Microfluidic devices and methods for using microfluidic devices

Abstract

To provide a microfluid device in which a substrate and a membrane can be sufficiently bonded even if they are non-adhesive.SOLUTION: A microfluid device 1 is formed by laminating a first plastic substrate 11, a membrane 13 and a second plastic substrate 12, where the substrate 11 is equipped with a first flow channel 111, the substrate 12 is equipped with a second flow channel 121, the first flow channel 111 and the second flow channel 121 are separated by the membrane 13, the substrate 11, the substrate 12 and the membrane 13 are made of non-adhesive plastic, the substrate 11 and the membrane 13 are adhered by a first adhesive sheet 14, the substrate 12 and the membrane 13 are adhered by a second adhesive sheet 15, a flow channel 141 is provided at a position corresponding to the first flow channel 111 in the first adhesive sheet 14, and a flow channel 151 is provided at a position corresponding to the second flow channel 121 in the second adhesive sheet 15.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microfluidic device used for reproducing the tissue structure of organs and evaluating pharmacokinetics and the like.

Background Art

[0002] In the development of pharmaceuticals and the like, if the pharmacokinetics in humans cannot be predicted until non-clinical trials for drug candidate substances, and the toxicity is revealed in clinical trials, resulting in the suspension of development, the research and development costs up to that point will be wasted. That is, in non-clinical trials, cell assays, animal experiments, etc. are conducted. However, in cell assays, the blood flow in the living body cannot be reproduced, the expression of cell-specific functions is insufficient, and there is a problem that pharmacokinetics cannot be evaluated. Also, in animal experiments, due to species differences, the results of pharmacokinetics do not always match those in humans, and there is a problem that it is difficult to predict human pharmacokinetics.

[0003] In such a situation, in recent years, organ chips have been expected to solve these various problems and improve the prediction accuracy of human pharmacokinetics. An organ chip is one that reproduces the tissue structure of an organ in a microfluidic device. There have already been proposed a lung chip that models the structure of the lung, one that models the structure of enterohepatic circulation by combining a small intestine model and a liver model, and ones that model the structure of the kidney such as a glomerulus model and a proximal tubule model.

[0004] Here, in a conventional in vitro culture system using a culture dish or the like, the culture environment is quasi-static, there is no reproduction of blood flow, and the supply of oxygen and nutrients and the removal of waste products depend only on diffusion. Therefore, it has been difficult to conduct tests considering cell-specific functions and interactions between organs. In contrast, organ chips allow for the reproduction of blood flow through pump delivery, and enable the adjustment of oxygen and nutrient supply based on flow rate. This makes it possible to suitably conduct tests that include specific cell functions and inter-organ interactions. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication No. 2007-525667 [Patent Document 2] Japanese Patent Publication No. 2008-8880 [Non-patent literature]

[0006] [Non-Patent Document 1] Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device, Yu-suke Torisawa, Bor-han Chueh, Dongeun Huh, Poornapriya Ramamurthy, Therese M.Roth, Kate F. Barald and Shuichi Takayama, Received 18th, December 2006, Accepted 27th March 2007, First published as an Advance Article on the web 20th April 2007, DOI:10.1039 / b618439a [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Microfluidic devices used in organ chips include monolayer culture devices that culture only one type of cell, and co-culture devices that allow the culture of two types of cells via a membrane. Microfluidic devices for co-culture are constructed by stacking two substrates and a membrane. Each substrate has a channel (microchannel) formed within it, and the membrane separates each channel, allowing the same or different cells to be cultured in each channel.

[0008] Conventionally, microfluidic devices for co-culture have used substrates made of PDMS (polydimethylsiloxane, silicone rubber) and membranes made of PET (polyethylene terephthalate). While such microfluidic devices have excellent adhesion between the substrate and membrane, they have the problem of low-molecular-weight components adsorbing onto the PDMS. In other words, when injecting pharmaceuticals containing low-molecular-weight compounds as active ingredients into microfluidic devices, the low-molecular-weight compounds are adsorbed onto the PDMS, resulting in a decrease in the concentration of the pharmaceutical in the flow path and making accurate evaluation impossible.

[0009] Furthermore, some microfluidic devices for co-culture used PET (polyethylene terephthalate) for both the plastic substrate and the membrane. However, because PET exhibits autofluorescence, microfluidic devices using PET as the substrate had the problem of not being practical for real-world use.

[0010] Therefore, the inventors attempted to construct a microfluidic device using a plastic substrate made of COP (cycloolefin polymer) and a membrane made of PET (polyethylene terephthalate). However, since COP and PET are non-adherent to each other, there was a problem in that the substrate and membrane could not be easily joined.

[0011] Here, a conventional microfluidic device is shown in Figure 4. As shown in Figure 4(1), this microfluidic device 100 is constructed by stacking an upper substrate 110, a membrane 130, and a lower substrate 120, with an upper channel 1101 formed in the upper substrate 110 and a lower channel 1201 formed in the lower substrate 120 being separated by the membrane 130. Such microfluidic devices 100 have a problem in that, as shown in Figure 4(2), there is a gap between the substrates, which can cause liquid leakage.

[0012] While heat sealing or ultrasonic welding could be considered to fill the gaps between these substrates, these methods have the problem of deforming the microchannels that are formed in the substrates of microfluidic devices. Another option is to use adhesive to fill the gaps between the substrates, but if adhesive is used, it needs to be applied very precisely, and if it is not applied properly, there is a problem that the microchannels will become blocked. Furthermore, when the substrate and membrane materials were different, there was a problem in that it was difficult to obtain sufficient bonding strength unless the bonding was done properly.

[0013] Furthermore, plastic substrates are typically formed by injection molding. The surface of a plastic substrate obtained by injection molding has irregularities on the order of several micrometers due to mold precision and shrinkage during molding. In such microfluidic devices, a gap will be created between the plastic substrate and the membrane. One approach to fill the gap between the plastic substrate and the membrane is to apply pressure in the stacking direction of the microfluidic device. However, compared to soft materials like PDMS, plastic is a rigid material, and therefore cannot deform enough to fill the gaps of several micrometers on the surface of the plastic substrate, resulting in the problem of leakage.

[0014] In this case, Patent Document 1 discloses a microfluidic device using COP for both the substrate and the membrane. In this microfluidic device, since the substrate and membrane are made of the same material, the bonding between the substrate and the membrane is good, but because the membrane is smaller than the flow path, a gap is created between the substrate and the membrane, which could lead to leakage. Furthermore, Patent Document 2 discloses a method of laminating multiple substrates by applying adhesives or tacks to them. However, this method requires high precision in application, and there is a problem that the flow channels will become blocked if the application is not done accurately.

[0015] Furthermore, Non-Patent Document 1 discloses a configuration in which PDMS is used for the substrate and polycarbonate is used for the membrane, and the adhesion of silicone rubber is used for bonding the substrate. However, this method could not solve the bonding problem when the substrate and membrane were not adhesive to each other.

[0016] Therefore, the inventors diligently conducted research and developed a microfluidic device capable of solving the above problem. In other words, the present invention aims to provide a microfluidic device in which a substrate and a membrane can be sufficiently bonded even if they are not adhesive to each other, and a method for using the microfluidic device. [Means for solving the problem]

[0017] To achieve the above object, the microfluidic device of the present invention is a microfluidic device formed by laminating a first plastic substrate, a membrane, and a second plastic substrate. The first plastic substrate has a first flow path, and the second plastic substrate has a second flow path. The first flow path and the second flow path are separated by the membrane. The first plastic substrate, the second plastic substrate, and the membrane are made of plastics that are non-adhesive to each other. The first plastic substrate and the membrane are adhered by a first adhesive sheet, and the second plastic substrate and the membrane are adhered by a second adhesive sheet. A flow path is provided at a position corresponding to the first flow path in the first adhesive sheet, and a flow path is provided at a position corresponding to the second flow path in the second adhesive sheet.

[0018] Also, it is preferable that in the microfluidic device of the present invention, the membrane covers the entire first flow path and the second flow path. Also, it is preferable that in the microfluidic device of the present invention, the first plastic substrate, the first adhesive sheet, and the membrane are configured to include a liquid feeding hole for feeding liquid to the first flow path or the second flow path. Also, it is preferable that in the microfluidic device of the present invention, the first plastic substrate, the first adhesive sheet, the membrane, the second adhesive sheet, and the second plastic substrate are configured to include positioning holes for laminating them.

[0019] Furthermore, it is preferable that in the microfluidic device of the present invention, the first flow path and the second flow path are configured to be used for co-culturing the same or different types of cells. Also, it is preferable that in the microfluidic device of the present invention, the first plastic substrate and the second plastic substrate are made of cycloolefin polymer, polymethyl methacrylate, or cycloolefin copolymer, and the membrane is made of polyethylene terephthalate, polycarbonate, or polytetrafluoroethylene.

[0020] Further, the method of using the microfluidic device of the present invention is a method of injecting a culture medium and cells into the first flow path and the second flow path using the above microfluidic device, and co-culturing the same or different types of cells in the first flow path and the second flow path.

Effect of the Invention

[0021] According to the present invention, it is possible to provide a microfluidic device in which a substrate and a membrane can be sufficiently joined even if they are non-adhesive to each other, and a method of using the microfluidic device.

Brief Description of the Drawings

[0022] [Figure 1] It is a schematic diagram showing a constituent member of the microfluidic device according to an embodiment of the present invention. [Figure 2] It is a schematic diagram showing the configuration of the microfluidic device according to an embodiment of the present invention. [Figure 3] It is a schematic diagram showing a partial cross section of the microfluidic device according to an embodiment of the present invention. [Figure 4] It is a schematic diagram showing a partial cross section of the same device as the constituent member of the conventional microfluidic device.

Modes for Carrying Out the Invention

[0023] Hereinafter, embodiments of the microfluidic device of the present invention and a method of using the microfluidic device will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments.

[0024] The microfluidic device of the present embodiment is a device for forming a biological tissue having a plurality of cell layers composed of adherent cells, and can be used as a so-called organ chip. Specifically, as shown in FIGS. 1 and 2, the microfluidic device of the present embodiment is configured by laminating an upper substrate (first plastic substrate) 11, a membrane 13, and a lower substrate (second plastic substrate) 12.

[0025] The upper substrate 11 is provided with an upper channel (first channel) 111, and the lower substrate 12 is provided with a lower channel (second channel) 121, with the upper channel 111 and the lower channel 121 separated by a membrane 13. The upper substrate 11 and the lower substrate 12, and the membrane 13 are made of non-adhesive plastics.

[0026] In this embodiment, the specific materials of the upper substrate 11, the lower substrate 12, and the membrane 13 are not particularly limited, but for example, COP (cycloolefin polymer) can be used as the material for the upper substrate 11 and the lower substrate 12, and PET (polyethylene terephthalate) can be suitably used as the material for the membrane 13.

[0027] In other words, using PDMS (polydimethylsiloxane) or PET as the substrate presents problems such as the adsorption of low-molecular-weight components onto the substrate and the presence of autofluorescence in the substrate, which may degrade the performance of microfluidic devices. In contrast, the microfluidic device 1 of this embodiment prevents such problems from occurring by using COP for the substrate and PET for the membrane. Furthermore, since the membrane is generally very thin (for example, about 10 μm), the problem of autofluorescence does not occur even when PET is used for the membrane.

[0028] For the upper substrate 11 and lower substrate 12, transparent resins are preferred for observing cells. Besides COP, other materials that can be used for the upper substrate 11 and lower substrate 12 include, for example, PMMA (polymethyl methacrylate) and COC (cycloolefin copolymer).

[0029] Furthermore, it is preferable that the material of the membrane 13 is such that cells adhere to the membrane during cell culture. Besides PET, other materials that can be used for the membrane 13 include, for example, PC (polycarbonate) and PTFE (polytetrafluoroethylene). In addition, it is preferable to perform surface treatment (plasma treatment, collagen treatment) on these materials to improve their cell adhesion properties.

[0030] The upper substrate 11 and the membrane 13 are bonded together by a first adhesive sheet 14, and the lower substrate 12 and the membrane 13 are bonded together by a second adhesive sheet 15. As shown in Figure 1, a channel is provided at the position corresponding to the upper channel 111 in the first adhesive sheet 14, and a channel is also provided at the position corresponding to the lower channel 121 in the second adhesive sheet 15.

[0031] As mentioned above, if a substrate material such as COP, which does not have problems with low molecular weight adsorption or autofluorescence, is used, and a membrane material such as PET is used, the substrate and membrane will not adhere to each other, making it difficult to easily bond the substrate and membrane together. Therefore, in the microfluidic device 1 of this embodiment, the upper substrate 11 and the membrane 13 are bonded together by a first adhesive sheet 14 having a channel at a position corresponding to the upper channel 111, and the lower substrate 12 and the membrane 13 are bonded together by a second adhesive sheet 15 having a channel at a position corresponding to the lower channel 121 in the second adhesive sheet 15, thereby enabling sufficient bonding between the substrate and the membrane.

[0032] In other words, the microfluidic device 1 of this embodiment, by bonding the substrate and the membrane 13 using such a first adhesive sheet 14 and a second adhesive sheet 15, can eliminate the problem of deformation of the microchannels that occurs when bonding by heat sealing or ultrasonic welding, and the problem of blockage of the microchannels that occurs when bonding using an adhesive.

[0033] Furthermore, in this embodiment, it is preferable that the membrane 13 of the microfluidic device 1 covers the entire upper channel 111 and the lower channel 121. In other words, in the conventional microfluidic device shown in Figure 4, the membrane 13 covers only the channel portion that forms biological tissue, which creates a gap between the upper substrate 111 and the lower substrate 120. This gap can cause liquid leakage from the channel near the liquid delivery hole. Figure 4(2) shows a partial cross-section of the center of a conventional microfluidic device, obtained by cutting vertically along the center of the long axis, and illustrates the state in which gaps are present on both sides of the membrane 130 between the upper substrate 110 and the lower substrate 120.

[0034] In contrast, according to the microfluidic device 1 of this embodiment, as shown in Figures 1 and 2, the membrane 13 covers the entire upper channel 111 and the lower channel 121. Figure 3 shows a partial cross-section of the center of the microfluidic device 1 of this embodiment shown in Figure 2, obtained by cutting vertically along the center of the long axis, and shows a state in which there is no gap between the upper substrate 11 and the lower substrate 12. Thus, in the microfluidic device 1 of this embodiment, there is no gap between the upper substrate 11 and the lower substrate 12, and no liquid leakage occurs from the flow path.

[0035] Furthermore, the membrane 13 covers the entire upper channel 111 and the lower channel 121, and the membrane 13 is bonded to each substrate with the first adhesive sheet 14 and the second adhesive sheet 15. By providing channels corresponding to each substrate in these adhesive sheets, the channels are prevented from becoming blocked, and the substrate and the membrane 13 are sufficiently bonded.

[0036] Furthermore, as shown in Figure 1, the microfluidic device 1 of this embodiment preferably has liquid delivery holes in the upper substrate 11, the first adhesive sheet 14, and the membrane 13 for delivering liquid to the upper channel 111 or the lower channel 121. In other words, as shown in the figure, it is preferable to have a liquid delivery hole 112 in the upper substrate 11, and to have liquid delivery holes in the first adhesive sheet 14 and membrane 13 at positions corresponding to this liquid delivery hole 112.

[0037] By configuring the microfluidic device 1 of this embodiment in this way, it is possible to appropriately deliver culture media, reagents, etc., to the upper channel 111 or the lower channel 121 through these liquid delivery holes. In this embodiment of the microfluidic device 1, the shapes of the upper channel 111 and the lower channel 121, and the arrangement of the fluid delivery holes are not limited to Figure 1 and can be changed as appropriate.

[0038] Furthermore, as shown in Figure 1, the microfluidic device 1 of this embodiment preferably has positioning holes (through holes for positioning) in each of the upper substrate 11, first adhesive sheet 14, membrane 13, second adhesive sheet 15, and lower substrate 12 for stacking them. In other words, as shown in the figure, it is preferable to have a positioning hole 113 in the upper substrate 11 and a positioning hole 123 in the lower substrate 12, and to have positioning holes in the first adhesive sheet 14, membrane 13, and second adhesive sheet 15 at positions corresponding to these positioning holes.

[0039] By configuring the microfluidic device 1 of this embodiment in this way, each substrate, each adhesive sheet, and the membrane 13 can be precisely positioned and stacked, resulting in no difference in the cell culture area between microfluidic devices and enabling accurate evaluation. Furthermore, each layer and the microfluidic device 1 can be fixed in place by inserting pins or the like into the positioning holes. In Figure 1, four positioning holes are formed in each layer, but the number of positioning holes is not limited to this; it may be three or fewer, or five or more.

[0040] Furthermore, in this embodiment, it is preferable that the upper channel 111 and the lower channel 121 of the microfluidic device 1 are used for co-culturing the same or different types of cells. The cells cultured using the microfluidic device 1 of this embodiment can be, for example, induced pluripotent stem cells (such as iPS cells) or embryonic stem cells (ES cells). The type of biological tissue to be created is not particularly limited, and various types can be used, such as biological tissue in a proximal tubule model that includes tissue composed of tubular epithelial cells and tissue composed of vascular endothelial cells, as well as biological tissue in glomerular models, small intestine models, liver models, and lung models.

[0041] The method of using the microfluidic device of this embodiment is characterized by using the microfluidic device 1 described above to inject culture medium and cells into the upper channel 111 and the lower channel 121, thereby co-culturing the same or different types of cells in the upper channel 111 and the lower channel 121. With this method of using microfluidic devices, the substrate and membrane are sufficiently bonded even if they are not adhesive to each other, making it possible to properly form biological tissue in each channel. By recreating the tissue structure of organs within a microfluidic device in this way, it becomes possible to precisely control the cell culture environment and favorably express cellular functions that closely resemble those found in vivo.

[0042] As described above, according to this embodiment, it is possible to provide a microfluidic device in which low molecular weights do not adhere to the substrate, the substrate does not self-illuminate, and the substrate and membrane can be sufficiently bonded even if they are not adhesive to each other, as well as a method for using the same.

[0043] The present invention is not limited to the embodiments described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, the shape of the flow channels and the arrangement of the fluid delivery holes and positioning holes in a microfluidic device are not limited to those shown in Figure 1, and can be changed to various other shapes as appropriate. [Industrial applicability]

[0044] The present invention can be suitably used in cases such as forming biological tissue using microfluidic devices. [Explanation of Symbols]

[0045] 1. Microfluidic Devices 11. Upper circuit board (first plastic circuit board) 111 Upper channel (first channel) 112 Liquid feed hole 113 Positioning holes 12 Lower substrate (second plastic substrate) 121 Lower channel (second channel) 123 Positioning holes 13 Membrane 14 First adhesive sheet 141 Channel 15. Second adhesive sheet 151 channel

Claims

1. A microfluidic device comprising a first plastic substrate, a membrane, and a second plastic substrate stacked together, The first plastic substrate is provided with a first channel, and the second plastic substrate is provided with a second channel, and the first channel and the second channel are separated by the membrane. The first plastic substrate, the second plastic substrate, and the membrane are made of plastics that are non-adherent to each other. The first plastic substrate and the membrane are bonded together by a first adhesive sheet, the second plastic substrate and the membrane are bonded together by a second adhesive sheet, a channel is provided in the first adhesive sheet at a position corresponding to the first channel, and a channel is provided in the second adhesive sheet at a position corresponding to the second channel. The membrane covers the entirety of the first channel and the second channel. The first plastic substrate and the second plastic substrate are made of a cycloolefin polymer, and the membrane is made of polyethylene terephthalate. A microfluidic device characterized by the following features.

2. The microfluidic device according to claim 1, characterized in that the first plastic substrate, the first adhesive sheet, and the membrane are provided with liquid delivery holes for delivering liquid to the first channel or the second channel.

3. The microfluidic device according to claim 1 or 2, characterized in that the first plastic substrate, the first adhesive sheet, the membrane, the second adhesive sheet, and the second plastic substrate are provided with positioning holes for stacking them.

4. The microfluidic device according to any one of claims 1 to 3, characterized in that the first channel and the second channel are used for co-culturing the same or different types of cells.

5. A method for using a microfluidic device, characterized by injecting a culture medium and cells into the first channel and the second channel using the microfluidic device described in claims 1 to 4, thereby co-culturing the same or different types of cells in the first channel and the second channel.

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