Microfluidic device, microfluidic unit, and microfluidic module

Abstract

To enable the easy, quick and low-cost manufacture of microfluidic systems configured according to an evaluation target and an evaluation purpose.SOLUTION: The present invention relates to a microfluidic device having a microfluidic unit with a fluid flow path formed therein. The microfluidic unit is constructed by interconnecting multiple microfluidic modules with predetermined external shapes corresponding to the shapes of the microfluidic unit divided into multiple sections. Each of the multiple microfluidic modules has a partial fluid flow path that constitutes a part of the fluid flow path. At least one of the multiple microfluidic modules is a culture module with an organ holding chamber located midway through the partial fluid flow path. The multiple microfluidic modules are detachably connected by interconnecting the partial fluid flow paths with joints.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microfluidic device, a microfluidic unit constituting the microfluidic device, and a microfluidic module constituting the microfluidic unit. [Background technology]

[0002] A microfluidic chip is constructed by bonding two substrates, for example, made of glass, PDMS (polydimethylsiloxane), resin, or the like. Microscale grooves measuring 1 mm or less in width and depth are formed on the bonding surfaces of one or both substrates, and a microchannel is formed at the bonded portion by bonding the two substrates. Microfluidic chips are also manufactured by various methods, such as directly fabricating a microchannel structure within a substrate using 3D stereolithography technology or fabricating a microchannel structure within a substrate by hardening a liquid substrate containing a sacrificial material and then removing the sacrificial material. Such microfluidic chips are attracting attention as a fundamental technology in the fields of regenerative medicine, cell therapy, drug discovery, diagnosis, and treatment.

[0003] One application of microfluidic chips in the medical field is a microfluidic system called "organ-on-a-chip," which incorporates organs such as the heart, lungs, liver, and vascular system into a microfluidic chip (Non-Patent Document 1). In this system, a space where the organ is incorporated is formed on the microchannel, and by supplying drugs to the organ through the microchannel, it is possible to evaluate the pharmacological action and toxicity of the drug on the organ, or to investigate the reaction of the organ to a specific compound.

[0004] In the human body, no single organ functions independently; multiple organs function while interacting with one another. Because the interactions between organs are complex, a microfluidic system has been proposed that allows multiple organs to be incorporated into a single microfluidic chip (Non-Patent Document 2). This system incorporates multiple spaces within the microfluidic chip, along with microchannels connecting these spaces. This allows for comprehensive evaluation of the effects of drugs and other substances on multiple organs, as well as the effects of upstream organs on downstream organs. Another proposed microfluidic system involves stacking multiple microfluidic chips in multiple layers and irreversibly bonding them together using quantum beams (Non-Patent Document 3, Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO 2020 / 195361 [Patent Document 2] International Publication No. WO 2020 / 004646 [Non-patent literature]

[0006] [Non-Patent Document 1] Jia-Wei Yang, et al., "Organ-on-a-Chip: Opportunities for Assessing the Toxicity of Particulate Matter", Frontiers in Bioengineering and Biotechnology, 8, 519 (2020) [Non-patent document 2] Chak Ming Leung, et al., "A guide to the organ-on-a-chip", Nature Reviews Methods Primers, 2, 33 (2022) [Non-patent document 3] Tomoko Gowa Oyama, et al., "A simple method for production of hydrophilic, rigid, and sterilized multi-layer 3D integrated polydimethylsiloxane microfluidic chips", Lab Chip, 2020, 20, 2354-2363 Summary of the Invention [Problem to be solved by the invention]

[0007] The microfluidic system disclosed in Non-Patent Document 2 requires that a space and microchannels for accommodating organs be formed in advance inside the microfluidic chip according to the type, number, and combination of organs to be evaluated. Furthermore, the microfluidic systems disclosed in Non-Patent Document 3 and Patent Document 1 require that microfluidic chips be prepared according to the type and number of organs to be evaluated, and that these microfluidic chips be stacked and joined in an appropriate order. Thus, conventional microfluidic systems have to be manufactured each time according to the type and number of organs to be evaluated and the purpose of the evaluation, resulting in problems such as high costs due to custom manufacturing and the time and effort required for the manufacturing process.

[0008] The problem to be solved by the present invention is to make it possible to easily manufacture, in a short time, and at low cost, a microfluidic system configured in accordance with the evaluation target and evaluation purpose. [Means for solving the problem]

[0009] The present invention, which has been made to solve the above problems, is a microfluidic device having a microfluidic unit having a fluid flow path formed therein, the microfluidic unit is configured by interconnecting a plurality of microfluidic modules each having a predetermined outer shape corresponding to a shape obtained by dividing the microfluidic unit into a plurality of parts, the plurality of microfluidic modules each have a partial fluid flow path that constitutes a part of the fluid flow path, at least one of the plurality of microfluidic modules is a culture module having an organ holding chamber in the middle of the partial fluid flow path, The plurality of microfluidic modules are characterized in that the partial fluid flow paths are connected to each other by joints, so that the microfluidic modules are detachably connected to each other.

[0010] In the microfluidic device according to the present invention, the ends of the partial fluid channels of the microfluidic modules are connected in sequence with joints to link the microfluidic modules together to form a microfluidic unit. At this time, the microfluidic modules are linked so that the culture module is positioned according to the type of organ to be held in the organ holding chamber of the culture module, the purpose of evaluating the organ, etc. In this way, a microfluidic unit according to the intended use of the microfluidic device is fabricated.

[0011] The organ holding chamber of the culture module may contain organs such as the heart, lungs, liver, and vascular system obtained by cell culture using a device or instrument other than the microfluidic device of the present invention, and the organ holding chamber may contain cells, cell masses, tissues, and organs differentiated and proliferated from stem cells called organoids and culture them therein.

[0012] Since the multiple microfluidic modules are detachably connected by joints that connect the partial fluid channels, the multiple microfluidic modules can be separated from the microfluidic unit by removing the joints. Therefore, when the use of the microfluidic device for a certain purpose is finished, the multiple microfluidic modules can be disassembled and the microfluidic unit can be reassembled.

[0013] A microfluidic unit can be constructed by connecting an appropriate number of microfluidic modules, two or more. It is also possible to prepare various types of microfluidic modules, each with different shapes of partial fluid channels inside, in excess of the number of microfluidic modules constituting the microunit, and select and connect appropriate microfluidic modules to construct the microfluidic unit. Since each of the multiple microfluidic modules has a predetermined external shape corresponding to the shape of the microfluidic unit divided into multiple parts, the microfluidic unit can be constructed using only some of the microfluidic modules. However, among the microfluidic modules, a culture module having a partial fluid channel and an organ holding chamber can also be used alone as a microfluidic unit.

[0014] In the microfluidic device of the present invention, it is preferable that the organ holding chamber has an upper opening that opens onto the upper surface of the culture module, and that the organ holding chamber is provided with a cap that is attached to the upper opening.

[0015] The microfluidic device configured as described above can easily accommodate organs such as the heart, lungs, liver, and vascular system obtained by cell culture using another device or instrument in the organ holding chamber of the culture module, and can easily culture the cells and tissues accommodated in the organ holding chamber.

[0016] In the microfluidic device of the present invention, when the organ holding chamber has an upper opening and is equipped with a cap that is attached to the upper opening, it is preferable that the microfluidic device further comprises an air vent having one end that opens at a position different from the upper opening on the upper surface of the culture module and the other end that opens to the side of the organ holding chamber.

[0017] When the cap is opened and closed, there is a risk that the culture medium, cultured cells, or organoids in the organ holding chamber of the culture module may be pushed into the inflow or outflow path due to air pressure, or that the culture medium etc. pushed into the inflow or outflow path may be pushed into the inflow or outflow path of the microfluidic module connected next to it. However, by providing the air hole, such situations can be avoided.

[0018] Furthermore, in the microfluidic device of the present invention, it is preferable that the plurality of microfluidic modules include unit modules consisting of at least two identical unit shapes, and it is further preferable that the plurality of microfluidic modules include two or more unit modules and one or more multi-unit modules consisting of two to six unit modules connected together.

[0019] When multiple microfluidic modules include two or more unit modules of the same shape, or when multiple microfluidic modules include two or more unit modules and one or more multi-unit modules, the degree of freedom in arranging the multiple microfluidic modules that make up the microfluidic unit is increased.

[0020] Here, for example, when the shape of the unit module (unit shape) is a cube or a rectangular parallelepiped, examples of the shape of the multi-unit module include a cube and a rectangular parallelepiped. Also, when the shape of the unit module (unit shape) is a regular triangular prism, examples of the shape of the multi-unit module include a regular triangular prism, a rhombic prism, a trapezoidal prism, and a hexagonal prism. Furthermore, when the shape of the unit module (unit shape) is a quarter cylinder, examples of the shape of the multi-unit module include a semi-cylinder and a cylinder.

[0021] In the microfluidic device of the present invention, It is preferable that the plurality of microfluidic modules include at least one of the culture module, a control module having a liquid delivery pump, mixer, check valve, etc., provided at the inlet end of the partial fluid flow path or midway along the partial fluid flow path, and at least one analysis module having an analysis unit provided midway along the partial fluid flow path or at the outlet end of the partial fluid flow path.

[0022] According to the microfluidic device having the above configuration, by combining a culture module with at least one of a control module and an analysis module to form a microfluidic unit, it is possible to easily produce an appropriate microfluidic unit according to the intended use of the microfluidic device.

[0023] In addition, in the microfluidic device of the present invention, The unit module is a rectangular parallelepiped or cubic shape surrounded by six sides, It is preferable that the partial fluid flow path of the unit module is branched into two to six paths, and the ends of the partial fluid flow paths are open on two to six faces selected from the six faces. With the above configuration, the partial fluid flow paths of the unit modules can be easily connected by joints.

[0024] One of the partial fluid flow paths of the unit module is an inlet flow path, and the inlet flow path may be connected to the organ-holding chamber at a lower part of the organ-holding chamber. With the above configuration, culture medium and the like can be supplied to the organ held in the organ holding chamber from the lower part of the organ holding chamber.

[0025] The microfluidic device may further include a frame in which all of the microfluidic modules that make up the microfluidic unit are housed. According to the above configuration, the microfluidic unit can be easily transported while maintaining the connection state of the multiple microfluidic modules. In addition, since the position of each microfluidic module can be accurately adjusted, it becomes easy to analyze the organs held in the organ holding chamber using a plate reader or the like. [Effects of the Invention]

[0026] According to the present invention, since the microfluidic module is made to a fixed standard shape, not only can the microfluidic module be manufactured at low cost, but also a microfluidic system configured according to the type and number of organs to be evaluated and the purpose of evaluation can be manufactured easily, in a short time, and at low cost. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is a perspective view showing a first embodiment of a microfluidic device according to the present invention. [Figure 2] FIG. 1 is a perspective view of a microfluidic device showing the frame removed from the microfluidic unit. [Figure 3] Longitudinal cross section of a microfluidic device. [Figure 4] Top view of the microfluidic device. [Figure 5] Longitudinal cross section (a) and transverse cross section (b) of the microfluidic module. [Figure 6] FIG. 1 is a perspective view of the inside of a microfluidic module. [Figure 7] (a) is a plan view of a microfluidic unit in which microfluidic modules are arranged and connected in a different configuration, and (b) is a perspective view of the frame. [Figure 8] FIG. 10(a) is a plan view of a microfluidic unit in which microfluidic modules are arranged and connected in yet another configuration, and FIG. 10(b) is a perspective view of the frame. [Figure 9] Photographs showing examples (a) to (d) of fabricated microfluidic units. [Figure 10] Photographs showing the results of a liquid transfer experiment (Experiment 1) using a microfluidic unit. [Figure 11]These are photographs showing the results of an experiment (Experiment 2) in which the microfluidic module was separated from the microfluidic unit and recombined. (1) is a photograph showing the removal of the rightmost microfluidic module from the microfluidic unit after Experiment 1 was completed. (2) is a photograph showing the construction of a new microfluidic unit by connecting a new microfluidic module to the right of the removed microfluidic module. (3) is a photograph showing the connection of a syringe pump to the new microfluidic unit. (4) is a photograph showing the operation of the syringe pump to supply MilliQ water to the microfluidic unit. [Figure 12] (1) is a photograph of the microfluidic module used, (2) to (6) are microscope images, (2) is a phase contrast image of the seeded cells, (3) is a superposition of the three images below, (4) is the Hoechst staining result, (5) is the Calcein staining result, and (6) is the PI staining result. [Figure 13] 1(a) to 1(c) are diagrams showing modified examples of the microfluidic module. [Figure 14] FIG. 10 is a longitudinal cross-sectional view of a modified microfluidic device. [Figure 15] FIG. 1 is a diagram showing a schematic configuration of a microfluidic device 100 according to a second embodiment. [Figure 16] 1A is a perspective view showing the inside of a solution tank module, and FIG. 1B is a longitudinal cross-sectional view showing the inside of the solution tank module. [Figure 17] 1A is a perspective view showing the inside of a check valve module, FIG. 1B is a longitudinal cross-sectional view, FIG. 1C is a partially enlarged view showing the outlet flow path when the flow path is blocked, and FIG. 1D is a partially enlarged view showing the outlet flow path when the flow path is open. [Figure 18] 1A is a perspective view showing the inside of a cross-flow channel module, and FIG. 1B is a cross-sectional view showing the inside of the cross-flow channel module. [Figure 19] (a) is a perspective view and (b) is a longitudinal cross-sectional view showing the inside of an ELISA module. [Figure 20] Photographs showing an example of the fabrication of a microfluidic unit for ELISA. [Figure 21] Photograph (a) and graph (b) showing the experimental results. [Figure 22]FIG. 1 shows an example of a microfluidic unit composed of a unit module and multiple unit modules. [Figure 23] FIG. 10 is a diagram showing another example of a microfluidic unit composed of a unit module and a multi-unit module. DETAILED DESCRIPTION OF THE INVENTION

[0028] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a microfluidic device, a microfluidic unit, and a microfluidic module according to the present invention will be described with reference to the drawings.

[0029] [First embodiment] 1 and 2 are schematic perspective views of the microfluidic device of this embodiment, Fig. 3 is a longitudinal side view of the microfluidic device, and Fig. 4 is a top view of the microfluidic device. In the drawings, the actual shapes are appropriately simplified and the actual scale is appropriately changed to make the features of each part easier to understand. Also, the left and right in Fig. 1 are the left and right, respectively, of the microfluidic device.

[0030] The microfluidic device 1 is a microfluidic system including a horizontally elongated rectangular parallelepiped microfluidic unit 10 and a rectangular frame 20 that holds the microfluidic unit 10. The microfluidic unit 10 is configured by connecting a plurality of (four in this example) square pillar-shaped microfluidic modules 30 that are arranged in a line.

[0031] The frame 20 is made of metal or plastic and is composed of an outer frame 201 that corresponds to the overall planar shape of the microfluidic unit 10 (rectangular in this example) and partition plates 202 that divide the interior of the outer frame 201 into four compartments each having a shape that corresponds to the planar shape of the microfluidic modules 30 (approximately square in this example). The microfluidic unit 10 is held in the frame 20 by inserting the bottoms of the four microfluidic modules 30 that make up the unit into the four compartments of the frame 20. Due to the above-mentioned configuration of the frame 20, the microfluidic unit 10 held in the frame 20 maintains the connection between the four microfluidic modules 30. Furthermore, because the microfluidic unit 10 is held in the frame 20, it is possible to prevent the connection between the four microfluidic modules 30 from being released when the microfluidic unit 10 is transported.

[0032] Although not shown, the microfluidic device 1 is also equipped with other components, such as a culture medium storage tank, a liquid delivery pump that supplies the culture medium stored in the culture medium storage tank to the fluid flow path within the microfluidic unit 10, a reagent tank, and a waste liquid tank that collects waste liquid such as culture medium discharged from the fluid flow path within the fluidic unit 10.

[0033] The microfluidic module 30 is composed of a transparent rectangular pillar-shaped substrate 301 having a hollow space inside. To express the transparency of the substrate 301, the hollow space inside the substrate 301 is indicated by a thin solid line in Figures 1 and 2. The substrate 301 is made of a transparent and highly biocompatible material such as silicone resin, glass, or PDMS.

[0034] Figures 5(a) and 5(b) show a longitudinal cross-sectional view and a transverse cross-sectional view, respectively, of the microfluidic module 30. Figure 6 shows a perspective view of the interior of the microfluidic module 30, with Figure 6(e) being a perspective view of the microfluidic module 30 seen from above, Figure 6(a) to 6(d) being perspective views seen from the directions of arrows a to d in Figure 6(e), and Figure 6(f) being a cross-sectional view of the organ holding chamber 302 taken along a diagonal line on the top surface of the microfluidic module. The configuration of the microfluidic module 30 will be described in detail using Figures 5 and 6.

[0035] The hollow part of the microfluidic module 30 includes a cylindrical organ-holding chamber 302 formed near the center inside the base 301, flow paths 303-306 formed between the organ-holding chamber 302 and the four side surfaces of the base 301, and an air vent 307. The flow paths 303-306 correspond to the partial fluid flow paths of the present invention. The bottom surface of the organ-holding chamber 302 is located slightly above the lower surface of the base 301, and its upper portion opens at the upper surface of the base 301. The microfluidic module 30 has a cap 308 that is detachably attached to the upper opening of the organ-holding chamber 302. The upper opening of the organ-holding chamber 302 is closed by attaching the cap 308. One end of the air vent 307 opens at a position different from the upper opening on the upper surface of the base 301 (the upper surface of the microfluidic module 30 that functions as a culture module) at a position different from the upper opening, and the other end opens at a side surface of the organ-holding chamber 302. This air hole 307 is a thin tube for releasing air from inside the organ-holding chamber 302 to the outside when the organ-holding chamber 302 is closed by the cap 308, and is formed between the side surface of the organ-holding chamber 302 and the upper surface of the base 301.

[0036] Two of the four channels 303-306 are outlet channels (hereinafter referred to as outlet channels 303 and 304), and the remaining two channels are inflow channels (hereinafter referred to as inflow channels 305 and 306). In this embodiment, the outlet channels 303 and 304 are formed between two adjacent side surfaces of the base 301 and the organ holding chamber 302, and the inflow channels 305 and 306 are formed between another two adjacent side surfaces of the base 301 and the organ holding chamber 302. The ends of the outlet channels 303 and 304 and the inflow channels 305 and 306 on the side surfaces of the base 301 all open at corresponding positions on each side surface (specifically, slightly above the center in the vertical direction and near the center in the horizontal direction on each side surface). Note that the inflow and outflow sides of the microfluidic module 30 may be reversed, in which case the inflow channels are 303 and 304, and the outflow channels are 305 and 306.

[0037] The ends of the outflow channels 303 and 304 on the organ holding chamber 302 side are connected to the organ holding chamber 302 at approximately the same height as the ends on the side of the base 301. On the other hand, the ends of the inflow channels 305 and 306 on the organ holding chamber 302 side are connected near the bottom of the organ holding chamber 302. Therefore, the inflow channels 305 and 306 are made up of crank-shaped channels made up of a channel extending from the end on the side of the base 301 in parallel with the top surface of the base 301, a channel extending downward along the side of the organ holding chamber 302, and a channel parallel to the top surface of the base 301 connected near the bottom of the organ holding chamber 302.

[0038] The ends of the outflow channels 303, 304 and the inflow channels 305, 306 on the side of the base 301 have slightly larger inner diameters than the other parts, and a convex joint 401 and a concave joint 402 are fitted into these ends. The convex joint 401 consists of an insertion portion 4011 that inserts into the base 301 and a protrusion 4012 that protrudes out of the base 301, and a through-hole 4013 that penetrates the insertion portion 4011 and the protrusion 4012. The insertion portion 4011 consists of a tubular portion 4011a and a flange portion 4011b. The convex joint 401 is inserted into the ends of the outflow channels 303, 304 with the tubular portion 4011a facing the organ holding chamber 302, and is thereby attached to the base 301 with the insertion portion 4011 inserted into the base 301.

[0039] The concave joint 402 is made of an annular member, and is attached to the base 301 by inserting it into the ends of the inflow channels 305, 306. At this time, the entire concave joint 402 is fitted into the base 301. The inner diameter of the through hole 4023 that passes through the concave joint 402 is set to a size that allows the protrusion 4012 of the convex joint 401 to fit into it.

[0040] With the above configuration, the protrusion 4012 of the convex joint 401 of each microfluidic module 30 can be fitted into the through-hole 4023 of the concave joint 402 of another microfluidic module 30. This allows the two microfluidic modules 30 to be coupled together with the outflow channel 303 or the outflow channel 304 connected to the inflow channel 305 or the inflow channel 306.

[0041] Specifically, four microfluidic modules 30 are arranged in a row with their outflow channels 303, 304 located on the right side and front side and their inflow channels 305, 306 located on the left side and rear side, and of two adjacent microfluidic modules 30, the protrusion 4012 of the convex joint 401 of the left microfluidic module 30 is fitted into the through-hole 4023 of the concave joint 402 of the right microfluidic module 30, thereby connecting the four microfluidic modules 30 and forming the microfluidic unit 10 as shown in Fig. 1. The concave joint 402 located on the left side of the leftmost microfluidic module 30 is connected to a culture medium storage tank via a tube 501, and the protrusion 4012 of the convex joint 401 located on the right side of the rightmost microfluidic module 30 is connected to a waste liquid tank via a tube 502. In addition, a cap 60 and a plug 61 (shown only in Figure 4) are attached to the convex joint 401 located on the front side of each microfluidic module 30 and the concave joint 402 located on the back side, respectively, to close the flow path.

[0042] As a result, a fluid flow path consisting of the flow paths 303-306 of the four microfluidic modules 30 and having four organ holding chambers 302 in the middle is formed inside the microfluidic unit 10. When a culture medium is supplied to the microfluidic unit 10 from the tube 501 with a predetermined organ contained in each of the four organ holding chambers 302 of the microfluidic unit 10, the culture medium flows through the fluid flow path, passes through the four organ holding chambers 302 in order along the way, and is then discharged through the tube 502.

[0043] Specifically, the culture medium supplied from the culture medium storage tank through the tube 501 flows into the organ holding chamber 302 from the inlet channel 305 of the leftmost microfluidic module 30 of the four microfluidic modules 30 constituting the microfluidic unit 10, then flows into the organ holding chamber 302 through the outlet channel 303 of that microfluidic module 30 and the inlet channel 305 of the second microfluidic module 30 from the left. Thereafter, the culture medium similarly flows into the organ holding chamber 302 of the third microfluidic module 30 from the left and the organ holding chamber 302 of the fourth (rightmost) microfluidic module 30 in this order, and then is discharged from the outlet channel 303 of the fourth microfluidic module 30 through the tube 502 to the waste liquid tank.

[0044] The above-described flow of culture medium is the same whether tube 501 is connected to the inlet flow channel 305 or 306 of the leftmost microfluidic module 30, or whether tube 502 is connected to the outlet flow channel 303 or 304 of the rightmost microfluidic module 30.

[0045] In this embodiment, each microfluidic module 30 is configured so that the ends of the inflow channels 305, 306 and the outflow channels 303, 304 on the side of the base 301 are located near the vertical center of the side of the base 301. Furthermore, the ends of the outflow channels 303, 304 on the organ holding chamber 302 side are connected to the organ holding chamber 302 at approximately the same position as the ends of the base 301. Therefore, the culture solution flowing into the organ holding chamber 302 from the inflow channels 305, 306 is stored in the organ holding chamber 302 up to a height corresponding to the vertical center of the side of the base 301. When the amount of culture solution flowing into the organ holding chamber 302 from the outflow channels 303, 304 exceeds this height, the excess culture solution flows out of the organ holding chamber 302 through the outflow channels 303, 304. At this time, since the inflow channels 305 and 306 are connected near the bottom of the organ holding chamber 302, the culture medium stored in the organ holding chamber 302 flows out of the outflow channel 303 as if being pushed out by the new culture medium flowing into the organ holding chamber 302 from the inflow channel 305. In other words, the culture medium in the organ holding chamber 302 can be replaced with fresh culture medium.

[0046] Furthermore, in the microfluidic unit 10, the stopper 60 can be removed from the concave joint 402 that is closed by the stopper 60, and a drug, chemical substance, etc. can be introduced into the organ holding chamber 302 through the stopper 60 to examine the effects of the drug, chemical substance, etc. on the organ housed in the organ holding chamber 302. In this case as well, since the inflow channels 305, 306 are connected near the bottom of the organ holding chamber 302, the drug, chemical substance, etc. introduced into the organ holding chamber 302 can come into contact with the organ.

[0047] In the microfluidic module 30 of the above embodiment, the ends of the channels are exposed on all side surfaces of the base 301, and all ends are located at corresponding positions on each side surface. Therefore, the four microfluidic modules 30 can be connected in a line with all the four modules aligned in the same direction, as in the microfluidic unit 10 shown in FIG. 1 , or the microfluidic modules 30 can be connected in any orientation. Furthermore, instead of connecting the four microfluidic modules 30 in a line, the microfluidic unit 10A can be constructed by connecting four microfluidic modules 30 in two rows (2 × 2), as shown in FIG. 7(a). In this case, a frame 20A such as that shown in FIG. 7(b) can be prepared. The bottoms of the four microfluidic modules 30 can be inserted into the four compartments of the frame 20A, respectively, to maintain the four microfluidic modules 30 connected in a 2 × 2 arrangement.

[0048] 7(a), tubes 501 are connected to concave joints 402 attached to the ends of the inflow channels located on the right side of the two microfluidic modules 30 located on the right side, and tubes 502 are connected to convex joints 401 attached to the end of the outflow channel located on the left side of the single microfluidic module 30 located on the lower left side. In this way, the number of inflow channels for introducing culture medium into the microfluidic unit 10A and the number of outflow channels for discharging waste liquid may be different.

[0049] 8(a), a microfluidic unit 10B may be constructed by connecting eight microfluidic modules 30 in two rows of four (4 × 2). For such a microfluidic unit 10B, a frame 20B as shown in FIG. 8(b) may be prepared, and in this case too, the eight microfluidic modules 30 can be maintained connected in a 4 × 2 arrangement.

[0050] [Manufacturing example] Figure 9 shows photographs of a microfluidic unit manufactured by connecting two-inflow, two-outflow microfluidic modules in various configurations. The base of the microfluidic module in this example is made of transparent PDMS and is configured as a rectangular parallelepiped with four sides of 10 mm on each side of the top and bottom surfaces and a height of 16.5 mm. The convex joints attached to the base are made of a hard resin such as an epoxy-based photocurable resin or ABS resin, while the concave joints are made of a soft resin such as silicone. Because the base is transparent, organs placed in the organ holding chamber can be observed in their original state using an optical microscope or the like.

[0051] Next, the following experiment was carried out using the microfluidic module constituting the microfluidic unit of the manufacturing example shown in Figure 9. The results will be described.

[0052] [Experiment 1: Liquid transfer experiment] In Experiment 1, a microfluidic unit was used, which consisted of four microfluidic modules (two inflow, two outflow modules) connected in a straight line. A frame was attached to the bottom of the microfluidic unit, and the bottoms of the four microfluidic modules were inserted into the four compartments of the frame, respectively, to maintain the connection of the four microfluidic modules (see Figure 10).

[0053] The microfluidic unit consisted of four connected microfluidic modules, with the inlet channel located at the left end and the outlet channel located at the right end. One end of a 2 mm outer diameter, 1 mm inner diameter silicone tube (manufactured by AS ONE Corporation) was connected to a concave joint attached to the end of the left-most inlet channel. The other end of the silicone tube was connected to a 50 mL syringe (manufactured by Terumo Corporation) filled with MilliQ water dyed with food coloring. The 50 mL syringe was mounted on a syringe pump (manufactured by YMC Co., Ltd.). A cap was attached to the top opening of the organ holding chamber, the air vent was blocked with a pin, and the convex and concave joints attached to the ends of the fluid channels, except for the inlet channel at the left end and the outlet channel at the right end, were blocked with caps and stoppers. MilliQ water was then pumped into the microfluidic unit by operating the syringe pump at a flow rate of 500 μL / min.

[0054] Figure 10 is a photograph showing the microfluidic unit 30 seconds after the start of fluid transfer. As can be seen from this photograph, MilliQ water was delivered into the microfluidic unit from the inlet channel of the microfluidic module on the far left, and was discharged from the outlet channel of the microfluidic module on the far right without leaking from the connections between the microfluidic modules or gaps in the caps.

[0055] [Experiment 2: Separation and recombination of microfluidic units] In Experiment 2, a microfluidic unit was used that was constructed by connecting a microfluidic module removed from the microfluidic unit after Experiment 1 to another new microfluidic module. Specifically, the rightmost microfluidic module was removed from the microfluidic unit shown in Figure 10 (Figure 11(1)), and a new two-inlet, two-outlet microfluidic module was connected to the right of this microfluidic module (Figure 11(2)), thereby constructing a microfluidic unit consisting of two two-inlet, two-outlet microfluidic modules (Figure 11(3)).

[0056] In this microfluidic unit, the end of the inlet channel is located on the left side surface of the left microfluidic module, and a 50 mL syringe mounted on a syringe pump is connected to the concave joint at that end via a silicone tube with an outer diameter of 2 mm and an inner diameter of 1 mm. The convex joint at the end of the outlet channel is located on the right side surface of the microfluidic module on the right side of the microfluidic unit, and the concave and convex joints at the ends of the inlet and outlet channels, located on the front and rear sides of the two microfluidic modules, are closed with caps or plugs, the organ holding chambers are closed with caps, and the air vents are closed with pins.

[0057] The syringe pump was driven at a flow rate of 500 μL / min to deliver MilliQ water from the syringe into the microfluidic unit configured as described above. Figure 11(4) is a photograph showing the state of the microfluidic unit 10 seconds after the start of delivery. As can be seen from the photographs in Figures 11(1) to (4), no liquid leakage occurred when the microfluidic module was removed from the microfluidic unit in which Experiment 1 was performed. Furthermore, when a new microfluidic unit was constructed and liquid was delivered to the microfluidic unit, there was no leakage of MilliQ water from the connections between the microfluidic modules or gaps in the caps, and it was observed that MilliQ water was accumulating in the organ holding chamber of the microfluidic module on the right.

[0058] [Experiment 3: Cell culture test in a microfluidic module] The organ holding chamber of the microfluidic module was irradiated with plasma for 30 seconds using a tabletop vacuum plasma device (product name: YHS-R, manufactured by Sakigake Semiconductor Co., Ltd.) to hydrophilize the interior of the organ holding chamber. The squamous cell carcinoma cell line HSC-1 was cultured at 1.0 × 10 cells / well in DMEM medium (Dulbecco's modified Eagle's medium, manufactured by Invitrogen) containing 20% ​​FBS (fetal bovine serum, manufactured by Gibco). 5A cell suspension was prepared by adding 100 μL of the cell suspension to the organ holding chamber. The microfluidic module was placed in a 37°C, 5% CO incubator (manufactured by Panasonic Holdings Co., Ltd.) and cultured for 72 hours.

[0059] After culturing, the old medium was removed from the organ holding chamber, and a cell staining dye mixed medium was prepared by adding Calcein AM (Dojindo Laboratories, Inc. (DOJINDO)), PI solution (Dojindo Laboratories, Inc. (DOJINDO)), and Hoechst solution (Dojindo Laboratories, Inc. (DOJINDO)) to DMEM medium (Invitrogen) containing 20% ​​FBS (Gibco). Hoechst is a dye that stains cell nuclei, Calcein stains live cells, and PI stains dead cells. The Hoechst-stained areas indicate the location of each cell.

[0060] 100 μL of each of the three dye mixed media was placed in the organ holding chamber of a separate microfluidic module and left to stand in a 5% CO2 incubator at 37°C for 20 minutes. After that, the microfluidic module was removed from the 5% CO2 incubator, and the cells in the cell holding chamber were observed under a microscope (Olympus).

[0061] Figure 12 (1) shows a photograph of the microfluidic module (bright-field image; this image was taken with an Xperia 1 III SO-51B (Sony Group Corporation)), and (2) shows a phase-contrast image of the squamous cell carcinoma cell line HSC-1 cultured in the well. (3) shows a superimposed image of (4) to (6), and (4) to (6) show microscopic images of the squamous cell carcinoma cell line HSC-1 stained with Hoechst, calcein, and PI, respectively. As can be seen from these figures, almost all of the squamous cell carcinoma cell line HSC-1 cells cultured in the organ holding chamber were stained with calcein, with very few stained with PI. This suggests that stable cell culture can be achieved using the microfluidic module.

[0062] [Modification of the microfluidic module] The above-mentioned microfluidic module 30 (see Figures 1 to 8) has its outlet flow channels 303 and 304 arranged between two adjacent side surfaces on one side of the base 301 and the organ holding chamber 302, and its inlet flow channels 305 and 306 arranged between two adjacent side surfaces on the other side of the base 301 and the organ holding chamber 302, but can be modified as follows.

[0063] In the modified example illustrated in Figure 13(a), the microfluidic module 30 has its outlet flow channels 303 and 304 arranged between two opposing side surfaces of one side of the base 301 and the organ holding chamber 302, and its inlet flow channels 305 and 306 arranged between two opposing side surfaces of the other side of the base 301 and the organ holding chamber 302. In the modification shown in FIG. 13(b), the microfluidic module 30 has three outlet channels 303A and one inlet channel 305A (one inlet, three outlet type module). In a modification shown in FIG. 13(c), the microfluidic module 30 has one outlet channel 303B and three inlet channels 305B (a three inlet, one outlet module).

[0064] 13(a) to 13(c), like the microfluidic module 30 shown in Fig. 1 etc., the ends of the flow channels are exposed on all of the side surfaces of the base 301, and all of the ends are located at corresponding positions on each side surface. Therefore, a microfluidic unit can be constructed by appropriately combining the microfluidic module 30 shown in Fig. 1 etc. and the microfluidic modules 30 shown in Fig. 13(a) to 13(c).

[0065] Furthermore, although not shown, the microfluidic module is not limited to a configuration in which the total number of inflow and outflow channels formed inside the base is four, but can also be configured as a configuration in which the total number of inflow and outflow channels is three (a 1-inflow, 2-outflow module, a 2-inflow, 1-outflow module), or a configuration in which the total number of inflow and outflow channels is two (a 1-inflow, 1-outflow module). In the case of a 1-inflow, 2-outflow module and a 2-inflow, 1-outflow module, there is no channel between one of the four side surfaces of the base and the organ-holding chamber, and a closed structure is formed in which only a concave joint is installed to insert the convex joint of the adjacent microfluidic module and close the convex joint. In the case of a 1-inflow, 1-outflow module, there is no channel between two of the four side surfaces of the base and the organ-holding chamber, and a closed structure similar to that described above is formed.

[0066] [Modifications of the microfluidic device] Each of the multiple microfluidic modules 30 constituting the microfluidic unit of the first embodiment described above had an organ holding chamber 302 in the middle of the fluid flow path formed inside the base 301, but it is sufficient that at least some of the multiple microfluidic modules constituting the microfluidic unit have an organ holding chamber, and the other microfluidic modules do not need to have an organ holding chamber.

[0067] 14, three microfluidic modules 30 each having an organ holding chamber 302 and one microfluidic module 31 having only a fluid flow path 70 (i.e., no organ holding chamber) are connected in a row. In the following description, the microfluidic modules 30 and 31 will be referred to as the culture module 30 and the fluid flow path module 31, respectively.

[0068] The fluid channel module 31 is located second from the left in the microfluidic unit 10C, and can be used as a channel member for connecting the culture modules 30 on either side of it. In the example shown in Fig. 14, the fluid channel 70 of the fluid channel module 31 has a shape that branches from the center of the base 301 in five directions: front-to-back, left-to-right, and up-to-down, but it may also have a shape that branches in six directions: front-to-back, left-to-right, and up-to-down, or a shape that branches in two directions (straight pipe, L-shape, etc.), three directions (T-shape, etc.), or four directions (cross-shape, etc.).

[0069] As shown in FIG. 14, the culture module 30 can be configured such that a scaffold hydrogel 80 is held in advance in the organ holding chamber 302. For example, the hydrogel disclosed in Patent Document 2 (International Publication No. WO 2020 / 004646) can be used as the scaffold hydrogel 80. Holding such a scaffold hydrogel 80 in the organ holding chamber 302 allows the organ to be cultured simply by placing the organ in the organ holding chamber 302. Furthermore, the organ holding chamber 302 may contain not only the scaffold hydrogel 80 but also organoids held in the scaffold hydrogel 80.

[0070] [Second embodiment] 15 shows a schematic configuration of a microfluidic device 100 according to a second embodiment of the present invention. The microfluidic device 100 includes a microfluidic unit 110 for ELISA (Enzyme-Linked Immunosorbent Assay), three ring pumps 111, 112, and 113, and a control unit 114 that controls the ring pumps 111 to 113.

[0071] The ELISA microfluidic unit 110 includes one incubation module 130 , three solution tank modules 132 , three check valve modules 133 , one cross fluid transport module 134 , and one ELISA module 135 .

[0072] Since the culture module 130 has the same configuration as the culture module 30 described above, the description thereof will be omitted, and the configurations of the modules 132 to 135 will be described in detail below. Modules 132 to 135 are common to culture modules 30, 130 and fluid channel module 31 in that they are configured from a transparent rectangular pillar-shaped base 131 having a hollow portion therein. On the other hand, base 131 of modules 132 to 135 differs from base 301 of culture modules 30, 130 and fluid channel module 31 in that it is configured by joining a plurality of base components each having a shape obtained by dividing base 131 into a plurality of parts in the height direction. The shape of the base components obtained by dividing base 131 at which point is determined by the shape of the hollow portion of each module.

[0073] [Solution Tank Module 132] 16(a) and 16(b), the solution tank module 132 has a cylindrical solution tank 1322 located at the center inside the base 131, and an inlet channel 1323 and an outlet channel 1324 connected to two opposing locations on the side surface near the bottom of the solution tank 1322. The upper end of the solution tank 1322 opens on the upper surface of the base 131, and a cap 1325 is detachably attached to the upper end. The cap 1325 has an air hole 1326 that penetrates in the vertical direction and a lid 1327 that closes the upper opening of the air hole 1326. The air hole 1326 is used to release air from inside the solution tank 1322 when the cap 1325 is attached to the upper end of the solution tank 1322, and after the cap 1325 is attached to the upper end of the solution tank 1322, the air hole 1326 is closed by the lid 1327.

[0074] The upper portions of inflow channel 1323 and outflow channel 1324 are bent at a right angle toward the side surface of base 131, with their ends opening at the side surface. A convex joint 401 and a concave joint 402 are fitted into the upper portions of inflow channel 1323 and outflow channel 1324, respectively. In the first and second embodiments, convex joint 401 is fitted into the end of the outflow channel, and concave joint 402 is fitted into the end of the inflow channel, but in this embodiment, the convex joint 401 is fitted into the end of the inflow channel, and concave joint 402 is fitted into the end of the outflow channel.

[0075] The base 131 is formed by joining four base parts. The four base parts, from bottom to top, constitute a first section extending from the bottom surface of the base 131 to the bottom surface of the solution tank 1322, a second section extending from the bottom surface of the solution tank 1322 to the height position of the lower part of the convex joint 401, a third section extending from the height position of the lower part of the convex joint 401 to the height position of the lower part of the cap 1325, and a fourth section extending from the height position of the lower part of the cap 1325 to the upper surface of the base 131. The base part of the first section is made of a plate-shaped part, and part of the solution tank 1322 and parts of the inlet flow path 1323 and outlet flow path 1324 are formed in the base part of the second section. The base part of the third part is formed with part of the solution tank 1322 and the upper parts of the inlet flow path 1323 and outlet flow path 1324, and the base part of the fourth part is formed with a cap insertion space at the upper part of the solution tank 1322 and a space into which the flange portion 4011b of the convex joint 401 fits.

[0076] By dividing the base 131 into four base parts, the solution tank 1322, the outflow channel 1323, and the inflow channel 1324, which are hollow portions, can be easily formed.

[0077] [Check valve module 133] The check valve module 133 corresponds to the control module of the present invention. As shown in Figures 17(a) and (b), the check valve module 133 has a U-shaped fluid flow path 1331 formed inside the base 131. Like the fluid flow path module 31, the check valve module 133 does not have an organ holding chamber. The fluid flow path 1331 has an intermediate flow path 1332 with a semicircular vertical cross section located at the bottom inside the base 131, an inflow flow path 1333 with a rectangular cross section extending upward from one end of the intermediate flow path 1332, and an outflow flow path 1334 extending upward from the other end of the intermediate flow path 1332.

[0078] Figures 17(c) and (d) are partial enlarged views of the outflow channel 1334. As shown in these figures, the outflow channel 1334 consists of an upper channel 13341 with a rectangular cross section, a lower channel 13342 with a circular cross section, and a funnel-shaped portion 13343 located between the upper channel 13341 and the lower channel 13342. The funnel-shaped portion 13343 is a cylindrical member with a hemispherical bottom, and the upper end of the lower channel 13342 is connected to the center of the hemispherical bottom. Inside the funnel-shaped portion 13343, a stainless steel spherical bead 13344 with an outer diameter larger than the inner diameter of the lower channel 13342 is accommodated. When no fluid is flowing through the fluid channel 1331, the bead 13344 is located on the bottom surface of the funnel-shaped portion 13343 and blocks the lower channel 13342 connected to the bottom surface. On the other hand, when a fluid flows in the direction indicated by arrow A1 in the fluid flow path 1331, the flow pushes up the beads 13344, opening the opening at the upper end of the lower flow path 13342 connected to the bottom surface of the funnel-shaped portion 13343. This allows the fluid in the fluid flow path 1331 to flow smoothly.

[0079] On the other hand, when fluid attempts to flow in the direction indicated by arrow A2 within fluid flow path 1331, beads 13344 are pressed against the bottom surface of funnel-shaped portion 13343, blocking the opening at the upper end of lower flow path 13342. This prevents fluid from flowing in the direction indicated by arrow A2 within fluid flow path 1331. In other words, when there is no fluid flow or fluid pressure is applied in the opposite direction to the desired direction, beads 13343 block the flow path, thereby preventing backflow.

[0080] In addition, in the check valve module 133, a convex joint 401 and a concave joint 402 are fitted into the ends of the inlet flow path 1333 and the outlet flow path 1334, respectively. Furthermore, like the solution tank module 132, the base 131 of the check valve module 133 is also made up of multiple base parts. The same is true for the other modules described below.

[0081] [Cross-flow module 134] The cross-shaped fluid transport channel module 134 is a microfluidic module for allowing a solution that has flowed in from three different directions to flow out from one direction. As shown in FIG. 18, the cross-shaped fluid transport channel module 34 has a configuration in which a cross-shaped fluid channel 1341 is formed inside the base 131. Like the fluid channel module 31, the cross-shaped fluid transport channel module 134 does not have an organ holding chamber. The fluid channel 1341 is formed inside the base 131 substantially parallel to the upper surface of the base 131. The fluid channel 1341 has branch channels 1342 with circular cross sections that extend in all directions from a central intersection toward the four side surfaces of the base 131. Three of the four branch channels 1342 have convex joints 401 fitted at their ends, and one has a concave joint 402 fitted at its end. The three branch channels 1342 fitted with the convex joints 401 function as inflow channels, and the one branch channel 1342 fitted with the concave joint 402 functions as an outflow channel.

[0082] The four branch paths 1342 may have the same or different inner diameters. If the four branch paths 1342 have the same inner diameter, there is a possibility of backflow due to the flow rate from the three branch paths 1342 that serve as inflow paths to the single branch path 1342 that serves as an outflow path. In this case, backflow can be prevented by connecting the above-mentioned check valve module 133 to the convex joint 401 of the cross liquid transport path module 134.

[0083] [ELISA Module 135] 19(a) and 19(b), the ELISA module 135 includes a recess 1351 having a rectangular cross section formed in the upper part of the base 131, a recess 1352 formed in the lower part of the base 131, a neodymium magnet 1353 fitted in the recess 1352, and a fluid flow path 1354 formed in the base 131 so as to pass between the recess 1351 and the recess 1352.

[0084] The recess 1351 opens in the upper surface of the base 131, and the bottom surface of the recess 1351 is located about one-quarter of the height of the base 131 from the upper surface (i.e., the recess 1351 has a depth of about three-quarters the height of the base 131). The recess 1352 opens in the lower surface of the base 131, and its upper bottom surface is located lower than the bottom surface of the recess 1351. The fluid flow path 1354 consists of an intermediate flow path 1355 that passes between the bottom surface of the recess 1351 and the upper bottom surface of the recess 1352, and an inlet flow path 1356 and an outlet flow path 1357 that extend upward from both ends of the intermediate flow path 1355. The upper ends of the inlet flow path 1356 and the outlet flow path 1357 open on opposing side surfaces of the base 131, and a convex joint 401 and a concave joint 402 are fitted into these upper ends, respectively.

[0085] A plurality of magnetic beads 1358 are arranged in the intermediate flow channel 1355. An antibody for detecting a target protein is attached to the surface of the magnetic beads 1358. The magnetic beads 1358 are retained in the intermediate flow channel 1355 due to the magnetic attractive force of a neodymium magnet 1353 fitted in the recess 1352, even when a liquid flows through the intermediate flow channel 1355.

[0086] [ELISA Microfluidic Unit 110] The ELISA microfluidic unit 110 is configured as follows. The convex joint 401 of the ELISA module 135 is connected to the concave joint 402 of the cross liquid transport channel module 134, and the three convex joints 401 of the cross liquid transport channel module 134 are each connected to the concave joints 402 of the check valve module 133. Furthermore, the convex joints 401 of two of the three check valve modules 133 are each connected to the concave joints 402 of the solution tank modules 132, and the convex joint 410 of one check valve module 133 is connected to the concave joint 402 of the culture module 130. Furthermore, the convex joint 401 of one of the two solution tank modules 132 is connected to the concave joint 402 of another solution tank module 132.

[0087] With the above configuration, the ELISA microfluidic unit 110 has a first path through which the solution (reagent) contained in the solution tank 1322 of each of the two solution tank modules 132 is supplied to the ELISA module 135 through the check valve module 133 and the cross liquid supply path module 134, a second path through which the solution (reagent) contained in the solution tank 1322 of one solution tank module 132 is supplied through the check valve module 133 and the cross liquid supply path module 134, and a third path through which the supernatant of the culture solution held in the organ holding chamber of the culture module 130 is supplied through the check valve module 133 and the cross liquid supply path module 134.

[0088] The discharge portions of ring pumps 111 to 113 are connected to the convex joints 401 of the upstream solution tank module 132 of the first path, the solution tank module 132 of the second path, and the culture module 130 of the third path, and when the ring pumps 111 to 113 are driven, reagents and culture supernatant are supplied to the ELISA module 135 through each path.

[0089] [Manufacturing example] 20 is a photograph showing a manufacturing example of an ELISA microfluidic unit 110. The base of each module constituting the ELISA microfluidic unit 110 in this manufacturing example is formed from transparent PDMS, as in the manufacturing example of the first embodiment, and is configured in the shape of a rectangular parallelepiped with four sides of 10 mm on each of the top and bottom surfaces and a height of 16.5 mm. By forming the base from PDMS, the base parts can be joined together by plasma treating the joining surfaces of the base parts without using adhesive.

[0090] The convex joint is made of a hard resin such as an epoxy-based photocurable resin or an ABS resin, and the concave joint is made of a soft resin such as silicone. Because the substrate is transparent, the magnetic beads 1358 held in the intermediate flow channel 1355 can be observed with a fluorescent microscope or the like by removing the neodymium magnet 1353 after the reaction has been carried out.

[0091] [Experiment 4: Microfluidic unit for ELISA] Next, ring pumps 111 to 113 and a control unit 114 were connected to the ELISA microfluidic unit 110 shown in Figure 20 to construct the microfluidic device 100, and the following experiment was performed using the microfluidic device 100. The ring pumps 111 to 113 were manufactured by Aquatec Co., Ltd. (product number: RP-QIIIB1.5S-P35C-DC3VS). The control unit was a motor controller manufactured by Aquatec Co., Ltd. (product number: RE-C500).

[0092] Prior to the experiment, human iPS-derived cardiac organoids were placed in the organ holding chamber of the culture module 130, and 150 μL of control medium (medium containing 30 mM acrylamide) was added. The culture module 130 was then placed in a 5% CO2 incubator (manufactured by Panasonic Holdings Co., Ltd.) at 37°C and cultured for 24 hours. The culture module 130 after culture was used to construct the ELISA microfluidic unit 100.

[0093] The solution tanks 1322 of the two solution tank modules 132 of the first path both contained an albumin solution (PBS / 1% BSA), and the solution tank 1322 of the solution tank module 132 of the second path contained a secondary antibody solution. Furthermore, antibody beads (TOTAL GAPDH Magnetic Beads (46-667)) manufactured by Sigma-Aldrich were placed in the intermediate flow path 1355 of the ELISA module 135 as magnetic beads 1358.

[0094] The ring pump 112 was driven at 100 μL / min for 2 minutes to deliver 150 μL of culture medium from the culture module 130 to the ELISA module 135. After delivery, the system was left standing at room temperature for 2 hours. Next, the ring pump 111 was driven at 100 μL / min for 5 minutes to deliver a PBS / 1% BSA solution from the two solution tank modules 132 to the ELISA module 130, thereby cleaning the fluid flow path 1354. Thereafter, the ring pump 113 was driven at 100 μL / min for 1.5 minutes to deliver 100 μL of a PBS / 1% BSA solution containing a secondary antibody (GAPDH (14C10) Rabbit mAb (#3906), manufactured by Cell Signaling Technology) from the solution tank modules 132 to the ELISA module 135. After delivery, the system was left standing at room temperature for 1 hour, allowing a secondary antibody reaction to occur in the ELISA module 135.

[0095] After the secondary antibody reaction, the ring pump 111 was driven at 100 μL / min for 5 minutes to pump PBS / 1% BSA solution from the two solution tank modules 132 to the ELISA module 135, cleaning the fluid flow path 1354. Thereafter, the ELISA module 135 was removed from the ELISA microfluidic unit 110, and observation was performed under a fluorescence microscope (BZ-X, manufactured by Keyence).

[0096] The observation results are shown in Figure 21. In this experiment, GAPDH contained in the culture medium after 24 hours of culture of cardiac organoids was detected. When cell death occurs, GAPDH contained in the dead cells is released into the culture medium. Therefore, in this experiment, GAPDH should be detected only in the culture medium containing acrylamide, a cytotoxic component. As shown in the photograph in Figure 21(a) and the graph in (b), a significant increase in GAPDH was detected only when the acrylamide-containing culture medium was pumped into the ELISA module 135. This result suggests that the ELISA microfluidic unit 100 used in this experiment can be used to detect target proteins.

[0097] [Variations] Although not shown, a microfluidic module (corresponding to the control module of the present invention) may be configured in such a manner that a closed space and a flow path extending from the closed space to the outer surface of the base are formed inside the base of the microfluidic module, and a liquid transfer pump, a mixer, and the like are housed in the closed space. Furthermore, a microfluidic module (corresponding to the analysis module of the present invention) may be configured in such a manner that an analysis unit corresponding to a test kit such as a PCR test kit, as well as an ELISA test kit, is housed in the closed space. By constructing a microfluidic unit from such a microfluidic module and the above-described microfluidic modules 30 and 31, a microfluidic device having a pump, a mixer, and an analysis unit within the microfluidic unit can be configured.

[0098] 22(a) to 22(c), a microfluidic unit can be configured from a unit module 130, which is a microfluidic module having a square shape in plan view, and a multi-unit module 131, which is a microfluidic module formed by combining a plurality of such unit modules. Specifically, FIG. 22(a) shows a microfluidic unit formed from four unit modules 130 and one multi-unit module 131 formed by combining two of the unit modules 130, FIG. 22(b) shows a microfluidic unit formed from six unit modules 130 and one multi-unit module 131 formed by combining three of the unit modules 130, and FIG. 22(c) shows a microfluidic unit formed from four unit modules 130 and one multi-unit module 131 formed by combining four of the unit modules 130. However, the number of unit modules 130, the number of multi unit modules 131, and the relationship between the shape of the multi unit module and the shape of the unit module are not limited to the examples shown in FIGS. 22(a) to 22(c).

[0099] Furthermore, the shapes of the unit module 130 and the multi-unit module 131 are not limited to rectangular parallelepiped (quadratic prism), but can be a pillar with a fan shape in plan view, as shown in Figures 23(a-1) and (a-2), or a pillar with a triangular shape in plan view, or a diamond shape or trapezoid shape in plan view, as shown in Figures 23(b-1) and (b-2), or a pentagonal pillar, not shown.

[0100] [Aspect] It will be apparent to those skilled in the art that the above-described exemplary embodiments are examples of the following aspects.

[0101] (Item 1) One aspect of the present invention is a microfluidic device having a microfluidic unit having a fluid flow path formed therein, the microfluidic unit is configured by interconnecting a plurality of microfluidic modules each having a predetermined outer shape corresponding to a shape obtained by dividing the microfluidic unit into a plurality of parts, the plurality of microfluidic modules each have a partial fluid flow path that constitutes a part of the fluid flow path, at least one of the plurality of microfluidic modules is a culture module having an organ holding chamber in the middle of the partial fluid flow path, The plurality of microfluidic modules are detachably connected by connecting the partial fluid flow paths to each other through joints.

[0102] According to the microfluidic device of paragraph 1, a microfluidic unit is fabricated by connecting the ends of the partial fluid channels of a plurality of microfluidic modules in order with joints to link the plurality of microfluidic modules. At this time, by linking the plurality of microfluidic modules so that the culture module is positioned according to the type of organ to be held in the organ holding chamber of the culture module, the purpose of evaluating the organ, etc., a microfluidic unit according to the purpose of use of the microfluidic device can be fabricated easily and in a short time.

[0103] (Item 2) The microfluidic device according to item 2 is the microfluidic device according to item 1, The organ holding chamber has an upper opening that opens on the upper surface of the culture module, and is characterized by being equipped with a cap that is attached to the upper opening.

[0104] (Item 3) The microfluidic device according to item 3 is the microfluidic device according to item 2, The culture module is characterized by further comprising an air vent having one end opening at a position different from the upper opening on the top surface thereof and the other end opening to the side of the organ holding chamber.

[0105] (Item 4) The microfluidic device according to item 4 is the microfluidic device according to any one of items 1 to 3, wherein the plurality of microfluidic modules include unit modules consisting of at least two identical unit shapes.

[0106] (Item 5) The microfluidic device according to item 5 is the microfluidic device according to item 4, wherein the plurality of microfluidic modules include two or more unit modules and one or more multi-unit modules each consisting of two to six unit modules connected together.

[0107] (Item 6) The microfluidic device according to item 6 is the microfluidic device according to any one of items 1 to 5, the plurality of microfluidic modules It includes at least one of the culture modules, a control module having a liquid supply pump or mixer or the like provided at the inlet end of the partial fluid flow path or midway along the partial fluid flow path, and at least one analysis module having an analysis unit provided midway along the partial fluid flow path or at the outlet end of the partial fluid flow path.

[0108] (Item 7) The microfluidic device according to item 7 is the microfluidic device according to item 4 or 5, The unit module is rectangular parallelepiped or cubic, The partial fluid flow path of the unit module branches into two to six paths, and the ends of the partial fluid flow paths open on two to six outer surfaces selected from the six outer surfaces of the unit module.

[0109] (Item 8) The microfluidic device according to item 8 is the microfluidic device according to any one of items 1 to 7, One of the partial fluid flow paths of the culture module is an inlet flow path, which is connected to the organ holding chamber at a lower part of the organ holding chamber.

[0110] (Item 9) The microfluidic device according to item 9 is the microfluidic device according to any one of items 1 to 8, further comprising: The microfluidic unit includes a frame in which all the microfluidic modules that make up the microfluidic unit are housed.

[0111] (Item 10) The microfluidic device according to item 10 is the microfluidic device according to any one of items 1 to 9, Organoids are housed in the organ holding chamber of the culture module. With this type of microfluidic device, organoids are already housed in the organ holding chambers of the culture modules, eliminating the need to prepare organoids. Furthermore, organ-on-chips can be easily constructed by connecting multiple culture modules, each containing multiple types of organoids in its own organ holding chamber, to form a microfluidic unit. In this case, organoids can be cultured in the organ holding chambers of each of the multiple culture modules, and then the multiple culture modules can be connected to form a microfluidic unit. Therefore, organ-on-chips incorporating multiple types of organoids with different culture periods and culture media can be easily constructed.

[0112] (Item 11) The microfluidic unit according to item 11 comprises: 11. A microfluidic unit constituting the microfluidic device according to any one of items 1 to 10.

[0113] (12) The microfluidic module according to the 12th paragraph comprises: A microfluidic module constituting the microfluidic unit according to item 11.

[0114] (Item 13) The microfluidic device according to Item 13 is a microfluidic module according to Item 12, an organ-holding chamber provided midway along the partial fluid flow path; Organoids are housed in the organ holding chamber.

[0115] (Item 14) The microfluidic module according to Item 14 is the microfluidic module according to Item 12, an organ-holding chamber provided midway along the partial fluid flow path; The organ holding chamber may contain a hydrogel scaffold.

[0116] (Item 15) The microfluidic module according to Item 15 is the microfluidic module according to Item 12, an organ-holding chamber provided midway along the partial fluid flow path; The organ holding chamber may contain a scaffold hydrogel, and the organoid may be held in the scaffold hydrogel.

[0117] The hydrogel disclosed in Patent Document 2 (International Publication No. WO 2020 / 004646) can be used as the scaffold hydrogel. This hydrogel has a cell proliferation activity of 100 pg / mL or less, calculated as fibroblast growth factor-1 activity, a modulus of elasticity of 0.1 to 500 kPa, and a radiation-crosslinked structure of a hydrophilic polymer, the hydrophilic polymer containing one or more components selected from the group consisting of gelatin and artificial proteins. By storing this hydrogel as a scaffold hydrogel in an organ holding chamber, it is possible to stably control gene expression during cell culture while reducing proliferation activity caused by unidentified growth factors contaminated in the scaffold raw material. [Explanation of symbols]

[0118] 1...Microfluidic device 10...Microfluidic unit 20, 20A, 20B...frame 30...Microfluidic module 301...Base 302...Organ holding room 303, 304...Outlet flow path 305, 306...inlet flow passage 307...Air vent 308...Cap 401...Convex joint 402...Concave joint 130...Unit module 131...Multi-unit module 70...Fluid flow path 80...Hydrogel for scaffolding

Claims

1. A microfluidic device having a microfluidic unit having a fluid flow path formed therein, the microfluidic unit is configured by interconnecting a plurality of microfluidic modules each having a predetermined outer shape corresponding to a shape obtained by dividing the microfluidic unit into a plurality of parts, the plurality of microfluidic modules each have a partial fluid flow path that constitutes a part of the fluid flow path, at least one of the plurality of microfluidic modules is a culture module having an organ holding chamber in the middle of the partial fluid flow path, A microfluidic device, wherein the plurality of microfluidic modules are detachably connected by connecting the partial fluid flow paths to each other through joints.

2. 2. The microfluidic device according to claim 1, wherein the organ holding chamber has an upper opening that opens onto the upper surface of the culture module, and is provided with a cap that is attached to the upper opening.

3. The microfluidic device according to claim 2, further comprising an air vent having one end opening at a position different from the upper opening on the top surface of the culture module and the other end opening to a side surface of the organ holding chamber.

4. The microfluidic device according to claim 1 , wherein the plurality of microfluidic modules include unit modules each having at least two identical unit shapes.

5. 5. The microfluidic device according to claim 4, wherein the plurality of microfluidic modules include two or more of the unit modules and one or more multi-unit modules each having a shape in which two to six of the unit modules are connected together.

6. the plurality of microfluidic modules At least one of the culture modules; 6. The microfluidic device according to claim 1, further comprising at least one of a control module having a liquid delivery pump or a mixer provided in the partial fluid flow path, and an analysis module having an analysis unit provided in the partial fluid flow path.

7. The unit module is rectangular parallelepiped or cubic, 6. The microfluidic device according to claim 4 or 5, wherein the partial fluid flow path of the unit module branches into two to six paths, and the ends of the partial fluid flow paths open in two to six outer surfaces selected from six outer surfaces of the unit module, respectively.

8. 6. The microfluidic device according to claim 1, wherein one of the partial fluid flow paths of the culture module is an inlet flow path, and the inlet flow path is connected to the organ holding chamber at a lower part of the organ holding chamber.

9. The microfluidic device according to any one of claims 1 to 5, further comprising: A microfluidic device comprising a frame in which all of the microfluidic modules constituting the microfluidic unit are housed.

10. The microfluidic device according to any one of claims 1 to 5, wherein an organoid is contained in the organ holding chamber of the culture module.

11. A microfluidic unit constituting the microfluidic device according to any one of claims 1 to 5.

12. A microfluidic module constituting the microfluidic unit according to claim 11.

13. 13. The microfluidic module of claim 12, an organ-holding chamber provided midway along the partial fluid flow path; A microfluidic module, wherein the organoid is contained in the organ holding chamber.

14. 13. The microfluidic module of claim 12, an organ-holding chamber provided midway along the partial fluid flow path; A microfluidic module in which a scaffold hydrogel is contained in the organ-holding chamber.

15. 13. The microfluidic module of claim 12, an organ-holding chamber provided midway along the partial fluid flow path; A microfluidic module in which a scaffold hydrogel is contained in the organ holding chamber and an organoid is held in the scaffold hydrogel.

Images