JP2007020486A - Microchip and method for evaluating cell using the microchip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchip capable of accurately and simply reproducing and evaluating various interactions between cells through a cellular secretory substance outside a living body, and to provide a method for evaluating a cell using the microchip. <P>SOLUTION: This microchip includes a first unit chip having a first base plate on which a first cell holding part for holding the cell and a first channel for circulating a solution in the first cell holding part are formed and a second unit chip having a second base plate on which a second cell holding part for holding the cell and a second channel for circulating the solution in the second cell holding part are formed, wherein the first unit chip and the second unit chip are detachably and attachably joined to each other, so as to make the first channel and the second channel communicate with each other. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microchip for evaluating cells and a cell evaluation method using the microchip, and more particularly to evaluation of cell-cell interaction via a cell secretory substance.

  Human and animal living bodies are composed of various types of cells, and these various cells perform various functions while performing complex interactions with each other in the living body.

  For example, when a living body is infected with bacteria or viruses, certain immune system cells (eg, lymphocytes) present in the blood sense the infection and send a specific substance (eg, cytokine) to the blood. Secreted in. Then, when other types of immune system cells that have come into contact with the secretory substance are activated, bacteria and viruses are eliminated.

  Further, for example, in the process of wound healing, a cell that senses the occurrence of a wound secretes a specific substance (for example, a growth factor). Then, by promoting the growth of cells around the wound site in contact with the secretory substance, the wound site is repaired and regenerated.

  As described above, in vivo, complex and sophisticated cell-cell interactions are performed through various substances secreted by various cells.

  Therefore, if the mechanism of cell-cell interaction via cell-secreting substances can be reproduced in vitro, the mechanism will be elucidated to develop new drugs and produce cells used in so-called regenerative medicine. It is expected that this can be done efficiently.

As a method for evaluating the interaction between cells via such a cell secretory substance, conventionally, for example, a membrane made of PTFE (Poly Tetra Fluoro Ethylene) having a large number of micropores formed in a predetermined culture vessel is used. There was a method of culturing two types of cells through the medium (for example, Non-Patent Document 1).
S. Murakami, H .; Ijima, T .; Ono, K .; Kawakami; The International Journal of Artificial Organs, Vol. 27, no. 2, 118-126 (2004)

  However, in the conventional evaluation method, since two types of cells are physically separated by a membrane having a predetermined thickness, a substance secreted by cells cultured on one side of the membrane can be separated on the opposite side of the membrane. It was difficult to efficiently contact the cultured cells.

  Further, in the conventional evaluation method described above, the number of cells that can be cultured at one time through the membrane is limited to two types, and therefore complicated by the combination of various cells that are actually performed in vivo. It has been difficult to reproduce and evaluate various cell-cell interactions.

  The present invention has been made in view of the above problems, and a microchip capable of accurately and simply reproducing and evaluating various cell-cell interactions via cell secretory substances in vitro and the microchip One of the objects is to provide a cell evaluation method using the.

  In order to solve the above-mentioned conventional problems, a microchip according to an embodiment of the present invention includes a first cell holding unit for holding cells, and a first flow path for circulating a solution through the first cell holding unit. A first unit chip having a first substrate on which is formed, a second cell holding part for holding cells, and a second channel for circulating a solution through the second cell holding part, A second unit chip having a second substrate formed, wherein the first unit chip and the second unit chip are attached to and detached from each other so as to communicate the first channel and the second channel. It is connected to be possible.

  The first cell holding part and the second cell holding part may have a cell adhesive surface.

  In addition, the first unit chip has a first connecting portion in which a flow path communicating with the first flow path is formed, and the second unit chip is formed with a flow path communicating with the second flow path. The first connection part and the second connection part may be directly connected so as to communicate the first flow path and the second flow path. .

  In addition, the connecting member includes a needle-shaped connecting member having a flow path, and one end of the connecting member is punctured into the first substrate, and the other end of the connecting member is punctured into the second substrate. The first flow path and the second flow path may be communicated with each other via a member flow path.

  Further, a plurality of unit chip sets including the first unit chip and the second unit chip may be provided.

  The first substrate and the second substrate may have the same outer shape.

  Moreover, it is good also as a cell being hold | maintained at said 1st cell holding part and said 2nd cell holding part. In this case, the cells held in the first cell holding unit and the cells held in the second cell holding unit may be cells having different attributes.

  The cell evaluation method according to an embodiment of the present invention includes a preparation step of preparing the microchip, and the first cell holding portion of the microchip prepared in the preparation step to the second cell holding portion. A contacting step in which the substance secreted by the cells held in the first cell holding part is brought into contact with the cells held in the second cell holding part by circulating the solution; An evaluation step of evaluating the response of the cells held in the second cell holding portion, which is brought into contact with a substance secreted by the cells held in the cell holding portion.

  Further, in the evaluation step, as a response of the cells held in the second cell holding unit, at least one of the form of the cells, the number of the cells, or the amount of the predetermined substance secreted by the cells is used. It may be evaluated.

  According to the present invention, simulation that accurately and easily reproduces complex cell-cell interactions that are performed in vivo in vitro, and rapid and simple screening of cell secretory substances that cause the cell-cell interactions. It is possible to provide a microchip and a cell evaluation method that enable the above.

  Hereinafter, a microchip according to an embodiment of the present invention and a method for evaluating an interaction between cells using the microchip will be described.

  First, an outline of a microchip according to the present embodiment (hereinafter referred to as the present microchip) will be described.

  One of the features of the present microchip is that a cell unit chip having a substrate on which a cell holding part for holding cells and a channel for flowing a solution through the cell holding part are formed. It is to include more than one. The plurality of cell unit chips included in the present microchip can communicate with each other between the cell holding portions by allowing the flow paths to communicate with each other.

  For this reason, in this microchip, for example, the solution is circulated from the cell unit chip that holds the first cell in the cell holding part to the other cell unit chip that holds the second cell in the cell holding part. The substance secreted into the solution by the first cell can be brought into direct contact with the second cell.

  That is, in this microchip, a substance secreted by a cell held in one cell unit chip among a plurality of cell unit chips is efficiently brought into contact with a cell held in another cell unit chip. Therefore, for example, it is possible to accurately and simply reproduce the interaction between cells via a small amount of a cell secretory substance that is actually performed in a living body.

  Further, one of the features of this microchip is that a plurality of cell unit chips are detachably connected to each other.

  That is, the microchip can be configured by connecting a plurality of cell unit chips prepared separately from each other, and if necessary, the connection of the plurality of cell unit chips can be released and again connected to each other. It can be separated into separate single cell unit chips.

  For this reason, for example, after evaluating a predetermined cell-cell interaction by circulating a solution between a plurality of cell unit chips included in the microchip, a desired cell-cell interaction among the cell unit chips is performed. By selectively separating a part of the cell unit chips in which the cell was observed, or by further connecting another cell unit chip to this microchip, the further cell-cell interaction can be evaluated. .

  That is, since the plurality of cell unit chips included in the microchip can be arbitrarily connected or separated from each other, in the cell evaluation method using the microchip, for example, recombination of the cell unit chips is sequentially performed. By doing so, it is possible to quickly and easily evaluate a chain-cell interaction between a plurality of cells in various combinations.

  Further, for example, the present microchip may have a plurality of unit chip sets in which a plurality of cell unit chips are connected in series, and the plurality of unit chip sets may be arranged in parallel to each other.

  In this case, for example, in each unit chip set included in this microchip, by causing the cell-cell interaction to occur under different conditions, the conditions for reproducing the desired cell-cell interaction and the desired cell-cell interaction Screening relating to the interaction between cells, such as specifying a cell secretory substance involved in the interaction, can be performed quickly and easily.

  Next, specific contents of the microchip will be described with reference to the drawings. FIG. 1 shows an example of a cell unit chip (hereinafter referred to as a type I cell unit chip 1) included in the microchip and a connection for connecting the type I cell unit chip 1 to another cell unit chip. Examples of members (hereinafter referred to as type I connecting members 20) are shown.

  This type I cell unit chip 1 has a cell holding part 11 formed as a bottomed depression on an upper surface 10a of a substrate 10 formed as a plate-like body, one end opened to the cell holding part 11, and the other end An inflow path 12 and an outflow path 13 formed as a bottomed groove opening in a part of the side surface 10b of the substrate 10, an opening on the substrate side surface 10b side of the inflow path 12 and the substrate side surface 10b side of the outflow path 13 The inflow side connection part 14 and the outflow side connection part 15 which were each formed as a hollow with a bottom in each opening part.

  The cell holder 11 of this type I cell unit chip 1 has a cell holder bottom surface 11a used as a culture substrate for culturing cells, and a side surface 11b surrounding the cell holder bottom surface 11a.

  The type I connecting member 20 is molded separately from the type I cell unit chip 1 and has an outer casing 21 that can be fitted to the inflow side connecting portion 14 or the outflow side connecting portion 15 of the type I cell unit chip 1. And a communication path 22 that penetrates the inside of the casing 21.

  The communication path 22 of the type I connecting member 20 is in the state where the type I connecting member 20 is fitted to the inflow side connecting portion 14 or the outflow side connecting portion 15 of the type I cell unit chip 1. Alternatively, it is formed so as to communicate with the inflow path 12 or the outflow path 13 that opens to the outflow side connecting portion 15. Although FIG. 1 shows a rectangular parallelepiped type I connecting member 20, the present invention is not limited to this. For example, it may be formed in a cylindrical shape or the like. The connecting portion 15 is formed so as to be able to fit with this type I connecting member 20.

  The substrate 10 of the type I cell unit chip 1 and the housing 21 of the type I connecting member 20 can be formed of any material depending on the purpose. For example, polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, Synthetic resins such as polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate, polyvinyl, silicone resins such as PDMS (poly (dimethylsiloxane)), synthetic rubbers such as EPDM (Ethylene Propylene Diene Monomer), natural rubber Metal materials such as glass, ceramic, and stainless steel can be suitably used.

  Moreover, it is preferable that at least one of the substrate 10 and the housing 21 has an appropriate elasticity so that high adhesion can be achieved when they are fitted to each other, and is formed of, for example, a silicon-based resin such as PDMS. A thing can be used suitably.

  Further, the transparency of the substrate 10 or the housing 21 may be any of transparent, translucent, or opaque. For example, at least a part of the substrate 10, particularly the cell holding unit bottom surface 11a for holding cells, In order to optically observe the cell, it is preferably transparent or translucent.

  In addition, the cell holding part 11, the inflow path 12, the outflow path 13, the inflow side connection part 14, the outflow side connection part 15, the communication path 22 and the like are arbitrarily selected according to the material of the substrate 10 or the casing 21. For example, drilling using a machining center or the like, optical fine processing using a laser or the like, etching, embossing, or the like is used on the substrate 10 or in the housing 21. Or can be formed at the time of molding the substrate 10 or the casing 21 using injection molding, press molding, stereolithography or the like.

  Further, the cell holding part 11, the inflow path 12, the outflow path 13, the inflow side connection part 14, the outflow side connection part 15 and the like are formed on the surface of the substrate 10 having a predetermined thickness by using the above-described molding method. It can be formed as a bottomed recess or groove having a depth smaller than the thickness, or on the substrate 10, the cell holding part 11, the inflow path 12, the outflow path 13, the inflow side connection part 14, and the outflow side connection part 15. Can be formed by bonding another member separate from the substrate 10 on one side of the substrate 10 to form a bottom surface. In addition, as this bottom member, a plate-like body or a film having a predetermined thickness formed of any material that is the same as or different from that of the substrate 10 can be used.

  Since at least a part of the cell holding portion bottom surface 11a is used as a base material for culturing cells, the cell holding portion bottom surface 11a is formed with surface characteristics and surface shapes suitable for holding cells, for example, as a surface exhibiting cell adhesiveness. It is formed. Here, the surface exhibiting cell adhesion is, for example, a charged state or hydrophilicity / hydrophobicity suitable for cell adhesion in a predetermined solution, or a substance capable of cell adhesion is immobilized. The surface. When cells settle on this cell-adhesive surface in solution and remain in contact with the surface for a predetermined time, for example, the shape of the cell is gradually changed from spherical to relatively flat over time. It changes into a shape and adheres to the surface in a so-called extended state.

  Specifically, the cell-adhesive cell holding portion bottom surface 11a is formed, for example, by forming the cell holding portion 11 as a depression using the molding method as described above, thereby exposing the surface of the substrate 10 exposed as the bottom surface of the depression. Forming itself (ie, the surface of the material itself of the substrate 10) or on the exposed surface as a surface on which a cell adhesive substance or a derivative thereof obtained from a living body or artificially synthesized is immobilized. Can do.

  Here, examples of the cell adhesive substance immobilized on the cell holding portion bottom surface 11a to form the cell adhesive surface include, for example, proteins and sugar chains existing in the cell membrane of cells held on the cell holding portion bottom surface 11a. A substance capable of reversibly or irreversibly binding to a specific cell surface molecule (for example, integrin or sugar chain receptor) can be suitably used.

  That is, as a cell adhesive substance obtained from a living body, for example, a so-called extracellular matrix such as collagen, fibronectin, laminin, proteoglycan, an antibody that recognizes a cell surface molecule as an antigen, or a derivative thereof can be used. For example, a substance obtained by binding an arbitrary functional group or molecular chain to the cell adhesive substance by a predetermined method (for example, covalently binding using a condensation reaction or the like) can be used.

  Examples of the synthesized cell adhesion substance include a specific amino acid sequence exhibiting cell adhesion (for example, arginine / glycine / aspartic acid (so-called RGD) sequence) and a specific sugar chain sequence (for example, galactose side). A compound containing a chain or the like) can be used, and as a derivative thereof, a substance obtained by binding an arbitrary functional group, molecular chain or the like to the cell adhesive substance by a predetermined method can be used.

  As a method for fixing these cell adhesive substances or derivatives thereof on the cell holding portion bottom surface 11a, any method may be used depending on the chemical characteristics of the cell holding portion bottom surface 11a, the cell adhesive material, or the like. For example, by drying an aqueous solution of a cell adhesive substance or the like on the cell holding part bottom surface 11a, or in an aqueous solution of the cell adhesive substance or the like, the functional group of the cell adhesive substance or the like and the cell holding part A cell-retaining substance or the like is removed by causing a chemical reaction (eg, a condensation reaction between a carboxyl group and an amino group) with a functional group on the bottom surface 11a to form a covalent bond. It can be physically or chemically immobilized on the bottom surface 11a.

  In addition, for example, when it is necessary to efficiently supply oxygen or the like to the cells held on the cell holding portion bottom surface 11a, the cell holding portion bottom surface 11a is formed of a material having a relatively high gas permeability. It is preferable to do.

  That is, for example, the substrate 10 itself or the bottom member to be bonded to the substrate 10 as the cell holding unit bottom surface 11a is formed on the cell holding unit bottom surface 11a by using a silicon-based resin such as PDMS. Oxygen or the like in the outside air can be supplied to the cells.

  The cells held in the cell holding part 11 of the microchip are not particularly limited, and the type of animal, organ, tissue, disease, differentiation stage, proliferation ability, expression level of a specific gene, culture substrate, etc. Any one can be selected and used regardless of its attribute, such as adhesiveness to, for example, liver, pancreas, kidney, nerve, skin, blood of animals such as humans, pigs, dogs, rats, mice, etc. Primary cells (including undifferentiated so-called stem cells) collected from organs and tissues such as embryos, embryonic ES cells (Embryonic Stem Cells), established cell lines, or artificial manipulations such as genetic modification thereof Or the like can be used.

  Moreover, in each cell holding | maintenance part 11, the cell of a single attribute among these cells can be hold | maintained, or two or more types of cells from which an attribute mutually differs can be mixed and hold | maintained by arbitrary ratios. .

  In addition, on the cell holding portion bottom surface 11a, cells are two-dimensionally bonded (so-called single layer) and held, or cells are three-dimensionally assembled to form a spherical shape, a rod shape, a tube shape, or the like. The tissue body can be held. Such two-dimensional or three-dimensional cell morphology is, for example, the surface characteristics of the cell holder bottom surface 11a (degree of cell adhesion, regular pattern arrangement of the cell adhesion region, etc.), three-dimensional shape, etc. (predetermined shape) It can be arbitrarily controlled by a regular pattern arrangement of the depressions.

The shape and size of the substrate 10 can be arbitrarily set according to the use and operability of the microchip. For example, when the substrate 10 is formed as a rectangular plate as shown in FIG. In this case, it is preferable to set the range of several mm square to about several tens of cm square. Further, the cell holding portion bottom surface 11a or the cell adhesive surface formed on the cell holding portion bottom surface 11a can be set to any size as long as one or more cells can be held. It is preferable to be in the range of about several tens of μm ² to several tens of cm ² . The depth of the cell holding part 11 (that is, the height of the cell holding part side surface 11b) is preferably in the range of several tens of μm to several mm. In addition, the width and depth of the inflow path 12 and the outflow path 13 are not particularly limited as long as the solution can be circulated in the cell holding unit 11 that communicates with the inflow path 12 and the outflow path 13, for example, in the range of several tens of μm to several mm. Is preferred. If these dimensions are larger than the above preferred range, for example, the size of the microchip becomes too large, so that the operability is reduced, and the cell distribution in the cell holding part 11 and the flow of fluid are affected. Due to the occurrence of bias, there may be a problem that stable cell-cell interaction is not performed. On the other hand, when these dimensions are smaller than the above preferred range, for example, the size of the present microchip is too small, so that the operability is lowered, the processing of the substrate 10 and the like becomes difficult, and the inflow path 12 and the outflow Problems such as an increase in fluid flow resistance in the passage 13 and the like may occur.

  In FIG. 2, the present micro, which is configured by connecting two type I cell unit chips (hereinafter referred to as a first unit chip 1 </ b> A and a second unit chip 1 </ b> B, respectively) via a type I connecting member 20. An example of a chip is shown. In the following description, when a plurality of similar parts are indicated, for example, 1A and 1B are distinguished by attaching different uppercase alphabets to the same numeral, but it is not necessary to distinguish between them. Only numbers are shown.

  3 shows the microchip along the SS line that cuts the cell holding part 11, the inflow path 12, the outflow path 13, and the type I connecting member 20 of the type I cell unit chip 1 shown in FIG. A cross-sectional view is shown.

  In the microchip shown in FIGS. 2 and 3, the type I connecting member 20 straddles the substrate 10A of the first unit chip 1A and the substrate 10B of the second unit chip 1B, and flows out of the first unit chip 1A. The first unit chip 1A and the second unit chip 1B are detachably connected to each other by being press-fitted into the connecting part 15A and the inflow side connecting part 14B of the second unit chip 1B. Further, the outflow path 13A of the first unit chip 1A and the inflow path 12B of the second unit chip 1B communicate with each other through the communication path 22 of the type I connecting member 20.

  As shown in FIG. 3, the cell holding portion bottom surfaces 11aA and 11aB are formed with a step so as to be positioned below the inflow channel bottom surfaces 12aA and 12aB and the outflow channel bottom surfaces 13aA and 13aB. That is, the cell holding part 11 is formed as a bottomed depression that is further recessed from the inflow path 12 and the outflow path 13.

  As shown in FIG. 3, the microchip has a canopy 16 that covers the substrate upper surface 10 a (see FIG. 1) and the type I connecting member 20. The canopy 16 is formed separately from the substrate 10 and the type I connecting member 20 and is fitted to a part of the type I connecting member 20 to cover the cell holding part 11, the inflow path 12, and the outflow path 13. ing.

  The canopy 16 can be formed of any material according to the purpose. For example, polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, polymethacrylate A plate-like body or film molded using a synthetic resin such as polyvinyl, a silicon-based synthetic resin such as PDMS, a synthetic rubber such as EPDM, a natural rubber, a metal material such as glass, ceramic, or stainless steel. Can be used. The canopy 16 is not shown except for a sectional view.

  4 and 5 show another example of the type I connecting member 20 included in the microchip. The type I connecting member 20 shown in FIG. 4 straddles the substrate 10A of the first unit chip 1A and the substrate 10B of the second unit chip 1B, and the outflow side connecting portion 15A of the first unit chip 1A and the second unit chip 1B. And the top cover 16B of the first unit chip 1A and the top cover 16B of the second unit chip 1B are in contact with the two top covers 16A and 16B. Further, the type I connecting member 20 shown in FIG. 5 is fitted to the two canopies 16A and 16B from above as in the example shown in FIG. 4, but the first unit chip 1A and the second unit chip 1B are different from each other. Not mated.

  FIG. 6 shows another example of the cell unit chip included in the microchip (hereinafter referred to as type II cell unit chip 3). As shown in FIG. 6, this type II cell unit chip 3 is an example of a cell unit chip having three or more flow paths 32, 33, 36 communicating with one cell holding unit 31. That is, this type II cell unit chip 3 has one end connected to the cell holding part 31 in addition to the inflow path 32, the inflow side connecting part 34, the outflow path 33, and the outflow side connecting part 35 communicating with the cell holding part 31. It has a bottomed groove recovery channel 36 formed in the substrate upper surface 30a as a third channel that communicates with the other end and opens to a part of the substrate side surface 30b. Further, the substrate side surface 30b of the recovery channel 36 is provided. The opening on the side has a recovery side connecting portion 37 formed in the same shape as the inflow side connecting portion 34 and the outflow side connecting portion 35.

  In this type II cell unit chip 3, for example, a solution is caused to flow from the inflow path 32 to the cell holding unit 31, and the solution is allowed to flow out from the outflow path 33. By flowing the solution out of the flow path 36 or by flowing the solution into the cell holding part 31 from both the inflow path 34 and the outflow path 35 and outflowing the solution from the recovery flow path 36, the cell holding part 31 The solution or cells inside can be recovered.

  Next, an example of a cell evaluation method using the microchip (hereinafter referred to as the evaluation method) will be described. Here, two types of cells having different attributes (hereinafter referred to as a first cell X and a second cell Y, respectively) are used depending on a substance secreted by the first cell X (hereinafter referred to as a stimulating substance P). An example will be described in which the culture conditions under which the second cells Y are activated are screened and only the second cells Y activated by the stimulating substance P are selectively recovered.

  FIG. 7 shows an example of the present microchip used in the present evaluation method. That is, as shown in FIG. 7, this microchip includes a type I cell unit chip 1 (hereinafter referred to as the first unit chip 1 in this example) and a type II cell unit chip 3 (hereinafter referred to as the first unit chip 1 in this example). Two unit chips 3) have three unit chip sets that are connected in series so that they can communicate with each other through a type I connecting member 20 and are detachable. Chip sets are arranged in parallel with each other. Note that a stopper 23 that closes the opening of the recovery flow path 36 is press-fitted into the recovery-side connecting portion 37 (see FIG. 6) of each second unit chip 3 in order to prevent the solution from flowing out of the recovery flow path 36. Has been.

  This evaluation method includes a unit chip preparation step, a pre-culture step, a unit chip connection step, a cell stimulation step, a cell evaluation step, a unit chip separation step, and a post-separation processing step.

  In the unit chip preparation step, a plurality of cell unit chips, which are used to configure the present microchip and are molded separately from each other, are prepared. That is, in this unit chip preparation step, three first unit chips 1 and three second unit chips 3 constituting the present microchip shown in FIG. 7 are prepared.

  Here, the first cell holding portion bottom surface 11a (see FIG. 1) of each first unit chip 1 and the second cell holding portion bottom surface 31a (see FIG. 6) of each second unit chip 3 are attached to the cells to be held. It has a suitable surface. That is, at least a part of each first cell holding part bottom surface 11a has cell adhesion suitable for adhesion of the first cell X, and at least a part of each second cell holding part bottom surface 31a adheres to the second cell Y. Cell adhesion suitable for In addition, the cell adhesiveness of these 1st cell holding | maintenance part bottom faces 11a and the cell adhesiveness of the 2nd cell holding | maintenance part bottom face 31a may be the same, and may mutually differ.

  In the pre-culture process, cells are held in each cell unit chip prepared in the unit chip preparation process. That is, in this pre-culture step, for example, a predetermined amount of a solution in which the first cells X are dispersed at a predetermined density is placed in each first cell holding unit 11 and the second cells Y are dispersed at a predetermined density. Of the first cell X and the second cell Y under a predetermined condition (for example, in an atmosphere of 5% carbon dioxide / 95% air, humidity 100%, 37 ℃), pre-culture is held for a predetermined time. In this preculture, the first cells X adhere to the cell adhesive surface of the first cell holding portion bottom surface 11a, and the second cells Y adhere to the cell adhesive surface of the second cell holding portion bottom surface 31a.

  In this microchip, as a solution for dispersing cells or circulating in the cell holders 11 and 31, appropriate salts and nutrients necessary for maintaining the survival state and function of the cells are appropriately used. An arbitrary solution such as an aqueous solution containing a concentration can be appropriately selected and used. For example, a culture solution obtained by adding an antibiotic or the like to a basal medium such as DMEM (Dulbecco's Modified Eagle's Medium) or a so-called physiological Saline solution or the like can be used. Moreover, arbitrary growth factors (for example, epidermal growth factor, nerve growth factor, etc.), cytokines (for example, interleukin, interferon, etc.) etc. can also be added and used for this solution.

  In addition, this pre-culture is performed for each cell unit chip 1, 3 while circulating the solution through the inflow channels 12, 32 and the outflow channels 13, 33 in the cell holders 11, 31 holding the cells. Alternatively, it can be carried out by a so-called batch method without circulating the solution. In the case where the pre-culture is performed in a batch mode, for example, the inflow side connecting portion 34, the outflow side connecting portion 35, and the recovery channel 36 of each cell unit chip 1 and 3 are provided with a stopper 23 as shown in FIG. Can be prevented from leaking out of the cell unit chips 1 and 3 from the inflow channels 12 and 32, the outflow channels 13 and 33, and the recovery channel 36.

  Further, this pre-culture can be performed for each unit chip set including a plurality of cell unit chips, or can be performed after the present microchip in which a plurality of unit chip sets are connected to each other is configured. That is, this pre-culture, for example, after configuring three unit chip sets in which the first unit chip 1 and the second unit chip 3 are connected one by one in the unit chip connecting step described later, Alternatively, as shown in FIG. 7, this can be performed in the present microchip in which three unit chip sets are arranged in parallel.

  In this pre-culture, cells such as culture time, presence / absence of solution flow, operation conditions such as solution composition, cell type, density, culture form (two-dimensional monolayer, three-dimensional tissue, etc.) to be retained The culture conditions such as the holding conditions may be the same for all the cell unit chips 1, 3 or the unit chip set, or may be different among at least some of the cell unit chips 1, 3 or the unit chip set.

  In the unit chip connecting step, the plurality of cell unit chips prepared in the unit chip preparing step are detachably connected to each other to constitute the present microchip. That is, in this unit chip connection step, for example, one first unit chip 1 in which the first cells X are pre-cultured in the pre-culture step, and one second unit chip 3 in which the second cells Y are pre-cultured, 7 are manufactured in series, which are detachably connected to each other via a type I connecting member 20, and the three unit chip sets are arranged in parallel to produce the microchip shown in FIG. To do.

  The parallel connection between the plurality of unit chip sets is, for example, along the outer shape of the entire microchip (in the example shown in FIG. 7, the outer shape of the entire plurality of unit chips arranged in parallel to each other). This can be done by preparing a structure having a rectangular frame and placing the plurality of unit chip sets in parallel in the frame-like structure.

  In addition, for example, a plurality of connecting portions (for example, hook shapes that engage or fit with each other) that can be connected to each other are provided on the substrates 10 and 30 of the cell unit chips 1 and 3, and the like. The microchip can also be configured by connecting the connecting portions between the cell unit chips 1 and 3. In this case, for example, the microchip first connects three first unit chips 1 to each other and three second unit chips 3 to each other in parallel, and the three first unit chips 1 connected in parallel to each other. By connecting the three second unit chips 3 connected in parallel with each other in series, or after configuring three unit chip sets, the three unit chip sets are connected in parallel to each other. can do.

  In this unit chip connecting step, for example, when the type I connecting member 20 shown in FIG. 3 is used, the type I connecting member 20 is fitted to the first unit chip 1 and the second unit chip 3. After that, the canopy 16 is put on the top. Further, for example, in the case shown in FIG. 4, the first unit chip 1 and the second unit chip 3 are arranged in series, and after the canopy 16 is installed, the type I connecting member 20 is covered from above, The type I connecting member 20 is fitted to the first unit chip 1, the second unit chip 3, and the canopy 16. Further, for example, in the case shown in FIG. 5, the first unit chip 1 and the second unit chip 3 are arranged in series, and after the canopy 16 is further installed, the type I connecting member 20 is covered from above, The type I connecting member 20 is fitted to the canopy 16.

  Moreover, in this unit chip connection process, in order to distribute | circulate a solution for every unit chip set contained in this microchip, the distribution system containing this microchip is comprised. That is, for example, one end of each first unit chip 1 included in the microchip shown in FIG. 7 is connected to a reservoir holding a predetermined amount of solution, and a part thereof is connected to the pump device. While connecting the connected tube etc., the tube for making the said solution flow out from this microchip is connected with the outflow side connection part 35 of each 2nd unit chip | tip 3. FIG.

  In the cell stimulation step, the solution is circulated through the microchip produced in the unit chip connection step to cause cell-cell interaction. That is, in this cell stimulation step, for each unit chip set included in the microchip, one upstream cell unit chip among the plurality of cell unit chips included in each unit chip set is connected to the other downstream side. By causing the solution to flow through the cell unit chip, the cells secreted into the solution by the cells held in the upstream cell unit chip are brought into contact with the cells held in the downstream cell unit chip. .

  Specifically, for example, in the present microchip shown in FIG. 7, the pump device connected to the tube connected to the inflow side connecting portion 14 of each first unit chip 1 is driven, and is indicated by an arrow in FIG. 7. As described above, a predetermined flow rate of the solution in the reservoir to which the tube is connected from the first inflow path 12 of the first unit chip 1 into the first cell holding unit 11 holding the first cell X. Let it flow in.

  At least a part of the solution that contains the stimulating substance P secreted by the first cell X by the solution that has flowed into the first inflow path 12 from the outside of the microchip and that is held in the first cell holding unit 11, It is pushed out from the first outflow path 13 to the second unit chip 3 through the type I connecting member 20. Then, the solution containing the stimulating substance P flows into the second cell holding part 31 from the second inflow path 32 of the second unit chip 3, so that the stimulating substance P is held in the second cell holding part 31. It comes into contact with the second cell Y. The solution held in the second cell holding unit 31 is supplied from the second outlet channel 33 of the second unit chip 3 to the second unit at a predetermined flow rate at which the solution flows into the first unit chip 1. It flows out of the chip 3 (that is, out of the y-knitted chip set).

  Further, in this cell stimulation step, cells such as the type, density, culture form (two-dimensional monolayer, three-dimensional tissue, etc.) held in the first unit chip 1 or the second unit chip 3 are used. The same or different stimulation conditions such as holding conditions, composition of the solution to be circulated (concentration of nutrients, concentration of dissolved oxygen, etc.) and flow rate, and circulation conditions such as the time for which the solution is circulated, etc. As such, it can be set arbitrarily.

  That is, for example, in the present microchip shown in FIG. 7, the density of the first cells X held in each of the three first unit chips 1 (adhered per unit area of the adhesive surface of the cell holding unit 11). The number of first cells X) is different from each other, and the densities of the second cells Y held in the three second unit chips 3 are set to be the same, and other stimulation conditions are the same. . In this case, in the present microchip, different cell-cell interactions according to the density of the first cells X held in the first unit chip 1 can be realized.

  In the cell evaluation step, the response of the cell to the cell secretory substance is evaluated for at least some of the cells contacted with the cell secretory substance in the cell stimulation step. As the response of the cell, an arbitrary index can be selected. For example, the shape of the cell or cell tissue body such as the outer shape, size, number, etc., the substance expressed in the cell (protein such as enzyme) , Lipids, DNA (Deoxy Ribo Nucleic Acid), RNA (Ribo Nucleic Acid, etc.), types and amounts of substances secreted by cells can be evaluated.

  Specifically, in this cell evaluation step, for example, for each unit chip set included in the present microchip shown in FIG. 7, stimulation held by the second unit chip 3 and secreted by the first cell X in the cell stimulation step About the 2nd cell Y which contacted the substance P, the form is evaluated. That is, for example, this microchip is placed on a sample stage of a microscope apparatus such as a phase-contrast microscope or a fluorescence microscope, and second cells held in the second cell holding unit 31 of the second unit chip 3. The form of Y is optically observed.

  In this cell evaluation step, for example, it is evaluated whether or not the cell to be evaluated shows a predetermined response. That is, for example, when it is known that when the second cell Y is activated, the shape thereof becomes distorted, it is known that the second cell unit chip 3 is provided for each unit chip set. It is observed whether or not the retained second cell Y shows the predetermined morphological change, and whether or not the second cell Y is activated is evaluated.

  Further, for example, when it is known that the activated second cell Y secretes a predetermined substance (hereinafter referred to as response substance Q), the second cell holding the second cell Y is used. The solution in the holding unit 31 flows out of the microchip from the second outflow path 33 of the second unit chip 3 and is collected, and the presence or concentration of the response substance Q contained in the collected solution is evaluated. Thus, it can be determined whether or not the second cell Y is activated.

  In the unit chip separation step, based on the evaluation results obtained in the cell evaluation step, a plurality of unit chip sets included in the microchip or a part of the cell unit chips 1 and 3 included in each unit chip set are separated. The target is determined and separated from the microchip.

  That is, for example, in the cell evaluation step, of the second cells Y held by the three second unit chips 3A, 3B, 3C constituting the microchip shown in FIG. A predetermined morphological change indicating an activated state is observed in the second cell Y held in the cell holding part 31A and the second cell Y held in the second cell holding part 31B of the second unit chip 3B. In the case of being performed, the two second unit chips 3A and 3B in which the activated second cells Y exhibiting the predetermined morphological change are held, or the two second unit chips 3A and 3B are included. Separate one unit chip set from the microchip.

  In the post-separation processing step, predetermined processing is performed on the cell unit chips 1 and 3 or the unit chip set separated in the unit chip separation step. That is, in this post-separation processing step, for example, the activation is performed from the two second unit chips 3A and 3B selectively retained in the unit chip separation step and holding the activated second cells Y. Collect the second cells Y.

  In this case, for example, as shown in FIG. 8, the stopper 23 (see FIG. 7) is removed from the collection-side connecting portion 37 of the second unit chip 3 separated from the present microchip shown in FIG. 7 as a single cell unit chip. As shown by the arrows in FIG. 8, the second cell Y is detached from the second cell holding portion bottom surface 31 a into the second cell holding portion 31 from both the second inflow passage 32 and the second outflow passage 33. A recovery solution (for example, a solution containing a proteolytic enzyme such as trypsin) is allowed to flow, and the recovery solution is allowed to flow out of the recovery flow path 36, whereby the recovery solution is dispersed in the recovery solution. The second cell Y is collected.

  In this post-separation processing step, for example, at least a part of the present microchip used in the cell evaluation step, the cell unit chips 1 and 3 or the unit chip set separated in the unit chip separation step, By additionally connecting the unit chips, the microchip can be reconfigured, and further cell evaluation can be performed using the reconfigured microchip.

  That is, for example, among the second cells Y evaluated to be activated in the cell evaluation step, the third cells Z (cells having different attributes from the first cells X and the second cells Y) are activated. A substance secreting a substance (hereinafter referred to as stimulating substance R) is specified, and the specified second cell Y is selectively recovered.

  In this case, for example, in each of the two second unit chips 3A and 3B holding the activated second cells Y separated and activated in the unit chip separation step, a third unit chip holding the third cell Z (see FIG. Two unit chip sets formed by connecting the second unit chip 3 and the third unit chip in series with each other in series are connected in parallel to each other to reconfigure the microchip. To do.

  And, for example, in each unit chip set included in the reconfigured microchip, the solution is circulated from the second unit chip 3 to the third unit chip in the same manner as in the cell stimulation step described above, and the second cell Y After the secreted stimulating substance R is brought into contact with the third cell Z, whether or not the third cell Z is activated is evaluated in the same manner as in the cell evaluation step described above.

  As a result, for example, when a predetermined activation state is observed in the third cell Z in only one third unit chip among the two third unit chips included in the reconfigured microchip, The second unit chip 3 connected to the third unit chip holding the activated third cells Z is separated from the microchip, and the second unit chip 3 held by the separated second unit chip 3 is separated. Cells Y can be collected.

  That is, in this case, a stimulus secreted by the first cell X by realizing a chain-cell interaction between the first cell X, the second cell Y, and the third cell Z in this microchip. The second cell Y activated by the substance P and having the ability to secrete the stimulating substance R that activates the third cell Z can be selectively recovered.

  As described above, in the cell evaluation method using the present microchip, a plurality of cell unit chips holding cells having different attributes can be appropriately connected to each other and separated from each other. Various cell-cell interactions can be evaluated quickly, accurately and simply.

  In the microchip shown in FIG. 7, since the outer shapes of the cell unit chips 1 and 3 constituting the microchip (in this case, the outer shapes of the substrates 10 and 30) are all the same, they are recombined with each other. Can be performed easily.

  FIG. 9 shows still another example of a cell unit chip included in the microchip (hereinafter referred to as a type III cell unit chip 4). The type III cell unit chip 4 shown in FIG. 9 is provided at the end of the inflow part 42 and the outflow part 43 opposite to the side communicating with the cell holding part 41 (that is, the part opened to a part of the side surface of the substrate 40). Each has an inflow side connection portion 44 and an outflow side connection portion 47.

  The inflow side connection portion 44 and the outflow side connection portion 47 have a communication passage 46 that communicates with the inflow passage 42 and a communication passage 47 that communicates with the outflow passage 43, respectively, and are formed integrally with the substrate 40.

  Moreover, the inflow side connection part 44 and the outflow side connection part 47 are shape | molded by the shape which can be connected mutually. That is, in the example shown in FIG. 9, the inflow side connecting portion 44 has a protruding convex portion 48 at its tip, and the outflow side connecting portion 45 has a recess that can be fitted with the convex portion 46 at its tip. A receiving hole 49 is formed as follows.

  FIG. 10 shows an example of the present microchip in which a plurality of type III cell unit chips 4 are configured by directly connecting the inflow side connection portion 44 and the outflow side connection portion 45 to each other. In the microchip shown in FIG. 10, the receiving hole 49 (see FIG. 9) of the outflow side connection portion 45A of one type III cell unit chip 4A is inserted into the inflow side connection portion 44B of the other type III cell unit chip 4B. When the convex portion 48 (see FIG. 9) is press-fitted, the two type III cell unit chips 4A and 4B are connected to each other so as to allow the solution to flow and to be detachable. In addition, the inflow side connection portion 44 and the outflow side connection portion 45 of the type III cell unit chip 4 may be formed integrally with the substrate 40, or after being formed separately from the substrate 40, the substrate 40. It may be fixed to.

  In addition, the inflow side connection portion 44 and the outflow side connection portion 45 can handle the present microchip integrally in a state where a plurality of type III cell unit chips 4 are connected to each other, for example, Like the substrate 40, the microchip is preferably formed of a material having relatively high rigidity so that the microchip can be carried like a single substrate.

  The present microchip including the type III cell unit chip 4 is formed separately as the type I connecting portion 20 because the inflow side connecting portion 44 and the outflow side connecting portion 45 are provided integrally with the substrate 40. Compared with the case where the connected member is used, operations such as attachment and detachment between the type III cell unit chips 4 can be easily performed.

  FIG. 11 shows still another example of the cell unit chip included in the microchip (hereinafter referred to as a type IV cell unit chip 5), the type IV cell unit chip 5 and other cell unit chips. An example of a connecting member (hereinafter referred to as a type II connecting member 60) formed into a needle shape to connect the two.

  In the type IV cell unit chip 5 shown in FIG. 11, the end of the inflow path 52 and the outflow path 53 opposite to the side that opens to the cell holding portion 51 is not open to the side surface of the substrate 50. The cell holding part 51, the inflow path 52, and the outflow path 53 are formed as one closed recess that communicates with each other.

  The type IV cell unit chip 5 has an inflow side puncture portion 54 having a predetermined thickness between the inflow channel 52 and the side surface of the substrate 50, and between the outflow channel 53 and the other side surface of the substrate 50. Has an outflow side puncture portion 55 having a predetermined thickness.

  On the other hand, the type II connecting member 60 shown in FIG. 11 is formed separately from the type IV cell unit chip 5 and is connected to the inside of a needle-shaped housing 61 with sharpened ends, and a communication path that penetrates the housing 61. 62 is formed.

  FIG. 12 shows an example in which the type IV cell unit chip 5 and the type II connecting member 60 are connected to connect the type IV cell unit chip 5 shown in FIG. 11 and another cell unit chip. That is, as shown in FIG. 12, by piercing and penetrating one needle-shaped tip of the type II connecting member 60 into the outflow side puncture portion 55 (see FIG. 11) of the type IV cell unit chip 5, The communication path 62 of the punctured type II connecting member 60 and the outflow path 53 whose one end is blocked by the outflow side punctured portion 55 communicate with each other so that the solution can flow. As a result, the outflow path 53 of the type IV cell unit chip 5 opens to a part of the side surface of the substrate 50 via the communication path 62 of the punctured type II connection member 60.

  FIG. 13 shows an example of the present microchip in which two type IV cell unit chips 5 are detachably connected in series with each other via a type II connecting member 60. In the present microchip shown in FIG. 13, one end of the type II connecting member 60 is inserted into the outflow side puncture portion 55 (see FIG. 11) of one type IV cell unit chip 5A, and the type II connecting member When the other end of 60 is inserted into the inflow side puncture portion 54 (see FIG. 11) of the other type IV cell unit chip 5B, the outflow path 53A of the one type IV cell unit chip 5A and the other The type IV cell unit chip 5B is in fluid communication with the inflow path 52B of the type IV cell unit chip 5B through the communication path 62 (see FIG. 12) of the type II connection member 60.

  In the microchip shown in FIG. 13, the inflow side puncture portion 54 (see FIG. 11) of one type IV cell unit chip 5A and the outflow side puncture portion 55 (see FIG. 11) of the other type IV cell unit chip 5B 11), type II connecting members 63A and 63B having only one end formed into a needle shape are inserted, and the solution is allowed to flow into the microchip through the type II connecting members 63A and 63B. Or can be drained.

  In the type IV cell unit chip 5, when the type II connecting member 60 inserted into the puncture parts 54 and 55 of the type IV cell unit chip 5 is removed from the puncture parts 54 and 55. As shown in FIG. 11 again, the inflow path 52 and the outflow path 53 return to the state where they are blocked by the puncture portions 54 and 55 (that is, they are not opened on the side surface of the substrate 50).

  That is, the portion to be punctured 54, 55 of the type IV cell unit chip 5 can pierce the type II connecting member 60, and after the type II connecting member 60 once inserted is pulled out. Again, the inflow passage 52 and the outflow passage 53 are formed of a material having moderate elasticity so that the tips of the inflow passage 52 and the outflow passage 53 are blocked. Specifically, the puncture portions 54 and 55 can be formed of an elastomer made of a crosslinked body such as a silicon-based resin, a urethane resin, natural rubber, or synthetic rubber. In this case, the entire substrate 50 (see FIG. 11) can be formed of the elastic material as described above, and only the portions to be punctured 54 and 55 can be formed of the elastic material. That is, for example, by inserting an elastic member molded separately from the substrate 50 into the positions of the punctured portions 54 and 55 of the substrate 50, only the punctured portions 54 and 55 of the substrate 50 are made of an elastic material. Can be formed.

  The portions to be punctured 54 and 55 may be formed of the same material as that of the substrate 50 as a part of the substrate 50, or formed separately from the substrate 50 and using the same or different material as that of the substrate 50. Then, it may be fixed to the substrate 50 so as to block the tips of the inflow path 52 and the outflow path 53.

  As described above, in the present microchip including the plurality of type IV cell unit chips 5 connected by the needle-shaped type II connecting member 60, the connection and separation of the plurality of type IV cell unit chips 5 are held in the cell. It can be easily and repeatedly performed without leaking the solution or cells held in the part 51 or the like and maintaining a sterile state.

  FIG. 14 shows still another example of the cell unit chip included in the microchip (hereinafter referred to as type V cell unit chip 7). A type V cell unit chip 7 shown in FIG. 14 includes a square substrate 70, one cell holding portion 71 formed near the center of the substrate 70, and the cell holding portion 71. And four flow paths 72, 73, 74, 75 extending toward the side surfaces. These four flow paths 72, 73, 74, and 75 are closed at the tip opposite to the side opened to the cell holding part 71, similar to the inflow part 52 and outflow part 53 of the type IV cell unit chip 5 described above. Yes.

  FIG. 15 includes three unit chip sets formed by connecting three type V cell unit chips 7A, 7B, and 7C in series via type II connecting members 60A and 60B, and the three unit chip sets. Shows an example of the present microchip configured by being arranged in parallel with each other. In this microchip, for each unit chip set, as shown by the arrow in FIG. 15, the most upstream type V cell unit chip 7A, the central type V cell unit chip 7B, and the most downstream type V cell unit chip. By causing the solution to flow to 7C, a chain-cell interaction between cells held in each type V cell unit chip 7 can be performed.

  Further, the outer shapes of the plurality of type V cell unit chips 7 constituting this microchip (in this case, the shape of the substrate 70) are all square, and from the cell holding portion 71 of each type V cell unit chip 7 Since three or more (in this case, four) flow paths extend, a part of the plurality of type V cell unit chips 7 included in the microchip is separated, or another part is added to the microchip. The microchip can be reconfigured in various and simple ways, such as by additionally connecting the type V cell units 7.

  Further, as shown in FIG. 16, a needle-shaped type II connecting member 63B is punctured into the substrate 70B of some of the type V cell unit chips 7B constituting the present microchip, and is closed in the distribution state shown in FIG. By opening the one flow path 74B that has been formed in a part of the side surface of the substrate 70B, as shown by an arrow in FIG. 16, a solution, a cell, or the like can be selectively transferred only from the type V cell unit chip 7B. It can be recovered.

  In this case, since the inserted type II connecting member 63B is removed from the type V cell unit chip 7B, the outflow path 74B communicating with the type II connecting member 63B is blocked again. Thereafter, further interaction between cells can be evaluated, for example, through circulation as shown in FIG.

  FIG. 17 shows an example of the present microchip including a connecting member (hereinafter referred to as a type III connecting member 80) that can connect a plurality of type I cell unit chips 1 in parallel to each other. In the present microchip shown in FIG. 17, two type I cell unit chips 1A and 1B are connected in parallel to each other on the upstream side of the type III connecting member 80, and via the type III connecting member 80, Two downstream type I cell unit chips 1C and 1D are connected in parallel to each other downstream of the type III connecting member 80 so as to face the two upstream type I cell unit chips 1A and 1B, respectively. . The upstream type I cell unit chips 1A and 1B and the downstream type I cell unit chips 1C and 1D connected in series via the type III connecting member 80 are connected to the type III connecting member 80. The solutions are in fluid communication with each other via a passage (not shown). By using such a type III connecting member 80, the entire microchip can be handled more integrally, so that the operability is improved.

  FIG. 18 shows an example of the present microchip including a unit chip having no cell holding portion (hereinafter referred to as a flow path unit chip 9). The microchip shown in FIG. 18 has only a flow path for circulating the solution, receives the solution from the two upstream type I cell unit chips 1A and 1B connected to one, and mixes the solution, It has a branched flow path for allowing it to flow out to one downstream type I cell unit chip 1C connected to the other.

  That is, the flow path unit chip 9 includes a first inflow path 91 formed on the substrate 90 so as to communicate with the outflow path 13A of one upstream type I cell unit chip 1A, and the one upstream type I. A second inflow path 92 formed to communicate with the outflow path 13B of the other upstream type I cell unit chip 1B connected in parallel with the cell unit chip 1A, and the first inflow path 91 and the second inflow path 92 and the merging channel 93 formed so as to communicate with the inflow channel 12C of the downstream type I cell unit chip 1C.

  For this reason, in the present microchip shown in FIG. 18, for example, as shown by the arrow in FIG. 18, the first cells are secreted from the first upstream type I cell unit chip 1A holding the first cells. A solution containing the second substance secreted by the second cell from the second upstream type I cell unit chip 1B holding the second cell while allowing the solution containing the first substance to flow into the first inflow passage 91. A downstream type I cell unit that holds a third cell with a mixed solution containing the first substance and the second substance after flowing into the second inflow path 92 and mixing the solution in the merging channel 93 By flowing into the chip 1C, the first substance and the second substance can be brought into direct contact with the third cell at the same time.

  Note that the microchip according to the present invention and the cell evaluation method using the microchip are not limited to those shown in the above examples. That is, for example, the shape, number and arrangement of the cell unit chip and the substrate of the flow path unit chip, the cell holding part, the flow path, etc., and the shape, number, attachment position, etc. of the connecting members and connecting parts that connect the unit chips. Can be set arbitrarily. That is, for example, in the above-described example, the case where the type V cell unit chip 7 having the square substrate 70 is connected by the needle-shaped type II connecting members 60A and 60B has been described. The type I connecting member 20 shown, the inflow side connecting portion 44 and the outflow side connecting portion 45 shown in FIG. 9 and the like, the type III connecting member 80 shown in FIG. It may be connected by a connecting member having a shape. In addition, as a member that connects the unit chips constituting the microchip, for example, a member that can transport a solution such as a tube can be used.

It is a perspective view about an example of a type I cell unit chip and a type I connection member which constitute a microchip concerning one embodiment of the present invention. It is a top view about an example of a microchip containing a type I cell unit chip concerning one embodiment of the present invention. It is sectional drawing along the SS line | wire of the microchip shown in FIG. It is sectional drawing about the other example of the microchip containing the type I cell unit chip | tip which concerns on one Embodiment of this invention. It is sectional drawing about the further another example of the microchip containing the type I cell unit chip | tip which concerns on one Embodiment of this invention. It is a perspective view about an example of the type II cell unit chip which constitutes the microchip concerning one embodiment of the present invention. It is a top view about an example of a microchip including a type I cell unit chip and a type II cell unit chip according to an embodiment of the present invention. It is a top view about an example of a type II cell unit chip according to an embodiment of the present invention. It is a top view about an example of a type III cell unit chip according to an embodiment of the present invention. It is a top view about an example of a microchip containing a type III cell unit chip concerning one embodiment of the present invention. It is a top view about an example of a type IV cell unit chip and a type II connecting member constituting a microchip according to an embodiment of the present invention. It is a top view about an example at the time of connecting a type IV cell unit chip and a type II connection member concerning one embodiment of the present invention. It is a top view about an example of a microchip containing a type IV cell unit chip concerning one embodiment of the present invention. It is a top view about an example of a type V cell unit chip constituting a microchip according to an embodiment of the present invention. It is a top view about an example of a microchip containing a type V cell unit chip concerning one embodiment of the present invention. FIG. 16 is a top view showing a case where a solution or a cell is collected from some type V cell unit chips included in the microchip shown in FIG. 15. It is a top view about an example of a microchip including a type III connecting member according to an embodiment of the present invention. It is a top view about an example of a microchip including a flow path unit chip according to an embodiment of the present invention.

Explanation of symbols

  1, 3, 4, 5, 7 cell unit chip, 9 flow path unit chip, 10, 30, 40, 50, 70, 90 substrate, 11, 31, 41, 51, 71 cell holding unit, 20, 60, 80 Connecting member, 44, 45 connecting portion.

Claims (10)

A first unit chip having a first substrate on which a first cell holding unit for holding cells and a first flow path for circulating a solution through the first cell holding unit are formed;
A second unit chip having a second substrate on which a second cell holding part for holding cells, and a second flow path for circulating a solution in the second cell holding part,
Including
The first unit chip and the second unit chip are detachably connected to each other so as to communicate the first flow path and the second flow path.
A microchip characterized by that. The first cell holding part and the second cell holding part have a cell adhesive surface,
The microchip according to claim 1. The first unit chip has a first connecting portion in which a flow path communicating with the first flow path is formed,
The second unit chip has a second connecting part in which a channel communicating with the second channel is formed,
The first connection part and the second connection part are directly connected so as to communicate the first flow path and the second flow path,
The microchip according to claim 1 or 2, characterized in that: Including a needle-shaped connecting member having a flow path;
One end of the connection member is punctured into the first substrate, and the other end of the connection member is punctured into the second substrate, whereby the first flow path and the first flow path are connected via the flow path of the connection member. Two channels are in communication,
The microchip according to claim 1 or 2, characterized in that: A plurality of unit chip sets including the first unit chip and the second unit chip;
The microchip according to any one of claims 1 to 4, wherein the microchip is characterized in that The first substrate and the second substrate have the same outer shape.
The microchip according to any one of claims 1 to 5, wherein Cells are held in the first cell holding part and the second cell holding part,
The microchip according to any one of claims 1 to 6, characterized in that: The cells held in the first cell holding unit and the cells held in the second cell holding unit are cells having different attributes.
The microchip according to claim 7. A preparation step of preparing the microchip according to claim 7 or 8,
Distributing the solution from the first cell holding part of the microchip prepared in the preparation step to the second cell holding part, the substance secreted by the cells held in the first cell holding part, A contact step of contacting the cells held in the second cell holding unit;
An evaluation step of evaluating a response of a cell held in the second cell holding portion, in which a substance secreted by a cell held in the first cell holding portion is contacted in the contact step;
A cell evaluation method comprising: In the evaluation step, as a response of the cell held in the second cell holding unit, at least one of the form of the cell, the number of the cells, or the amount of the predetermined substance secreted by the cells is evaluated. ,
The cell evaluation method according to claim 9.

Images