JP7262042B2 - ADAPTER, CLOSED CHAMBER, CELL CULTURE DEVICE, AND ADAPTER MANUFACTURING METHOD - Google Patents

Description

TECHNICAL FIELD The present invention relates to an adapter, a closed chamber, a cell culture apparatus, and a method of manufacturing an adapter that can easily control the flow of fluid in a container.

In recent years, as regenerative medicine using stem cells, for example, treatment of liver cirrhosis, blood diseases, and myocardial infarction, construction of blood vessels, regeneration of bones and corneas, and securing of skin for transplantation are considered. In regenerative medicine, target cells or organs are grown from stem cells or the like in a culture dish and transplanted to humans. Recently, angiogenesis has been performed from bone marrow-derived stem cells, which has been successful in treating angina pectoris, myocardial infarction, and the like.

Here, in the conventional cell culture apparatus, the culture medium in the culture dish is periodically changed to proliferate the cultured cells. In this cell culture device, cells are greatly stimulated when the culture solution is replaced, and waste products are discharged into the culture solution due to the metabolic activity of the cells, which stresses and damages the cells. I had a problem with it.

Therefore, Patent Literature 1 discloses a method in which cell culture is performed by feeding and draining the culture solution without replacing the culture solution.

Utility Model Registration No. 3196673

However, in the case of perfusion culture, in which the culture solution is fed and drained without replacing the culture solution, the flow in the culture vessel is uneven due to the effects of the meniscus, liquid volume, and wall resistance. Become. Here, cells differ in the amount of gene expression and adhesion area depending on the strength of shear stress, and there is a possibility that the properties of cells will vary depending on the strength of shear stress. Therefore, if the flow in the culture vessel is non-uniform, different shear stresses are generated in the culture vessel, which poses a problem in obtaining reproducibility of changes over time in living cell culture.

Therefore, in Patent Document 1, an adapter (drop lid) is fitted in the culture vessel to eliminate the effects of the meniscus and liquid volume, but the flow in the culture vessel is still uneven.

On the other hand, in fields such as cell culture, there are cases where it is desired to observe cell chemotaxis and growth rate depending on the concentration distribution. In this case, it is preferable that the flow rate can be controlled so that the concentration distribution per unit time of the culture solution in the cell vessel has a desired concentration distribution.

The present invention has been made in view of the above, and an object of the present invention is to provide an adapter, a closed chamber, a cell culture device, and a method of manufacturing the adapter that can easily control the flow of fluid in a container. .

In order to solve the above-described problems and achieve the object, an adapter according to the present invention provides a container dish having a bottomed cylindrical body with an open top and a cross-sectional shape corresponding to the internal cross-sectional shape of the cylindrical body. for forming a closed space by being fitted into the inside of the container plate from the top of the container plate, wherein the sealed space is defined by the bottom surface of the adapter and the bottom and side surfaces of the container plate. A space formed between and through which a fluid flows, comprising an injection communication port for injecting the fluid from the outside and a discharge communication port disposed at a position facing the injection communication port for discharging the fluid from the sealed space to the outside. is formed on the peripheral edge, and the bottom surface has a flow velocity adjusting portion formed with a continuous or discontinuous concave or convex portion from the outlet opening of the injection communication port to the discharge communication port. characterized by

Further, in the adapter according to the present invention, in the above invention, the flow rate adjusting portion has a predetermined length from the outlet opening of the injection communication port toward the discharge communication port.

Moreover, the adapter according to the present invention is characterized in that, in the above invention, a plurality of the flow velocity adjusting portions are formed.

Moreover, the adapter according to the present invention is characterized in that, in the above invention, the flow rate adjusting portion is a combination of one or more concave portions or convex portions having different lengths, widths or depths.

Further, in the adapter according to the present invention, in the above-described invention, the flow rate adjusting portion has a width and/or a depth that changes from the inlet communication port toward the discharge communication port.

Further, in the adapter according to the present invention, in the above invention, the flow rate adjusting portion is a linear concave portion extending from the outlet opening of the injection communication port on the bottom surface to both sides along the side surface of the cylindrical body for a predetermined length. It is characterized by

Further, in the adapter according to the present invention, in the above invention, the flow rate adjusting portion is a linear concave portion or a linear convex portion formed on the bottom surface between the outlet opening of the injection communication port and the discharge communication port. characterized by being

Further, in the adapter according to the present invention, in the above invention, a flange that engages with the upper end portion of the cylindrical body and defines the depth of the sealed space is provided on the upper outer peripheral edge of the lid main body portion. It is characterized by

Moreover, the adapter according to the present invention is characterized in that, in the above invention, the container plate and the adapter are cylindrical.

Further, in the adapter according to the present invention, in the above invention, the lid main body is a bottomed elastic cylinder formed of an elastic member and having a shape that rubs against the inner side surface of the container plate, A rigid member press-fitted into the elastic cylinder is provided, and the lid main body is fixed to the container tray by pressing against the inner side surface of the container tray by the rigid member.

In the adapter according to the present invention, in the above invention, a rigid injection nozzle and a rigid discharge nozzle are press-fitted into the injection communication port and the discharge communication port, respectively.

Further, the adapter according to the present invention is characterized in that, in the above invention, the rigid member is a rigid ring.

Further, the adapter according to the present invention is characterized in that, in the above invention, the lid main body is made of PDMS or silicon rubber, and the rigid member is made of acrylic resin.

A closed chamber according to the present invention includes a container plate that is a bottomed cylindrical body with an open top, and an adapter according to any one of the above inventions that corresponds to the container plate. characterized by

Further, in the cell culture apparatus according to the present invention, cultured cells are arranged in a closed space within the closed chamber according to the above invention, and a liquid necessary for growing or maintaining the cultured cells is supplied, and the liquid inside the closed chamber is A cell culture apparatus for continuously culturing the cultured cells by discharging the liquid, comprising: a reservoir tank for storing the liquid supplied to the closed space; and a waste liquid tank for storing the liquid discharged from the closed space. and a pump for supplying the liquid in the reservoir tank to the closed space.

Moreover, the cell culture apparatus according to the present invention is characterized in that, in the above invention, a bubble removing mechanism is provided between the pump and the closed chamber.

Further, in the method of manufacturing an adapter according to the present invention, a container plate having a bottomed cylindrical body with an open top has a cross-sectional shape corresponding to the internal cross-sectional shape of the cylindrical body, and the upper part of the container plate A manufacturing method of an adapter for forming a closed space by being fitted into the inside of the container plate from the base, wherein the diameter is larger and deeper than the manufacturing container plate having an outer shape corresponding to the inner shape of the container plate. A double-faced tape corresponding to the size of the bottom surface of the manufacturing container plate is attached to the top surface of the bottom of the mold forming container plate, and an injection communication port outlet for injecting fluid from the outside is provided on the bottom surface of the manufacturing container plate. for generating a flow velocity adjusting portion that forms a continuous or discontinuous concave or convex portion with respect to a discharge communication port that is disposed at a position facing the injection communication port from the opening and that discharges the fluid from the sealed space to the outside. an affixing step of affixing a flow velocity adjusting portion forming member and then affixing the bottom surface of the production container plate onto the double-sided tape; A mold forming step of injecting a mold forming resin to the depth of the manufacturing container plate and hardening it, removing the manufacturing container plate while leaving the flow rate adjusting part forming member on the double-sided tape, and removing the mold a bottom portion forming step of injecting an adapter forming resin to a predetermined depth onto the flow rate adjusting portion forming member surrounded by the forming resin and curing the resin to form a bottom portion of the adapter; After disposing the members and further injecting the adapter-forming resin between the cylindrical member and the mold-forming resin to the depth of the mold-forming container dish and hardening, the cylindrical member and the an adapter main body producing step of removing the mold forming container plate on which the mold forming resin is formed to form an adapter main body, forming the injection communication port and the discharge communication port in the adapter main body to form the adapter; and an adapter generating step for generating.

According to the present invention, since the bottom surface of the lid main body has the flow velocity adjusting portion formed with a continuous or discontinuous concave portion or convex portion from the outlet opening of the injection communication port to the discharge communication port on the bottom surface, the container can be The flow of fluid within can be easily controlled.

FIG. 1 is a diagram showing a schematic configuration of a cell culture device including an adapter that is an embodiment of the present invention. FIG. 2 is a cross-sectional view of the adapter shown in FIG. 1 taken along the line AA. 3 is a cross-sectional view of the adapter shown in FIG. 3 taken along the line BB. 4 is an exploded cross-sectional view of the culture vessel including the adapter shown in FIG. 1. FIG. FIG. 5 is an explanatory diagram showing the state of cultured cells in a closed space. 6 is a plan view showing the configuration of the adapter shown in FIG. 1. FIG. FIG. 7 is a plan view showing the configuration of an adapter that is Modification 1. FIG. FIG. 8 is a diagram showing a linear flow velocity distribution in the Y direction along line CC in FIGS. 6 and 7. FIG. FIG. 9 is a diagram showing the linear flow velocity distribution in the Y direction along line DD in FIGS. 6 and 7. FIG. FIG. 10 is a diagram showing temporal changes in the ratio (exchange ratio) of the concentration at the wall surface to the concentration at the center of the adapter shown in FIG. FIG. 11 is a process diagram showing the manufacturing process of the adapter. FIG. 12 is a plan view showing the configuration of an adapter that is Modification 2. FIG. FIG. 13 is a plan view showing the configuration of an adapter that is Modification 3. FIG. FIG. 14 is a plan view of an adapter that is Modification 4. FIG. FIG. 15 is a cross-sectional view of the adapter shown in FIG. 14 along line EE. FIG. 16 is a plan view of an adapter that is Modification 5. FIG. FIG. 17 is a cross-sectional view of the adapter shown in FIG. 16 taken along line DD. FIG. 18 is a diagram showing a linear flow velocity distribution in the Y direction along line CC in FIGS. 14 and 16. FIG. FIG. 19 is a diagram showing the linear flow velocity distribution in the Y direction along line DD in FIGS. 14 and 16. FIG. FIG. 20 is a cross-sectional view of the adapter of modification 6 taken along line DD. FIG. 21 is a cross-sectional view of the adapter of modification 7 taken along line DD. FIG. 22 is a cross-sectional view of the adapter of modification 7 taken along line EE. FIG. 23 is a cross-sectional view of the adapter of modification 8 taken along line EE. FIG. 24 is a plan view of an adapter of modification 10. FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments for carrying out the present invention will be described with reference to the accompanying drawings.

<Overall composition>
FIG. 1 is a diagram showing a schematic configuration of a cell culture device 100 including an adapter 11 that is an embodiment of the present invention. FIG. 2 is a cross-sectional view of the adapter 11 shown in FIG. 1 taken along line AA. 3 is a cross-sectional view of the adapter 11 shown in FIG. 3 taken along the line BB. Furthermore, FIG. 4 is an exploded cross-sectional view of the culture vessel 10 including the adapter 11 shown in FIG.

As shown in FIGS. 1 to 4, the cell culture apparatus 100 has a culture container 10 as a closed chamber in which a closed space E1 is formed, a reservoir tank 30 and a waste liquid tank 33. The culture container 10 forms an enclosed space E1, and the cultured cells 20 are placed in the enclosed space E1. The reservoir tank 30 supplies the liquid S, which is a fluid necessary for growing or maintaining the cultured cells 20, to the closed space E1 through the pipe LA. A pump 31 and a bubble removing mechanism 32 are arranged on the pipe LA in order from the reservoir tank 30 side. The pump 31 is, for example, a peristaltic pump, and sends a minute flow rate of the liquid S in the reservoir tank 30 into the sealed space E1. The air bubble removal mechanism 32 removes air bubbles that may be generated by the pumping of the pump 31 . For removing the bubbles, for example, a PVDF (Poly Vinylidene Di-Fluoride) film is used. The liquid S discharged from the sealed space E1 is stored in the waste liquid tank 33 via the pipe LB.

<Configuration of culture vessel>
The culture container 10 has a container plate 1 which is a bottomed cylindrical body with an open top, and a cross-sectional shape corresponding to the internal cross-sectional shape of the cylindrical body of the container plate 1 with respect to the container plate 1. and an adapter 11 for fitting into the inside of the container plate 1 from the top to form a closed space E1. The container plate 1 and the adapter 11 are bottomed cylindrical bodies. The closed space E1 is formed between the bottom surface of the adapter 11 and the bottom and side surfaces of the container plate 1. As shown in FIG. A cultured cell 20 is placed in the closed space E1. The closed space E1 is filled with a liquid S necessary for growing or maintaining the cultured cells 20 and perfused.

The adapter 11 has a lid main body 2, an injection nozzle 3a, a discharge nozzle 3b and a rigid member 4. As shown in FIG. The lid main body 2 is made of an elastic member such as silicone rubber containing PDMS (Polydimethylsiloxane). The lid main body 2 has an injection communication port 2a for injecting the liquid S to be perfused from the outside (pipe LA) into the sealed space E1, and is arranged at a position facing the injection communication port 2a to allow the liquid S to flow from the sealed space E1 to the outside. A discharge communication port 2b for discharging to (pipe LB) is formed in the periphery.

As shown in FIGS. 2 and 3, the bottom surface of the lid main body 2 is provided with a groove extending from the outlet opening of the injection communication port 2a to both sides along the side surface of the container plate 1 for a predetermined length. Adjustment parts 21 and 22 are formed. As will be described later, the flow velocity adjusting portions 21 and 22, which are grooves, are continuous or discontinuous concave or convex portions on the bottom surface of the lid main body 2 from the outlet opening of the inlet communication port 2a to the discharge communication port 2b. An example.

The injection nozzle 3a, the discharge nozzle 3b, and the rigid member 4 are made of rigid material such as acrylic resin, for example. As shown in FIGS. 1 and 4, the injection nozzle 3a and the discharge nozzle 3b are press-fitted into the injection communication port 2a and the discharge communication port 2b, respectively. This makes it possible to easily and reliably attach the pipe LA to the injection nozzle 3a and the pipe LB to the discharge nozzle 3b.

The rigid member 4 is a rigid ring, as shown in FIGS. As shown in FIG. 4, the diameter d1 of the rigid member 4 is larger than the diameter d2 of the internal space 2d. The lid main body 2 is fixed to the container plate 1 by . In addition, the rigid member 4 may be provided with a reinforcing member in the rigid ring.

A flange 2c is formed on the outer peripheral edge of the upper portion of the lid main body 2. The flange 2c engages with the upper end portion of the cylindrical body of the container plate 1 and defines the depth of the sealed space E1.

The injection nozzle 3 a and the discharge nozzle 3 b are attached by press fitting before the lid main body 2 is press fitted into the container tray 1 or after the lid main body 2 is press fitted into the container tray 1 .

As shown in FIG. 5, by perfusing the sealed space E1 with the liquid S, the cultured cells 20 arranged in the sealed space E1 are supplied with the nutrients and additive factors in the liquid S. , waste products and physiologically active substances are discharged and collected in the waste liquid tank 33 as waste liquid.

In this embodiment, the meniscus in the closed space E1 is removed by fixing the bottom surface of the lid main body 2, and the liquid volume becomes constant. Moreover, the wall resistance is reduced by the flow velocity adjusting portions 21 and 22, which are grooves provided on the bottom surface of the lid body portion 2, and the liquid S supplied from the outlet opening of the injection communication port 2a is directed toward the discharge communication port 2b side. , and flows toward the discharge communication port 2b side at a uniform flow rate. As a result, the strength of the shear stress becomes uniform within the closed space E1, the difference in gene expression level and adhesion area becomes small, and the properties of the cultured cells 20 are less likely to vary. This makes it possible to obtain reproducibility of changes over time in cultured cells.

In the present embodiment, the lid main body 2 is transparent, has a thin central bottom surface, and the rigid member 4 is also ring-shaped. Visibility is good, and changes over time can be observed.

Further, when the lid main body 2 is made of PDMS, it has good gas permeability, especially oxygen permeability, so that oxygen can be supplied to living cultured cells.

<Equalization control of flow>
6 is a plan view showing the configuration of the adapter 11 shown in FIG. 1. FIG. Moreover, FIG. 7 is a plan view showing the configuration of an adapter 11' that is Modification 1. As shown in FIG. The adapter 11 extends from both sides (+X direction and -X direction) of the injection communication port 2a along the side surface of the container plate 1, that is, the lid main body 2, with respect to the center of the lid main body 2 at 90 degrees. Existing flow velocity adjusting portions 21 and 22 are formed. The flow velocity adjusting portions 21 and 22 have a rectangular cross section and a depth of 0.5 mm. The liquid volume (depth) of the sealed space E1 is 1 mm. The adapters 11 and 11' correspond to the container dish 1 with a culture dish having a diameter of 35 mm.

On the other hand, the adapter 11' further forms flow velocity adjusting portions 23 and 24, which are grooves, inside the flow velocity adjusting portions 21 and 22, respectively. Other configurations are the same as those of the adapter 11 .

FIG. 8 is a diagram showing a linear flow velocity distribution in the Y direction along line CC in FIGS. 6 and 7. FIG. In FIG. 8, the solid line curve L1 shows the linear flow velocity distribution when the flow velocity adjusting parts 21 to 24 are not provided, and the rough broken line curve L2 shows the case where the grooved flow velocity adjusting parts 21 and 22 shown in FIG. 6 are provided. , and a fine broken line curve L3 shows the linear flow velocity distribution when the flow velocity adjusting portions 21 to 24, which are grooves shown in FIG. 7, are provided. FIG. 9 is a diagram showing the linear flow velocity distribution in the Y direction along line DD in FIGS. 6 and 7. In FIG. In FIG. 9 as well, the solid curve L11 shows the linear flow velocity distribution when the flow velocity adjusting parts 21 to 24 are not provided, and the rough dashed curve L12 shows the flow velocity adjusting parts 21 and 22, which are the grooves shown in FIG. A fine dashed line curve L13 indicates the linear flow velocity distribution when the flow velocity adjusting portions 21 to 24, which are grooves shown in FIG. 7, are provided.

As shown in FIGS. 8 and 9, the linear flow velocity increases at the center (near "0" in the X direction) when the flow velocity adjusting section is not provided. In particular, in FIG. 8, the linear flow velocity at the center when no flow velocity adjusting section is provided is about twice the linear flow velocity when the flow velocity adjusting sections 21 and 22 or the flow velocity adjusting sections 21 to 14 are provided. current velocity. On the other hand, when the flow velocity adjusting units 21 and 22 or the flow velocity adjusting units 21 to 14 are provided, the linear flow velocity has a flat linear flow velocity distribution in the central portion. In addition, the linear flow velocity distribution when the four flow velocity adjusting units 21 to 14 are provided has a flatter linear flow velocity distribution in the central portion than the linear flow velocity distribution when the two flow velocity adjusting units 21 and 22 are provided. It is Therefore, when the flow velocity adjusting units 21 and 22 or the flow velocity adjusting units 21 to 14 are provided, the difference in strength of shear stress in the closed space E1 becomes small.

<Temporal equalization>
FIG. 10 is a diagram showing the change over time of CP2/CP1, which is the ratio (exchange ratio) of the concentration CP2 at the wall position P2 to the concentration CP1 at the center position P1 of the adapter 11 shown in FIG. The exchange ratio is obtained by observing the concentration of the liquid S that is diluted by injecting a methylene blue solution into water as the liquid S to be supplied. That is, the lighter the methylene blue color of the liquid S, the better the mixing with the liquid S in the closed space E1. The color density of this methylene blue is obtained based on the color image.

In FIG. 10, the solid line indicates the change over time of the adapter 11 provided with the flow rate adjusting portions 21 and 22, and the broken line indicates the change over time of the drop lid without the groove. The horizontal axis indicates time (hour), which indicates changes over time for 12 hours.

As shown in FIG. 10, the replacement ratio CP2/CP1 does not converge to 1 when the flow rate adjusting unit is not provided, but when the flow rate adjusting units 21 and 22 are provided, the replacement ratio CP2/CP1 after 12 hours is , converging to 1. That is, the density CP1 at the center position P1 of the adapter 11 and the density CP2 at the wall position P2 are the same, and there is no density difference in the X direction. As a result, regardless of the position in the closed space E1, the cultured cells 20 grow uniformly.

In addition, when the flow path adjusting portion is provided at the bottom of the container plate 1, a special and dedicated container plate (dish) must be produced. Since the channel adjustment part is provided on the adapter 11 side, a general-purpose commercial dish can be used repeatedly, and a closed chamber having various desired channel adjustment parts can be easily formed for the same general-purpose commercial dish. Obtainable.

Further, in the above-described embodiment, the ring-shaped rigid member 4 is used to press the side surface to reliably form the closed space E1. The sealed space E1 may be formed using a member that does. Furthermore, although the rigid member 4 is made of acrylic resin as an example, it is not limited to this and may be made of metal or the like. Moreover, the rigid member 4 may be an expansion member that expands after being attached to the lid body 2 as long as it can press the side surface. The expansion member may be any material that expands upon application of a physical quantity such as heat or ultraviolet rays. Moreover, the rigid member 4 may not be used, and the lid main body portion 2 itself may be a member that expands.

Furthermore, in the above embodiment, the pump 31 is provided between the reservoir tank 30 and the culture container 10, and the liquid in the reservoir tank 30 is pressure-fed to the culture container 10 side and flowed to the waste liquid tank 33 side. However, not limited to this, a pump may be provided between the culture container 10 and the waste liquid tank 33 to suck the liquid in the culture container 10, thereby introducing the liquid in the reservoir tank 30 into the culture container 10. good too.

<Manufacturing method of the adapter>
Here, a simple manufacturing method of the adapter 11 will be described. 11A and 11B are process diagrams showing the manufacturing process of the adapter 52 corresponding to the adapter 11. FIG. First, as shown in FIG. 11A, a manufacturing container plate 41 having an outer shape corresponding to the inner shape of the container plate 1 is prepared. Also, a die-forming container plate 42 having a larger radius and a deeper depth than the manufacturing container plate 41 is prepared. The manufacturing container plate 41 is, for example, a 35 mm culture plate, and the mold forming container plate 42 is, for example, a 60 mm culture plate.

Then, a double-faced tape 44 corresponding to the size of the bottom surface of the manufacturing container plate 41 is attached to the bottom upper surface of the mold forming container plate 42, and the outlet opening of the injection communication port 2a is attached to the bottom surface of the manufacturing container plate 41. A flow velocity adjusting part forming member 43 for forming a flow velocity adjusting part that forms a continuous or discontinuous concave or convex part is attached to the discharge communication port 2b, and then the bottom surface of the manufacturing container plate 41 is attached with double-sided tape. 44 (FIG. 11(a)).

After that, between the manufacturing container plate 41 and the mold forming container plate 42, the mold forming resin Q1 is injected to the depth of the manufacturing container plate 41 and hardened to form the mold forming resin 45. (FIG. 11(b)). The mold forming resin Q1 is, for example, an epoxy resin.

Thereafter, the manufacturing container plate 41 is removed while the flow rate adjusting portion forming member 43 remains on the double-sided tape 44, and the adapter forming resin Q2 is applied onto the flow rate adjusting portion forming member 43 surrounded by the mold forming resin 45. is injected to a predetermined depth and hardened to form the adapter forming resin 46 which is the bottom of the adapter 52 (FIG. 11(c)). The adapter-forming resin Q2 is PDMS, for example.

After that, the cylindrical member 47 is placed on the center of the bottom of the adapter 52 (adaptor-forming resin 46). This cylindrical member 47 is, for example, a cylinder made of aluminum. The height of the cylindrical member 47 is higher than the mold forming container plate 42 . Then, between the cylindrical member 47 and the mold forming resin 45, the adapter forming resin Q2 is injected to the depth of the mold forming container plate 42 and hardened to form the adapter forming resin 48 (FIG. 11(d)).

After that, the cylindrical member 47 and the mold forming container plate 42 on which the mold forming resin 45 is formed are removed to generate the adapter forming resin 48 which is the main body of the adapter. An inlet 2a and an outlet 2b for the resin 48 are formed to produce the adapter 52 (FIG. 11(e)).

According to this adapter manufacturing method, the adapter 52 can be manufactured simply by using a ready-made culture dish.

<Modification 2>
In the above embodiment and Modification 1, the container plate 1 and the adapters 11, 11' are cylindrical, but in Modification 2, as shown in FIG. there is That is, the adapter 111 of Modified Example 2 has the lid body portion 102 provided with a wall surface forming a rectangular portion between two semi-cylindrical shapes. Also in Modification Example 2, the bottom surface of the lid body portion 102 is provided with flow velocity adjusting portions 21 and 22 of grooves on both sides of the injection communication port 2a.

<Modification 3>
In the above embodiment and modification 1, the container plate 1 and the adapters 11 and 11' are cylindrical. It has a cylindrical shape. In Modification 3, the bottom surface of the lid main body 202 is provided with flow velocity adjusting portions 221 and 222 of grooves corresponding to the flow velocity adjusting portions 21 and 22 on both sides of the injection communication port 2a. Unlike the flow rate adjusting sections 21 and 22, the flow rate adjusting sections 221 and 222 do not have an arc shape, but have a shape along the elliptical wall surface.

It should be noted that the shape of the lid main body can be any shape corresponding to the container plate 1, without being limited to the second and third modifications. For example, the container plate 1 may have a square tubular shape. In this case, the groove may be formed along the wall surface, may be arcuate, or may have a radius of curvature that changes in the X direction.

<Modification 4>
FIG. 14 is a plan view of an adapter 311 that is Modification 4. FIG. 15 is a cross-sectional view of the adapter 311 shown in FIG. 14 taken along line EE. As shown in FIGS. 14 and 15, the flow rate adjusting portion 25 of the adapter 311 is a groove that passes through the central position P1 of the adapter 311 and is linearly formed between the inlet communication port 2a and the discharge communication port 2b. be. This makes it possible to further increase the linear flow velocity in the central portion in the Y direction.

<Modification 5>
FIG. 16 is a plan view of an adapter 411 that is Modified Example 5. FIG. 17 is a cross-sectional view of the adapter 411 shown in FIG. 16 taken along line DD. As shown in FIGS. 16 and 17, the flow rate adjusting portion 26 of the adapter 411 is linearly formed between the inlet communication port 2a and the discharge communication port 2b through the central position P1 of the adapter 411. It is a protrusion. As a result, the linear flow velocity in the central portion in the Y direction can be further reduced.

FIG. 18 is a diagram showing a linear flow velocity distribution in the Y direction along line CC in FIGS. 14 and 16. FIG. FIG. 19 is a diagram showing the linear flow velocity distribution in the Y direction along line DD in FIGS. 14 and 16. In FIG. As shown in FIGS. 18 and 19, when the grooved flow velocity adjusting portion 25 is provided, a flow velocity distribution in which the central linear flow velocity is further increased can be obtained (curves L4 and L14). Further, as shown in FIGS. 18 and 19, when the linear protrusion flow velocity adjusting portion 26 is provided, it is possible to obtain a flow velocity distribution that slows down the central linear flow velocity (curves L5 and L15).

<Modification 6>
FIG. 20 is a cross-sectional view of an adapter 511 of modification 6 taken along line DD. As shown in FIG. 20, in Modification 6, a flow velocity adjusting section 26 is provided in addition to the flow velocity adjusting sections 21 and 22 .

<Modification 7>
FIG. 21 is a cross-sectional view of an adapter 611 of modification 7 taken along line DD. As shown in FIG. 21, in Modification 6, in addition to the flow rate adjusting sections 21 and 22, a flow rate adjusting section 25' corresponding to the flow rate adjusting section 25 is provided. The width of the flow velocity adjusting portion 25 ′ is formed to be larger than the width of the flow velocity adjusting portion 25 . Thereby, the central linear flow velocity can be further increased. As shown in FIG. 21, the concave portion or convex portion of the flow velocity adjusting portion may have a relative relationship with respect to the reference surface SS of the bottom portion of the adapter. are formed.

<Modification 8>
FIG. 22 is a cross-sectional view of the adapter 711 of Modification 7 taken along line EE. As shown in FIG. 22, in Modification 8, the depth of the groove of the flow velocity adjusting portion 25 is made shallower in the Y direction. In this manner, the depth and height of the flow velocity adjusting portion may be changed in the longitudinal direction. Thereby, the linear flow velocity distribution in the Y direction can be controlled.

<Modification 9>
FIG. 23 is a cross-sectional view of an adapter 811 of modification 8 taken along line EE. As shown in FIG. 23, in the flow velocity adjusting portion 25b of Modified Example 9, the grooves of the flow velocity adjusting portion 25 are not formed continuously in the Y direction, but are formed discontinuously. The flow velocity can also be adjusted by forming such discontinuous recesses or protrusions.

<Modification 10>
FIG. 24 is a plan view of an adapter 911 of Modification 10. FIG. As shown in FIG. 24, the flow velocity adjusting portions 21' and 22' of Modification 10 are arranged at positions corresponding to the flow velocity adjusting portions 21 and 22, and the groove width is increased as the distance from the injection communication port 2a increases. I'm trying In this manner, the flow velocity can be adjusted even by changing the width of the concave portion or the convex portion in the longitudinal direction (extending direction) of the flow velocity adjusting portion.

In addition, in the above-described embodiment and modifications 1 to 10, the cross-sectional shape of the flow velocity adjusting portion is all rectangular. There may be.

Further, the width of the flow rate adjusting portion is the same as the diameter of the outlet opening of the injection communication port 2a, but the width is not limited to this, and may be widened or narrowed.

Furthermore, in the above-described embodiment and modified example 1, the container plate 1 of the 35 mm dish has been described as an example, but the present embodiment is not limited to this and can be applied to various container plates. For example, it can be applied to each well of a 60 mm dish, a 100 mm dish, and a 96 well plate.

Further, in the above embodiment and Modifications 1 to 10, the closed chamber having the adapter used in the cell culture apparatus 100 is described as a closed chamber, but the size of the closed chamber is arbitrary. It can also be used for observation of

It should be noted that each configuration illustrated in the above-described embodiment and modified examples is functionally schematic, and does not necessarily need to be physically configured as illustrated. That is, the form of distribution/integration of each device is not limited to the illustrated one, and all or part of them can be functionally or physically distributed/integrated in arbitrary units according to various loads and usage conditions. Can be configured. Moreover, each component of the above-described embodiment or modified example can be appropriately combined.

1 container plate 2, 102, 202 lid main body 2a injection communication port 2b discharge communication port 2c flange 2d internal space 3a injection nozzle 3b discharge nozzle 4 rigid member 10 culture vessel 11, 11', 52, 111, 211, 311, 411 . Manufacturing container plate 42 Mold forming container plate 43 Flow rate adjusting part forming member 44 Double-sided tape 45 Mold forming resin 46, 48 Adapter forming resin 47 Cylindrical member 100 Cell culture device CP1, CP2 Concentration d1, d2 Diameter E1 Sealing Space LA, LB Piping P1, P2 Position S Liquid

Claims (14)

It has a cross-sectional shape corresponding to the internal cross-sectional shape of the cylindrical body, and is fitted into the container plate from the top to form a sealed space. an adapter for forming,
The sealed space is a space formed between the bottom surface of the adapter and the bottom surface and side surfaces of the container plate through which fluid flows,
An injection communication port for injecting the fluid from the outside and a discharge communication port disposed at a position facing the injection communication port and for discharging the fluid from the sealed space to the outside are formed in the peripheral edge, and the injection communication port is formed on the bottom surface. A lid main body portion having a flow velocity adjusting portion forming continuous or discontinuous recesses and/ or protrusions from the outlet opening of the communication port to the discharge communication port ,
The continuous or discontinuous concave portion of the flow rate adjusting portion is a linear concave portion extending from the outlet opening of the injection communication port on the bottom surface to both sides for a predetermined length along the side surface of the cylindrical body,
The continuous or discontinuous convex portion of the flow rate adjusting portion is a linear convex portion that passes through the center of the lid main body on the bottom surface and is formed linearly between the inlet and discharge ports. An adapter characterized by: 2. The adapter according to claim 1 , wherein a plurality of said flow velocity adjusting parts are formed. 3. The adapter according to claim 2 , wherein the flow rate adjusting portion is one or more combinations of recesses and/ or protrusions of different lengths, widths or depths. 4. The adapter according to any one of claims 1 to 3 , wherein the flow rate adjusting portion varies in width and/or depth from the injection communication port toward the discharge communication port. 5. A flange according to any one of claims 1 to 4 , characterized in that an upper outer peripheral edge of the lid main body is provided with a flange that engages with the upper end of the cylindrical body and defines the depth of the closed space. one described adapter. The adapter according to any one of claims 1 to 5 , wherein the container plate and the adapter are cylindrical. The lid main body is a bottomed elastic cylinder formed of an elastic member and having a shape that rubs against the inner side surface of the container plate,
Having a rigid member press-fitted into the elastic cylinder,
The adapter according to any one of claims 1 to 6 , wherein the lid main body is fixed to the container tray by pressing against the inner side surface of the container tray by the rigid member. 8. The adapter according to claim 7 , wherein a rigid injection nozzle and a rigid discharge nozzle are press-fitted into said inlet connection and said outlet connection, respectively. 9. Adapter according to claim 7 or 8 , characterized in that the rigid member is a rigid ring. The lid body is made of PDMS or silicon rubber,
The adapter according to any one of claims 7 to 9 , wherein the rigid member is acrylic resin. a container plate having an open top and a bottomed cylindrical body;
an adapter according to any one of claims 1 to 10 , corresponding to the container plate;
A closed chamber comprising: 12. The cultured cells are arranged in a closed space within the closed chamber according to claim 11 , and a liquid necessary for growing or maintaining the cultured cells is supplied, and the liquid in the closed chamber is discharged to continue the cultured cells. A cell culture device for culturing by
a reservoir tank for storing the liquid to be supplied to the sealed space;
a waste liquid tank for storing the liquid discharged from the closed space;
a pump that supplies the liquid in the reservoir tank to the closed space;
A cell culture device comprising: 13. The cell culture apparatus according to claim 12 , wherein a bubble removing mechanism is provided between said pump and said closed chamber. It has a cross-sectional shape corresponding to the internal cross-sectional shape of the cylindrical body, and is fitted into the container plate from the top to form a sealed space. A method of manufacturing an adapter for forming, comprising:
Both sides corresponding to the size of the bottom surface of the manufacturing container plate on the upper surface of the bottom of the mold forming container plate having a radius larger and deeper than the manufacturing container plate having an external shape corresponding to the internal shape of the container plate A tape is attached to the bottom surface of the manufacturing container plate, and the outlet opening of the injection communication port for injecting the fluid from the outside is arranged at a position facing the injection communication port, and the fluid is discharged to the outside from the sealed space. A flow velocity adjusting part forming member for forming a flow velocity adjusting part that forms a continuous or discontinuous concave or convex part is attached to the discharge communication port, and then the bottom surface of the manufacturing container plate is placed on the double-sided tape. a pasting step of pasting;
a mold forming step of injecting a mold forming resin between the manufacturing container plate and the mold forming container plate to the depth of the manufacturing container plate and curing the resin;
The manufacturing container plate is removed while the flow rate adjusting portion forming member remains on the double-sided tape, and the adapter forming resin is applied to a predetermined depth on the flow rate adjusting portion forming member surrounded by the mold forming resin. a bottom forming step of injecting and curing to form the bottom of the adapter;
A cylindrical member was placed on the center of the bottom of the adapter, and the adapter-forming resin was injected between the cylindrical member and the mold-forming resin to the depth of the mold-forming container plate and cured. an adapter main body producing step of producing an adapter main body by removing the cylindrical member and the mold forming container plate on which the mold forming resin is formed;
an adapter producing step of producing the adapter by forming the injection communication port and the discharge communication port in the adapter main body;
A method of manufacturing an adapter, comprising:

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