Liquid feed system for perfusion culture

Abstract

To provide a liquid feed system for perfusion culture which can be compactly and simply constructed without increasing tubing connections even when the number of samples is increased.　A stretchable membrane 57 separates a fluid chamber 7 on the plastic molded body 3 side from an individual air chamber 41 on the combined structure 33 side to form a membrane pump 59. A pair of check valves (V1, V2) are provided in the fluid chamber 7. The individual air chamber 41 communicates with a common air chamber 55. Since the stretchable membranes 57, 57,... receive the same air pressure by pushing air from the common air chamber 55 out from or back into the individual air chamber 41 side, all the individual flow path structures create the same circulating perfusion culture conditions while being independent of each other. Thus, the circulating perfusion culture units 1 can provide pump action independently of each other for a plurality of individual fluid structures from a single air drive source and the system as a whole can be unitized, allowing the system as a whole to be configured simply and compactly while minimizing the tubing connections.

Description

Perfusion culture fluid delivery system

[0001] The present invention relates to a fluid delivery system in a perfusion culture system for perfusion culture of cells and the like.

[0002] In recent years, in the field of drug discovery, a microphysiological system (MPS), which is a cellular assay platform that reproduces the in vivo environment in vitro, has been attracting attention as an alternative to animal testing.

[0003] Japanese Patent Application Laid-Open No. 2021-153520

[0004] Therefore, as described in Patent Document 1, peripheral equipment such as flow channels, pumps, and tanks must be provided around the culture vessel containing the sample as a liquid delivery system for each sample. Increasing the number of samples (= n number) not only increases the number of peripheral equipment, but also complicates the control required to coordinate conditions between samples. This can lead to increased size and cost. If the system becomes too large, it may no longer be possible to incorporate it into existing, commonly used culture vessels. Furthermore, since the system is connected to the peripheral equipment via tubes, increasing the number of samples makes handling even more complicated, reducing convenience.

[0005] The present invention was made in response to the above-mentioned conventional problems, and its object is to provide a novel and useful fluid delivery system for perfusion culture that can be constructed compactly and simply without increasing the number of tubing connections, even when the number of samples is increased.

[0006] The present invention has been made to solve the above-mentioned problems, and provides an air-driven fluid delivery system for perfusion culture that includes a membrane pump composed of individual air chambers and fluid chambers separated by a stretchable membrane, and a pair of check valves in the fluid chambers. Driving air is pushed into and returned from the individual air chambers, causing the stretchable membranes to reciprocate, and this reciprocating motion opens and closes the pair of check valves, thereby delivering perfusion fluid from the fluid chambers. The system is an independent, multiple-connected fluid delivery system for perfusion culture, characterized in that multiple individual fluid delivery systems are provided, each consisting of an individual air chamber and a fluid chamber, and each individual air chamber is connected to a common air chamber on the drive source side.

[0007] Preferably, the molded body on the perfusion culture side has a recess formed on the upper surface side and a pair of communication holes connecting the recess on the upper surface side with the lower surface side, and the recess on the upper surface side forms a fluid chamber for a membrane pump, and the pair of communication holes form a pair of check valves, thereby forming multiple individual liquid supply systems in the molded body.

[0008] Preferably, a culture vessel setting section is provided on the molded body on the perfusion culture side, and is connected to an individual liquid supply system so that liquid can be supplied to the sample in the culture vessel set in the culture vessel setting section. Preferably, the set culture vessel is open on the top or closed with a transparent surface so that it can be observed under a microscope. Preferably, a perfusion fluid storage chamber is formed in the molded body on the perfusion culture side, and is connected to the fluid chamber and the culture vessel setting section to form a circulation flow path.

[0009] Preferably, the culture vessel setting section is formed using a recessed portion open upward on the molded body on the perfusion culture side, and a recessed groove is formed on the underside of the molded body, forming a circulation flow path connecting a pair of communication holes, the culture vessel setting section, and the perfusion fluid storage chamber. Preferably, a lower molded body and an upper molded body are joined to form a cavity therebetween, and the lower molded body and the upper molded body are provided with an individual air chamber side structure in which through holes communicating with and opening to the cavity are formed, the cavity being configured as a common air chamber, and the through holes of the lower molded body being configured as individual air chambers. Preferably, an engagement recess is formed on the upper surface of the molded body on the perfusion culture side, and a recess forming a fluid chamber is formed on the concave bottom surface of the engagement recess, and the air chamber side structure is fitted into the engagement recess via a stretchable membrane, thereby creating a membrane pump in which the individual air chamber and the fluid chamber are separated by the stretchable membrane. Preferably, the inner surface of the culture vessel setting section is designed to match the shape of the culture vessel.

[0010] Preferably, the culture vessel setting unit is composed of luer fittings that are respectively attached to a pair of through-holes formed through the underside of the molded body on the perfusion culture side, and the culture vessel to be set has a pair of female luer fittings that are respectively fitted into the pair of luer fittings. Preferably, the culture vessel is composed of microslides, and is provided with a holder that fixes multiple microslides so that they can be fitted into the molded body on the perfusion culture side. Preferably, the holder is fitted to the microslides with some play, so that the position of the female luer of the microslide can be adjusted.

[0011] Preferably, a flexible tube having one end connected to the through hole of the common air chamber and the other end closed, and a reciprocating pressing mechanism for compressing and restoring the tube, are used to push and return the driving air from the common air chamber to the plurality of individual air chambers.

[0012] The perfusion culture solution delivery system of the present invention integrates multiple independent solution delivery systems into a compact unit by simply combining molded bodies, and is driven by a single drive source, simplifying control. This eliminates the need for bulky systems even when the number of samples is increased. Furthermore, the set section for the culture vessels that contain the samples can also be integrated into the unit.

[0013] 1. A perspective view from above of a circulating perfusion culture unit according to a first embodiment of the present invention. 1. An exploded perspective view of the circulating perfusion culture unit of FIG. 1. 2. A cross-sectional view of the plastic molded body for perfusion culture of FIG. 1. 3. A cross-sectional view in a direction different from that of FIG. 3. 4. An exploded perspective view of the combined structure of FIG. 1. 5. A cross-sectional view of the circulating perfusion culture unit of FIG. 1. 6. An explanatory diagram of the pump action of the circulating perfusion culture unit of FIG. 1. 7. An explanatory diagram of the circulation path of the perfusion fluid of the circulating perfusion culture unit of FIG. 1. 8. An example is shown which includes a plurality of circulating perfusion culture units of FIG. 1. 9. An exploded perspective view of a circulating perfusion culture unit according to a second embodiment of the present invention. 10. An exploded top view and a cross-sectional view of FIG. 11. 11. An exploded perspective view of the holder side of FIG. 11. 12. An exploded perspective view of the holder side of FIG. 11 with a presser plate placed on it. 13. An exploded perspective view of the plastic molded body being lifted and supported by the microslide of FIG. 11. 14. An exploded perspective view of the plastic molded body of FIG. 11 with the stretch membrane removed from the back surface of the plastic molded body. 18 is an exploded perspective view of the plastic molded body of FIG. 17, where (1) is a view from above and (2) is a view from below. 19 is an explanatory diagram of the circulation path of the perfusion fluid of the circulating perfusion culture unit of FIG. 11, where (1) is a longitudinal cross-sectional view and (2) is a top view. 19 is a cross-sectional perspective view of FIG. 11.

[0014] A circulating perfusion culture unit 1 according to a first embodiment of the present invention is described with reference to the drawings. This unit is equipped with a parallel-branch type perfusion culture solution supply system equipped with a culture vessel setting section corresponding to each solution supply system, enabling independent circulating perfusion culture of multiple samples. As shown in Figures 1 and 2, the circulating perfusion culture unit 1 is constructed by simply assembling transparent polycarbonate plastic molded bodies into an integrated unit, with the lower portion comprising a plastic molded body 3 for perfusion culture. The plastic molded body 3 is a flat rectangular block, and its rectangular upper surface 3a is formed with a downwardly recessed groove 5. The groove 5 extends parallel to the longitudinal direction at its midpoint, with both ends open at opposing short edges. When viewed longitudinally, the groove 5 has a horizontally elongated rectangular cross section, and is positioned symmetrically in the left-right direction.

[0015] Circular recesses 7, 7, ... are formed in the concave bottom surface 5a of this groove portion 5 at regular intervals in the longitudinal and lateral directions, recessing downward. Four are formed in the longitudinal direction and two are formed in the lateral direction, resulting in an arrangement of eight circular recesses 7, 4 x 2 = 8. Each circular recess 7 is shaped like a shallow dish, with the concave bottom surface 7a being a circular flat surface parallel to the underside of the plastic molded body 3 and the concave circumferential surface 7b being a circumferential surface perpendicular to the underside of the plastic molded body 3.

[0016] A pair of grooves 9, 9 are connected to each circular recess 7 at a position perpendicular to the radial direction. The grooves 9 have a square cross section, and their bottom surfaces are parallel to the upper surface 3a and open upward. The grooves 9, 9 extend parallel to the longitudinal direction of the grooved streak portion 5. The groove depth of the grooves 9 is the same as that of the circular recess 7, but the groove width is sufficiently narrow compared to the diameter of the circular recess 7. As shown in Figures 3 and 4, a communication hole 11 constituting the upstream check valve (V1) is connected to the tip of one of the grooves 9. This communication hole 11 extends in the vertical direction, and its cross section perpendicular to the extension direction is circular, but a coaxial step surface is formed in the middle to form a small-diameter portion, and a ball 13 is housed in the upper large-diameter portion 11a so as to be movable up and down. The inclined step surface between the large diameter portion 11a and the small diameter portion serves as a valve seat 11b, and when the ball 13 is seated on the valve seat 11b by the flow of the perfusion fluid K, it closes the opening of the valve seat 11b.

[0017] The other groove 9 is connected to a communicating hole 15 that constitutes a downstream check valve (V2) at its tip. Like communicating hole 11, this communicating hole 15 extends vertically. Its cross section perpendicular to the direction of extension is circular, but a coaxial step is formed in the middle, resulting in a small-diameter section at the top. A ball 13 is housed in a large-diameter section 15a at the bottom, allowing it to move vertically. The inclined step between the large-diameter section 15a and the small-diameter section forms a valve seat 15b. When the ball 13 seats on the valve seat 15b due to the flow of perfusion fluid K, it closes the opening of the valve seat 15b.

[0018] The circular recess 7 and the pair of check valves (V1, V2) are aligned laterally along the transverse direction of the groove portion 5 of the plastic molded body 3. The following description focuses on the left circular recess 7 and the pair of check valves (V1, V2). As shown in FIG. 2 , a notch 17 is formed in the left plastic molded body 3 across the left concave side surface 5b. This notch 17 extends vertically and penetrates the plastic molded body 3, opening not only to the concave side surface 5b of the groove portion 5, but also to the concave side surface 5b of the groove portion 5. When viewed from above, the arc-shaped inner circumferential surface 19 of the notch 17 and the concave side surface 5b are connected in an Ω shape. As shown in FIG. 5 , the inner circumferential surface 19 has a coaxial step formed in the middle in the vertical direction, narrowing the lower side, forming an inner circumferential surface 19a with a circular cross section. Furthermore, the step surface 19b is slightly elevated above the concave bottom surface 5a of the groove portion 5. A step is also formed coaxially on the upper end side to form a step surface 19c, and the upper end side is slightly enlarged.

[0019] Furthermore, a through hole 21 is formed. This through hole 21 extends vertically and penetrates the substrate, and its cross section perpendicular to the extending direction is circular. The cross section of the through hole 21 is smaller than the cross section of the notched portion 17.

[0020] An upwardly recessed inverted groove 23 is formed on the lower surface 3b of the plastic molded body 3. This inverted groove 23 is configured similarly to the groove 9. The inverted groove 23 is connected to the lower ends of the communicating holes 11 and 15, and also to the notched portion 17 and the through-hole 21. Therefore, as shown in FIG. 4 , the communicating holes 11, 15, the notched portion 17, and the through-hole 21 form a U-shape, with the nodes being the communicating holes. The communicating holes 11 and 15 are connected to the grooves 9, 9 at their upper ends, and a circular recess 7 is formed between the pair of grooves 9, 9. The individual flow path structure for perfusion culture configured as described above is also formed on the right side, equipped with a circular recess 7 and a pair of check valves (V1, V2), for a total of eight in the plastic molded body 3. These structures are physically separated from each other.

[0021] Reference numeral 25 denotes a silicone-based pressure-sensitive adhesive film. This pressure-sensitive adhesive film 25 is superimposed and adhered to the entire underside 3b of the plastic molded body 3. Therefore, the pressure-sensitive adhesive film 25 closes the missing portion 17 and the lower opening of the through-hole 21 of the individual flow path structure, forming a bottom. The missing portion 17 side forms a culture vessel setting section 27, and the through-hole 21 side forms a perfusion fluid storage chamber 29. The lower opening of the inverted groove 23 is also closed to form a flow path 31, resulting in an overall circulation structure. When the underside 3b of the plastic molded body 3 is viewed with the pressure-sensitive adhesive film 25 made transparent, a circulation flow path is configured as shown in FIG. 9 , and the check valves V1 and V2 ensure that the perfusion fluid K flows in the normal direction from the circular recess 7 to the culture vessel setting section 27, the perfusion fluid storage chamber 29, and the circular recess 7.

[0022] The air-driven side of the plastic molded body 3 is a combination structure 33. As shown in Figure 6, this combination structure 33 is formed by joining a lower mold plastic molded body 35 and an upper mold plastic molded body 43, and is a rectangular block shape with a square cross section. The lower mold plastic molded body 35 is a flat, rectangular plate, and its upper surface is raised one step from the inner edge 37 to form a plate surface portion 39 similar to a rectangle. Through holes 41 are formed in the plate surface 39, penetrating the plate surface in the vertical direction. These through holes 41 have a circular cross section, and a coaxial step is formed in the middle of the vertical direction, widening the lower side. Thus, the lower portion 41b is wider laterally than the upper portion 41a, and the horizontal shape of this lower portion 41b is approximately the same as the circular recess 7 of the plastic molded body 3 on the fluid chamber side. Eight through holes 41 are formed at regular intervals in a 4 x 2 arrangement in the longitudinal direction x lateral direction of the rectangular plate surface portion 39.

[0023] The upper mold plastic molded body 43 also has a flat, rectangular plate-like shape, and its underside is stepped in two steps along its edge, forming an edge portion 45, an intermediate portion 47, and a rectangular, similar plate portion 49. A pair of disk-shaped protrusions 51, 51 protrude downward from the plate portion 49 at intervals, each at a position that is symmetrical when the plate portion 49 is divided into two longitudinal halves. The underside of the disk-shaped protrusions 51 is flat and is substantially flush with the underside of the intermediate portion 47. A through hole 53 is formed on one short edge of the upper mold plastic molded body 43. This through hole 53 has a circular cross section and extends linearly. One end opens across the stepped side of the boundary between the plate portion 49 and the intermediate portion 47 and the plate portion 49, and the other end opens to the outside of the upper mold plastic molded body 43.

[0024] As shown in FIG. 7 , the lower mold plastic molded body 35 and the upper mold plastic molded body 43 are joined together with their respective edges aligned to form an integrated combined structure 33, with the edge 45 of the upper mold plastic molded body 43 overlapping the edge 37 of the lower mold plastic molded body 35. In this state, the middle portion 47 overlaps the plate surface portion 39 internally, with the edges engaging with each other. A cavity 55 is formed between the plate surface portions 49 and 39. This cavity 55 opens to the outside through a through-hole 53. The cavity 55 serves as a common air chamber. The lower surface of the disk-shaped protrusion 51 also overlaps the plate surface portion 39, so the common air chamber 55 is divided by the two disk-shaped protrusions 51. The through-hole 41 serves as an individual air chamber and communicates with the common air chamber 55.

[0025] The combined structure 33 is fitted into the grooved streak portion 5 of the plastic molded body 3 on the fluid chamber side and integrated with it, so that when viewed longitudinally, the edges are aligned and the top surface is nearly flush. The lateral opening of the missing portion 17 is closed by the combined structure 33. Reference numeral 57 denotes a stretchable membrane made of silicone rubber. This stretchable membrane 57 is formed to be approximately the same size as the recessed bottom surface 5a of the grooved streak portion 5, and in the fitted state described above, it is sandwiched between the recessed bottom surface 5a and the underside of the combined structure 33. For each individual flow path structure, the lower portion 41b of the through-hole 41 faces the circular recess 7 via the stretchable membrane 57, thereby forming a membrane pump 59 separated by the stretchable membrane 57, with the circular recess 7 side serving as a fluid chamber and the through-hole 41 side serving as an individual air chamber. The membrane pump 59 and a pair of check valves (V1, V2) allow the perfusion fluid K to circulate in the order membrane pump 59 →culture vessel setting section 27 →perfusion fluid reservoir chamber 29 →membrane pump 59.

[0026] The plastic molded bodies 3, 35, and 43 of the circulating perfusion culture unit 1 are made of rigid polycarbonate, and the ball 13 is made of nylon. A tube connection (not shown) is provided in the through-hole 53 on the common air chamber 55 side, and one end of a flexible silicone tube 61 is connected to the tube 61. The other end of the tube 61 is closed. The tube 61, the common air chamber 55, and the individual air chambers 41, 41, 41, etc. are connected and filled with air. As shown in FIG. 1 , a roller pump 63 is attached to the middle of the tube 61. The tube 61 is sandwiched between the roller of the roller pump 63 and a circumferential receiving member. As the roller rotates and revolves, the tube 61 is intermittently squeezed and compressed, forcing the air inside the tube 61 toward the common air chamber 55. When the compression is released, air is returned from the common air chamber 55. This configuration conforms to the configuration described in Japanese Patent Application Laid-Open No. 2022-100769, and the roller pump 63 is controlled by a controller.

[0027] The culture vessel setting section 27 accommodates a commercially available cell culture insert 65, a type of culture vessel, in a set state. This cell culture insert 65 is composed of a bottom portion 65a and a plastic peripheral wall portion 65b, and is cup-shaped with an opening at the top. Multiple hanging hooks 65c for hanging are connected to the upper end of the peripheral wall portion 65b and protrude outward. The bottom portion 65a has a frame that connects to the peripheral wall portion 65b, and the inside of the frame is made of a membrane. This membrane has holes that allow the culture medium to pass through but not cells. The cell culture insert 65 is a commercially available "Falcon (registered trademark)" and is usually used by hanging it and setting it in a Transwell well plate.

[0028] The culture vessel setting section 27 is designed for a cell culture insert 65, and as shown in Figure 5, the underside of the peripheral wall section 65b of the cell culture insert 65 is loosely fitted into the inner peripheral surface 19a, and the latching claws 65c are suspended and supported with the latching claws 65c resting on the stepped surface 19c. The lower surface of the bottom section 65a is one step higher than the groove top surface 23a of the inverted groove 23.

[0029] A predetermined amount of perfusion fluid K is supplied to each of the individual flow path structures, filling the flow paths 31 with the perfusion fluid K. The liquid level in the culture vessel setting section 27 can be adjusted according to the sample, and is set, for example, to a level where the bottom surface 65a of the cell culture insert 65 is immersed so that the perfusion fluid K is delivered, i.e., perfused, to the sample in the culture insert 65. The provision of the perfusion fluid reservoir chamber 29 suppresses changes in flow rate and ensures pulsation.

[0030] When the controller is driven, air is pushed out from the common air chamber 55 to the individual air chambers 41, 41, ... and returned. The disk-shaped protrusions 51 make it easy for air to reach even the corners of the common air chamber 55, thereby uniforming the air flow rate within the individual air chambers 41, 41, .... As shown in Figure 8, when the air in the fluid chamber 7 is returned in the direction of the black arrow, the stretchable membrane 57 expands upward, increasing the volume of the fluid chamber 7 and attempting to suck in the perfusion fluid K through the pair of check valves V1, V2, but this is blocked on the downstream side by the check valve action, and the perfusion fluid K is sucked in from the upstream side as shown by the white-framed arrow and stored in the fluid chamber 7. When air is subsequently forced in as shown by the black arrow, the stretchable membrane 57 expands downward, reducing the volume of the fluid chamber 7 and attempting to discharge the perfusion fluid K through the pair of check valves V1, V2, but this is blocked upstream by the check valve action, and the perfusion fluid K in the fluid chamber 7 is discharged downstream as shown by the white-framed arrow. Because the stretchable membranes 57, 57, ... of the individual flow path structures are subjected to the same air pressure, all of the individual flow path structures, while independent of one another, achieve the same circulatory perfusion culture conditions.

[0031] In this way, in the circulating perfusion culture unit 1, a single air drive source can pump a plurality of individual fluid structures independently of one another, and since the entire unit is constructed as a unit, the entire unit can be constructed simply and compactly while minimizing tubing connections. ２ It can also be installed inside an incubator.

[0032] A circulating perfusion culture unit 71 according to a second embodiment of the present invention will be described with reference to the drawings. As shown in Figures 11, 12, and 13, the culture vessel for the circulating perfusion culture unit 71 is a "microslide 73" manufactured by Ibidi, which has two female luer bars 73b, 73b spaced apart from each other and protruding from the upper surface of a flat, rectangular body 73a. A plurality of these microslides 73 are fixed to a holder 75 in a parallel arrangement with their long edges adjacent to each other.

[0033] As shown in Figures 14 and 15, the holder 75 is shaped like a guide frame. A circular flange 77a extends inward from the lower edge of the rectangular fitting hole 77, and the micro-slide 73 is placed on this flange 77a. A protrusion 77b extends inward from the upper edge of the hole 77 on one short side, forming a fitting groove between the flange 77a and the upper flange 77a. Furthermore, a shallow recess 77c extends outward from the hole edge 77 on the other short side, forming a recess 77c that is one step lower than the upper surface. This recess 77c extends across the hole edges 77 of the multiple fitting holes. A plurality of circular through-holes 77d are formed at intervals in the recess 77c. The fitting hole is formed to provide some play with respect to the micro-slide 73, allowing the micro-slide 73 to be placed smoothly on the flange 77a. Furthermore, the longitudinal play of the fitting hole is set to be relatively large, allowing the micro-slide 73 to be displaced within the range of the hole edge.

[0034] A rectangular presser plate 79 is placed on the recessed surface 77c. The widthwise dimension of this presser plate 79 is set larger than that of the recessed surface 77c, and a fitting groove is formed between the portion of the presser plate 79 that extends into the hole edge and the lower flange 77a. The presser plate 79 also has a plurality of spaced-apart through-holes 79a. These through-holes 79a are oval holes that are elongated in the longitudinal direction of the micro-slide 73 placed on it. However, the hole size in the direction perpendicular to the longitudinal direction is the same as the hole size of the through-hole 77d. The degree of extension of the presser plate 79 into the fitting hole can be adjusted by moving the through-hole 79a while communicating with the through-hole 77d. Furthermore, by passing a countersunk head screw 81 through the through-holes 79a and 77d from below and tightening it with a knurled nut 83, the presser plate 79 is fixed to the holder 75 with the end of the micro-slide 73 sandwiched between them. Therefore, no matter where the micro-slide 73 is located within its movable range, both short edges of the micro-slide 73 are set in the fitting grooves.

[0035] A transparent polycarbonate plastic molded body 85 for perfusion culture is placed above the microslide 73. As shown in FIG. 16 , the plastic molded body 85 is a flat rectangular block. Its top surface 85a is formed with downwardly recessed circular recesses 87, 87, ..., spaced at regular intervals along the longitudinal direction and near one long edge. A pair of grooves 89, 89 are connected to the circular recess 87. These correspond to the circular recess 7 and groove 9 in the first embodiment. A communication hole 91 is connected to the tip of one of the grooves 89. This communication hole 91 extends straight downward and opens to the bottom surface 85b. Two stepped surfaces are formed along the way, reducing its diameter toward the top. The small cylindrical side of a tapered luer fitting 93 is fitted into the larger diameter portion of the communication hole 91, and the base end of a rubber duckbill-type check valve 95 is fixed to the top end of the luer fitting 93. The luer fitting 93 is fitted into a collar 97 at the large diameter portion of the communication hole 91 , and the check valve 95 is located in the upper half of the medium diameter portion of the communication hole 91 .

[0036] A communicating hole 99 communicates with the tip of the other recessed groove 89. This communicating hole 99 extends downward to the middle, then changes direction almost vertically away from the communicating hole 91, continues extending horizontally, and opens at the side surface 85c. It also branches midway toward the side surface 85c, and a branch hole 101 extends upward and opens at the top surface 85a. A single stepped surface is formed in the communicating hole 99, and the diameter increases toward the top. The base end of a check valve 95 is fixed to the stepped surface 99a on the upper edge of this communicating hole 99. The side surface 85c is also sealed with a bolt 103. The check valve 95 on the communicating hole 91 side allows passage upward, and the check valve 95 on the communicating hole 99 side allows passage downward.

[0037] Reference numeral 105 denotes a perfusion fluid reservoir, which is a rectangular recess deeply recessed downward from the upper surface 85a of the plastic molded body 85. The perfusion fluid reservoir 105 is formed near the other long side edge of the plastic molded body 85 and faces the circular recess 87 in the short direction. A communication hole 107 is formed in the bottom surface of the perfusion fluid reservoir 105. This communication hole 107 extends downward and opens at the lower surface 85b. The perfusion fluid reservoir 105 and the branch hole 101 are connected by a grooved channel 109 that is shallowly recessed downward from the upper surface 85a. This grooved channel 109 is located near one short side edge of the upper surface 85a. Similar to the communication hole 91, a luer fitting 93 is fitted into the communication hole 107. The communication holes 91 and 107 and the luer fittings 93 attached to them respectively constitute a culture vessel setting section.

[0038] As described above, the positions of the female luers 73b, 73b on the microslide 73 are adjustable, and the luer fittings 93, 93 are fitted to the pair of female luers 73b, 73b of the microslide 73 in accordance with the luer fittings 93, 93, respectively. This connects the flow path on the microslide 73 to the communication holes 91, 107, and supports the plastic molded body 85 in an elevated position. When the plastic molded body 85 is removed from the microslide 73, the microslide 73 is pulled upward. However, since the microslide 73 is fixed to the holder 75, it will not come off the holder 75. This engagement completes the circulation flow path configuration, and the perfusion fluid K flows in the forward direction from the perfusion fluid reservoir chamber 105 to the microslide 73, the circular recess 87, and then to the perfusion fluid reservoir chamber 105. A total of six individual flow path structures for perfusion culture configured in this manner are formed along the longitudinal direction of the plastic molded body 85. These structures are physically spaced apart.

[0039] Between the opposing perfusion fluid reservoir chamber 105 and the circular recess 87, the plastic molded body 85 is hollowed out in the vertical direction to form a hollowed-out portion 111. In addition, a plurality of mounting holes 113 are formed between the individual flow path structures, penetrating them in the vertical direction.

[0040] A flat, transparent, polycarbonate plastic molded body 115 for use as a lid is disposed above the plastic molded body 85. This plastic molded body 115 has a rectangular shape similar to that of the plastic molded body 85, and is positioned above with its edges aligned. As shown in Figures 17 and 18, the plastic molded body 115 also has a plurality of mounting holes 117, which are arranged as described above and face the mounting holes 113 from above and below. The plastic molded body 115 and the plastic molded body 85 are integrated by inserting a flathead screw 119 into the mounting holes 113 and 117 from below and tightening it with a knurled nut 121. A plurality of cutout portions 123 are also formed, and the cutout portions 123 face the cutout portions 111 from above and below.

[0041] A thin groove 125 extends along one long side edge on the top surface 115a of the plastic molded body 115. Both longitudinal ends of the groove 125 are closed. A ring-shaped step is provided, with the opening above the step surface 125a being larger. A through-hole 126 is formed penetrating from the side surface 115c of the long side edge toward the groove 125. This through-hole 126 opens into the groove 125 below the step surface 125a. A tube connection 128 (Figure 11) is attached to this through-hole 126. A plurality of through-holes 127 are formed in the bottom surface 125b of the groove 125, penetrating the plate surface in the vertical direction. Each through-hole 127 has a circular cross section, with a coaxial step formed midway between them in the vertical direction, widening the lower side. The shape of this lower portion is approximately the same as the circular recess 87 of the plastic molded body 85 on the fluid chamber side.

[0042] When the plastic molded body 115 is fixed to and integrated with the plastic molded body 85 as described above, the through hole 127 faces the circular recess 87. A lid (not shown) is attached to the step surface 125a of the groove portion 125, and the groove portion 125 is closed.

[0043] The plastic molded body 115 also has a through-hole 131 that penetrates the plate surface in the vertical direction and opens toward the perfusion fluid reservoir chamber 105. An air filter 133 is attached to this through-hole 131. Furthermore, a shallow concave portion 135 that is one step lower than the top surface 115a extends along the other long side edge of the top surface 115a of the plastic molded body 115. A plurality of ports 137 are formed in the bottom surface of the concave portion 135, penetrating the plate surface in the vertical direction. These ports 137 also open toward the perfusion fluid reservoir chamber 105. Valves similar to the check valves 95 are attached to the ports 137, utilizing the stepped surface 137a. The edge surface of the concave portion 135 on the peripheral side is concave, so that even when the concave portion 135 is closed by the port cover 139, the port 137 can be exposed by sliding it beyond the peripheral side.

[0044] Reference numeral 141 denotes a stretchable membrane made of silicone rubber. This stretchable membrane 141 is sandwiched between the lower surface 115b of the plastic molded body 115 and the upper surface 85a of the plastic molded body 85. The stretchable membrane 141 has notches 143 formed in locations corresponding to the mounting holes 117, hollowed-out portions 123, through-holes 131, and ports 137 of the plastic molded body 85, corresponding to their respective shapes. For each individual flow path structure, the lower part of the through-hole 127 faces directly opposite the circular recess 87 via this stretchable membrane 141, thereby forming a membrane pump 145 separated by the stretchable membrane 141, with the circular recess 87 side serving as a fluid chamber and the through-hole 127 side serving as an individual air chamber.

[0045] Furthermore, the groove portion 125 forms a common air chamber, which communicates with the individual air chambers formed by the through-holes 127, 127, .... As in the first embodiment, air is pushed toward the common air chamber 125 via the tube 61 and the roller pump 63 through the tube connection portion 128 of the through-hole 126 on the common air chamber 125 side, and air is returned from the common air chamber 125 side to the tube connection portion 128. This operates the membrane pump 145, and as in the first embodiment, as shown in FIG. 19 , the perfusion fluid K circulates by flowing along the circulation flow path configuration as indicated by the arrows in the following direction: perfusion fluid reservoir 105 → microslide 73 (female luer 73b → main body 73a → female luer 73b) → communication hole 91 → fluid chamber (circular recess) 87 → communication hole 99 → branch hole 101 → groove flow path 109 → perfusion fluid reservoir 105.

[0046] In this circulating perfusion culture unit 71, the main body 73a of the microslide 73 is exposed upward through the hollowed-out portions 111 and 123, allowing for microscopic observation. ２ Gases such as these are taken into the perfusion fluid storage chamber 105. Furthermore, the concave portion 131 is normally closed by a port cover 139, but the port cover 139 can be shifted and a pipette can be inserted into the valve to replace the culture medium.

[0047] In this way, the circulating perfusion culture unit 71 according to the second embodiment can also provide pumping action to a plurality of individual fluid structures independently from one another from a single air drive source, and since the entire unit can be made into a unit, the entire unit can be constructed simply and compactly while minimizing tubing connections. ２ In the first embodiment, the check valve V uses a ball 13, which requires high precision cutting of the communication holes 11 and 15 to prevent valve leakage, but in the second embodiment, a duckbill-type check valve 95 is used, so such precision cutting is not required.

[0048] Although the embodiments of the present invention have been described in detail above, the specific configuration is not limited to these embodiments, and design changes within the scope of the present invention are also included. For example, in the first embodiment, the culture vessel setting section 27 may be configured to accommodate samples placed on a plate such as a glass plate. Furthermore, in the second embodiment, if strength is an issue, the upper surface 115a of the plastic molded body 115, excluding the concave portion 135, and the side surface facing the through-hole 127 may be covered with an L-shaped stainless steel lid. In this case, the port cover 139 can be made of magnetic stainless steel to prevent the port cover 139 from slipping. Furthermore, while the roller pump 63 is used to vary the air pressure, other types of mechanisms may be used as long as they can vary the air pressure. Furthermore, the perfusion fluid K may be directly discharged after passing through the culture vessel setting section without circulating through the circulation path.

[0049] Furthermore, as shown in Figure 10, a structure in which the common air chambers 55, 55, 55 of multiple circulating perfusion culture units 1, 1, 1 are connected to a single large common air chamber 67, and a roller pump 63 is connected to this large common air chamber 67 can also be considered.

[0050] DESCRIPTION OF SYMBOLS 1...Circulating perfusion culture unit 3...Plastic molded body 3a...Upper surface 3b...Lower surface 5...Concave rib portion 5a...Concave bottom surface 5b...Concave side surface 7...Circular recess (fluid chamber) 7a...Concave bottom surface 7b...Concave peripheral surface 9...Concave groove 11...Communicating hole 11a...Large diameter portion 11b...Valve seat 13...Ball 15...Communicating hole 15a...Large diameter portion 15b...Valve seat 17...Notched portion 19...Inner peripheral surface 19a...Inner peripheral surface 19b...Step surface 19c...Step surface 21...Through hole 23...Inverted concave groove 23a...Groove top surface 25...Pressure-sensitive adhesive film 27...Culture vessel setting portion 29...Perfusion fluid storage chamber 31...Flow path 33...Combined structure 35...Lower mold plastic molded body 37...Edge portion 39...Plate surface portion DESCRIPTION OF SYMBOLS 41...Through-hole (individual air chamber) 41a...Upper part 41b...Lower part 43...Upper mold plastic molding 45...Edge part 47...Middle part 49...Plate surface part 51...Disc-shaped protrusion 53...Through-hole 55...Cavity (common air chamber) 57...Stretchable membrane 59...Membrane pump 61...Tube 63...Roller pump 65...Cell culture insert 65a...Bottom part 65b...Peripheral wall part 65c...Latching claw 67...Large common air chamber V1, V2...Check valve K...Perfusion fluid 71...Circumferential perfusion culture unit 73...Microslide 73a...Main body 73b...Female luer 75...Holder 77...Hole edge 77a...Flange 77b...Projection piece 77c...Concave surface 77d...Through-hole 79...Pressure plate 79a...Through-hole 81...Countersunk head screw 83... Knurled nut 85... Plastic molded body 85a... Upper surface 85b... Lower surface 85c... Side surface 87... Circular recess (fluid chamber) 89... Groove 91... Communication hole 93... Luer fitting 95... Check valve 97... Collar 99... Communication hole 99a... Step surface 101... Branch holeDESCRIPTION OF SYMBOLS 103...Bolt 105...Perfusion fluid storage chamber 107...Communicating hole 109...Grooved flow path 111...Cutout portion 113...Mounting hole 115...Plastic molded body 115a...Upper surface 115b...Lower surface 115c...Side surface 117...Mounting hole 119...Flat head screw 121...Knurled nut 123...Cutout portion 125...Grooved portion (common air chamber) 125a...Step surface 125b...Bottom surface 126...Through hole 127...Through hole (individual air chamber) 128...Tube connection portion 131...Through hole 133...Air filter 135...Concave portion 137...Port 137a...Step surface 139...Port cover 141...Stretchable membrane 143...Notch 145...Membrane pump

Claims

1. An air-driven fluid supply system for perfusion culture, which comprises a membrane pump composed of individual air chambers and fluid chambers separated by a stretchable membrane, and a pair of check valves in the fluid chambers, and in which driving air is pushed into and returned from the individual air chambers to cause the stretchable membranes to reciprocate, and this reciprocating motion opens and closes the pair of check valves to supply perfusion fluid in the fluid chambers, the system being an independent, multiple-connected fluid supply system for perfusion culture, characterized in that multiple individual fluid supply systems are provided, each consisting of an individual air chamber and a fluid chamber, and each individual air chamber is connected to a common air chamber on the drive source side.

2. A fluid supply system for perfusion culture as described in claim 1, characterized in that a molded body on the perfusion culture side has a recess formed on the top surface and a pair of communication holes connecting the recess on the top surface with the bottom surface, and the recess on the top surface forms a fluid chamber for a membrane pump, and the pair of communication holes form a pair of check valves, thereby forming a plurality of individual fluid supply systems in the molded body.

3. A fluid supply system for perfusion culture according to claim 2, characterized in that a culture vessel setting section is provided on the molded body on the perfusion culture side, and the system is connected to an individual fluid supply system so that fluid can be supplied to the sample in the culture vessel set in the culture vessel setting section.

4. A fluid supply system for perfusion culture according to claim 3, characterized in that the upper side of the set culture vessel is open or closed with a transparent surface so that it can be observed under a microscope.

5. A fluid supply system for perfusion culture according to claim 4, characterized in that the perfusion fluid storage chamber is formed by the molded body on the perfusion culture side, and is connected to the fluid chamber and the culture vessel setting section to form a circulation flow path.

6. A fluid supply system for perfusion culture according to claim 5, characterized in that the culture vessel setting section is formed using a recessed portion that is open above the molded body on the perfusion culture side, and a groove is formed on the underside of the molded body, and the groove forms a circulation flow path that connects the pair of communication holes, the culture vessel setting section, and the perfusion fluid storage chamber.

7. A fluid supply system for perfusion culture according to claim 6, characterized in that a lower molded body and an upper molded body are joined together to form a cavity therebetween, and the system is provided with an individual air chamber side structure in which through holes communicating with and opening into the cavity are formed in the lower molded body and the upper molded body, and the cavity is configured as a common air chamber, and the through holes in the lower molded body are configured as individual air chambers.

8. A fluid supply system for perfusion culture according to claim 7, characterized in that an insertion recess is formed on the upper surface of the molded body on the perfusion culture side, and a recess constituting a fluid chamber is formed on the concave bottom surface of said insertion recess, and the structure on the air chamber side is inserted into said insertion recess via a stretchable membrane, thereby creating a membrane pump in which individual air chambers and fluid chambers are separated by said stretchable membrane.

9. A fluid supply system for perfusion culture according to claim 8, characterized in that the inner surface of the culture vessel setting section is designed to fit the shape of the culture vessel.

10. A fluid supply system for perfusion culture as described in claim 5, characterized in that the culture vessel setting section is composed of luer fittings attached to a pair of through-holes formed through the underside of the molded body on the perfusion culture side, and the culture vessel to be set is equipped with a pair of female luers that are fitted into the pair of luer fittings, respectively.

11. A fluid supply system for perfusion culture according to claim 10, characterized in that the culture vessel is made up of microslides, and a holder is provided for fixing a plurality of microslides so that they can be fitted into the molded body on the perfusion culture side.

12. A fluid supply system for perfusion culture as described in claim 11, characterized in that the holder is fitted to the microslide with some play, making it possible to adjust the position of the female luer of the microslide.

13. A fluid supply system for perfusion culture as described in claim 1, characterized in that it comprises a flexible tube having one end connected to the through-hole of the common air chamber and the other end closed, and a reciprocating pressing mechanism that compresses and restores the tube, thereby forcing and returning driving air from the common air chamber to the multiple individual air chambers.

Images