JPWO2020003504A1 - Perfusion culture system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a perfusion culture system which is compact and suitable for eliminating the risk of failure by realizing labor saving of a worker and ensuring cell quality. SOLUTION: A perfusion culture system A10 has a closed culture container unit 1 having a fluid inlet 131 and a fluid outlet 141, a culture solution container 2 capable of accommodating a culture solution, and the inside thereof is adjusted to a negative pressure. The vacuum vessel unit 3 and the first flow path 41, one end of which can communicate with the inside of the culture vessel unit 1 via the fluid inflow port 131, and the other end of which can communicate with the inside of the culture solution container 2. A second flow path 42 is provided, one end of which can communicate with the inside of the culture vessel unit 1 via the fluid outlet 141, and the other end of which can communicate with the inside of the vacuum vessel unit 3.

Description

The present invention relates to a system for culturing cells and biological tissues, and more particularly to a perfusion culture system capable of supplying a new culture solution to a closed culture vessel.

The evolution of cell culture technology is indispensable for the practical application of regenerative medicine. For example, a cell culture system that combines a robot that performs operations for culturing cells and a plurality of devices has been developed for CPC (Cell Processing Center) for mass production of cell preparations (for example). , Patent Document 1).

In addition to CPC for production purposes, new cell culture techniques are required. For example, in a medical facility where transplant surgery is performed, for the purpose of maintaining and culturing the delivered cells, it is a small scale that can be installed in the field, and even people who do not have specialized cell culture skills such as doctors and medical staff can easily operate it. A capable culture system is required. It is important to be able to ensure the quality of cells while considering labor saving so as not to interfere with medical work. In addition, even in general small and medium-sized laboratories that conduct research and development of various cell products, there is a need to easily and effortlessly perform cell culture with existing equipment and to ensure cell quality. high.

On the other hand, a perfusion culture device has been developed that eliminates the trouble of manually exchanging the culture medium (medium), automatically supplies the culture solution at a constant speed, and at the same time extracts the same amount of the culture solution (for example, patent). See Reference 2). However, since these perfusion culture systems basically include a pump drive unit and a control controller with a power source for exchanging the culture medium and controlling the culture medium, the equipment and facilities have become large. Further, even in the case of a miniaturized device, there is a problem that there is a risk of failure of the electric system by bringing it into an incubator which is a harsh and humid environment.

International Publication WO2016 / 170623 JP-A-2017-79633

The present invention has been conceived under such circumstances, and provides a perfusion culture system that is compact and suitable for eliminating the risk of failure by realizing labor saving of workers and ensuring cell quality. The main task is to do.

In order to solve the above problems, the following technical means are adopted in the present invention.

The perfusion culture system provided by the present invention includes a closed culture container unit having a fluid inlet and a fluid outlet, a culture solution container capable of accommodating a culture solution, and a vacuum container whose inside is adjusted to a negative pressure. The unit and the first flow path, one end of which can communicate with the inside of the culture vessel unit via the fluid inlet, and the other end of which can communicate with the inside of the culture solution container, and one end of the fluid flow. It is provided with a second flow path that can communicate with the inside of the culture vessel via an outlet and the other end can communicate with the inside of the vacuum vessel unit.

In a preferred embodiment, at least one of the first flow path and the second flow path is provided with a constricted portion.

In a preferred embodiment, the constriction is a columnar body through which micropores penetrate.

In a preferred embodiment, the constriction is a microchannel tip.

In a preferred embodiment, the culture vessel unit and the culture solution container are connected to each other in a closed state by being provided in the first flow path, and the culture container unit and the culture solution container are closed to each other. The separable first connecting means and the second flow path are provided so that the culture vessel unit and the vacuum vessel unit are communicated and connected in a closed state, and the culture vessel unit and the vacuum vessel unit are sealed to each other. A separable second connection means is provided.

In a preferred embodiment, a plurality of the culture solution containers are provided, and the first flow path is a first main trunk road communicating with the culture container unit, and a plurality of first channels, each of which communicates with the culture solution container. A branch road is included, and the end of the first main trunk road and the end of each of the plurality of first branch roads are connected, and any one of the plurality of first branch roads and the first main trunk are connected. A first switching means capable of switching the flow path so as to communicate with the road is provided.

In a preferred embodiment, a plurality of the vacuum vessel units are provided, and the second flow path has a second main trunk road communicating with the culture vessel unit and a plurality of second branches each communicating with the vacuum vessel unit. Including a road, the end of the second main road and the end of each of the plurality of second branch roads are connected, and any one of the plurality of second branch roads and the second main road are connected to each other. A second switching means capable of switching the flow path is provided so that the flow paths can be switched.

In a preferred embodiment, the vacuum vessel unit comprises a bottomed tube having an open end and a stopper that closes the open end, and is attached to the other end of the second flow path. The stopper is provided with a puncture needle capable of puncturing.

In a preferred embodiment, the culture container unit is attached to a bottomed tubular container body having an opening at one end in the first direction, and the inside of the container body by closing the opening. The attachment has an attachment that seals a space, and the attachment has a fluid inlet that leads to the outside at one end, a fluid introduction flow path that leads to the inner space that is sealed at the other end, and the fluid that leads to the outside at one end. It is provided with an outflow port and a fluid outlet flow path leading to the inner space in which the other end is sealed.

In a preferred embodiment, the attachment has a protrusion that fits inside the container body and projects towards the other side of the first direction.

In a preferred embodiment, the culture medium is hermetically sealed, flexible and variable in volume.

In a preferred embodiment, the second flow path allows the flow of fluid from the culture vessel unit side to the vacuum vessel unit side, and allows the fluid from the vacuum vessel unit side to the culture vessel unit side. A check valve is provided to block the flow of water.

In a preferred embodiment, the inner surface of the culture vessel unit includes a flat cell culture surface, and the cell culture surface is subjected to surface treatment for improving cell adhesion.

Other features and advantages of the present invention will become more apparent with the detailed description given below with reference to the accompanying drawings.

It is a schematic block diagram which shows 1st Embodiment of the perfusion culture system which concerns on this invention. It is an exploded view of the perfusion culture system shown in FIG. It is a top view which shows an example of a culture container unit. It is sectional drawing which follows the IV-IV line of FIG. It is sectional drawing which follows the VV line of FIG. It is an exploded perspective view of the culture vessel unit shown in FIG. It is the same cross-sectional view as FIG. 5, and shows the state which contained the contents in the culture vessel unit. It is a schematic block diagram which shows the 2nd Embodiment of the perfusion culture system which concerns on this invention. It is a top view which shows another example of a constriction part.

Hereinafter, preferred embodiments of the present invention will be specifically described with reference to the drawings.

1 to 6 show a first embodiment of the perfusion culture system according to the present invention. The perfusion culture system A10 of the present embodiment includes a culture vessel unit 1, a culture solution container 2, a vacuum vessel unit 3, a first flow path 41, a second flow path 42, a first connection means 51, and the like. A second connecting means 52 and a narrowed portion 6 are provided. Although the details will be described later, the perfusion culture system A10 supplies the new culture solution contained in the culture solution container 2 to the culture container unit 1 and collects the old culture solution in the culture container unit 1 into the vacuum container unit 3. It is for doing.

As shown in FIGS. 1 and 4 to 6, in the present embodiment, the culture container unit 1 includes a container body 11 and an attachment 12. As the container body 11, for example, an existing open culture container such as a dish (Petri dish) or a well plate is used, and in the present embodiment, the case where the container body 11 is a dish is shown. The container body 11 has a bottomed cylindrical shape having an opening 110 at the upper end (one side end in the first direction), and has a side plate 111 and a bottom plate 112.

The upper surface of the bottom plate 112 is a flat cell culture surface for culturing cells. The cell culture surface (upper surface of the bottom plate 112) is appropriately subjected to surface treatment for improving cell adhesion, if necessary. Examples of the surface treatment include corona discharge treatment and plasma treatment (hydrophilization treatment).

The attachment 12 is attached to the container body 11 and is for closing the opening 110 of the container body 11 and sealing the inner space of the container body 11. In this embodiment, the attachment 12 includes a base member 12A and a seal member 12B.

The base member 12A has a bottom wall 121, a cylindrical side wall 122 extending upright from the peripheral edge of the bottom wall 121, and a flange 123. The bottom wall 121 fits inside the side plate 111 in the container body 11 at a distance from the side plate 111, and protrudes downward (the other side in the first direction). The bottom wall 121 corresponds to the protruding portion referred to in the present invention. The flange 123 extends radially outward from the upper end of the side wall 122 and has an annular shape. The base member 12A having such a configuration overlaps all the openings 110 of the container body 11 in a plan view.

The container body 11 and the base member 12A are made of, for example, a translucent or transparent hard plastic material. As the plastic material, for example, polystyrene, methylpentene, polycarbonate, cycloolefin polymer, cycloolefin copolymer, and other materials having transparency are preferably used, but the plastic material is not limited thereto.

In the present embodiment, at least the bottom plate 112 of the container body 11 and the bottom wall 121 of the base member 12A are transparent. As a result, the bottom plate 112 and the bottom wall 121 are portions where the inner space of the container body 11 can be visually recognized from the outside.

The seal member 12B is, for example, a rubber molded product, and has a cylindrical portion 125 extending vertically, a flange portion 126, and an annular protrusion 127, as shown in FIGS. 4 and 5. The seal member 12B is interposed between the side plate 111 of the container body 11 and the side wall 122 or the flange 123 of the base member 12A. The cylindrical portion 125 is fitted onto the side wall 122 of the base member 12A. The natural inner diameter of the cylindrical portion 125 is slightly smaller than the outer diameter of the side wall 122, whereby the cylindrical portion 125 (seal member 12B) is pressed and held by the side wall 122 (base member 12A). The flange portion 126 extends radially outward from the upper end of the cylindrical portion 125 and has an annular shape. The flange portion 126 overlaps with the flange 123 of the base member 12A in a plan view. The annular protrusion 127 projects radially outward from the outer circumference of the cylindrical portion 125, and is in pressure contact with the inner peripheral surface of the side plate 111 when the attachment 12 is attached to the container body 11. With such a configuration, the base member 12A and the seal member 12B (attachment 12) seal the inner space of the container body 11.

The sealing member 12B is made of a flexible and elastic material. Examples of the material constituting the seal member 12B include silicone rubber, natural rubber, urethane rubber, and elastomer resin. Although details will be described later, considering the contact between the sealing member 12B and the contents (for example, a culture solution), the material of the sealing member 12B is more preferably a medical grade silicone rubber having no cytotoxicity and biocompatibility. .. Regarding the hardness of the seal member 12B, for example, the rubber hardness is preferably about 20 to 40 degrees.

In the present embodiment, the attachment 12 has a fluid introduction flow path 13 and a fluid lead-out flow path 14. The fluid introduction flow path 13 is a flow path for introducing a fluid such as a culture solution from the outside to the inside of the culture container unit 1, and is composed of holes and grooves formed in the seal member 12B in the present embodiment. More specifically, the fluid introduction flow path 13 has one end as a fluid inflow port 131 leading to the outside and the other end communicating with the inner space of the container body 11 which is sealed.

The fluid lead-out flow path 14 is a flow path for leading out the fluid from the inside to the outside of the culture vessel unit 1. In the present embodiment, the fluid lead-out flow path 14 is provided on the opposite side of the fluid introduction flow path 13 with the center of the attachment 12 interposed therebetween in a plan view. The fluid lead-out flow path 14 is composed of holes and grooves formed in the seal member 12B, one end of which is a fluid outlet 141 leading to the outside, and the other end of which leads to the inner space of the container body 11 which is sealed. ing.

The culture solution container 2 shown in FIG. 1 is a closed container body for accommodating a replacement culture solution. The culture solution container 2 is provided with a lead-out port 21 for leading out the contents. In the present embodiment, the culture solution container 2 is preferably configured to be flexible and changeable in volume. Examples of such a culture solution container 2 include a bellows-shaped container, a film-shaped container, a syringe, a laminated peeling bottle, and the like. In this embodiment, the case where the culture solution container 2 is a bellows-shaped container is shown.

The inside of the vacuum vessel unit 3 is adjusted to a negative pressure state, and the culture solution or the like inside the culture vessel unit 1 is recovered. In the present embodiment, the vacuum container unit 3 includes a pipe body 31 and a plug 32. The tubular body 31 has a bottomed cylindrical shape having an open end 311 at the tip. The plug 32 closes the opening end 311, and the inside of the sealed tube 31 is adjusted to a negative pressure state. A part of the stopper 32 is made of a rubber material capable of puncturing, for example, a puncture needle. As the vacuum container unit 3 having such a configuration, for example, a general-purpose blood collection tube can be used.

The first flow path 41 is a fluid flow path that connects the culture container unit 1 and the culture solution container 2, and is composed of, for example, a flexible tube. One end of the first flow path 41 can communicate with the inside of the culture vessel unit 1 via the fluid inflow port 131. The other end of the first flow path 41 can communicate with the inside of the culture solution container 2 via the outlet 21.

The first connection means 51 shown in FIG. 1 is provided in the first flow path 41, and connects the culture container unit 1 and the culture solution container 2 in a closed state. Further, the first connecting means 51 can separate the culture container unit 1 and the culture solution container 2 in a sealed state (see FIG. 2). The first connecting means 51 having such a configuration is realized by, for example, a luer lock connection structure having a female luer connector 511 and a male luer connector 512. In the configuration shown in FIGS. 1 and 2, the first flow path 41 is a partial flow path 411 connected to the culture vessel unit 1 (fluid inflow port 131) and a partial flow path connected to the culture solution container 2 (outlet port 21). It is composed of a road 412. Then, for example, a female luer connector 511 is provided at the tip of the partial flow path 411, and a male luer connector 512 is provided at the tip of the partial flow path 412.

The second flow path 42 is a fluid flow path connecting between the culture container unit 1 and the vacuum container unit 3, and is composed of, for example, a flexible tube. One end of the second flow path 42 can communicate with the inside of the culture vessel unit 1 via the fluid outlet 141. A puncture needle 43 capable of puncturing the stopper 32 of the vacuum container unit 3 is attached to the other end of the second flow path 42. The other end of the second flow path 42 can communicate with the inside of the vacuum container unit 3 (tube 31) via the puncture needle 43.

The second connecting means 52 shown in FIG. 1 is provided in the second flow path 42, and connects the culture container unit 1 and the vacuum container unit 3 in a closed state. Further, the second connecting means 52 can separate the culture vessel unit 1 and the vacuum vessel unit 3 from each other in a sealed state (see FIG. 2). The second connecting means 52 having such a configuration is realized by, for example, a luer lock connection structure having a female luer connector 521 and a male luer connector 522. In the configuration shown in FIGS. 1 and 2, the second flow path 42 is composed of a partial flow path 421 connected to the culture vessel unit 1 (fluid outlet 141) and a partial flow path 422 connected to the vacuum vessel unit 3. To. Then, for example, a female luer connector 521 is provided at the tip of the partial flow path 421, and a male luer connector 522 is provided at the tip of the partial flow path 422.

The puncture needle 43 described above is attached to the end of the partial flow path 422. Further, the puncture needle 43 is surrounded by a tubular holder 44 to prevent danger. The inner diameter of the holder 44 is larger than the outer diameter of the pipe body 31, and as can be understood from FIGS. 1 and 2, the pipe body 31 (vacuum container unit 3) is inserted into the holder 44 to insert the plug 32. It is possible to safely puncture the puncture needle 43.

The narrowed portion 6 is provided in at least one of the first flow path 41 and the second flow path 42. The narrowed portion 6 has a portion where the cross section of the flow path is narrowed, and the flow rate of the fluid passing through the narrowed portion 6 per unit time (hereinafter, simply referred to as “flow rate”) is adjusted to be desired. Is what you do.

In the present embodiment, the narrowed portion 6 is composed of a columnar body having micropores penetrating in the longitudinal direction. A fluid (for example, about 0.02 to 0.05 mm) that passes through the narrowed portion 6 by appropriately selecting the pore diameter (for example, about 0.02 to 0.05 mm) and the length of the micropore (dimension in the longitudinal direction: about 5 to 20 mm). The flow rate of the culture solution, etc.) can be adjusted. The narrowed portion 6 having such a configuration can be manufactured by extrusion molding or firing using a ceramic material such as zirconia, and a fine through hole having a high aspect ratio and high accuracy can be obtained. As shown in FIGS. 1 and 2, in the present embodiment, the narrowed portion 6 is provided in the partial flow path 422 of the second flow path 42. The narrowed portion 6 may be provided with a tapered portion or a large diameter portion at its end so as to facilitate connection with the partial flow path 422 (flexible tube) and make it difficult to separate.

Next, the usage and operation of the perfusion culture system A10 will be described.

The perfusion culture system A10 accommodates cultured cells and a culture solution in a closed culture container unit 1 configured by using an open culture container (container body 11), and the culture solution is maintained in a cell culture state. Used to make a replacement. The cultured cells and the culture solution contained in the container body 11 (culture container unit 1) are not particularly limited.

In cell culture using the perfusion culture system A10, first, the cells to be cultured are seeded in the container body 11 (culture container unit 1), and the inside of the culture container unit 1 is filled with the culture solution. The cells may be seeded by removing the attachment 12 from the container body 11 and directly injecting the culture solution containing the cells into the container body 11, but it is also possible to use the perfusion culture system A10. ..

Although detailed illustration will be omitted, when cells are seeded using the perfusion culture system A10, a culture medium container 2 containing a culture medium containing the cells to be cultured is prepared, and the culture medium container 2 is used. Connect to the culture vessel unit 1. Further, a syringe is prepared in place of the vacuum vessel unit 3, and the syringe is connected to the culture vessel unit 1. Then, by pulling the piston to make the inside of the syringe a negative pressure, the culture solution (including cells) in the culture solution container 2 is guided into the culture container unit 1. FIG. 7 shows a state in which the inside of the container body 11 (culture container unit 1) is filled with the culture solution M1. After filling the inside of the culture vessel unit 1 with the culture solution, the connection between the culture vessel unit 1 and the culture solution container 2 and the connection between the culture vessel unit 1 and the syringe are separated from each other.

The cells in the culture container unit 1 (container body 11) adhere to the cell culture surface (upper surface of the bottom plate 112) over time, and proliferation begins. When the upper surface of the bottom plate 112 is subjected to the above-mentioned surface treatment for improving cell adhesion, the cells adhere to the cell culture surface more reliably. Therefore, such a configuration is preferable for the proper progress of cell culture in the culture vessel unit 1.

In the present embodiment, the bottom plate 112 of the container body 11 and the bottom wall 121 of the base member 12A are transparent. Thereby, it is possible to observe the situation inside the culture vessel unit 1 from either the upper portion or the lower portion of the culture vessel unit 1 through the transparent bottom wall 121 or the bottom plate 112. With such a configuration, irradiation light can be transmitted regardless of whether the light source is above or below, and cells can be observed using various types of microscopes such as an optical microscope and a phase-contrast microscope.

In the present embodiment, the bottom wall 121 of the base member 12A projects downward. By arranging the bottom wall 121 at a low position in this way, the culture solution in the culture vessel unit 1 comes into contact with the surface of the bottom wall 121. As a result, the liquid surface of the culture solution becomes flat due to the effect of underwater eyeglasses, diffuse reflection of irradiation light is suppressed, and the effect of facilitating focusing of the microscope can be expected.

With respect to the culture solution in the culture vessel unit 1, nutrients decrease as the culture cells proliferate, and waste products excreted from the culture cells increase. Therefore, it is necessary to replace the culture solution in the culture vessel unit 1 as appropriate. When exchanging the culture solution in the culture container unit 1 during cell culture, a partial flow having a culture solution container 2 containing the replacement culture solution, a vacuum container unit 3, a constriction portion 6, and a puncture needle 43. Road 422 and is prepared (see FIG. 2). Then, the first connecting means 51 between the culture vessel unit 1 and the culture solution container 2 and the second connecting means 52 between the culture vessel unit 1 and the vacuum vessel unit 3 are separated from each other (FIG. FIG. 2) to the connected state as shown in FIG.

Here, the culture container unit 1 and the culture solution container 2 communicate with each other via the first flow path 41, and the culture container unit 1 and the vacuum container unit 3 communicate with each other via the second flow path 42. Then, the old culture solution in the culture vessel unit 1 moves into the vacuum vessel unit 3 via the second flow path 42 by the suction force of the vacuum vessel unit 3 (tube 31) whose inside is in a negative pressure state. The new culture solution in the culture solution container 2 moves into the culture container unit 1 via the first flow path 41. When the exchange of the culture solution in the culture vessel unit 1 is completed, the first connecting means 51 and the second connecting means 52 are returned from the connected state to the separated state.

According to the perfusion culture system A10 of the present embodiment, it is possible to exchange the culture solution in the culture vessel unit 1 without using an electric device such as a controller or a power source. Therefore, the perfusion culture system A10 can be miniaturized without worrying about failure.

In the perfusion culture system A10, the culture solution in the culture vessel unit 1 can be exchanged by a one-touch operation such as changing the first connection means 51 and the second connection means 52 from the separated state to the connected state as described above. In addition, the culture solution can be exchanged in a short time by selecting the pore size and length of the constricted portion 6, or the culture solution is slowly supplied to the culture system (culture container unit 1) in a constant supply amount over several days. At the same time, it is also possible to withdraw the same amount of the culture solution from the culture vessel unit 1 (so-called perfusion culture). Further, the culture solution exchange work can be performed while the culture vessel unit 1 is closed, and the risk of contamination can be avoided. Therefore, according to the perfusion culture system A10, it is possible to reduce the operational burden on the operator and ensure the quality of cells when exchanging the culture solution.

As described above, when the culture vessel unit 1 and the vacuum vessel unit 3 are communicated with each other, the old culture in the culture vessel unit 1 is performed by the suction force of the vacuum vessel unit 3 (tube 31) whose inside is in a negative pressure state. The liquid moves into the vacuum vessel unit 3. Here, by appropriately selecting the degree of vacuum (pressure) inside the tube 31 in a negative pressure state, the suction amount of the culture solution into the tube 31 (for example, for 5 ml, 10 ml, etc.) is determined in advance. It is possible to keep it.

A narrowed portion 6 is provided in the second flow path 42 connecting the culture container unit 1 and the vacuum container unit 3. According to the configuration including the narrowed portion 6, the flow rate of the culture solution exchange can be adjusted. By providing such a narrowed portion 6, the exchange time of the culture solution can be appropriately set within a range of, for example, about 1 to 48 hours depending on the exchange amount of the culture solution. The flow rate suitable for exchanging the culture solution varies depending on the type of cultured cells, the amount of the exchanged culture solution, and the like, but according to the present embodiment, the flow rate at the time of exchanging the culture solution can be appropriately adjusted.

On the other hand, if the flow rate at the time of exchanging the culture solution is not changed when the exchange of the culture solution is repeated, it is possible not to provide the luer lock connection portion. In this case, as the second flow path 42, one end is connected to the culture vessel unit 1 (fluid outlet 141) and the puncture needle 43 is attached to the other end to prepare a series, and the puncture needle 43 is plugged 32. The exchange of the culture solution may be started by puncturing the cells. Although not shown, a check valve may be provided in the second flow path 42. The check valve allows the flow of fluid from the culture vessel unit 1 side to the vacuum vessel unit 3 side, and blocks the flow of fluid from the vacuum vessel unit 3 side to the culture vessel unit 1 side. According to such a configuration, it is possible to prevent the inconvenience that the old culture solution moved from the culture vessel unit 1 to the vacuum vessel unit 3 returns to the culture vessel unit 1 side.

The culture solution container 2 is flexible and can change in volume. According to such a configuration, as the culture solution in the culture solution container 2 moves to the culture container unit 1, the internal volume of the culture solution container 2 decreases, and the culture solution exchange is smoothly performed.

The attachment 12 has a bottom wall 121 (protruding portion), and the bottom wall 121 projects downward (the other side in the first direction) inside the container body 11. According to such a configuration, the storage space of the culture solution in the container main body 11 is reduced, and the amount of the culture solution used during cell culture can be reduced.

In the present embodiment, when the culture solution is exchanged, the old culture solution in the culture vessel unit 1 is collected inside the vacuum vessel unit 3 (tube 31). According to such a configuration, sampling of the collected culture solution can be easily performed.

After the cell culture using the perfusion culture system A10 is completed, the cultured cells in the culture vessel unit 1 are collected. The cultured cells may be collected by removing the attachment 12 from the container body 11 and directly peeling off the cultured cells in the container body 11, but it is also possible to use the perfusion culture system A10.

Although detailed illustration will be omitted, when collecting cultured cells using the perfusion culture system A10, a culture solution container 2 containing a buffer solution is prepared, and the culture solution container 2 is referred to as a culture vessel unit 1. hook up. Further, a syringe is prepared in place of the vacuum vessel unit 3, and the syringe is connected to the culture vessel unit 1. Then, by pulling the piston to make the inside of the syringe negative pressure, the buffer solution in the culture solution container 2 is guided into the culture container unit 1, and the old culture solution in the culture container unit 1 is washed away. Next, a culture solution container 2 containing an enzyme (for example, trypsin) for peeling the cultured cells from the container body 11 is prepared, and the culture solution container 2 is connected to the culture container unit 1. Further, the vacuum vessel unit 3 is prepared, and the vacuum vessel unit 3 is connected to the culture vessel unit 1. Then, the enzyme in the culture solution container 2 moves into the culture vessel unit 1, and the cultured cells in the culture vessel unit 1 are peeled off by the enzyme treatment. The detached cultured cells are collected in the vacuum vessel unit 3 via the second flow path 42. When the cultured cells are collected using the vacuum vessel unit 3 in this way, a mesh-shaped filter may be interposed in the partial flow path 422 on the vacuum vessel unit 3 side instead of the narrowed portion 6 described above. .. The filter removes unnecessary substances such as foreign substances and aggregated cells, and functions as a strainer.

FIG. 8 shows a second embodiment of the perfusion culture system according to the present invention. The perfusion culture system A20 of the present embodiment includes a culture vessel unit 1, a plurality of (three in the present embodiment) culture solution containers 2, a plurality of (three in the present embodiment) vacuum vessel units 3, and a first. It includes one flow path 41, a second flow path 42, a plurality of narrowed portions 6, and switching valves 7 and 8. In the drawings after FIG. 8, the same or similar elements as those in the above embodiment are designated by the same reference numerals, and the description thereof will be omitted as appropriate.

The perfusion culture system A20 of the present embodiment is significantly different from the above-mentioned perfusion culture system A10 in that it includes a plurality of culture solution containers 2 and a plurality of vacuum container units 3. Along with this, the connection structure between the culture solution container 2 or the vacuum container unit 3 and the culture container unit 1 is different from that of the above embodiment. The configurations of the culture vessel unit 1, the culture solution container 2, and the vacuum vessel unit 3 are substantially the same as those in the above embodiment.

In the present embodiment, the first flow path 41 includes a first main trunk road 415 and a plurality of (three in this embodiment) first branch roads 416. One end of the first main trunk road 415 communicates with the culture vessel unit 1 (fluid inflow port 131), and the other end of the first main trunk road 415 is connected to the switching valve 7. The plurality of first branch paths 416 correspond to each of the plurality of culture solution accommodating bodies 2, one end communicating with the culture solution accommodating body 2 (outlet port 21), and the other end being a switching valve 7. It is connected to the.

The switching valve 7 selectively communicates one of the plurality of culture solution containers 2 with the culture container unit 1. Although detailed illustration will be omitted, the switching valve 7 has a valve structure capable of switching the flow path so that any one of the plurality of first branch roads 416 and the first main trunk road 415 communicate with each other. It has an operation unit for manually switching the flow path. The switching valve 7 is an example of the first switching means referred to in the present invention. The switching valve 7 may have a structure capable of not communicating with any of the plurality of first branch paths 416.

In the present embodiment, the second flow path 42 includes a second main trunk road 425 and a plurality of (three in the present embodiment) second branch roads 426. One end of the second main trunk road 425 communicates with the culture vessel unit 1 (fluid outlet 141), and the other end of the second main trunk road 425 is connected to the switching valve 8. The plurality of second branch paths 426 correspond to each of the plurality of vacuum vessel units 3, one end communicating with the vacuum vessel unit 3 and the other end being connected to the switching valve 8. In the present embodiment, the plurality of narrowed portions 6 are provided corresponding to each of the plurality of second branch paths 426.

The switching valve 8 selectively communicates one of the plurality of vacuum vessel units 3 with the culture vessel unit 1. Although detailed illustration will be omitted, the switching valve 8 has a valve structure capable of switching the flow path so that any one of the plurality of second branch roads 426 and the second main road 425 communicate with each other. It has an operation unit for manually switching the flow path. The switching valve 8 is an example of the second switching means referred to in the present invention. The switching valve 8 may have a structure capable of not communicating with any of the plurality of second branch paths 426. In the configuration example shown in FIG. 8, the plurality of first branch paths 416 and the plurality of second branch paths 426 are each separable in the middle of the flow path. The first branch road 416 and the second branch road 426 are provided with connection portions 47 and 48 having, for example, a luer lock connection structure. With such a configuration, by separating the connecting portion 47, the culture container unit 1 and the culture solution container 2 can be separated from each other in a sealed state. Further, by separating the connecting portion 48, the culture vessel unit 1 and the vacuum vessel unit 3 can be separated from each other in a sealed state.

Next, the operation of the perfusion culture system A20 will be described.

The perfusion culture system A20 accommodates cultured cells and a culture solution inside a closed culture container unit 1 configured by using an open culture container (container body 11), and the culture solution is maintained in a cell culture state. Used to make a replacement.

The perfusion culture system A20 of the present embodiment includes a plurality of culture solution containers 2 and a plurality of vacuum container units 3. When the contents of the culture solution container 2 and the vacuum container unit 3 are relatively small, a plurality of culture solution containers 2 and a plurality of vacuum container units 3 are connected to the culture container unit 1 in advance, and the switching valve 7, By appropriately switching the flow path according to No. 8, the culture solution can be exchanged seamlessly.

The perfusion culture system A20 is also useful when it is necessary to exchange different types of cultures. That is, if different types of culture solutions are stored in the plurality of culture solution containers 2, different types of culture solutions can be smoothly supplied to the culture vessel unit 1 simply by appropriately switching the flow path with the switching valve 7. Can be done.

The perfusion culture system A20 is also useful when the flow rate of the culture solution exchange is changed during the culture solution exchange work. In this case, if a plurality of constricted portions 6 having different flow rates of the culture solution passing through are prepared, the flow rate of the culture solution exchange can be quickly changed only by appropriately switching the flow path with the switching valve 8. It is possible.

Although the specific embodiment of the present invention has been described above, the present invention is not limited to this, and various modifications can be made without departing from the idea of the invention. The specific configuration of each part of the perfusion culture system according to the present invention can be freely redesigned.

In the above embodiment, the case where the narrowed portion 6 is composed of a columnar body having micropores has been described, but the present invention is not limited to this. The narrowed portion may be configured by, for example, the microchannel chip shown in FIG. The microchannel chip 6A shown in the figure is configured by, for example, laminating a hydrophilic polyethylene terephthalate (PET) film on a plate material of dimethylpolysiloxane (PDMS) in which a groove-shaped microchannel 61 is formed by laser processing. To. Although the microchannel 61 meanders in FIG. 9, the flow rate at the time of exchanging the culture solution can be appropriately adjusted by appropriately setting the width and length of the microchannel 61.

Further, in the above embodiment, the case where the culture solution container 2 is hermetically sealed, flexible, and volume-changeable is described, but the present invention is not limited to this. The culture solution container may have a fixed volume structure. In the case of a fixed-volume culture solution container, the culture solution container is provided with small holes, and the small holes are provided with a filter (for example, a hepa filter) that captures fine particles in the air. According to such a configuration, when the old culture solution in the culture container unit moves into the vacuum container unit by the suction force of the vacuum container unit at the time of exchanging the culture solution, the inside of the culture solution is cleaned through the above filter. As air is drawn in, new culture medium in the culture medium container moves into the culture vessel unit.

A10, A20 Perfusion culture system M1 Culture solution 1 Culture container unit 11 Container body 110 Opening 111 Side plate 112 Bottom plate 12 Attachment 12A Base member 12B Seal member 121 Bottom wall (protruding part)
122 Side wall 123 Flange 125 Cylindrical part 126 Flange part 127 Circular protrusion 13 Fluid introduction flow path 131 Fluid inflow port 14 Fluid outflow flow path 141 Fluid outflow port 2 Culture solution container 21 Outlet port 3 Vacuum vessel unit 31 Tube body 311 Open end 32 Plug 41 1st flow path 411 Partial flow path 412 Partial flow path 415 1st main road 416 1st branch road 42 2nd flow path 421 Partial flow path 422 Partial flow path 425 2nd main road 426 2nd branch road 43 Penetration needle 44 Holder 47 Connection part 48 Connection part 51 First connection means 511 Female lure connector 512 Male lure connector 52 Second connection means 521 Female lure connector 522 Male lure connector 6 Constriction part 6A Micro flow path tip 61 Fine flow path 7 Switching Valve (first switching means)
8 Switching valve (second switching means)

Claims (13)

A closed culture vessel unit with a fluid inlet and fluid outlet,
A culture solution container capable of containing the culture solution and
A vacuum vessel unit whose inside is adjusted to negative pressure,
A first flow path in which one end can communicate with the inside of the culture vessel unit via the fluid inlet and the other end can communicate with the inside of the culture solution container.
A second flow path in which one end can communicate with the inside of the culture vessel unit via the fluid outlet and the other end can communicate with the inside of the vacuum vessel unit.
A perfusion culture system. The perfusion culture system according to claim 1, wherein a stenotic portion is provided in at least one of the first flow path and the second flow path. The perfusion culture system according to claim 2, wherein the narrowed portion is a columnar body through which micropores penetrate. The perfusion culture system according to claim 2, wherein the narrowed portion is a microchannel chip. A first connection means provided in the first flow path, capable of communicating and connecting the culture vessel unit and the culture solution container in a closed state, and separating the culture container unit and the culture solution container from each other in a closed state. When,
A second connecting means provided in the second flow path, in which the culture vessel unit and the vacuum vessel unit are communicated and connected in a closed state, and the culture vessel unit and the vacuum vessel unit can be separated from each other in a sealed state. The perfusion culture system according to any one of claims 1 to 4, further comprising. A plurality of the above-mentioned culture medium containers are provided.
The first flow path includes a first main trunk path communicating with the culture vessel unit, and a plurality of first branch paths communicating with each of the culture medium containing bodies.
The end of the first main trunk road and the end of each of the plurality of first branch roads are connected, and the flow is such that any one of the plurality of first branch roads and the first main trunk road communicate with each other. The perfusion culture system according to any one of claims 1 to 4, further comprising a first switching means capable of switching routes. Equipped with multiple vacuum container units
The second flow path includes a second main trunk path communicating with the culture vessel unit and a plurality of second branch passages each communicating with the vacuum vessel unit.
The end of the second main trunk road and the end of each of the plurality of second main trunk roads are connected, and the flow is such that any one of the plurality of second main trunk roads and the second main trunk road communicate with each other. The perfusion culture system according to any one of claims 1 to 4, and 6, further comprising a second switching means capable of switching routes. The vacuum vessel unit includes a bottomed tube having an open end and a stopper for closing the open end.
The perfusion culture system according to any one of claims 1 to 7, further comprising a puncture needle attached to the other end of the second flow path and capable of puncturing the plug. The culture container unit includes a bottomed tubular container body having an opening at one end in the first direction, and a bottomed tubular container body.
It has an attachment that is attached to the container body and closes the opening to seal the inner space of the container body.
The attachment includes a fluid inlet that leads to the outside at one end, a fluid introduction flow path that leads to the inner space that is sealed at the other end, a fluid outlet that leads to the outside at one end, and the other end that is sealed. The perfusion culture system according to any one of claims 1 to 8, further comprising a fluid lead-out flow path leading to an inner space. The perfusion culture system according to claim 9, wherein the attachment fits inside the container body and has a protrusion that protrudes toward the other side in the first direction. The perfusion culture system according to any one of claims 1 to 10, wherein the culture solution container is hermetically sealed, flexible, and variable in volume. A check valve that allows the flow of fluid from the culture vessel unit side to the vacuum vessel unit side and blocks the flow of fluid from the vacuum vessel unit side to the culture vessel unit side in the second flow path. The perfusion culture system according to any one of claims 1 to 11, wherein a valve is provided. The inner surface of the culture vessel unit includes a flat cell culture surface.
The perfusion culture system according to any one of claims 1 to 12, wherein the cell culture surface is subjected to a surface treatment for improving cell adhesion.

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