JP7078918B2 - Biochip and bioassay device and bioassay method - Google Patents

Description

The present invention relates to biochips and bioassay devices and bioassay methods, and more particularly to biochips and bioassay devices and bioassay methods that can be suitably used for cell culture and subsequent bioassays.

In the field of basic research and clinical research, for the purpose of analyzing biological samples such as cells, a method of evaluating the reaction between a biological sample and a specific compound while the biological sample is fixed to a support is used. .. For example, there is known a technique for evaluating the permeability of a component such as lipoic acid in vitro using a cell layer obtained by culturing an intestinal epithelial cell on a permeable membrane in a single layer (for example, Non-Patent Document 1).

The substance permeability test apparatus described in Non-Patent Document 1 includes an inner container and an outer container, and a permeable membrane is arranged at the bottom of the inner container. Then, the Caco-2 cell layer, which is a small intestinal epithelial model, is cultured on the permeable membrane so as to be a single layer, and then a solution containing a permeable sample is introduced into the luminal side of the permeable membrane to the basement membrane side. Measure the concentration of the permeation sample that permeates through.

Further, as a chip on which a biological sample is fixed, a biological functional chip (organ on a chip) such as an organ chip or a tissue chip is also known. Biological function chips are devices that reproduce the functions of organs by creating complex and dynamic microenvironments around cells, and devices that reproduce various organs have been developed. Biofunctional chips are expected to be used for toxicity tests, safety tests, biomarker identification, etc., which are difficult to perform directly on organs.

Conventionally, as an organ chip, a polymer chip provided with a microfluidic and a porous scaffold is known (see, for example, Patent Document 1). FIG. 5 is a schematic diagram showing the polymer chips described in this document. As shown in this figure, the conventional polymer chip 200 includes a porous scaffold 205, 210, a top cover plate 250, and a bottom cover plate 260. Microfluidic channels 230 and 240 are formed between the porous scaffolds 205 and 210 and the top cover plate 250, and microfluidic channels 220 are formed between the porous scaffolds 205 and 210 and the bottom cover plate 260.

Next, an assay using the polymer chip of this document will be described. To simulate the effect of liver metabolism on drug delivery and drug response via bloodstream, hepatocytes and cancer cells are first cultured on a porous scaffold. Specifically, stem cells and cancer cells are seeded in the porous scaffold 205 and the porous scaffold 210, respectively, and after the cells are attached, the medium is injected from the inlet 230 onto the upper surface of the porous scaffold 205. The medium exits the porous scaffold 205 from the lower surface of the porous scaffold 205 and flows into the microfluidic channel 220, flows into the porous scaffold 210, and flows into the microfluidic channel 240 from the upper surface. Subsequently, the reactivity of the cells to the drug is evaluated by delivering the drug through the same flow path as described above.

JP-A-2010-505393 (Claim 1, paragraphs 0033, 0043 to 0049, FIGS. 4, 5, etc.)

Makoto Shimizu et al., "(2) Material Permeation Experiment Method Using Caco-2 Cell Layer", 2008 Ministry of Agriculture, Forestry and Fisheries Subsidy Project (Food Industry Cluster Development Project) Food Functionality Evaluation Manual Collection No. III, Japan Food Science Industry Association

The conventional polymer chip described in Patent Document 1 has a step V1 in which the middle of the microfluidic channel 240 is lowered in the vertical direction. Therefore, the fluid such as the medium or the drug injected from the inlet 230 flows down vertically at the portion of the step V1, so that the flow velocity suddenly increases. Therefore, the cells on the surface of the porous scaffold 205 may be disturbed by the fluid due to the increased flow velocity, and the cells may be separated from the porous body.

An object of the present invention is to provide a biochip in which a biological sample on the surface of a support does not easily come off from the support. Another object of the present invention is to provide a bioassay apparatus and a bioassay method capable of performing a stable bioassay using such a biochip.

The present invention comprises a main body having a surface and an internal space, a support arranged in the internal space and capable of supporting a biological sample, and the main body has an opening provided in a part of the surface. It has a fluid introduction path that communicates from the opening toward the support, and the fluid introduction path has, at least in part, a tapered portion inclined from the surface side of the main body toward the support side. It is a biochip characterized by having.

As described above, since the above configuration includes the tapered portion inclined toward the support, the fluid is less likely to be disturbed and the fluid can be smoothly introduced.

In this case, it is preferable that the tapered portion is inclined at an inclination angle of 10 to 80 degrees with respect to the support.

As described above, when the inclination angle of the tapered portion is within the above range, the fluid can be smoothly introduced into the support.

Further, in the above case, the tapered portion is located on the opening side and is inclined at a first inclination angle with respect to the support, and the tapered portion is more than the first tapered portion. It is preferable to have a second tapered portion that is located on the support side and is inclined at a second inclination angle smaller than the first inclination angle with respect to the support.

As described above, in this configuration, the tapered portion has two stages, the inclination angle of the first tapered portion on the opening side is steep, and the inclination angle of the second tapered portion on the support side is gentler. Therefore, the fluid injected vertically from the opening hits the wall surface of the first tapered portion, the flow velocity is suppressed, and then the fluid moves to the support side through the gentle second tapered portion, so that the tapered portion is 1. It is possible to introduce the fluid into the support more smoothly than in the case of only one.

Further, in the above case, the opening is preferably circular in a plan view shape.

In this way, since the opening is circular, it becomes easy to insert a pipette tip or the like having a circular cross section to introduce a fluid.

Further, in the above case, it is preferable that the tapered portion has a V-shaped or U-shaped cross section in a direction perpendicular to the surface of the support.

As described above, since the cross-sectional shape of the tapered portion is V-shaped or U-shaped, the fluid can be smoothly flowed to the valley bottom along the V-shaped or U-shaped inclination.

Further, in the above case, it is preferable to provide a vent capable of exhausting the gas in the internal space.

In this way, by providing the vent, the gas in the internal space extruded by introducing the fluid can be discharged from the exhaust port.

Further, in the above case, the support is a sheet-shaped membrane, and the membrane is arranged at a position that divides the internal space into a first internal space and a second internal space, and the fluid introduction path. Is preferably communicated with the first internal space.

With this configuration, the membrane divides the space into a first internal space and a second internal space, so that various assays can be performed by introducing a fluid into both spaces.

Further, in this case, it is preferable that the main body includes a second fluid introduction path communicating with the second internal space.

With this configuration, it is possible to introduce a second fluid onto the opposite surface of the membrane and measure the concentration of the substance that has permeated the biological sample.

In the above case, it is preferable that the chip is a biological function chip.

The above biochip is particularly suitable for biological functional chips such as tissue chips and organ chips because the biological sample is not easily disturbed by the fluid.

The present invention is a bioassay apparatus for evaluating a biological sample using the biochip according to any one of the above, in which a medium containing cells is introduced from the fluid introduction path of the biochip to at least the support. It is provided with a cell culturing means for culturing the cells on one surface, and an analysis means for introducing a drug into the cells cultivated by the cell culturing means and analyzing the reaction between the cells and the drug. It is a bioassay device characterized by the above.

Since the biochip is not easily disturbed by the fluid, the above biochip can be suitably used for a bioassay device for cell culture.

The present invention is a bioassay method for evaluating a biological sample using the biochip according to any one of the above, wherein a medium containing cells is introduced from the fluid introduction path of the biochip to at least the support. It comprises a cell culturing step of culturing the cells on one surface, and an analysis step of introducing a drug into the cells cultivated in the cell culturing step and analyzing the reaction between the cells and the drug. It is a bioassay method characterized by the above.

Since the biochip is not easily disturbed by the fluid, the above biochip can be suitably used for cell culture and subsequent bioassay.

According to the present invention, it is possible to provide a biochip in which a biological sample on the surface of the support does not easily come off from the support. Further, according to the present invention, it is possible to provide a bioassay apparatus and a bioassay method capable of performing a stable bioassay using such a biochip.

It is a perspective view of the biochip which concerns on 1st Embodiment of this invention. It is a top view and a bottom view of the biochip of FIG. 1. It is sectional drawing in the arrow direction of the biochip of FIG. It is an enlarged view and the cross-sectional view which magnified the vicinity of an opening. It is a schematic cross-sectional view which shows the conventional biochip.

1. 1. Biochip Hereinafter, the biochip of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view showing a biochip 1 according to the first embodiment of the present invention. 2A is a top view of the biochip 1 of FIG. 1, and FIG. 2B is a bottom view of the biochip 1 of FIG. 3A is a cross-sectional view taken along the line A1-A2 of FIG. 2A, and FIG. 2B is a cross-sectional view taken along the line B1-B2 of FIG. 2B. 4 (a) is an enlarged view showing the region R1 around the opening of FIG. 2 (a) in an enlarged manner, and FIG. 4 (b) is a cross-sectional view taken along the line C1-C2 of FIG. 4 (a) and FIG. 4 (c). ) Is a cross-sectional view taken along the line D1-D2 of FIG. 4 (a).

This embodiment shows a microreactor of an organ-on-a-chip as an example of a biochip. Examples of the biological function chip include, but are not limited to, a tissue chip or an organ chip.

As shown in FIG. 1, the biochip 1 of the present embodiment includes an upper main body 3, a lower main body 5, and a membrane 7. The upper main body 3 and the lower main body 5 correspond to the main body of the present invention, and the membrane 7 corresponds to the support of the present invention.

The upper main body 3 of the present embodiment includes an upper substrate 10 and an upper cover plate 11. As shown in FIG. 2A, the upper substrate 10 is composed of a plate-shaped member having a substantially rectangular shape in a plan view. Further, in the central portion of the upper substrate 10, an upper opening 10a having a rectangular shape in a plan view is formed.

An upper cover plate 11 is provided on the surface of the upper substrate 10. The upper cover plate 11 is a member constituting the surface of the present invention. The upper cover plate 11 of the present embodiment is made of a transparent film-like member. As shown in FIGS. 3A and 3B, the space partitioned by the upper opening 10a of the upper substrate 10, the upper cover plate 11 and the upper surface 7a of the membrane 7 is the upper internal space 3a (first interior). Space).

The lower main body 5 of the present embodiment includes a lower substrate 20 and a lower cover plate 21. As shown in FIG. 2B, the lower substrate 20 is composed of a plate-shaped member having a substantially rectangular shape in a plan view. Further, in the central portion of the lower substrate 20, a lower opening 20a having a rectangular shape in a plan view is formed. The shape and dimensions of the lower substrate 20 are substantially the same as the shape and dimensions of the upper substrate 10.

A transparent lower cover plate 21 is provided on the surface of the lower substrate 20. The shape and dimensions of the lower cover plate 21 are substantially the same as the shape and dimensions of the upper cover plate 11. As shown in FIGS. 3A and 3B, the space partitioned by the lower opening 20a of the lower substrate 20, the lower cover plate 21 and the lower surface 7b of the membrane 7 is the lower internal space 5a (second interior). Space).

Both the upper substrate 10 and the lower substrate 20 can be made of any material. Examples of the material constituting these substrates include plastic, glass, metal, and ceramics. Examples of the plastic include polystyrene (PS), permanox (PMX), polyethylene (PE), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polycarbonate (PC), fluororesin and the like.

Both the upper cover plate 11 and the lower cover plate 21 can be made of any material. Examples of the material constituting these cover plates include plastic, glass, and ceramics. Examples of the plastic include polystyrene (PS), permanox (PMX), polyethylene (PE), polyethylene terephthalate (PET), polycarbonate (PC), fluororesin and the like.

Both the upper cover plate 11 and the lower cover plate 21 of the present embodiment are made of transparent members so that the membrane 7 and the like can be visually recognized. However, the upper cover plate 11 and the lower cover plate 21 are not limited to transparent ones, and may be translucent or opaque. The thicknesses of the upper cover plate 11 and the lower cover plate 21 are usually in the range of 10 μm to 1 mm, preferably in the range of 50 to 500 μm.

The membrane 7 of the present embodiment is a sheet-like member capable of supporting a biological sample. The membrane 7 has an internal space formed by the upper main body 3, the upper cover plate 11, the lower main body 5 and the lower cover plate 21 between the upper main body 3 and the lower main body 5, and the upper internal space 3a and the lower internal space 5a. It is arranged at the position to divide into. The membrane 7 is composed of an upper surface 7a and a lower surface 7b, and a biological sample (not shown) is placed on the upper surface 7a side.

The membrane 7 may be any as long as it can support the biological sample, and the material can be appropriately selected and used according to the type of the biological sample, the purpose of the assay, and the like. For example, when the biochip 1 is a biofunctional chip, polycarbonate, cellulose, collagen and the like can be exemplified as the material of the membrane 7. Examples of the type of the membrane 7 include a porous membrane and a semipermeable membrane. The thickness of the membrane 7 is usually in the range of 0.1 to 100 μm, preferably in the range of 1 to 50 μm. When the membrane 7 is porous, the pore size is usually in the range of 0.01 to 100 μm, preferably in the range of 0.1 to 50 μm.

Next, the openings and flow paths constituting the biochip 1 will be described. The upper cover plate 11 is provided with an opening 12 that penetrates the plate surface. As shown in FIG. 2A, the opening 12 is in the vicinity of one of the four corners of the upper substrate 10 partitioned by the upper internal space 3a, and is closer than the upper internal space 3a of the upper substrate 10. It is provided at the outer position. The opening 12 has a perfect circular shape in top view (same as in plan view in this embodiment). As described above, since the opening 12 has a circular top view shape, an instrument having a circular cross-sectional shape, for example, a pipette tip, is inserted into the opening 12 to introduce a fluid such as a medium or a chemical solution into the opening 12. It's getting easier.

A fluid introduction path 13 is formed from a region of the upper substrate 10 located below the opening 12. As shown in FIG. 3A, one end side (inlet side) of the fluid introduction path 13 communicates with the opening 12 and the other end side (outlet side) communicates with the upper internal space 3a. The fluid introduction path 13 is a flow path for introducing the fluid flowing in from the opening 12 into the upper internal space 3a.

The fluid introduction path 13 of the present invention has, at least in part, a tapered portion (that is, an inclined portion) inclined from the opening 12 side toward the membrane 7 side. Therefore, in the present invention, the biological sample or the like placed on the membrane 7 is less likely to be disturbed by the fluid, as compared with the conventional biochip having a fluid introduction path having a vertical step in the middle, and the membrane. Stable cell culture and bioassay can be performed on 7.

The term "at least a part" as used herein means that the tapered portion may be provided only in a part of the region of the fluid introduction path 13, and all of the tapered portions may be provided. In this embodiment, as will be described later, the first tapered portion 13a and the second tapered portion 13b are inclined in two stages. However, the present invention is not limited to this, and may be provided with only one tapered portion (inclined in one step) or may be provided with three or more tapered portions (inclined in three or more steps).

Further, as shown in FIG. 3A, the fluid introduction path 13 of the present embodiment has a first tapered portion 13a located on the opening 12 side and a second tapered portion located on the upper internal space 3a side. It has 13b. The first tapered portion 13a is tilted with respect to the membrane 7 at a first tilt angle θ1, and the second tapered portion 13b is tilted with respect to the membrane 7 at a second tilt angle θ2. The second tilt angle θ2 is smaller than the first tilt angle θ1 (that is, θ1> θ2).

It is preferable that the first inclination angle θ1 and the second inclination angle θ2 are both inclined at an inclination angle of 10 to 80 degrees with respect to the membrane 7. When the inclination angle is in this range, the inclination is neither too steep nor too gentle, so that the fluid can be stably introduced into the upper internal space 3a.

As described above, the fluid introduction path 13 of the present embodiment is inclined in two stages by the first tapered portion 13a and the second tapered portion 13b. Therefore, the flow velocity of the fluid flowing in from the opening 12 first becomes gentle in the first tapered portion 13a, and then the flow velocity further decreases in the second tapered portion 13b having a gentler inclination than the first tapered portion 13a. It becomes loose and is introduced into the upper internal space 3a. Therefore, as compared with the case where there is only one tapered portion, the fluid introduction path 13 of the present embodiment weakens the flow velocity of the fluid in two steps, so that the biological sample or the like placed on the membrane 7 is more dependent on the fluid. It is less likely to be disturbed and more stable cultures and assays can be performed.

Further, as shown in FIGS. 4 (b) and 4 (c), in the present embodiment, the first tapered portion 13a has a U-shaped cross-sectional shape, and the second tapered portion 13b crosses. The surface shape is V-shaped. As described above, since the cross-sectional shape of the tapered portion is V-shaped or U-shaped, the fluid can be smoothly flowed to the valley bottom along the V-shaped or U-shaped inclination. Further, if the cross-sectional shape of the tapered portion is V-shaped or U-shaped, the fluid can flow smoothly along the inclined valley bottom. Further, since the cross-sectional shape of the first tapered portion 13a is U-shaped, it is easy to insert the tip portion such as a pipette tip having a round tip shape into the fluid introduction path 13 from the opening 12.

Next, the ventilation holes 18 and the ventilation passages 19 will be described. A vent 18 penetrating the plate surface is provided at a position that is point-symmetrical to the position where the opening 12 is provided with respect to the center point in the plan view of the upper cover plate 11. As shown in FIG. 2A, the vent 18 is located diagonally near the corner where the opening 12 of the four corners of the upper substrate 10 partitioned by the upper internal space 3a is provided. It is provided at a position outside the upper internal space 3a of the upper substrate 10. The vent 18 has a perfect circular shape when viewed from above.

A ventilation path 19 is formed from a region of the upper substrate 10 located below the ventilation port 18. As shown in FIG. 3A, one end side (outlet side) of the ventilation passage 19 communicates with the ventilation port 18, and the other end side (inlet side) communicates with the upper internal space 3a. The ventilation path 19 is a path for exhausting the gas in the upper internal space 3a from the ventilation port 18. It is also possible to sample the fluid in the upper internal space 3a from the vent 18.

As shown in FIG. 3B, the upper cover plate 11 is provided with an upper fluid inlet 14 and an upper fluid outlet 16 penetrating the plate surface. As shown in FIG. 2A, the upper fluid inlet 14 is located near the short side of the four sides of the upper substrate 10 partitioned by the upper internal space 3a and on the side where the opening 12 is provided, and at the upper part. It is provided at a position outside the upper internal space 3a of the substrate 10. The upper fluid outlet 16 is provided at a position that is point-symmetrical to the position where the upper fluid inlet 14 is provided, with reference to the center point in the plan view of the upper cover plate 11. Both the upper fluid inlet 14 and the upper fluid outlet 16 have a perfect circular shape when viewed from above.

As shown in FIG. 3B, an upper vertical inflow passage 15 and an upper vertical outflow passage 17 are provided in the regions of the upper substrate 10 located below the upper fluid inlet 14 and the upper fluid outlet 16, respectively. There is. The upper vertical inflow path 15 and the upper vertical outflow path 17 are both circular holes penetrating the plate surface in the direction perpendicular to the plate surface of the upper substrate 10.

When the upper substrate 10 and the lower substrate 20 are overlapped with each other, the lower vertical inflow passage 24 and the lower vertical outflow passage 26 are provided in the regions corresponding to the upper fluid inlet 14 and the upper fluid outlet 16, respectively. A lower horizontal inflow path 25 is provided on the lower end side of the lower vertical inflow path 24. The lower horizontal inflow path 25 extends in the direction perpendicular to the lower vertical inflow path 24 and communicates with the lower internal space 5a. A lower horizontal outflow passage 27 is provided on the lower end side of the lower vertical outflow passage 26. The lower horizontal outflow passage 27 extends in the direction perpendicular to the lower vertical inflow passage 24 and communicates with the lower internal space 5a. Both the lower horizontal inflow passage 25 and the lower horizontal outflow passage 27 are provided at positions close to the membrane 7, and in the present embodiment, the thickness from the membrane 7 is 1/5 of the distance from the membrane 7 to the lower cover plate 21. It is thin to the extent. As described above, since the lower horizontal inflow passage 25 and the lower horizontal outflow passage 27 are provided at positions close to the membrane 7, the air in the lower internal space 5a can be easily discharged.

With such a configuration, when the upper substrate 10 and the lower substrate 20 are overlapped, the fluid introduced into the upper fluid inlet 14 is the upper vertical inflow passage 15, the lower vertical inflow passage 24, and the lower horizontal inflow passage. It passes through 25 and is introduced into the lower internal space 5a. Here, the upper vertical inflow path 15, the lower vertical inflow path 24, and the lower horizontal inflow path 25 correspond to the second fluid introduction path of the present invention. Further, the fluid flowing out from the lower internal space 5a passes through the lower horizontal outflow passage 27, the lower vertical outflow passage 26, and the upper vertical outflow passage 17, and is discharged from the upper fluid outlet 16. With such a configuration, a flow path for introducing and discharging the fluid in the lower internal space 5a is formed.

The biochip of the present invention can be used for various purposes using a biological sample, and is particularly suitable for use as a biological functional chip. Examples of the biological function imitated as a biological function chip include various organs such as lung, heart, small intestine, large intestine, liver, kidney, brain, and blood-brain barrier. Further, the biological sample used in the present invention is not particularly limited, and various biological samples can be used. Examples of biological samples include physiological samples such as cells and organs, as well as biochemical samples such as antibodies and enzymes.

In particular, when a biochip is used as a biological function chip, various biological samples are used depending on the biological function to be imitated. For example, in the case of mimicking the small intestine, examples of the biological sample include caco-2 cells derived from human colon cancer. In addition, as biological samples, human myocardial cells (hCMs), human liver cancer-derived cells (HepG2), human breast cancer-derived cells (MCF-7), normal human umbilical vein endothelial cells (HUVEC), iPS-derived differentiated cells, etc. are used. It can be exemplified.

2. 2. Biochip Manufacturing Method Next, a biochip manufacturing method will be described. The biochip 1 of the present embodiment can be manufactured by various methods depending on the shape, material, and the like of the members constituting the biochip 1. As a method for manufacturing the upper substrate 10, a photolithography method using a photocurable resin as a raw material and forming a desired shape by a photomask and exposure can be exemplified. Further, as another manufacturing method of the upper substrate 10, an injection molding method, an extrusion molding method, or the like in which a molten resin is used as a raw material and the molten resin is injected into a mold having a desired shape can be exemplified. Further, the upper substrate 10 may be manufactured by using a three-dimensional printer or the like. The lower substrate 20 can also be manufactured by the same method.

As a method for manufacturing the upper cover plate 11 and the lower cover plate 21, a method of punching a member processed into a flat plate shape or a film shape at a desired position by punching or the like can be exemplified. Further, the upper cover plate 11 and the lower cover plate 21 can also be manufactured by a method such as a photolithography method.

The upper main body 3 can be manufactured by bonding the upper substrate 10 and the upper cover plate 11 by a known bonding method. Similarly, the lower main body 5 can be manufactured by bonding the lower substrate 20 and the lower cover plate 21 by a known bonding method. Next, the membrane 7 is sandwiched between the upper main body 3 and the lower main body 5, and the upper main body 3 and the lower main body 5 are bonded to each other by a known bonding method to complete the biochip 1.

3. 3. Bioassay device and bioassay method Next, the bioassay device and bioassay method will be described. The biochip of the present invention can be used in various bioassays, and in particular, it can be suitably used as a biofunctional chip. Hereinafter, a bioassay apparatus and a bioassay method when the biochip of the present invention is used as a biofunctional chip will be described. Specifically, a bioassay device and a bioassay method for evaluating a reaction between a cell and a drug using a specific drug after using the cell as a biological sample and culturing on the membrane 7 will be described.

First, cells are seeded on the upper surface 7a of the membrane 7. The cells can be seeded by introducing the cell suspension from the opening 12 into the upper internal space 3a using a pipette or the like and attaching the cell suspension to the upper surface 7a of the membrane 7. Alternatively, before assembling the biochip 1, a cell suspension may be dropped onto the upper surface 7a of the membrane 7 in advance for seeding.

Next, the medium is injected through the opening 12. At this time, since the top view of the opening 12 is circular, it is preferable to use a pipette tip having a circular cross-sectional shape because the tip of the tip can be easily inserted into the opening 12. The injected medium passes through the fluid introduction path 13 and is introduced into the upper internal space 3a, but the flow velocity is relaxed at the two-step tapered portion of the first tapered portion 13a and the second tapered portion 13b. Therefore, the cells seeded on the upper surface 7a of the membrane 7 are less likely to be disturbed by the medium, and stable cell culture can be performed.

In particular, when monolayer cells such as small intestinal epithelial cells are cultured on the membrane 7, the cells on the membrane 7 are easily detached from the membrane 7 when the medium is introduced, which makes the culture difficult. However, in the present invention, since the fluid introduction path has a tapered portion, the cells are not easily disturbed by the medium, and the monolayer cells can be stably cultured.

When the medium is introduced into the upper internal space 3a, the air in the upper internal space 3a is pushed out into the ventilation passage 19 and exhausted from the ventilation port 18. The cells are cultured at a predetermined temperature, humidity, and time with the medium filled in the upper internal space 3a. The above step corresponds to the cell culture step of the present invention.

The biochip 1 of the present embodiment can be used as it is in the assay during or after cell culture. For example, the chemical solution is introduced from the opening 12 into the upper internal space 3a. As the chemical solution, one containing a drug and a medium for evaluation of toxicity and the like can be used. When the drug solution comes into contact with the cells, the reaction product is contained in the drug solution. By sampling a part of the chemical solution from the vent 18 and analyzing the components contained in the chemical solution by high performance liquid chromatography (HPLC) or the like, it is possible to analyze in detail the reactants due to the contact between the drug and the cells. can. The above process corresponds to the analysis process of the present invention.

On the other hand, the medium containing no drug is injected from the upper fluid inlet 14 and introduced into the lower internal space 5a through the upper vertical inflow passage 15, the lower vertical inflow passage 24, and the lower horizontal inflow passage 25. When the medium fills the lower internal space 5a, the cells come into contact with the medium via the membrane 7. Of the reactants of the drug and cells in the upper internal space 3a, the components that can permeate the membrane 7 permeate the membrane 7 and mix with the medium in the lower internal space 5a to form the lower horizontal outflow channel 27, the lower vertical outflow channel 26, It is discharged from the upper fluid outlet 16 through the upper vertical outflow passage 17. By analyzing the components contained in the discharged drug solution by HPLC or the like, it is possible to analyze the reactants due to the contact between the drug and the cells. In this embodiment, the cells are seeded only on the upper surface 7a of the membrane 7, but the seeding is not limited to this, and the cells can be seeded on the lower surface 7b or on the upper surface 7a and the lower surface 7b.

The bioassay device may be provided with a reservoir for accommodating the medium or the drug solution, a pump for inflowing / discharging the medium or the drug solution, a computer for controlling the flow rate, or the like. These devices correspond to the cell culture means of the present invention. In addition, the assay of the reaction between the cell and the drug is performed using an analyzer such as HPLC or a spectrophotometer. These devices correspond to the analytical means of the present invention. By using the bioassay device in this way, the assay using the biochip can be fully automated or semi-automated.

The drug used in the bioassay is not particularly limited, and various drugs can be used depending on the purpose of the assay. As the drug, for example, a drug such as an anticancer drug, a hormone, a fluorescent substance and the like can be exemplified.

1 Biochip, 3 Upper body (main body), 3a Upper internal space, 5 Lower main body (main body), 5a Lower internal space, 7 Membrane (support), 7a upper surface, 7b lower surface,
10 Upper substrate, 10a Upper opening, 11 Upper cover plate (surface), 12 Opening, 13 Fluid introduction path, 13a 1st taper, θ1 1st tilt angle, 13b 2nd taper, θ2 2nd Tilt angle, 14 upper fluid inlet, 15 upper vertical inflow channel (second fluid inlet), 16 upper fluid outlet, 17 upper vertical outflow channel, 18 vents, 19 vents, area around R1 opening,
20 lower substrate, 20a lower opening, 21 lower cover plate, 24 lower vertical inflow passage (second fluid introduction passage), 25 lower horizontal inflow passage (second fluid introduction passage), 26 lower vertical outflow passage, 27 lower horizontal Outflow channel,
200 Polymer Chips, 205 Porous Scaffolds, 210 Porous Scaffolds, 220 Microfluidic Channels, 230 Inlets, 240 Microfluidic Channels, 250 Top Cover Plates, 260 Bottom Cover Plates, V1 Steps

Claims (9)

The main body with surface and internal space,
A support that is placed in the internal space and can support a biological sample is provided.
The support is a sheet-shaped membrane, and the membrane is arranged at a position that divides the internal space into a first internal space and a second internal space.
The main body is
An opening provided in a part of the surface, a first fluid introduction path communicating from the opening to the first internal space, a vent, and the first internal space and the vent. The ventilation channels that communicate with each other,
A fluid inlet provided at a position different from the opening, a second fluid introduction path communicating from the fluid inlet to the second internal space, a fluid outlet, the second internal space, and the fluid outlet. With an outflow channel that communicates with ,
The first fluid introduction path has, at least in part, a tapered portion inclined from the surface side of the main body toward the support side.
The tapered portion is a biochip characterized in that the cross-sectional shape of the support in the direction perpendicular to the surface is V-shaped or U-shaped. The biochip according to claim 1, wherein the tapered portion is inclined at an inclination angle of 10 to 80 degrees with respect to the support. The tapered portion includes a first tapered portion that is located on the opening side and is inclined at a first inclination angle with respect to the support.
It is characterized by having a second tapered portion that is located closer to the support than the first tapered portion and is inclined at a second tilt angle smaller than the first tilt angle with respect to the support. The biochip according to claim 1. The biochip according to claim 1, wherein the opening is circular in a plan view shape. The biochip according to claim 1, wherein the biochip is a biological function chip. A bioassay device for evaluating a biological sample using the biochip according to claim 1.
A cell culture means for introducing a medium containing cells from the first fluid introduction path of the biochip and culturing the cells on at least one surface of the support.
A bioassay apparatus comprising: an analytical means for introducing a drug into the cells cultured by the cell culture means and analyzing the reaction between the cells and the drug. The cell culture means cultivates the cells on the surface of the support on the first internal space side, introduces the drug from the first fluid introduction path, and introduces the drug from the second fluid introduction path. Inject a medium that does not contain
The analytical means is characterized in that, among the reactants of the drug and the cells in the first internal space, the components that have permeated through the membrane and mixed with the medium in the second internal space are analyzed. The bioassay apparatus according to claim 6. A bioassay method for evaluating a biological sample using the biochip according to claim 1.
A cell culture step of introducing a medium containing cells from the first fluid introduction path of the biochip and culturing the cells on at least one surface of the support.
A bioassay method comprising an analysis step of introducing a drug into the cells cultured in the cell culture step and analyzing the reaction between the cells and the drug. In the cell culture step, the cells are cultured on the surface of the support on the first internal space side, the drug is introduced from the first fluid introduction path, and the drug is introduced from the second fluid introduction path. Inject the non-contained medium and
The analysis step is characterized in that, among the reactants of the drug and the cells in the first internal space, the components that have permeated through the membrane and mixed with the medium in the second internal space are analyzed. The bioassay method according to claim 8.

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