solutions for culturing tissues and organs have the property of easily releasing carbon dioxide (CO ₂ ), and when they come into contact with the outside air, they release CO ₂ and become alkaline. Such alkalinization of the culture solution can cause changes in the properties of tissues and organs or their death. For this reason, tissues and organs must usually be cultured in a CO ₂ incubator or glove box where the CO ₂ concentration is maintained at 5% and CO ₂ is forcibly added, which places significant restrictions on experimental operations and cannot be said to be easy to operate.

The present invention is intended to solve the above problems, and aims to provide a culture medium circulation device that achieves high versatility and low cost when verifying communication between multiple tissues or organs, and that eliminates the need for a _CO2 incubator or glove box, thereby improving operability.

The culture medium circulation device of the present invention is characterized in that it comprises a multi-well plate having a plurality of wells, a sealing lid for sealing each well, a flow path penetrating the sealing lid while maintaining the airtightness of the wells, for circulating the culture medium in a serial loop between the plurality of wells, and a single liquid delivery pump provided midway through the flow path for sucking up and delivering the culture medium.

The sealing lid preferably has a disk portion formed in a disk shape, a flange provided at the upper end of the disk portion, and an O-ring made of an elastic material that is wrapped around the disk portion, and when the disk portion is fitted into the well until the flange abuts against the upper edge of the well, the O-ring elastically deforms and adheres closely to the inner wall of the well, thereby ensuring the airtightness of the well.

The sealing lid has a plurality of through holes arranged perpendicular to the direction in which the disk portion expands, and is welded to the flow path forming body via the upper surface of the disk portion, and the flow path forming body is formed by welding together an upper flow path forming body and a lower flow path forming body, both of which are formed in a flat plate shape, and the lower flow path forming body has a groove communicating with the through holes on the surface facing the upper flow path forming body, and the flow path is preferably formed by the through holes and the grooves.

The culture medium circulation device of the present invention preferably further has a cell culture insert in the well, the sealing lid seals both the well and the cell culture insert, and the flow path is formed independently for each of the well and the cell culture insert.

The sealing lid is preferably made of a plate material fixed to the upper edge of the well so as to cover the well.

It is preferable that the flow path is composed of a pipe that penetrates the tight-fitting lid and is fixed thereto, and a tube that is connected to the upper end of the pipe.

According to the culture medium circulation device of the present invention, when verifying communication between multiple tissues or organs, no special chips or the like are required, and a commercially available multi-well plate can be used, thereby achieving high versatility and low cost. In addition, by sealing the wells with a sealing lid, _CO2 release from the culture medium due to contact with the outside air is suppressed, making a _CO2 incubator or glove box unnecessary and improving operability. In addition, evaporation of the culture medium in the wells can be suppressed, and further contamination between multiple samples can be prevented.

FIG. 1 is a side cross-sectional view of a culture medium circulating device according to an embodiment of the present invention. FIG. 2 is a side cross-sectional view of a well in which a cell culture insert is further placed in the culture medium circulation device. FIG. 1 is a side cross-sectional view of a culture medium circulating device according to a first embodiment of the present invention. FIG. 11 is a perspective view of a closed cell experimental apparatus utilizing a culture medium circulating device according to a second embodiment of the present invention. FIG. 11 is an exploded perspective view of the culture medium circulating device according to the second embodiment. FIG. 1A is a side cross-sectional view of the culture medium circulating device according to the second embodiment, and FIG. 1B is a cross-sectional view taken along line II in FIG. 1A is an exploded perspective view of a sealing lid constituting the culture medium circulation device according to the second embodiment, FIG. 1B is a perspective view of the sealing lid, and FIG. 1C is a perspective view showing a modified example of the sealing lid. 4A and 4B are cross-sectional side views showing the attachment of the sealing lid to a well. FIG. 11 is an exploded perspective view of a fluid delivery pump constituting the culture medium circulating device according to the second embodiment.

A culture medium circulation device according to an embodiment of the present invention will be described with reference to Figs. 1 and 2. As shown in Fig. 1, the culture medium circulation device 1 includes a sealing lid 2 that seals each well W in a multi-well plate P having a plurality of wells W, a flow path 3 that penetrates the sealing lid 2 while maintaining the airtightness of the wells W and circulates the culture medium M in a serial loop between the plurality of wells W, and a single liquid delivery pump 4 that is provided midway through the flow path 3 and delivers the culture medium M. Both the flow path 3 and the liquid delivery pump 4 have high airtightness and gas barrier properties, and are configured so that the culture medium M does not come into contact with the outside air.

In the illustrated example, the culture medium circulation device 1 further includes a reservoir 5 for culture medium M in the flow path 3 between the multi-well plate P and the liquid supply pump 4. The reservoir 5 is formed of a reagent bottle or the like capable of storing a larger volume of culture medium M than the well W, and is sealed by a sealing lid 51. The flow path 3 is provided penetrating the sealing lid 51 while maintaining the sealing ability of the reservoir 5. Each well W is, for example, a substantially cylindrical container with a bottom, and a sealing lid 2 is attached to the top opening.

The sealing lid 2 is made of a transparent material, and by attaching the sealing lid 2 to the well W, an airtight layer A is formed surrounded by the sealing lid 2 and the inner wall of the well W. In the illustrated example, four wells W are provided, and in the following description, they are referred to as wells W1, W2, W3, and W4 from the left side of FIG. 1. The flow path 3 is formed in a tubular shape through which the culture medium M can flow, and is composed of a flow path 3a connecting wells W1 and W2, a flow path 3b connecting wells W2 and W3, a flow path 3c connecting wells W3 and W4, a flow path 3d connecting well W4 and reservoir 5, and a flow path 3e connecting reservoir 5 and well W1 via liquid supply pump 4.

The flow path 3a has an outlet 31 at one end for discharging the culture fluid M from the well W1, and an inlet 32 at the other end for supplying the culture fluid M to the well W2. The flow paths 3b and 3c are configured in the same manner as the flow path 3a. The flow path 3d has an outlet 31 at one end for discharging the culture fluid M from the well W4, and an inlet 32 at the other end for supplying the culture fluid M to the reservoir 5. The flow path 3e has an outlet 31 at one end for discharging the culture fluid M from the reservoir 5, and an inlet 32 at the other end for supplying the culture fluid M to the well W1. The outlet 31 is disposed below the inlet 32. The liquid delivery pump 4 delivers the culture fluid M in one direction, and in the illustrated example, delivers the culture fluid M in the direction from the reservoir 5 toward the well W1.

Next, a method of using the culture medium circulation device 1 will be described. Wells W1-W4 are inoculated with culture medium M containing different cells. For example, well W1 is inoculated with culture medium M containing small intestine cells, well W2 with liver cells, well W3 with cardiac muscle cells, and well W4 with brain cells. After inoculation with culture medium M, each well W is sealed with a sealing lid 2. Culture medium M (culture medium) not containing cells is added to reservoir 5, which is then sealed with a sealing lid 51. At this time, it is preferable that the outlet 31 in each well W and reservoir 5 is immersed in culture medium M.

When the liquid supply pump 4 is operated in this state, first, the liquid supply pump 4 sucks up the culture fluid M in the reservoir 5 from the outlet 31 of the flow path 3e, and the amount of culture fluid M in the reservoir 5 decreases. Then, since the reservoir 5 is sealed by the sealing lid 51, the internal pressure of the reservoir 5 decreases, and to compensate for this decrease in internal pressure, the culture fluid M in the well W4 flows into the reservoir 5 through the flow path 3d. Then, as the amount of culture fluid M in the well W4 decreases, the internal pressure of the well W4 sealed by the sealing lid 2 decreases, and to compensate for this decrease in internal pressure, the culture fluid M in the well W3 flows into the well W4 through the flow path 3c. In the same manner, the culture fluid M in the well W2 flows into the well W3 through the flow path 3b, and the culture fluid M in the well W1 flows into the well W2 through the flow path 3a. The culture fluid M sucked up from the reservoir 5 is sent by the liquid supply pump 4 through the flow path 3e and flows into the well W1.

In this way, by sealing each well W and reservoir 5, the suction pressure from the liquid delivery pump 4 is transmitted to each well W and reservoir 5, creating an equal suction pressure in the wells W and reservoirs 5 connected in a serial loop. As a result, the culture fluid M delivered from the liquid delivery pump 4 circulates back to the liquid delivery pump 4 via the wells W1-W4 and reservoir 5. At this time, the liquid level of the culture fluid M in the wells W1-W4 and reservoir 5 is maintained at the height of the outlet 31.

By circulating the culture medium M in this way, for example, if a drug is added to well W1, the drug will also spread to the other wells W2-W4, making it possible to examine the effects of the drug in all wells W1-W4. In this case, if hormones or physiologically active substances are secreted from the cells in a specific well W in response to the drug, these hormones and physiologically active substances will also spread to the other wells W, making it possible to mimic communication between different tissues and organs in the living body.

According to the culture medium circulation device 1 configured as described above, a commercially available multi-well plate P can be used to verify communication between multiple tissues or organs. Therefore, there is no need to prepare a special microfluidic device chip or a specially designed multi-well plate, and high versatility and low cost can be achieved. In addition, since the culture medium M is circulated using one liquid delivery pump 4, the configuration is simpler than, for example, conventional techniques in which a liquid delivery pump is provided for each well, and installation and maintenance are easier. Furthermore, since the sealing lid 2 is made of a transparent material, cells can be observed microscopically from above through the sealing lid 2.

In addition, the well W is sealed with the sealing lid 2, and the flow path 3 and the liquid supply pump 4 are also configured so that the culture solution M does not come into contact with the outside air, so that _CO2 release from the culture solution M due to contact with the outside air is suppressed. Therefore, alkalization of the culture solution M is suppressed, and compulsory addition of _CO2 to the culture solution M using a _CO2 incubator or glove box is not required. As a result, alkalization of the culture solution M does not occur even when cells are cultured in normal air, and evaporation of the culture solution can be suppressed by sealing, so that the accuracy and operability of the experiment can be greatly improved.

As shown in FIG. 2, a cell culture insert (inner well) C may be further provided within the well W. The cell culture insert C has, for example, a bottom surface C1 made of a semipermeable membrane and a peripheral wall C2 made of plastic, and is formed in a cup shape with an open top. The bottom surface C1 has holes of a size that does not allow cells to pass through, but allows small molecules such as drugs, hormones, and biologically active substances to pass through.

When the cell culture insert C is provided, the sealing lid 2 seals both the well W and the cell culture insert C. This forms a first airtight layer A1 surrounded by the sealing lid 2, the inner wall of the well W, and the outer wall of the cell culture insert C, and a second airtight layer A2 surrounded by the sealing lid 2 and the inner wall of the cell culture insert C. The flow paths 3 are similar to those described in FIG. 1 and are formed independently for each of the well W and the cell culture insert C.

By providing such a cell culture insert C, for example, different cells can be inoculated into the first airtight layer A1 and the second airtight layer A2, respectively, and the culture medium M can be circulated while allowing communication between the cells.

Next, a culture medium circulation device according to a first embodiment of the present invention will be described with reference to FIG. 3. The culture medium circulation device 1a is an embodiment of the above-mentioned culture medium circulation device 1 with the sealing lid 2 and the flow path 3.

The sealing lid 2 of the culture medium circulation device 1a is made of a transparent plate material attached to the upper edge of the wells W so as to cover the wells W collectively, for example, a transparent acrylic plate attached to the upper edge of the wells W with double-sided tape. The flow path 3 is made of a pipe 33 that passes through the sealing lid 2 in the vertical direction and is attached, and a tube 34 connected to the upper end of the pipe 33. The pipe 33 and the tube 34 are made of, for example, a copper pipe and a PTFE tube, respectively. The pipe 33 is attached to the sealing lid 2 with, for example, an ultraviolet-curing adhesive.

Next, a culture medium circulation device according to a second embodiment of the present invention will be described with reference to Figs. 4 to 9. In the closed cell experimental device shown in Fig. 4, the culture medium circulation device 1b has a modular structure in which multiple closed lids 2, flow paths 3, and a peristaltic pump (registered trademark) 41 of the liquid delivery pump 4 are integrated. In the illustrated example, one culture medium circulation device 1b is arranged per row in a 24-hole plate P with 4 rows and 6 columns, and a total of up to four culture medium circulation devices 1b can be arranged at the same time.

The 24-well plate P is placed on a base 6 made of transparent glass and formed into a rectangular flat plate. The base 6 contains, for example, a transparent glass heater (not shown) and keeps the 24-well plate P at a constant temperature of 37 degrees. This eliminates the need for a thermostatic bath and makes it possible to monitor and observe the cells from below using an inverted microscope or the like.

A drive unit 42 for driving the peristaltic pump 41 is disposed on the side of the base 6. The drive unit 42 can be easily separated from the peristaltic pump 41, and in the illustrated example, the drive unit 42 of the culture medium circulation device 1b disposed in the first and third rows of the 24-hole plate P is disposed on the left side of the 24-hole plate P, and the drive unit 42 of the culture medium circulation device 1b disposed in the second and fourth rows of the 24-hole plate P is disposed on the right side of the 24-hole plate P. Because the drive unit 42 can be separated from the peristaltic pump 41, the disposability of the separated culture medium circulation device 1b can be improved.

As shown in Figures 5 and 6, the culture medium circulation device 1b includes a plurality of sealing lids 2 provided for each well W in a row of a 24-hole plate P, a flow path forming body 7 that holds the sealing lids 2 together and has a flow path formed therein, and a connecting pipe 8 that connects the flow path of the flow path forming body 7 to the peristaltic pump 41. The connecting pipe 8 is composed of a return pipe 81 for sending the culture medium from the flow path of the flow path forming body 7 to the peristaltic pump 41, and an outflow pipe 82 for sending the culture medium from the peristaltic pump 41 to the flow path of the flow path forming body 7.

As shown in Figures 7 and 8, the sealing lid 2 has a first disk portion 21 formed in a disk shape, a flange 22 provided on the upper end of the first disk portion 21, and an O-ring 23 made of an elastic material that is wrapped around the first disk portion 22. The sealing lid 2 also has a second disk portion 24 that is coaxial with the first disk portion 21 and attached to the lower surface of the first disk portion 21, and an O-ring 25 made of an elastic material that is wrapped around the second disk portion 24.

The diameter of the second disk portion 24 is smaller than the diameter of the first disk portion 21 and is configured to be approximately equal to the inner diameter of the cell culture insert C, which is formed into a cylindrical shape with a bottom. When the sealing lid 2 is attached to the cell culture insert C by fitting the second disk portion 24 into the upper opening of the cell culture insert C, the O-ring 25 elastically deforms and adheres to the inner wall of the cell culture insert C. This allows the sealing lid 2 to be detachably attached to the cell culture insert C, ensures the airtightness of the cell culture insert C, and forms the second airtight layer A2 (see FIG. 8(b)).

The sealing lid 2 also has a pair of first through holes 26a, 26b that are provided perpendicular to the direction in which the first disk portion 21 expands and penetrate only the first disk portion 21, and a pair of second through holes 27a, 27b that extend in the same direction as the first through holes 26a, 26b and penetrate both the first disk portion 21 and the second disk portion 24. These through holes 26a, 26b, 27a, 27b form part of the flow path 3 as described below, with the lower ends of the through holes 26a, 27a functioning as inlets 32 and the lower ends of the through holes 26b, 27b functioning as outlets 31.

Furthermore, the sealing lid 2 has first cylindrical portions 28a, 28b that are continuous with the first through holes 26a, 26b and protrude cylindrically from the upper surface of the first disk portion 21, and second cylindrical portions 29a, 29b that are continuous with the second through holes 27a, 27b and protrude cylindrically from the upper surface of the first disk portion 21. When the cell culture insert C is not used, the sealing lid 2 may be provided with only the first through holes 26a, 26b and the first cylindrical portions 28a, 28b, but without the second through holes 27a, 27b and the second cylindrical portions 29a, 29b (see FIG. 7(c)).

The diameter of the first disk portion 21 is slightly smaller than the inner diameter of the well W so that they can fit together (see Figs. 8(a) and 8(b)). To attach the airtight lid 2 to the well W, the first disk portion 21 and the second disk portion 24 are fitted into the well W until the flange 22 abuts against the upper edge of the well W. Then, the O-ring 23 elastically deforms and adheres to the inner wall of the well W, so that the airtight lid 2 is detachably attached to the well W and the airtightness of the well W is ensured to form the first airtight layer A1 (see Fig. 8(b)). In the illustrated example, the cell culture insert C is attached to the second disk portion 24, but the procedure for attaching the airtight lid 2 to the well W is the same even if the cell culture insert C is not attached.

As shown in Figures 5 and 6, five sealing lids 2 configured as described above are provided so as to be attached to the first to fourth and sixth wells W from the left in the second row of a 24-hole plate P. Note that from the left, the sealing lids are designated here as 2a, 2b, 2c, 2d, and 2e. The sealing lids 2a, 2b, 2c, and 2e each have only the first cylindrical portions 28a and 28b, while the sealing lid 2d has both the first cylindrical portions 28a and 28b and the second cylindrical portions 29a and 29b (see Figure 5).

The flow path forming body 7 is formed in a long rectangular flat plate shape extending along the row of the sealing lids 2a-2e, and is composed of an upper flow path forming body 71 and a lower flow path forming body 72 both formed in a flat plate shape. The upper flow path forming body 71 has a hole 71a into which the end 81a of the return pipe 81 on the flow path forming body 7 side fits, and a hole 71b into which the end 82a of the outflow pipe 82 on the flow path forming body 7 side fits, and which penetrates the upper flow path forming body 71 in the vertical direction. The end 81b of the return pipe 81 on the peristaltic pump 41 side is connected to a return port 43 for returning the culture solution from the flow path of the flow path forming body 7 to the peristaltic pump 41, and the end 82b of the outflow pipe 82 on the peristaltic pump 41 side is connected to an outflow port 44 for outflowing the culture solution from the peristaltic pump 41 to the flow path of the flow path forming body 7.

The lower flow path forming body 72 has holes 72a and 72b that penetrate the lower flow path forming body 72 in the vertical direction and into which the first cylindrical parts 28a and 28b of the tight-fitting lid 2a are fitted. Similarly, the lower flow path forming body 72 has holes 72c and 72d into which the first cylindrical parts 28a and 28b of the tight-fitting lid 2b are fitted, holes 72e and 72f into which the first cylindrical parts 28a and 28b of the tight-fitting lid 2c are fitted, holes 72g, 72h, 72i, and 72j into which the first cylindrical parts 28a and 28b and the second cylindrical parts 29a and 29b of the tight-fitting lid 2d are fitted, and holes 72k and 72l into which the first cylindrical parts 28a and 28b of the tight-fitting lid 2e are fitted.

The lower flow path forming body 72 has grooves 73 that connect holes 73a-73l in a predetermined pattern on the surface (top surface in the illustrated example) facing the upper flow path forming body 71. In the illustrated example, the grooves 73 are composed of groove 73a that connects holes 72b and 72c, groove 73b that connects holes 72d and 72e, groove 73c that connects holes 72f and 72g, groove 73d that extends from hole 72h to position 72m directly below hole 71a of the upper flow path forming body 71, and groove 73e that extends from position 72n directly below hole 71b of the upper flow path forming body 71 to hole 72a. In the illustrated example, holes 72i-l are not involved in the formation of groove 73.

The sealing lids 2a-2e are hermetically welded to the lower surface of the lower flow passage forming body 72 via the upper surface of the first disk portion 21, with the first cylindrical portions 28a, 28b and the second cylindrical portions 29a, 29b fitted into the holes 73a-l. The upper flow passage forming body 71 and the lower flow passage forming body 72 are hermetically welded to each other without filling the groove 73, so that the groove 73 becomes tubular with its upper part blocked by the upper flow passage forming body 71. As a result of these weldings, the first cylindrical portions 28a, 28b and the groove 73 of the sealing lids 2a-2e communicate with each other to form the flow passage 3. This hermetically welded welding is performed, for example, by high-frequency welding (high-frequency dielectric heating method).

High-frequency welding is a welding method that utilizes the phenomenon in which high-dielectric-constant materials are heated by applying electromagnetic waves (40 MHz) between electrodes with a wavelength close to that used in microwave ovens. When an electric field and a magnetic field are transmitted between the electrodes in rapid alternation, the electrical equilibrium state inside the material (dielectric) sandwiched between the electrodes is distorted, and charge separation occurs. When the frequency is fast, a sudden change called anomalous dispersion occurs in which the polarization of each cannot keep up with the change in the electric field, and the molecules that make up the dielectric undergo violent movements such as rotation, collision, vibration, and friction, and this energy becomes heat, causing internal heating of the dielectric. Using this phenomenon, a material with a high dielectric constant is sandwiched between the materials to be welded as a binder, and high-frequency waves are applied to the materials to be welded while they are sandwiched between the electrodes under pressure to heat and melt them. After that, the high-frequency waves are stopped while the pressure is still applied, and the materials are cooled, which causes the binder to solidify and the welding is completed. This high-frequency welding makes it possible to join the upper flow path forming body 71 and the lower flow path forming body 72 without blocking the groove 73 with a binder.

With the above configuration, when the liquid supply pump 4 is driven, the culture liquid passes through the liquid supply pump 4-outlet pipe 82-groove 73e-first through hole 26a of sealing lid 2a-first through hole 26b of sealing lid 2a-groove 73a-first through hole 26a of sealing lid 2b-first through hole 26b of sealing lid 2b-groove 73b-first through hole 26a of sealing lid 2c-first through hole 26b of sealing lid 2c-groove 73c-first through hole 26a of sealing lid 2d-first through hole 26b of sealing lid 2d-groove 73d-return pipe 81 and returns to the liquid supply pump 4 for circulation.

As shown in FIG. 9, the liquid delivery pump 4 has a peristaltic pump 41 that is rotationally driven by magnetic force, a drive unit 42 that has a magnet for rotationally driving the peristaltic pump 41 without contact, and an outer shell 45 that covers a flexible tube (not shown) that constitutes the peristaltic pump 41 and isolates the flexible tube from the outside air. The outer shell 45 is made of, for example, a resin with high gas barrier properties, and the peristaltic pump 41 is formed by welding.

By providing such an outer shell 45, even if the gas barrier property of the flexible tube is low, it is possible to suppress contact between the culture solution flowing through the flexible tube and the outside air, and suppress the release of _CO2 from the culture solution. Note that the gap between the outer shell 45 and the peristaltic pump 41 may be evacuated to further suppress the intrusion of outside air, or may be filled with an inert gas such as nitrogen or argon, or a liquid.

The present invention is not limited to the above embodiment, and various modifications are possible. Both the first and second examples can use a commercially available multi-well plate P, and the second example uses a dedicated flow path forming body 7, but both the first and second examples can be used in one multi-well plate P. Also, the sealing lid 2 shown in FIG. 7 is a more specific version of the sealing lid 2 shown in FIG. 2, and it goes without saying that the more specific sealing lid 2 can be applied to the configuration example of the culture medium circulation device 1 shown in FIG. 1.

Reference Signs List 1, 1a, 1b Culture medium circulating device 2, 2a-2e Sealing lid 21 (First) disk portion 22 Flange 23 O-ring 26a, 26b (First) through-hole 27a, 27b (Second) through-hole 3, 3a-3e Flow path 33 Pipe 34 Tube 4 Liquid delivery pump 41 Peristaltic pump 45 Shell 7 Flow path forming body 71 Upper flow path forming body 72 Lower flow path forming body 73, 73a-73e Groove C Cell culture insert P Multiwell plate W, W1-W4 Well

Claims (6)

A multi-well plate having a plurality of wells, a sealing cover for sealing each well,
a flow path penetrating the sealing lid while maintaining the sealing of the wells, for circulating the culture solution in a serial loop between the multiple wells;
A culture medium circulating device comprising: a liquid delivery pump that is provided midway along the flow path and that draws up and delivers the culture medium. The sealing lid has a disk portion formed in a disk shape, a flange provided on an upper end of the disk portion, and an O-ring made of an elastic member and wrapped around the disk portion,
The culture medium circulation device described in claim 1, characterized in that when the disk portion is inserted into the well until the flange abuts the upper edge of the well, the O-ring elastically deforms and adheres to the inner wall of the well, thereby ensuring airtightness of the well. the sealing lid has a plurality of through holes provided perpendicular to a direction in which the disk portion expands, and is welded to the flow path forming body via an upper surface of the disk portion;
The flow passage forming body is configured by welding together an upper flow passage forming body and a lower flow passage forming body, both of which are formed in a flat plate shape,
the lower flow passage forming body has a groove communicating with the through hole on a surface facing the upper flow passage forming body,
3. The culture medium circulating device according to claim 2, wherein the flow path is constituted by the through-holes and the grooves. Further comprising a cell culture insert in the well;
the sealing lid seals both the well and the cell culture insert;
4. The culture medium circulating device according to claim 1 , wherein the flow paths are formed independently for each of the wells and the cell culture insert . 2. The culture medium circulating device according to claim 1 , wherein the sealing lid is made of a plate material fixed to the upper edge of the well so as to cover the well . 6. The culture medium circulating device according to claim 5 , wherein the flow path is constituted by a pipe that penetrates through the sealing lid and is fixed thereto, and a tube that is connected to an upper end of the pipe .