JP2012235749A - Method for producing microchip, physical mask, and the microchip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a microchip having a scaffold factor disposed only on the bottom of a recess constituting a sealed chamber, a physical mask used for the method, and the microchip.SOLUTION: The method for producing the microchip 1 includes: a step A of preparing a substrate 2 having a recess that forms a cell culturing chamber 4 and a groove portion that forms a flow passage 11, which are formed on a surface thereof, a physical mask provided with a protruding portion which can be fitted in the recess, and a top plate 3 covering the recess and groove portion, and adsorbing a scaffold factor which promotes adhesion of cells to the surface of the substrate 2 and the recess and groove portion; a step B of fitting the protruding portion of the physical mask into the recess of the substrate 2 and covering at least part of the bottom surface of the recess by the protruding portion to protect the scaffold factor adsorbed to the bottom surface; a step C of removing the scaffold factor adsorbed to the outside of the covered bottom surface; a step D of removing the physical mask fitted into the recess; and a step E of joining the top plate 3 to the surface of the substrate 2 to lid the recess and groove portion.

Description

  The present invention relates to a microchip manufacturing method suitable for analysis using cell culture, a physical mask that can be used in the manufacturing method, and the microchip. More specifically, the present invention relates to a microchip manufacturing method in which a scaffolding factor that promotes cell adhesion is disposed, a physical mask that can be used in the manufacturing method, and the microchip.

With the recent development of microfabrication technology and Lab on a Chip-related technologies, various cell assays can be performed by culturing, analyzing, and manipulating cells in a fine structure configured in an array on a single chip. In addition, methods for efficiently performing cell manipulation have been reported. For example, Non-Patent Document 1 reported by the present inventors and Non-Patent Document 2 reported by an American research group describe a microchip capable of testing the cytotoxicity of a plurality of drugs at once.
Many of the previously reported microchips for cell assays are made of polydimethylsiloxane (PDMS), but the types of cells that can be directly adhered and cultured on PDMS are very limited. In order to realize an assay for various cells on-chip, a technique for constructing a culture environment suitable for individual cells on a micro scale is required. Further, in non-patent document 3 and non-patent document 4, it is often the case that a plurality of differentiation inducing factors composed of a scaffold factor and a humoral factor work in cooperation in the expression of cell functions in a living body. Have been described. Generally, in order to culture primary cultured cells collected from a living body in vitro, it is necessary to culture in a culture environment composed of appropriate scaffolding factors and liquid factors. In addition, in order to induce stem cells to differentiate into specific cells in vitro, it is necessary to cause a number of differentiation inducing factors to act as a complex system. Furthermore, it is reported in Non-Patent Document 5 that the differentiation-inducing ability of ES cells varies greatly depending on the cell line. Thus, the differentiation induction process of stem cells is complicated and diverse, and it is necessary to exhaustively search for the optimal culture environment for differentiating stem cells into specific cells for each cell line. However, in order to search for an optimum environment for culturing individual cells from a huge number of combinations of humoral factors and scaffolding factors, the conventional technology requires very complicated manual work and expensive robots. It was. In addition, it takes time to construct a large number of combinations of liquid factors and scaffolding factors by complicated manual work, and there is also a problem that reliability when a plurality of tests are performed tends to be lowered.

  In order to solve these problems related to cell culture, the present inventors developed a “microculture environment array chip” and reported it in Non-Patent Paper 6. The micro-culture environment array chip is a kind of perfusion culture micro-chamber array chip described in Non-Patent Document 1, in order to introduce a micro-chamber array, a cell suspension, and a medium containing four different humoral factors. The micro flow path and the introduction port are provided. This microchamber array is composed of a microchamber modified with three different cell adhesion proteins at the bottom and a non-modified microchamber. A total of four scaffolding factors are used as scaffolds for cell culture. As offered. Furthermore, by supplying the culture medium into the microchamber, 4 × 4 = 16 culture environments consisting of combinations of four types of humoral factors and four types of scaffolding factors contained in the medium are constructed on the microchamber array. it can. From these multiple culture environments, an environment suitable for cell culture can be screened.

S. Sugiura, J. Edahiro, K. Kikuchi, K. Sumaru, and T. Kanamori, Biotechnol. Bioeng., 100, 1156 (2008). Z. Wang, M.-C. Kim, M. Marquez and T. Thorsen, Lab Chip, 7, 740 (2007). M. A. Schwartz and M. H. Ginsberg, Nat. Cell Biol., 4, E65 (2002). K. M. Yamada and S. Even-Ram, Nat.Cell Biol., 4, E75 (2002). K. Osafune, et al., Nat. Biotechnol., 26, 313 (2008). K. Hattori, S. Sugiura, and T. Kanamori, Biotechnology J., 5, 463 (2010). K. Hattori, S. Sugiura and T. Kanamori, Lab Chip, 11, 212 (2011).

  In order to culture various cells in the chamber provided in the microchip, it is desirable to place a scaffolding factor such as a cell adhesion protein on the inner wall of the chamber. In this case, if a scaffold factor is disposed in a flow path other than the chamber, the cells adhere to the flow path, which causes a problem such as clogging of the flow path. Therefore, in the microchip manufacturing method disclosed in Non-Patent Document 7, when a chamber is formed by joining a top plate having a recess and a smooth substrate covering the recess, patterning of a scaffolding factor is performed by a stencil method. The scaffold factor is limited to the region of the substrate plane that faces the recess of the top plate. According to this method, since the scaffolding factor can be disposed only on the substrate plane constituting the bottom of the chamber, the cell suspension is allowed to flow into the microchip, whereby the bottom of the chamber in which the scaffolding factor is disposed. The cells can be adhered and cultured in a limited manner.

  However, when the above stencil method is used, a complex manufacturing process is performed in which the stencil and the substrate are aligned, the scaffolding factor is patterned, the stencil is peeled, and the top plate on which the substrate and the recess are formed is aligned and then bonded. There is a problem that requires a process. Further, in the above method, a scaffold factor cannot be arranged in the concave portion of the substrate constituting the chamber. The cells adhered to the smooth substrate constituting the bottom of the chamber are liable to receive the flow of liquid flowing through the flow path, and the physical pressure may affect the cell culture. For this reason, development of a microchip capable of culturing cells on the bottom surface of the concave portion constituting the sealed chamber is desired.

  The present invention has been made in view of the above circumstances, and a manufacturing method of a microchip in which a scaffold factor is arranged only on the bottom surface of a concave portion constituting a sealed chamber, a physical mask that can be used in the manufacturing method, and a microchip The issue is to provide

The manufacturing method of the microchip according to claim 1 of the present invention is a manufacturing method of a microchip in which a chamber for cell culture and a flow path connected to the chamber are formed, the recess serving as the chamber and the A substrate having a groove portion to be a flow path formed on the surface, a physical mask provided with a convex portion that can be inserted into the concave portion, and a top plate that covers the concave portion and the groove portion are prepared. Step A for adsorbing a scaffolding factor that promotes cell adhesion to the groove, and inserting the convex portion of the physical mask into the concave portion of the substrate, covering the bottom surface of the concave portion with the convex portion, A step B for protecting the scaffold factor adsorbed on the bottom surface, a step C for removing the scaffold factor adsorbed on a portion other than the covered bottom surface, a step D for removing the physical mask loaded in the recess, and the substrate The top plate is bonded to the surface, characterized in that it comprises a and a step E of the lid in the recess and the groove.
The manufacturing method of a microchip according to claim 2 of the present invention is the method of manufacturing a microchip according to claim 1, wherein the scaffolding factor other than the protected scaffolding factor is obtained by plasma-treating the surface and groove of the substrate in the step C. It is characterized by removing.
According to a third aspect of the present invention, there is provided a microchip manufacturing method according to the second aspect, wherein in the step C, the surface of the substrate and the surface of the top plate are activated by plasma treatment in the step C, and then in the step E, The joining is performed by pressing the activated surfaces together.
The method for producing a microchip according to claim 4 of the present invention is characterized in that, in any one of claims 1 to 3, the scaffold factor contains collagen, fibronectin, laminin, gelatin, or polylysine. .
A physical mask according to a fifth aspect of the present invention is a physical mask used in the method of manufacturing a microchip according to any one of the first to fourth aspects, wherein the physical mask protrudes from a surface of the substrate. At least including the convex portion arranged, the convex portion is columnar, the diameter is smaller than the diameter of the concave portion, the tip of the convex portion is a surface shape that can be in close contact with the bottom surface of the concave portion, The protruding height of the convex portion is longer than the depth of the concave portion.
The physical mask according to claim 6 of the present invention is characterized in that, in claim 5, the material of the physical mask is polytetrafluoroethylene.
The microchip according to claim 7 of the present invention includes a substrate on which a recess serving as a cell culture chamber and a groove serving as a flow channel connected to the chamber are formed, a top plate covering the recess and the groove, And a scaffold factor that promotes cell adhesion is disposed only on the bottom surface of the recess.

According to the microchip manufacturing method of the present invention, a microchip in which a scaffold factor is arranged only at the bottom of the chamber can be manufactured by a simple process.
According to the physical mask of the present invention, the convex portion protruding from the base is inserted into the concave portion serving as the chamber, and the surface of the convex portion is in close contact with the bottom surface of the concave portion, whereby the scaffolding factor at the bottom of the chamber is reduced. It can be physically masked and protected. As a result, the substrate surface other than the masked region can be subjected to physical or chemical treatment, and unnecessary scaffolding factors can be collectively removed from the substrate surface.
According to the microchip of the present invention, a cell suspension is allowed to flow into a sealed and sealed chamber through a flow path so that the cells adhere to the scaffolding factor at the bottom of the chamber and are cultured. Can do. Since the cells can be stably maintained by the scaffold factor, even when another solution is introduced into the chamber, there is no possibility that the cells are detached due to the momentum of the inflow.

It is a top view which shows the whole microchip of the 1st example of this invention. It is the top view to which the principal part of the microchip was expanded. It is sectional drawing to which the principal part of the microchip was expanded. It is a top view which shows the principal part of a microchip. It is typical sectional drawing of the board | substrate used for manufacture of a microchip, a physical mask, and a top plate. It is typical sectional drawing of the board | substrate which modified the surface with the scaffold factor. It is typical sectional drawing which shows the state which inserted the convex part of the physical mask in the recessed part of the board | substrate which modified the surface with the scaffold factor. It is typical sectional drawing which shows a mode that the plasma processing is performed in the state which inserted the convex part of the physical mask in the recessed part of the board | substrate which modified the surface with the scaffold factor. In FIG. 8, it is typical sectional drawing which shows a mode that the plasma processing is performed also about the board | substrate 3 simultaneously. It is typical sectional drawing of the manufactured microchip concerning this invention. FIG. 3 is a schematic cross-sectional view of a fixing device for loading and holding a physical mask 30 on a substrate 2. It is typical sectional drawing of an example of the physical mask concerning this invention. It is a perspective view which shows an example of the manufacturing method of the microchip concerning this invention. It is a perspective view which shows an example of the manufacturing method of the microchip concerning this invention. It is a perspective view which shows an example of the manufacturing method of the microchip concerning this invention. It is a photograph which shows a test result. It is a photograph which shows a test result. It is a photograph which shows a test result.

The present invention will be described below based on preferred embodiments with reference to the drawings.
<< Microchip >>
A microchip 1 which is a first example of the present invention is shown in FIGS.
FIG. 1 is a plan view showing the entire microchip 1, FIG. 2 is an enlarged plan view of the main part of the microchip 1, and FIG. 3 is an enlarged cross-sectional view of the main part of the microchip 1.
In the following description, the “longitudinal direction” of the microchip 1 means the direction of the arrow t in FIG. 1, and the “short direction” of the microchip 1 means the direction of the arrow u in FIG. .

As shown in FIGS. 2 and 3, the microchip 1 can be constituted by a flat substrate 2 and a flat top plate 3 superimposed on the flat substrate 2.
On the surface 2 a (surface 2 a) of the substrate 2 facing the top plate 3, a recess 5 is formed which becomes a chamber 4 in which a sample solution is introduced and cells are cultured.
The concave portion 5 includes a main space concave portion 5a having a circular shape in plan view and an outer peripheral space concave portion 5b formed on the outer peripheral side of the main space concave portion 5a so as to surround the main space concave portion 5a. The outer peripheral space recess 5b is formed shallower than the main space recess 5a.
As shown in FIG. 3, the chamber 4 includes a space in the main space recess 5a (main space portion 4a) and a space in the outer periphery space recess 5b (outer periphery space portion 4b).

As shown in FIGS. 1 and 2, the chambers 4 are two-dimensionally arranged to form a matrix composed of a plurality of rows and a plurality of columns.
In the illustrated example, a total of 64 chamber portions 4 are arranged in an array so as to form a matrix of 8 rows and 8 columns. The chambers 4 constituting the rows are arranged along the longitudinal direction (arrow t direction), and the chambers 4 constituting the columns are arranged along the short direction (arrow u direction). The chambers 4 are arranged at intervals.
Reference numerals 9A to 9H respectively indicate eight rows formed by the chamber section 4, and reference numerals 10A to 10H respectively indicate eight columns formed by the chamber section 4.

On the surface 2a of the substrate 2, an inlet channel 7 and an outlet channel 15 communicating with the recess 5 (chamber 4) are formed.
The inlet channel 7 includes a liquid supply channel 11 formed along the longitudinal direction, and a plurality of introduction channels 12 branched from the liquid supply channel 11 and communicating with the respective chambers 4. Since each of the passages 12 is connected to the chamber portion 4, the sample liquid from the storage portion 21 can be introduced into the chamber portion 4 from the liquid supply passage 11 through the introduction passage 12.

As shown in FIGS. 1 and 4, four inlet channels 7 are formed (referred to as inlet channels 7 </ b> A to 7 </ b> D), and each is connected to a different storage unit 21 (referred to as storage units 21 </ b> A to 21 </ b> D).
The liquid supply flow path 11A of the inlet flow path 7A extends between the first row 9A and the second line 9B, and the liquid supply flow path 11B of the inlet flow path 7B is between the third row 9C and the fourth row 9D. The liquid supply flow path 11C of the inlet flow path 7C extends between the fifth line 9E and the sixth line 9F, and the liquid supply flow path 11D of the inlet flow path 7D is the seventh line 9G and the eighth line. It extends between 9H.
The first introduction flow path 12A branched from the liquid supply flow path 11A of the inlet flow path 7A is connected to the chambers 4 of the first row 9A and the second row 9B, and branched from the liquid supply flow path 11B of the inlet flow path 7B. The second introduction flow path 12B is connected to the chambers 4 of the third row 9C and the fourth row 9D, and the third introduction flow path 12C branched from the liquid supply flow path 11C of the inlet flow path 7C The fourth introduction channel 12D connected to each chamber 4 in 9E and the sixth row 9F and branched from the liquid feeding channel 11D in the inlet channel 7D is connected to each chamber 4 in the seventh row 9G and the eighth row 9H. Has been.

As shown in FIG. 4, in the microchip 1, since the inlet channels 7A to 7D have the above-described configuration, the sample liquid 6A from the reservoir 21A is supplied from the inlet channel 7A to the first row 9A and second row 9B chambers. 4, the sample liquid 6B from the reservoir 21B can be introduced into the chamber 4 of the third row 9C and the fourth row 9D from the inlet channel 7B, and the sample liquid 6C from the reservoir 21C can be introduced into the fifth row 9E and the fifth row 9E. The sample liquid 6D from the reservoir 21D can be introduced into the chambers 4 of the seventh row 9G and the eighth row 9H.
Therefore, a desired sample solution can be introduced into each chamber 4 by introducing a desired sample solution into each reservoir.

As shown in FIG. 1 and FIG. 4, the outlet channel 15 has a plurality of outlet channels 13 communicating with each chamber 4 and an outlet channel 14 to which the plurality of outlet channels 13 are connected. The liquid in the chamber 4 can be discharged from the outlet channel 13 to the discharge unit 22 via the discharge channel 14. The discharge channel 14 is formed along the longitudinal direction.
As shown in FIG. 4, five outlet channels 15 are formed (exit channels 15A to 15E), and the outlet channel 14A of the outlet channel 15A is outside the first row 9A (upper side in FIG. 4). The outlet channel 15B of the outlet channel 15B extends between the second row 9B and the third row 9C, and the outlet channel 14C of the outlet channel 15C is formed of the fourth row 9D and the fifth row. 9D, the outlet channel 14D of the outlet channel 15D extends between the sixth row 9F and the seventh row 9G, and the outlet channel 14E of the outlet channel 15E is the eighth row 9H. It is formed on the outside (lower side in FIG. 4).

  As shown in FIG. 4, the outlet channel 13A of the outlet channel 15A is connected to the chamber 4 in the first row 9A, and the outlet channel 13B of the outlet channel 15B is connected to the second row 9B and the third row 9C. The outlet channel 13C of the outlet channel 15C is connected to the chamber 4, and the outlet channel 13D of the outlet channel 15D is connected to the chamber 4 of the fourth row 9D and the fifth row 9E. The outlet channel 13E of the outlet channel 15E is connected to the chamber 4 of the eighth row 9H.

The lead-out flow path 13 can have a smaller flow resistance than the introduction flow path 12 by making the sectional area larger than the introduction flow path 12 or shorter than the introduction flow path 12. As a result, it is possible to prevent the pressure in the chamber 4 from rising excessively.
In the illustrated example, the introduction channel 12 and the outlet channel 13 communicate with the outer peripheral space recess 5 b of the recess 5. It is preferable that the communication location of the introduction flow path 12 and the discharge flow path 13 is a position facing the main space recess 5a. By setting the positions so as to face each other, it becomes easier to sufficiently diffuse the sample liquid flowing in from the introduction flow path 12 into the chamber 4.

In the microchip 1 according to the present invention, a scaffold factor 8 is disposed on the bottom surface 5c of the recess 5 constituting the chamber 4 (see FIG. 3).
The scaffold factor 8 may be disposed in at least a part of the bottom surface 5c, or may be disposed in the entire region of the bottom surface 5c. Since cells adhere to the region where the scaffolding factor 8 is arranged, the larger the region, the larger the region where cells can be adhered and cultured, which is preferable.

  The bottom surface 5c of the chamber 4 does not necessarily need to be a flat plane, may be a curved surface, and may be provided with a step or a protrusion according to the application. The bottom surface 5c is preferably a shape that fits the surface shape (for example, a flat surface) in order to adhere to the surface shape of the tip of the physical mask described later.

In the recess 5 constituting the chamber 4, the scaffolding factor 8 may be disposed also on the side surface 5 d of the main space recess 5 a or the bottom surface of the outer peripheral space recess 5 b. However, since the culture environment (culture conditions) of the cells adhered to the bottom surface 5c and the culture environment (culture conditions) of the cells adhered to the side surface 5d are strictly different, when it is necessary to align the culture environment more accurately It is preferable to place the scaffolding factor 8 only on the bottom surface 5c.
As described above, in the microchip 1 according to the present invention, since the scaffolding factor 8 is not arranged in the inlet channel 7 and the outlet channel 15, the cells adhere to the channel and clog the channel. Can be prevented.

  The shape of the surface 3a (lower surface 3a) facing the substrate 2 of the top plate 3 constituting the chamber 4 is not particularly limited, and may be flat, for example. If it is flat, it can be easier to observe the inside of the chamber 4.

  As described above, the microchip 1 according to the present invention includes the substrate 2 on which the recess 5 serving as the cell culture chamber 4 and the groove R serving as the flow paths 12 and 13 connected to the chamber 4 are formed. A microchip having at least a top plate 3 that covers the concave portion 5 and the groove portion R, and a scaffolding factor 8 that promotes cell adhesion is disposed only on the bottom surface 5 c of the concave portion 5.

As a method for causing a cell suspension containing cells and a buffer solution to flow into the chamber 4 of the microchip 1, for example, it can be performed as follows.
First, the cell suspension is poured into the storage portion 21 and the discharge portion 22 side is set to a negative pressure, whereby the cell suspension is passed through the liquid supply passage 11 and the introduction passage 12 of the inlet passage 7. It can be pulled into the chamber 4. Next, the cell suspension is retained in the chamber 4, and the cells are settled on the bottom surface 5 c of the chamber 4. Within a predetermined time, the cells adhere to the scaffolding factor 8 disposed on the bottom surface 5c. Subsequently, by setting the discharge part 22 side to a negative pressure again, the buffer solution containing dead cells that have not adhered is discharged from the chamber 4 through the outlet channel 13 and the outlet channel 14 of the outlet channel 15. It can be discharged to the part 22. Furthermore, instead of the cell suspension, a culture solution containing nutrients and the like necessary for cell culture is introduced into the storage unit 21, and the discharge unit 22 side is continuously set to a negative pressure, whereby the bottom surface of the chamber 4 is obtained. Necessary nutrients and the like can be constantly supplied to the cells adhered to 5c. Moreover, the influence which the said chemical | medical agent has on the said cell can be investigated by including various chemical | medical agents in the said culture solution.

Here, a method of flowing the cell suspension through the introduction flow path 12 is shown. As shown in FIG. 2, since the introduction channel 12 is a relatively long and narrow channel, it may be difficult to pass the introduction channel 12 depending on the type and size of cells to be cultured. In this case, the cell suspension may be fed in the opposite direction to the above example, and the cells may be flowed into the chamber 4. That is, the cell suspension can be introduced into the discharge part 22, the storage part 21 can be under negative pressure, and the cell suspension can be drawn into the chamber 4 via the outlet channel 15. After the cells are adhered as in the above example, the buffer solution in which the cells are suspended can be discharged from the inlet channel 7.
Thus, the liquid feeding direction of the microchip 1 can be appropriately adjusted according to the application and purpose.

The microchip according to the present invention is not limited to the configuration of the microchip 1 shown in FIGS. The shape, size and number of the reservoir, the width, depth, path and number of the flow path, the shape, size and number of the chamber, and the like can be appropriately designed according to the use and purpose of the microchip.
As shown in FIG. 3, the chamber 4 of the microchip 1 is provided with an outer peripheral space 4b in addition to the main space 4a. By providing the outer peripheral space portion 4 b, it is easier to discharge all the gas originally filled in the chamber 4 to the outside of the chamber 4 when the liquid flows into the chamber 4.
Therefore, it is preferable that the outer peripheral space 4b is provided in addition to the main space 4 in the chamber of the microchip according to the present invention. Further, the chamber 4 may be configured by only the main space portion 4a without providing the outer peripheral space portion 4b.

In the microchip 1 according to the present invention, since the scaffolding factor 8 is disposed only on the bottom surface 5 c of the recess 5 constituting the chamber 4, it is possible to prevent cells from adhering to the inlet channel 7 and the outlet channel 15. Therefore, by using the microchip of the present invention, the cells to be tested can be adhered to the bottom surface 5c of the recess 5 in which the scaffolding factor 8 is disposed, and a test such as drug screening on the cultured cells can be performed. It can be carried out in a highly reliable culture environment (culture conditions).
One possible mechanism for the adhesion of cultured cells via a scaffolding factor is that adhesion proteins such as integrins and cadherins existing on the cell surface adhere to the scaffolding factor 8.

The cells cultured in the microchip 1 according to the present invention are not particularly limited, and for example, cells derived from animals including humans, cells derived from plants, cells derived from microorganisms and the like can be used according to the purpose.
Specific examples of cells that can be cultured include somatic stem cells such as hematopoietic stem cells, myeloid stem cells, neural stem cells, and skin stem cells, embryonic stem cells, and induced pluripotent stem cells. Also, leukocytes such as neutrophils, eosinophils, basophils, monocytes, lymphocytes (T cells, NK cells, B cells, etc.), platelets, erythrocytes, vascular endothelial cells, lymphoid stem cells, erythroblasts Examples thereof include blood cells such as myeloblasts, monoblasts, megakaryocytes and megakaryocytes, endothelial cells, epithelial cells, hepatocytes, pancreatic islet cells, and the like.

<< Microchip Manufacturing Method >>
A first embodiment of the microchip manufacturing method according to the present invention will be described with reference to FIGS. 5 to 10 are schematic views in which cross sections in the thickness direction of the substrate 2 and the top plate 3 constituting the microchip 1 are simplified.

  The first aspect of the microchip manufacturing method according to the present invention is an example of a manufacturing method of the microchip 1 in which the cell culture chamber 4 shown in FIG. 1 and the flow paths 12 and 13 connected to the chamber 4 are formed. And includes at least the following steps A to E.

First, as shown in FIG. 5, the first concave portion 5 that becomes the chamber 4, and the groove portion R that becomes the introduction channel 12 (inlet channel 7) and the outlet channel 13 (outlet channel 15) are formed on the surface 2 a. A substrate 2, a physical mask 30 provided with a convex portion 31 that can be inserted into the first concave portion 5, and a top plate 3 that covers the first concave portion 5 and the groove portion R are prepared.
In addition to the first recess 5 and the groove R, the substrate 2 is also formed with a second recess 5 ′ serving as a storage portion 21 and a third recess 5 ″ serving as a discharge portion 22. FIG. In order to achieve this, only one first recess 5 is drawn. Needless to say, a plurality of first recesses 5, second recesses 5 ′, third recesses 5 ″, and grooves R are formed as necessary. It's okay.
Further, the top plate 3 is provided with a through hole T at a position corresponding to the second recess 5 ′ and the third recess 5 ″.

  As the substrate 2, a substrate manufactured by a known method can be used. When the substrate 2 is made of resin, the substrate 2 in which the first to third recesses 5, 5 ′, 5 ″ and the groove portion R are collectively formed can be manufactured by, for example, a replica molding method. For example, a resist mask is formed by photolithography on the surface of a flat plate made of a material such as resin, glass, or silicon, and the first to third recesses 5, 5 ′, 5 are formed by an etching method such as RIE (Reactive Ion Etching). By forming the grooves R, the substrate 2 can be manufactured. In addition, methods such as laser processing, NC processing, optical modeling processing, injection molding processing, and nanoimprint processing can be appropriately used.

  The material of the board | substrate 2 and the top plate 3 is not restrict | limited in particular, For example, resin (plastic), glass, a silicon | silicone etc. can be used. Since it is easy to optically observe the cultured cells in the chamber 4, the material is preferably a transparent material, and specifically, resin and glass are preferable. Moreover, since the board | substrate 2 and the top plate 3 can be joined by activating the surface of the board | substrate 2 and the top plate 3 by plasma processing, and crimping | bonding the said surfaces, it is more preferable that the said material is resin. As the resin, a silicone resin {for example, polydimethylsiloxane (PDMS)}, an acrylic resin (for example, polymethyl methacrylate), and a styrene resin (for example, polystyrene) are more preferable because they have high transparency.

Since there are very few types of cells that can directly adhere to the surface of PDMS, it is possible to prevent cells from adhering to the flow path by using the substrate 2 made of PDMS. PDMS is suitable as a material for producing a microchip for cell culture because of its high oxygen permeability.
Therefore, PDMS is particularly preferable as the material of the substrate 2 and the top plate 3. Moreover, you may comprise only the location where the said cell suspension contacts among the board | substrate 2 and the top plate 3 by PDMS. The materials of the substrate 2 and the top plate 3 may be the same or different. For example, the substrate 2 can be made of PDMS and the top plate 3 can be made of glass.

A surface treatment such as a water repellent treatment may be performed on a region of the substrate 2 and the top plate 3 that is in contact with the cell suspension or the culture solution. In the place where the water repellent treatment has been performed, it is possible to suppress the adhesion of the cells and to further smooth the flow of the liquid such as the cell suspension or the culture solution. As the water-repellent treatment, a known fluororesin such as perfluoroalkyl group-containing silane (FAS) or polytetrafluoroethylene (PTFE) having a — (CF ₂ ) _n —CF ₃ group is used to coat the surface. This method can be exemplified.

[Process A]
As shown in FIG. 6, the step A of the first aspect is a scaffolding factor that promotes cell adhesion to the surface 2a, the first recess 5, the second recess 5 ′, the third recess 5 ″, and the groove R of the substrate 2. 8 is a step of adsorbing (arranging) 8.
The scaffold factor 8 is not particularly limited as long as it can promote the adhesion of cells to be introduced and can be cultured. For example, a scaffold containing a known cell adhesion protein can be used.

  Examples of the cell adhesive protein include collagen, fibronectin, laminin, gelatin, and polylysine. These cell adhesion proteins may be used alone or in combination. The polylysine is a peptide, but in the present specification and claims, both cell adhesion proteins and cell adhesion peptides are referred to as cell adhesion proteins without distinction. Examples of the cell adhesive peptide include hydrolyzed degradation products of cell adhesive proteins such as collagen, fibronectin, laminin, and gelatin, and chemically synthesized polylysine peptide chains.

  The cell adhesion protein contained in the scaffold factor 8 may affect the physiological activity of cells adhering thereto. For example, the cell does not proliferate in one cell adhesive protein, and the cell proliferates actively in another cell adhesive protein. Therefore, it is preferable that the cell adhesive protein to be used is appropriately selected in consideration of such effects.

The method for disposing the scaffolding factor 8 on the surface 2a, the recess 5 and the groove R of the substrate 2 is not particularly limited as long as the cell adhesion of the scaffolding factor 8 is not impaired. For example, it is physically or chemically adsorbed. A suitable method is mentioned. Specifically, a method in which a coating solution in which the scaffold factor 8 is suspended or dissolved is applied to the entire surface of the substrate 2 on the surface 2a side.
In the present invention, the place where the scaffolding factor 8 is finally disposed is only the bottom surface 5c of the first recess 5. However, in step A, it is preferably applied to the entire surface on the surface 2a side of the substrate 2 (see FIG. 6). ). This is because it is relatively difficult to apply only to the bottom surface 5c, and the application operation becomes complicated. On the other hand, the method of applying to the entire surface is relatively easy, and the operation of the application can be simplified.

Examples of the method for applying the coating solution on the entire surface include a spin coating method. Moreover, the method of immersing the board | substrate 2 in the said coating liquid is also mentioned. By performing ultrasonic treatment or the like during the immersion, it is possible to apply uniformly over the entire surface, except for bubbles that may adhere to the surface of the substrate 2.
By making the said scaffold factor 8 contact the surface 2a, the recessed part 5, and the groove part R, the said scaffold factor 8 can be made to adsorb | suck to the contact surface physically or chemically.

The contact time is not particularly limited, but a contact time of 1 minute or longer is preferable, a contact time of 5 minutes or longer is more preferable, and a contact time of 30 minutes or longer is more preferable. By setting the lower limit value or more, the adsorption can be made more reliable. The upper limit is not particularly limited. For example, if the upper limit is about 10 hours, the adsorption can be further ensured.
According to this physical adsorption, it can be adsorbed on the entire surface without substantially impairing the cell adhesion or physiological activity inherent to the scaffold factor 8.

  In addition to the scaffold factor 8, the coating solution may contain a polymer or the like that promotes the adsorption. It may be possible to perform the adsorption more reliably or more quickly. As the polymer, a known polymer may be used.

In order to adsorb the scaffold factor 8 on the entire surface, a method may be employed in which a polymer material is previously grafted to the entire surface and the scaffold factor 8 is bound to the entire surface via the polymer material.
Specifically, for example, a liquid containing acrylic acid is applied to the entire surface and photopolymerized to graft-bond polyacrylic acid to the entire surface, and then the scaffold factor 8 is brought into contact with the entire surface. A method (chemical adsorption) in which an amino group and a carboxyl group of polyacrylic acid are bonded by dehydration condensation can be employed.

  After the scaffolding factor 8 is adsorbed on the entire surface by applying the coating solution, it is preferable to remove the coating solution to remove moisture from the substrate 2 and to dry. This is because the water may get in the way in each operation in the subsequent process. As the drying method, a known method such as air blow or reduced pressure drying can be applied. During the drying, the scaffold factor 8 may be similarly dried.

[Process B]
As shown in FIG. 7, in the step B of the first aspect, the convex portion 31 of the physical mask 30 is inserted into the first concave portion 5 of the substrate 2, and at least a part of the bottom surface 5 c of the first concave portion 5 is formed by the convex portion 31. And protecting the scaffolding factor 8 adsorbed on the bottom surface 5c.

  In step B, as shown in FIG. 7, a front surface 31 a (hereinafter referred to as an end surface 31 a) of the convex portion 31 arranged so as to protrude from the surface 32 a of the base body 32 constituting the physical mask 30 is the first concave portion 5. It is preferable to be in a state of being pressed so as to be in close contact with the bottom surface 5c. In this state, the scaffolding factor 8 between the end surface 31a and the bottom surface 5c can be protected by blocking from the outside. In this state, it is preferable to perform the subsequent step C.

In the present invention, the shape of the physical mask 30 is not particularly limited, and the scaffolding factor 8 in which the convex portion 31 can be inserted into the first concave portion 5 and the convex portion 31 is disposed on the bottom surface 5 c of the first concave portion 5. Any device that can be protected from the outside can be used.
For example, as shown in FIGS. 5 to 9, the physical mask according to the present invention includes at least a base 32 and a convex portion 31 arranged so as to protrude from the surface 32 a of the base 32, and the convex portion 31 is cylindrical. The diameter l is smaller than the diameter L of the first recess 5, the tip of the projection 31 is a flat surface that can be in close contact with the bottom surface 5 c of the first recess 5, and the height h of protrusion of the projection 31 is A physical mask 30 that is longer than the depth H of the first recess 5 is preferred.

  The size in particular of the 1st recessed part 5 of the board | substrate 2 is not restrict | limited, For example, the diameter L can be 1000 micrometers-2000 micrometers, and the depth H can be 150 micrometers-300 micrometers. In this case, for example, the size of the columnar convex portion 31 of the physical mask 30 is such that the diameter m is 10 μm to 50 μm smaller than the diameter L of the chamber 4 and the height h is 10 μm to 50 μm larger than the depth H of the chamber 4. do it.

  The shape of the convex portion 31 of the physical mask 30 is not necessarily a cylinder, and is preferably a columnar shape such as an elliptical column, a quadrangular column, a pentagonal column, or a hexagonal column. What is necessary is just to select suitably according to the shape of the 1st recessed part 5, and the shape of the bottom face 5c. When the shape of the convex part 31 is columnar, it is good also as a shape which shaved a part of side surface of the pillar. For example, as shown in FIG. 12, the side surface 31b of the columnar convex portion 31 may be cut off. By removing the side surface 31b of the columnar convex portion 31, it is possible to more easily remove the scaffolding factor 8 disposed on the side surface 5d of the first concave portion 5 in the subsequent step C.

  The angle at which the convex portion 31 protrudes from the base body 32 is not particularly limited. Since the pressure load when pressing the convex portion 31 against the bottom surface 5 c of the first concave portion 5 is easy, the angle is preferably perpendicular to the surface 32 a of the base 2.

  As shown in FIG. 7, the height h at which the convex portion 31 protrudes from the surface 32 a of the base body 32 is preferably longer than the depth of the first concave portion 5 of the substrate 2. With this configuration, it becomes easier to bring the end surface 31 a of the convex portion 31 into close contact with the bottom surface 5 c of the first concave portion 5. Further, in the case of this configuration, the distance G between the surface 32a of the physical mask 30 and the surface 2a of the substrate 2 can be made larger than 0, and the scaffold factor 8 arranged in the region to be removed in the subsequent process C is physically It is possible to prevent the mask 30 from being covered.

The distance G is preferably as large as possible because it becomes easy to remove the scaffolding factor 8 disposed on the surface 2a and the groove R of the substrate in the subsequent step C. Although it depends on the area of the surface 2 a of the substrate 2 and the area of the surface 32 a of the physical mask 30, the lower limit of the distance G is usually 1 μm or more, preferably 5 μm or more, and more preferably 10 μm or more.
By setting it to the lower limit value or more, it becomes easier for the processing liquid such as the plasma in the plasma processing or the alkali solution or the acid solution in the chemical processing to enter the gap between the substrate 2 and the base 2 of the physical mask 30, It may be easier to remove the excess scaffolding factor 8 disposed other than the bottom surface 5 c of the first recess 5. The upper limit value of the distance G is not particularly limited, and can be, for example, about 50 μm to 5 mm.

  The area of the region where the end surface 31a of the convex portion 31 protects the bottom surface 5c of the first concave portion 5 is preferably as large as possible. Since cells can be cultured in the scaffold factor 8 placed in the protected region, the area that can be used for cell culture increases as the area of the region increases. For example, when the total area of the bottom surface 5c of the first recess 5 is 100%, the area to be protected is preferably 60% or more, more preferably 75% or more, and still more preferably 90% or more.

  When the number of the first recesses 5 provided on the surface 2a of the base body 2 is plural, in the base body 32 constituting the physical mask 30, a plurality of convex portions 31 are provided corresponding to the number of the first concave portions. It is preferable. The position where the plurality of convex portions 31 are provided on the base body 32 is preferably a position where the plurality of convex portions 31 respectively face the plurality of first concave portions 5 when the base body 32 is opposed to the surface 2 a of the substrate 2. With this configuration, the scaffold factor 8 disposed on each bottom surface 5 c of the plurality of first recesses 5 of the base 2 can be collectively protected by one physical mask 30.

  The shape of the base body 32 constituting the physical mask 30 is not particularly limited as long as the convex portion 31 is held and the convex portion 31 can be inserted into the first concave portion 5 of the substrate 2. A rod shape etc. are mentioned. When the base body 32 is a net-like shape, the convex portions 31 may be arranged at the intersections of the vertical bars and the horizontal bars that constitute the net. Moreover, when the base | substrate 32 is rod-shaped, the convex part 31 can be distribute | arranged to the desired position of the said bar | burr. These physical masks can be produced by machining using, for example, a resin substrate.

  The material (material) of the physical mask 30 is not particularly limited as long as it is a material that can withstand the process in the subsequent process C, and various resins can be applied, for example. The base 32 constituting the physical mask 30 shown in FIGS. 5 to 9 is made of flat polytetrafluoroethylene (PTFE), and the convex portion 31 is also made of PTFE. PTFE is preferable because it has resistance to plasma treatment and chemical treatment such as alkali or acid that can be used in the subsequent step C, and has rigidity, so that deformation of the substrate 2 during plasma treatment can be prevented. Therefore, the material of the physical mask according to the present invention is preferably PTFE.

By the way, as a method of fixing the convex portion 31 of the physical mask 30 in a state of being inserted into the first concave portion 5 of the substrate 2, for example, a fixing device 40 as shown in FIG. 11 can be used. FIG. 11 is a schematic cross-sectional view of the fixing device 40, the physical mask 30, and the base 2.
The fixing device 40 includes a fixing plate 41 and a pressing plate 42, and a concave fixing portion 45 capable of holding the substrate 2 is provided on the surface of the fixing plate 41. The base 2 is fixed in the fixing portion 45, and the upper surface 32 b of the base 32 of the physical mask 30 is pressed by the pressing plate 42 in a state where the convex portion 31 of the physical mask 30 is inserted into the first concave portion 5 of the base 2. The pressure for pressing the base body 32 by the pressing plate 42 can be adjusted by tightening the nuts 44 disposed on the bottom surface of the fixing plate 41 and the screws 43 disposed on the top surface of the pressing plate 42. A plurality of positions where the nut 44 and the screw 43 are arranged are preferably point-symmetric with respect to the center of the substrate 2. The pressing force by the pressing plate 42 can be evenly applied to the upper surface of the base 32 of the physical mask 30.

[Process C]
As shown in FIG. 8 or FIG. 9, the process C of the first aspect is a process of removing the scaffold factor 8 adsorbed on the substrate 2 other than the bottom surface 5 c of the first recess 5.
As shown in FIG. 8, the surface of the substrate 2 is plasma-treated in a state where the bottom surface 5c of the first recess 5 is protected by the physical mask 30, thereby modifying the scaffold factor 8 disposed other than the protected bottom surface 5c. Alternatively, it can be incinerated to lose the cell adhesion of scaffold factor 8.
Here, in the present invention, “removing the scaffolding factor” means removing the cell adhesion of the scaffolding factor and does not mean only removing the physical entity of the scaffolding factor completely. Absent.

  The method of removing the scaffold factor 8 in the step C is not particularly limited as long as it is a method capable of removing the scaffold factor 8 other than the region protected by the physical mask 30 while suppressing damage to the substrate 2 as much as possible. For example, plasma treatment or chemical treatment is a suitable removal method.

  As the working gas (processing gas) in the plasma processing, plasma such as argon gas, nitrogen gas, oxygen gas or the like can be used. The gas may be mixed with butadiene gas, carbon dioxide gas or the like. By mixing butadiene gas, a thin film can be formed on the surface 2a of the plasma-treated substrate 2, and the adhesion of the surface 2a can be further enhanced. Moreover, the said adhesiveness can also be adjusted by mixing a carbon dioxide gas.

Examples of the chemical treatment include surface treatment using an aqueous alkali solution such as NaOH and KOH, an aqueous acid solution such as hydrochloric acid and chromic acid, or a halogenated hydrocarbon solvent.
Further, as a method for removing the scaffold factor 8, corona discharge treatment, ozone treatment, UV irradiation treatment, electron beam irradiation treatment, surface treatment with flame, or the like may be applied.

  In step C, as a method for removing the scaffolding factor 8 other than the scaffolding factor 8 protected by the physical mask 30, a dry process that does not require a separate drying process is required, and damage to the substrate 2 can be minimized. Plasma treatment is preferred. By performing plasma processing on the surface 2a and the groove portion R of the substrate 2, the unnecessary scaffold factor 8 can be removed.

[Process D]
The process D of the first aspect is a process of removing the physical mask 30 inserted in the first recess 5.
By lifting the base 32 constituting the physical mask 30, the convex portion 31 that has been inserted into the first concave portion 5 and pressed against the bottom surface 5c may be pulled up. When the physical mask 30 is made of PTFE, it has excellent durability and can be used repeatedly.

[Process E]
As shown in FIG. 10, the process E of the first aspect is a process of bonding the top plate 3 to the surface 2 a of the substrate 2 and covering the first recess 5 and the groove R.
The method of joining the lower surface 3a of the top plate 3 to the surface 2a of the substrate 2 is not particularly limited. For example, a known method of joining the surface 2a of the substrate 2 by bonding the lower surface 3a by bonding is provided. The method is applicable.
Examples of the method for imparting the adhesive property include a method of applying an adhesive to the surface 2a of the substrate 2, a method of plasma-treating the surface 2a of the substrate 2.

When plasma processing is performed to remove the excess scaffold factor 8 in the preceding step C, the top plate 3 is placed in the plasma processing chamber, and simultaneously with the removal of the scaffold factor 8, the lower surface 3a of the top plate 3 is removed. Is preferably plasma-treated (see FIG. 9).
That is, in the microchip manufacturing method of the present invention, in step C, the surface 2a of the substrate 2 and the surface 3a (lower surface 3a) of the top plate 3 are activated by plasma treatment in step C, and then in step E, The bonding is preferably performed by pressure bonding the activated surfaces. According to this method, the excess scaffold factor 8 can be removed by the plasma treatment in the step C, and the pretreatment for joining the top plate 3 to the surface 2a of the substrate 2 can be performed. Simplification can increase production efficiency.

  The first concave portion 5 and the second concave portion 5 are formed by applying adhesiveness to the surface 2a of the substrate 2 and / or the lower surface 3a of the top plate 3 by the plasma treatment or the like, and pressing the surface 2a and the lower surface 3a together. ', The third concave portion 5 "and each groove portion R are sealed, and a storage portion 21, a chamber 4, a liquid feeding flow channel 11, an introduction flow channel 12, a discharge flow channel 13, a discharge flow channel 14 and a discharge portion 22 are provided. The microchip 1 can be manufactured (see FIG. 10). Since the top plate 3 is provided with through holes T at positions corresponding to the second recess 5 ′ and the third recess 5 ″, The storage unit 21 and the discharge unit 22 communicate with the outside through the through hole T.

The intensity and processing time of the plasma treatment vary depending on the substrate 2, the top plate 3, the scaffolding factor 8, and the type of plasma. For example, the substrate 2 and the top plate 3 are PDMS, and the scaffolding factor 8 is the cell adhesion property. When the protein is oxygen and oxygen plasma is used, the plasma treatment is preferably 100 W, preferably from 1 second to 30 seconds, more preferably from 1 second to 20 seconds, and even more preferably from 1 second to 10 seconds. By being above the lower limit value, the scaffold factor 8 can be sufficiently removed, and the bonding surface of the substrate 2 and the top plate 3 can be sufficiently modified to impart adhesiveness. By being below the upper limit, damage to the substrate 2 and the top plate 3 can be minimized.
Moreover, as the electric power in the said plasma processing, 100-200W is preferable. If it is at least the lower limit value of the power range, the surface can be more fully modified, and if it is at most the upper limit value, it is easy to suppress deformation of the substrate and the top plate to be processed. The pressure of the processing chamber for generating the plasma is preferably 5 to 10 Pa. The flow rate of the plasma working gas (processing gas) into the processing chamber is preferably 100 mL / min to 200 mL / min. For example, in the case of a PDMS substrate, it can be confirmed that the water contact angle on the surface is 25 to 40 °.

  The microchip manufacturing method according to the present invention may include processes or steps other than the processes A to E as long as they do not depart from the spirit of the present invention.

A substrate 2 having a plurality of first concave portions 5 and groove portions R arranged in an array on the surface was produced by photolithography and replica molding of PDMS (see FIG. 13).
The fine structure formed on the surface of the substrate 2 is composed of first recesses 5 serving as 64 independent chambers 4 (micro chambers) and groove portions R serving as a total of nine channels (micro channels). Yes. In addition, four second recesses 5 ′ serving as the cell introduction port 21 (reservoir 21) and four third recesses 5 ″ serving as the drug introduction port 22 (discharge unit 22) are formed. The cell suspension can be introduced into the microchamber, and four kinds of media containing different humoral factors can be perfused from the drug introduction port 22.

In the following procedure, the cell adhesion protein was patterned by oxygen plasma, and at the same time, the microchip 1 was assembled by plasma bonding.
1) The surface of the PDMS substrate 2 is irradiated with oxygen plasma under conditions of 100 W for 3 seconds to hydrophilize (activate) the substrate surface, and then the 100 μM collagen aqueous solution or fibronectin is applied to the entire surface of the first recess 5. The aqueous solution was applied, and the cell adhesion protein (scaffolding factor 8) was physically adsorbed on the substrate surface at 37 ° C. for 3 minutes.
2) A physical mask 30 made of Teflon (registered trademark) provided on the surface of the base 32 so that a convex portion 31 whose size conforms to the shape of the first concave portion 5 corresponds to the arrangement of the first concave portion 5 is used as a substrate. 2 and aligned with each other, each convex portion 31 was inserted into each first concave portion 5 (see FIG. 13).
3) A holder (fixed) (not shown) for fixing the substrate 2 and the physical mask 30 for the purpose of decomposing and removing the cell-adhesive protein adsorbed in the area other than the first recess 5 and activating the PDMS surface constituting the substrate 2 The device was fixed with an instrument) and irradiated with oxygen plasma at 100 W for 10 seconds (see FIG. 14). At this time, the top plate 3 was also irradiated with plasma.
4) The holder is disassembled, the physical mask 30 is taken, the base body 2 and the top plate 3 are bonded by pressure bonding, and each microchamber and the flow path are sealed, and the cell introduction port 21, the flow path 11, the chamber 4 and the microchip 1 provided with the drug introduction port 22 was produced (see FIG. 15).

  In order to observe the patterning state of scaffold factor 8 by the above method, patterning was performed by the above method using collagen labeled with FITC as a fluorescent dye or fibronectin labeled with TRITC. When the first concave portion 5 after patterning was observed with fluorescence, a circular spot having a diameter of about 1 mm was observed according to the pattern of the convex portion 31 of the physical mask 30, that is, only on the bottom portion 5 c of the microchamber 4, regardless of the type of cell adhesive protein. Thus, it was confirmed that the cell adhesion protein was immobilized (see FIG. 16).

FIG. 16A is a photograph of the microchamber 4 on which FITC-labeled collagen has been adsorbed observed with a fluorescence microscope, and FIG. 16B is a photograph after patterning by plasma processing. FITC-labeled collagen remains on the bottom surface 5c of the microchamber 4 after patterning without being removed. On the other hand, it is clear that the collagen arranged other than the bottom surface 5c is removed by the plasma treatment.
FIG. 16 (c) is a photograph of the microchamber 4 on which TRITC-labeled fibronectin is adsorbed observed with a fluorescence microscope, and FIG. 16 (d) is a photograph after patterning by plasma treatment. The result of patterning is the same as in the case of collagen.

  Four types of dye solutions were injected into the drug introduction port 22 of the microchip 1 and applied to the cell introduction port 21 through the microchamber 4 by applying a pressure of 20 to 100 kPa. The four dye solutions injected were introduced into each microchamber without leaking or mixing (see FIG. 17). This means that sufficient adhesion strength (bonding strength) between the substrate 2 and the top plate 3 was obtained to the extent that leakage of the liquid does not occur even when a liquid is flowed by applying pressure to the flow path. Means. Therefore, the cell adhesion protein other than the region protected by the physical mask 30 is sufficiently decomposed and removed by the oxygen plasma irradiation, and the surface 2a of the PDMS substrate under the removed cell adhesion protein layer is sufficiently active. It is clear that the adhesiveness necessary for joining was imparted.

  In the produced microchip 1, CHO cells and NIH3T3 cells were cultured. A microchamber 4 provided with collagen or fibronectin as the scaffold factor 8 was used. In the chamber 4 in which each scaffold factor 8 was arranged, each cell adhered to the bottom surface and advanced by 24 hours of static culture. Each cell was then grown by 48 hours of perfusion culture. The result is shown in FIG.

  FIG. 18 (a) shows CHO cells (24 hours later) in a microchamber not provided with scaffold factor 8, and FIG. 18 (b) shows NIH3T3 cells (24 hours later) in a microchamber not provided with scaffold factor 8. 18 (c, g) are CHO cells (24 hours and 48 hours later) in the microchamber 4 in which collagen is disposed, and FIG. 18 (d, h) is in the microchamber 4 in which collagen is disposed. NIH3T3 cells (24 hours and 48 hours later), and FIG. 18 (e, i) are CHO cells (24 hours and 48 hours later) in a microchamber provided with fibronectin, and FIG. 18 (f, j). Are NIH3T3 cells (24 hours and 48 hours later) in a microchamber with fibronectin.

  Neither CHO cells nor NIH3T3 cells adhered to the PDMS surface 2a without the scaffold factor 8 (FIGS. 18 (a, b)). On the other hand, when the scaffolding factor 8 was arranged by the patterning method, the cells adhered to the scaffolding factor 8 and proliferated. Therefore, it is clear that the scaffold factor 8 is protected by the physical mask 30 during the plasma treatment and does not lose its cell adhesion.

  By using the present invention, it is possible to easily produce a microchip that comprehensively analyzes the culture environment, and it is possible to individually and efficiently search for differentiation induction conditions for stem cells having different properties for each cell line It becomes. Therefore, the differentiation-inducing process when a patient model iPS cell is differentiated into a specific tissue to produce a disease model cell is dramatically improved. In the future, it is thought that it will lead to the realization of tailor-made regenerative medicine using cells and tissues that have been induced to differentiate under optimal conditions for each patient. In addition to stem cells, the function and fate of cells are determined through complex intracellular signal transduction that depends on the external environment composed of humoral factors and scaffolding factors. The new research tool realized by the present invention is expected to be used for elucidating the operating principle of such a complex life system.

DESCRIPTION OF SYMBOLS 1 ... Microchip, 2 ... Board | substrate, 2a ... Surface of board | substrate, 3 ... Top plate, 3a ... Lower surface of top plate, T ... Through hole, 4 ... Chamber, 4a ... Main space part, 4b ... Outer space part, 5 ... Recessed portion (first recessed portion), 5 '... second recessed portion, 5 "... third recessed portion, 5a ... main space recessed portion, 5b ... outer peripheral space recessed portion, 5c ... bottom surface of recessed portion, 5d ... side surface of recessed portion, R ... groove portion, H Depth of recess, L: Diameter of recess, 6, 6A-6D ... Sample solution, 7, 7A-7D ... Inlet channel, 8 ... Scaffolding factor, 11, 11A-11D ... Liquid feed channel, 12, 12A -12D: Introducing flow path, 13, 13A-13D: Deriving flow path, 14, 14A-14D: Discharging flow path, 15, 15A-15E: Outlet flow path, 21: Reserving section, 22: Discharging section, 30: Physical Mask 31, convex portion, 31 a, tip end surface (end surface), 32, base, 32 a, base surface (lower surface) 32b: upper surface of the substrate, h: height of the convex portion, l: diameter of the columnar convex portion, G: distance (gap distance) between the substrate surface and the substrate surface, P: plasma, 40: fixing device, 41: fixing Plate, 42 ... Presser plate, 43 ... Screw, 44 ... Nut, 45 ... Fixed part.

Claims (7)

A method for producing a microchip in which a chamber for cell culture and a flow path connected to the chamber are formed,
Prepare a substrate having a recess formed as the chamber and a groove serving as the flow path formed on the surface, a physical mask provided with a projection that can be inserted into the recess, and a top plate that covers the recess and the groove,
Adsorbing a scaffolding factor that promotes cell adhesion to the surface, recess and groove of the substrate; and
The step B of inserting the convex portion of the physical mask into the concave portion of the substrate, covering at least part of the bottom surface of the concave portion by the convex portion, and protecting the scaffold factor adsorbed on the bottom surface;
Step C for removing the scaffold factor adsorbed to other than the covered bottom surface;
Step D for removing the physical mask charged in the recess,
Bonding the top plate to the surface of the substrate and covering the recesses and grooves, and
A method for producing a microchip, comprising:   2. The method of manufacturing a microchip according to claim 1, wherein, in the step C, the scaffold factor other than the protected scaffold factor is removed by performing plasma treatment on a surface and a groove portion of the substrate. 3.   In the step C, after the surface of the substrate and the surface of the top plate are activated by plasma treatment, the bonding is performed by pressure bonding the activated surfaces in the step E. The method for producing a microchip according to claim 2.   The method for producing a microchip according to any one of claims 1 to 3, wherein the scaffold factor contains collagen, fibronectin, laminin, gelatin, or polylysine. It is a physical mask used in the manufacturing method of the microchip as described in any one of Claims 1-4,
Comprising at least a base and the convex portion arranged so as to protrude from the surface of the base;
The convex portion is columnar, and the diameter thereof is smaller than the diameter of the concave portion,
The tip of the convex portion has a surface shape that can be in close contact with the bottom surface of the concave portion,
A physical mask, wherein a height at which the convex portion protrudes is longer than a depth of the concave portion.   The physical mask according to claim 5, wherein a material of the physical mask is polytetrafluoroethylene. A microchip comprising at least a substrate on which a recess serving as a cell culture chamber and a groove serving as a flow channel connected to the chamber are formed, and a top plate covering the recess and the groove,
A microchip, wherein a scaffolding factor that promotes cell adhesion is disposed only on the bottom surface of the recess.