Perfusion culture unit

Abstract

To create agitation well so as to be applicable even to the culture of suspension-type cells while taking advantage of the features of perfusion culture.SOLUTION: When a lid 25 is placed on a container 3, a sealed culture container C is created in which a cell culture insert 37 housed in the container 3 and a concave surface portion 19 communicate at the top and bottom via a membrane of a bottom portion 37a. A hooked portion 53a is tilted from the axial direction of a peripheral wall portion 37b of the cell culture insert 37, and a culture medium flows out from the tip opening with force, creating a liquid flow in a culture medium pool (P) in which the hooked portion 53a is immersed. This liquid flow becomes an agitating flow and agitates the culture medium in the pool (P). Furthermore, since the culture medium is sucked in and collected from the concave surface portion 19 on the lower side of the membrane, cells floating on the upper side of the membrane are not sucked in when they are collected from a perfusion OUT hole 21.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] The present invention relates to a perfusion culture unit for perfusing and culturing cells and the like.

Background Art

[0002] There are various methods for culturing cells and the like. In the conventional static culture method, depending on the size of the specimen, nutrients and oxygen may not reach the cells deep within the specimen, resulting in necrosis. Adjacent specimens may stick together and tend to grow larger, and the risk of necrosis is always present. In contrast, as described in Patent Document 1, a bioreactor equipped with a stirring blade has been proposed. However, there is a risk that the cells may be damaged when the stirring blade moves and collides with the cells. Also, as described in Patent Document 2, a shaker has been proposed. A container is placed on a shaking table, and the shaking table is reciprocated. In currently commercially available types, the size of the shaking table is large, and installing it in a CO2 incubator takes up space. Furthermore, in the bioreactor type, the stirring blade moves within the container, and in the shaker type, the container itself vibrates. Therefore, not only is the replacement of the culture solution troublesome, but there are also concerns about adverse effects on cells and the like due to the renewal of the culture environment by the replacement of the culture solution.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] On the one hand, in recent perfusion culture methods, since the culture medium can be continuously updated, not only can the culture conditions be maintained constant for a long time and the adverse effects on cells and the like due to medium replacement be reduced, but it is also considered that nutrients and oxygen can be spread to the cells deep in the specimen by taking advantage of the perfusion momentum. However, in perfusion culture, usually, a pump system is used to continuously supply the culture medium to the culture system and continuously remove the culture supernatant outside the system. Therefore, there is a risk that floating-type cells may be sucked into a pipe or the like and discharged outside the system.

[0005] The present invention has been made by paying attention to the above-mentioned conventional problems, and by devising the structure of the perfusion culture unit, while taking advantage of the features of the perfusion culture method, it is applicable to the culture of floating-type cells, and an object of the present invention is to provide a novel and useful perfusion culture unit.

Means for Solving the Problems

[0006] The present invention has been made to solve the above problems, and it has a container with an upper opening and a concave surface portion provided on the inner bottom surface with the inner side recessed downward with respect to the outer side, a perfusion OUT hole provided through the concave surface portion of the container, a perfusion IN pipe inserted from the opening, and a stirring flow generation induction mechanism. When a cell culture insert is accommodated in the container in a state where the concave surface portion is closed at the bottom of the membrane of the cell culture insert and perfusion culture is performed, the cell culture insert and the concave surface portion become a culture container connected in the vertical direction through the bottom of the membrane. During perfusion culture, a stirring flow is generated in the culture medium accumulated in the cell culture insert by the stirring flow generation induction mechanism. It is a perfusion culture unit characterized by this.

[0007] Preferably, the opening of the perfusion IN pipe is immersed in the culture medium in a state inclined with respect to the axial direction of the inner surface of the cell culture insert, thereby inducing the generation of a stirring flow. Preferably, the inner surface of the cell culture insert is an inner peripheral surface, and the perfusion IN pipe opens in a tangential direction along the inner peripheral surface to induce the generation of a vortex flow.

[0008] Preferably, it is provided with a lid portion that closes the opening above the cell culture insert, and the perfusion IN pipe is passed through a perfusion IN hole provided through the lid portion. Preferably, an air release hole is provided through the lid portion, and a capped tube is inserted into the air release hole. Preferably, the lower surface of the lid portion is provided with a step such that the inner portion is lower than the annular outer portion. When the outer portion contacts the container and the opening of the container is closed, the inner portion contacts the cell culture insert and the opening of the cell culture insert is closed. Preferably, it is provided with an adapter for fixing the lid portion in a closed state with respect to the container. When the lid portion is fixed to the container by the adapter, the cell culture insert is also fixed to the lid portion inside the container, so that the unit can be installed in various orientations as a whole. Preferably, it is designed to be able to accommodate a commercially available cell culture insert in the container.

Effect of the Invention

[0009] According to the perfusion culture unit of the present invention, by devising the structure of the unit, it is applicable to the culture of floating-type cells while taking advantage of the continuous supply of the culture solution and the momentum of the liquid, which are characteristics of the perfusion culture method. Moreover, it can be designed to be of a size that can be installed in a CO2 incubator, and furthermore, it can be unitized by using a commercially available and inexpensive cell culture insert as the main part.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The perfusion culture unit 1 according to an embodiment of the present invention will be described with reference to the drawings. As shown in FIGS. 1 to 7, the container 3 of the perfusion culture unit 1 is made of polycarbonate and has a rigid specification. The container 3 is a substantially cylindrical container with a bottom and an opening at the top. The outer surface 7 of the side portion 5 has a pair of outer planes 7a, 7a facing each other across the axis, and the portion between the outer planes 7a and 7a forms an outer peripheral surface 7b. The inner surface 9 of the side portion 5 is coaxial with the outer peripheral surface 7b, but a step is provided coaxially on the opening side, and the inner peripheral edge of the inner peripheral surface 9b on the opening side is located outward with respect to the inner peripheral edge of the inner peripheral surface 9a continuous from the bottom 13 side. Also, the annular step surface 9c is a plane perpendicular to the axial direction. The annular upper end surface 11 of the side portion 5 forming the opening edge of the container 3 is a plane perpendicular to the axial direction.

[0012] The outer surface of the bottom 13 is flush and continuous with the outer surface 7 of the side portion 5. Also, with respect to the outer bottom surface 15 of the plane on the grounding side of the bottom 13, the inner bottom surface 17 that appears circular has an outer peripheral side that is a parallel plane, but a concave portion 19 that is recessed coaxially in a circular shape from the inner bottom surface 17 is formed at the center. The concave portion 19 is formed in a shallow and flat bowl shape, the concave bottom surface 19a is a plane parallel to the inner bottom surface 17, and the corner portion at the boundary between the concave bottom surface 19a and the concave side surface 19b is curved. The angle at the boundary between the concave side surface 19b and the inner bottom surface 17 is chamfered. Straddling the boundary between the concave bottom surface 19a and the concave side surface 19b of the concave portion 19, a perfusion OUT hole 21 is provided to penetrate in the radial direction. This perfusion OUT hole 21 has a circular cross-section and the same diameter. The perfusion OUT hole 21 appears as a groove 23 in the extension portion on the concave bottom surface 19a side. This groove 23 extends to about 1 / 4 of the diameter of the concave bottom surface 19a. The perfusion OUT hole 21 opens on the outer plane 7a on the outer surface 7 side of the side portion 5 of the container 3.

[0013] The lid portion 25 is made of polycarbonate and has a rigid specification. This lid portion 25 is in a flat plate shape, the upper surface is configured as a single plane, but there are irregularities on the lower surface. The end surface forming the contour of the lid portion 25 is composed of a thickness portion orthogonal to the upper surface. Also, functionally, it consists of a main body portion 27 and an engaging portion 29. When viewed from above, the main body portion 27 is slightly larger in a similar shape with respect to the circular size of the container 3 so that it can cover the opening of the container 3 by covering it from above. Also, a pair of engaging portions 29, 29 are continuously provided radially outward from the main body portion 27.

[0014] The pair of engaging portions 29, 29 are continuously provided in point symmetry. The concave curved surface 29a of this engaging portion 29 faces the circumferential direction. The main body portion 27 has an arc-shaped contour at the location where the engaging portions 29, 29 are not continuously provided, and the center of the circumference of which a part of the arc is located is the axis center of the lid portion 25. Also, the concave curved surface 29a of the engaging portion 29 has a U-shaped contour, and the bottom thereof is curved in an arc shape.

[0015] Regarding the lower surface of the lid portion 25, the lower surfaces on the engaging portions 29, 29 side are flat and parallel to the upper surface, but on the lower surface of the main body portion 27 side, a disk-shaped convex portion 31 is coaxially and continuously provided to create a step. The lower surface of the convex portion 31 is a surface parallel to the upper surface of the lid portion 25, and the step side surface is substantially perpendicular to the upper surface. The corner portions on the outer peripheral side of the convex portion 31 are chamfered. An annular and extremely shallow concave ring portion 33 is coaxially provided on the outer peripheral side of the circular lower surface of this convex portion 31 to create a step in the reverse direction. The upper surface of the concave ring portion 33 is a surface parallel to the upper surface of the lid portion 25, and the step side surface is substantially perpendicular to the upper surface.

[0016] A through hole 35 is provided penetrating the concave ring portion 33 in the axial direction. This through hole 35 has a circular cross-section and has a coaxial step in the middle with a changing diameter, and the hole size of the upper side 35a is smaller than the hole size of the lower side 35b. Also, the step is perpendicular to the axial direction. Two through holes 35 are provided, and they are opposed in the radial direction, and both are located near the edge of the concave ring portion 33. One through hole 35 is used as the perfusion IN hole 35A, and the other through hole 35 is used as the air release hole 35B.

[0017] A cell culture insert 37 is accommodated in the container 3. This cell culture insert 37 is composed of a bottom surface portion 37a and a peripheral wall portion 37b made of plastic, and has a cup shape that is open upward. A flange 37c for suspension is continuously provided at the upper end of this peripheral wall portion 37b. The inner side of the frame where the bottom surface portion 37a is continuous with the peripheral wall portion 37b is composed of a membrane, and this membrane has pores that do not allow cells to pass through but allow the culture solution to pass through. The cell culture insert 37 is a commercially available "Falcon (registered trademark)", and usually, it is used by being suspended and set in a well plate of a transwell.

[0018] A thin-walled annular rubber packing 39 with an adjusted size is placed on the inner bottom surface 17 of the container 3, and a thin-walled annular rubber packing 41 with an adjusted size is also fitted into the concave ring portion 33 of the lid portion 25. Both are made of silicone rubber. The cell culture insert 37 is housed in the container 3, and the frame side of the lower surface of the bottom portion 37a is grounded to this rubber packing 39. The hole size of the inner peripheral surface 9a of the container 3 is set so that the peripheral wall portion 37b of the cell culture insert 37 can be smoothly inserted. The flange 37c comes directly above the stepped surface 9c with a slight gap therebetween. When this flange 37c approaches or contacts the inner peripheral surface 9b, the cell culture insert 37 is naturally housed coaxially with respect to the container 3, and the concave portion 19 is closed by the bottom portion 37a of the cell culture insert 37 via the rubber packing 39.

[0019] In this housed state, when the lid portion 25 is placed over the container 3, the convex disk portion 31 enters into the inner peripheral surface 9b, and the rubber packing 41 is grounded to the flange 37c. When only the self-weight of the lid portion 25 is applied, a slight gap is formed between the lower surface of the lid portion 25 and the upper end surface 11 of the side surface portion 5 of the container 3, and the lid portion 25 can move downward by the amount of this gap. Thus, when the lid portion 25 is pressed against the container 3, the rubber packings 39 and 41 are compressed in the vertical direction, and the opening of the cell culture insert 37 is closed in a liquid-tight manner. Also, the boundary between the frame side of the bottom portion 37a of the cell culture insert 37 and the concave edge of the concave portion 19 on the grounded side is also closed in a liquid-tight manner via the rubber packing 39. Therefore, as shown in FIG. 7, a sealed type culture container C is created in which the cell culture insert 37 and the concave portion 19 communicate with each other vertically via the membrane of the bottom portion 37a. As this pressing and fixing means, an adapter 43 is used.

[0020] In this adapter 43, a flat square base plate 45 is provided for placing the container 3. A shallow circular concave portion 45a is provided on the upper surface of this base plate 45. Also, tapered holes 45b, 45b that are reduced in diameter upward for inserting dish screws are formed at opposite corner portions, respectively. A dish screw 47 can be inserted into this tapered hole 45b from below and tightened with a nut 49 with a knurled surface. Therefore, after inserting socket screws 47, 47 into the tapered holes 45b, 45b respectively and placing the container 3 on the base plate 45, when lowering the lid portion 25, the circumferential positions of the pair of engaging portions 29, 29 are adjusted, and the screw shafts of the socket screws 47, 47 are held by the respective concave curved surfaces 29a, 29a, thereby preventing the lid portion 25 from moving in the circumferential direction.

[0021] Then, by attaching and tightening nuts 49, 49 to the screw shafts of the respective socket screws 47, 47, the lower surface of the nut 49 presses against the upper surface of the engaging portion 29, so that the rubber packings 39, 41 are compressed in the vertical direction, and the above-described hermetically sealed culture container C is created. And its sealed state is stably maintained. Also, since the container 3 side is positioned by the concave surface portion 45a on the base plate 45 side and the lid portion 25 side is positioned by the holding relationship between the engaging portion 29 and the socket screw 47, the lid portion 25 is closed at a position where it is exactly coaxial with respect to the container 3.

[0022] Stainless pipes 51, 53 are inserted into the perfusion OUT hole 21 and the perfusion IN hole 35A. On the perfusion OUT hole 21 side, one end side of the pipe 51 is inserted near the boundary of the groove 23 and is fitted in a liquid-tight manner. The pipe 53 has a small diameter, and the tip side is bent substantially at a right angle to form a hook portion 53a. Also, a gasket 55 is externally fitted to the pipe 53. On the perfusion IN hole 35A side, the pipe 53 is penetrated so that the hook portion 53a comes to the lower side, and is fitted in a liquid-tight manner through the gasket 55. The hook portion 53a of the pipe 53 extends parallel to the tangential direction of the inner surface surrounded by the peripheral wall portion 37b of the cell culture insert 37, and its tip opening is located near the inner surface of the peripheral wall portion 37b. A silicon flexible tube 57 is inserted into the air release hole 35B in a liquid-tight manner. One end side of this flexible tube 57 extends to the middle of the through hole 35b of the lid portion 25. A cap 59 is attached to the portion drawn out outside the lid portion 25, and the air release hole 35B can be opened and closed.

[0023] The perfusion culture unit 1 is configured as described above. As shown in Fig. 9, if a perfusion culture system is constructed in a CO2 incubator, flexible silicon tubes 61 and 63 for recovery and supply, respectively, are connected to pipes 51 and 53, and a liquid delivery system is established in the direction indicated by the arrow by a tube pump 65. Therefore, the culture solution is taken out from the culture solution reservoir 67, passes through the flexible tube 63, and is introduced into the cell culture insert 37 above the culture vessel C. Further, it passes through the membrane, passes through the flexible tube 61 from the lower concave portion 19, and is returned to the culture solution reservoir 67. Also, the tube pump 65 is under the control of a pump control controller 69 and is adjustable in flow rate.

[0024] During perfusion culture, the culture solution is stored up to the liquid level that has risen inside the cell culture insert 37 beyond the membrane, that is, below the culture vessel C, and a pool (P) is formed. The tip opening of the hook portion 53a is immersed in the pool (P). Note that by opening the cap 59 of the tube 57 inserted into the air release hole 35B, the height of the liquid level can be adjusted according to the type of specimen, etc. In any case, perfusion culture is carried out with the opening of the hook portion 53a immersed in the pool (P). The inflow rate and the outflow rate are adjusted, and the height of the liquid level is always maintained constant. When the specimen S is introduced into the cell culture insert 37, in the case of the adherent type, it adheres to the membrane, and in the case of the floating type, it floats in the culture solution.

[0025] The hook portion 53a is inclined from the axial direction of the peripheral wall portion 37b of the cell culture insert 37, and the culture solution flows out vigorously from the tip opening thereof, so that a liquid flow is generated in the pool (P). Since the tip opening of the hook portion 53a faces the tangential direction along the inner surface of the peripheral wall portion 37b of the cell culture insert 37, it becomes a vortex as shown by the arrow in Fig. 8. This vortex becomes a stirring flow and stirs the culture solution in the pool (P). That is, a flow similar to that generated by moving a stirring blade is generated. However, since there is no object having a physical size like a stirring blade moving, there is no risk of damaging the cells. Further, since it is a rectified flow rather than a turbulent flow, collisions between cells can be significantly prevented even in floating type cells such as organoids. Furthermore, since the culture solution is sucked in and recovered from the concave portion 19 below the membrane, the cells floating above the membrane are not sucked in when being recovered from the perfusion OUT hole 21.

[0026] The perfusion culture unit 1 can be made compact in its configuration and can be installed in plural in a CO2 incubator. Also, since the culture container C is of a sealed type, as shown in Fig. 10, it can be freely selected to be placed horizontally or vertically. In this case, the stirring direction of the specimen S such as cells will change. The tubes 57, 61, 63 are inexpensive and can be replaced each time. Also, since the lid portion 25 and the container 3 can be autoclaved, the risk of contamination by miscellaneous bacteria can be reduced.

[0027] As described above in detail regarding the embodiments of the present invention, the specific configuration is not limited to this embodiment, and even design changes and the like within the scope not departing from the gist of the present invention are included in the invention. For example, the flow direction of the culture solution in the pool (P) is adjusted by the inner surface shape and size of the peripheral wall portion 37b of the cell culture insert 37 and the position and orientation of the tip opening of the pipe 53. In the above-described embodiment, the generated eddy current mainly has a lateral flow. However, for example, if the orientation of the tip opening of the pipe 53 is downward, a vertical flow, that is, a flow in the vertical direction can also be generated in contact. Further, since it is sealed, the momentum of the flow can also be adjusted. Therefore, according to the type of the specimen S and the purpose of culturing, the type of the flow and its momentum can be easily optimized.

[0028] Although the tube pump 65 is used, it is only necessary to ensure the liquid feeding function, and other types of pumps may also be used. Further, it may be discharged directly to the outside from the perfusion OUT hole 21 without flowing back to the culture solution reservoir 67.

Explanation of Reference Numerals

[0029] 1... perfusion culture unit, 3... container, 5... side surface 7... outer surface, 7a... outer flat surface, 7b... outer peripheral surface 9... inner surface, 9a... inner peripheral surface (bottom side), 9b... inner peripheral surface (opening side) 9c... stepped surface, 11... upper end surface, 13... bottom 15... outer bottom surface, 17... inner bottom surface, 19... concave portion 19a... concave bottom surface, 19b... concave side surface, 21... perfusion OUT hole 23... groove, 25... lid portion, 27... main body portion 29... engaging portion, 29a... concave curved surface, 31... convex disk portion 33... concave ring portion, 35a, 35b... through holes, 35A... perfusion IN hole 35B... air release hole, 37... cell culture insert 37a... bottom surface portion, 37b... peripheral wall portion, 37c... flange 39, 41... rubber packings, 43... adapter, 45... base plate 45a... concave portion, 45b... tapered hole, 47... dish screw 49... nut, 51... pipe, 53... pipe 53a... hook-shaped portion, 55... gasket, 57... tube 59…Cap 61…Tube (recovery) 63…Tube (supply) 65…Tube pump 67…Culture solution reservoir 69…Pump control controller C…Culture vessel P…Pool (of culture solution) S…Specimen

Claims

1. A container with an open top and a concave portion provided on the inner bottom surface, where the inner side is recessed downward with respect to the outer side, a perfusion OUT hole provided through the concave portion of the container, a perfusion IN pipe inserted from the opening, and a stirring flow generation induction mechanism. When a cell culture insert is accommodated in the container and perfusion culture is performed with the concave portion closed at the bottom of the membrane of the cell culture insert, the cell culture insert and the concave portion form a culture container connected in the vertical direction through the bottom of the membrane. During perfusion culture, a perfusion culture unit characterized in that a stirring flow is generated in the culture solution accumulated in the cell culture insert by the stirring flow generation induction mechanism.

2. In the perfusion culture unit according to Claim 1, The opening of the perfusion IN pipe is immersed in the culture solution in a state inclined with respect to the axial direction of the inner surface of the cell culture insert, thereby inducing the generation of a stirring flow. A perfusion culture unit characterized by this.

3. In the perfusion culture unit according to Claim 2, The inner surface of the cell culture insert is an inner circumferential surface, and the perfusion IN pipe opens in a tangential direction along the inner circumferential surface to induce the generation of a vortex flow. A perfusion culture unit characterized by this.

4. In the perfusion culture unit according to any one of Claims 1 to 3, It is provided with a lid portion for closing the opening above the cell culture insert, and the perfusion IN pipe is passed through a perfusion IN hole provided through the lid portion. A perfusion culture unit characterized by this.

5. In the perfusion culture unit according to Claim 4, An air release hole is provided through the lid portion, and a tube with a cap is inserted into the air release hole. A perfusion culture unit characterized by this.

6. In the perfusion culture unit according to claim 4, The lower surface of the lid is provided with a step, and the inner part is lower than the annular outer part. When the outer part contacts the container and the opening of the container is closed, the inner part contacts the cell culture insert and the opening of the cell culture insert is closed. A perfusion culture unit characterized by this.

7. In the perfusion culture unit according to claim 6, It is provided with an adapter for fixing the lid in a closed state with respect to the container. When the lid is fixed to the container by the adapter, the cell culture insert is also fixed to the lid inside the container, so that the unit as a whole can be installed in various orientations. A perfusion culture unit characterized by this.

8. In the perfusion culture unit according to claim 1, A perfusion culture unit characterized in that a commercially available cell culture insert can be designed to be accommodated in a container.

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