JP5322229B2 - Microchip and culture condition search method - Google Patents

Description

  The present invention relates to a microchip for biochemical tests used for efficiently performing cell culture, biochemical analysis, chemical reaction, and the like, and a culture condition searching method using the same.

Conventional cell assay methods that have been performed using microplates are being automated, and corresponding 384-well and 1,536-well plates are also commercially available.
However, in the 1,536-well plate, it is difficult to handle trace liquids and control evaporation from the surface, and 384 wells per plate are said to be the limit of integration in order to perform reliable analysis. And reliable cell assay methods are required.
On the other hand, with the development of microfabrication technology and Lab on a Chip related technology in recent years, various types of cells can be cultured, analyzed, and manipulated in a microstructure configured in an array on a single chip. Methods for efficiently performing cell assays and cell manipulations have been reported. For example, Non-Patent Document 1 by the present inventor and Non-Patent Document 2 by an American group describe a microchip capable of testing the cytotoxicity of a plurality of drugs at once.
On the other hand, many of the previously reported microchips for cell assays are made of polydimethylsiloxane (PDMS), but the number of cells that can be cultured on the surface made of PDMS is 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.
In addition, in many cases, it is known that a plurality of differentiation-inducing factors composed of a scaffold factor and a humoral factor work cooperatively in the expression of cell functions in a living body (Non-Patent Document 3, Non-patent document 4).
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.
Further, in order to induce differentiation of stem cells into specific cells in vitro, it is necessary to cause a number of differentiation inducing factors to act as a complex system.
Furthermore, it has been reported that the differentiation-inducing ability of ES cells varies greatly from cell line to cell line (see Non-Patent Document 5).
As described above, the differentiation induction process of stem cells is complicated and diverse, and it is necessary to exhaustively search for an optimal culture environment for differentiating stem cells into specific cells for each cell line.

Sugiura S., Edahiro J., Kikuchi K., Sumaru K., and Kanamori T.:Cell Culture Microchamber Array with Independent Perfusion Channel for Parallel Drug Toxicity Assay, The proceedings of microTAS 2007, Chemical and Biological Microsystems Society, pp1321-1323 (2007). Wang Z., Kim M.-C., Marquez M. and Thorsen T .: High-Density Microfluidic Arrays for Cell Cytotoxicity Analysis, Lab Chip, 7, 740-745 (2007). M. A. Schwartz and M. H. Ginsberg, Nat.Cell Biol., 4, E65-E68 (2002). K. M. Yamada and S. Even-Ram, Nat.Cell Biol., 4, E75-E76 (2002). K. Osafune, et al., Nat. Biotechnol., 26, pp. 313-315, (2008).

As described above, in many cultured cells, it is necessary to search for a culture environment composed of a scaffold factor and a humoral factor, but in order to comprehensively search for an appropriate culture environment using conventional culture techniques, Very complicated manual work and expensive robots are required. In addition, there is also a problem that the reliability is likely to be lowered due to a complicated and requiring a plurality of tests.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microchip and a culture condition search method capable of realizing an efficient and reliable culture condition search at low cost.

In order to solve the above problems, the present invention provides the following configuration.
The present invention provides a microchip in which a plurality of chamber portions for cell culture are formed, wherein the plurality of chamber portions are arranged so as to form a matrix composed of a plurality of rows and a plurality of columns, The inner surface of the chamber part belonging to some columns is modified with the first scaffolding factor that promotes cell adhesion, and the inner surface of the chamber unit belonging to at least some of the other columns promotes cell adhesion. A chamber part that is modified with a second scaffold factor and belongs to a part of the plurality of rows is connected to a first introduction flow channel for introducing a liquid containing a first liquid factor, A chamber portion belonging to at least some of the rows is connected to a second introduction flow channel for introducing a liquid containing a second liquid factor, and a main plate portion having a chamber recess serving as the chamber portion, In the chamber recess Moiety to provide a microchip that is configured by a substrate portion are overlapped with a flat facing surface is modified with the scaffold factor.
The microchip of the present invention branches from the first introduction flow path to the chamber portion belonging to at least a part of the rows other than the row including the chamber portion to which the first and second introduction flow channels are connected. It may be possible to introduce a liquid containing the first and second liquid factors supplied by the first branched flow path and the second branched flow path branched from the second introduction flow path.
In the present invention, the first and second chambers belonging to at least some of the rows other than the row including the chamber portion into which the liquid containing the first and second liquid factors can be introduced. A blank flow channel into which a liquid that does not contain a liquid factor can be introduced may be connected.
As the scaffold factor, one selected from collagen, fibronectin, laminin, gelatin, and polylysine can be used.
As the humoral factor, one selected from an activator, a suppressor, a growth factor, a hormone, and a cytokine can be used.
In the microchip of the present invention, it is preferable that at least the surface modified with the scaffolding factor is made of polydimethylsiloxane or glass.
In the microchip of the present invention, it is preferable that at least a part of the inner surface of the chamber portion is made of polydimethylsiloxane.

  The culture condition search method of the present invention is a method for searching for culture conditions using the microchip, wherein a target cell is placed in a chamber part belonging to a row modified with the first and second scaffold factors. Adhering and introducing the liquid containing the first and second liquid factors into the chamber part belonging to the row to which the first and second introduction flow paths are connected, respectively, and the cell state in each chamber part It is the culture condition search method to compare.

According to the present invention, chamber portions arranged in a matrix are modified with a predetermined scaffold factor for each column, and an introduction flow path for introducing a predetermined liquid factor for each row is connected to these chamber portions. As such, many combinations of scaffold and humoral factors can be tested at once.
For this reason, it is possible to exhaustively test the culture conditions of the target cells. For example, it is possible to search for optimum environmental conditions for inducing differentiation of stem cells into specific cells.
Since a large number of combinations can be realized at a time, an efficient and reliable condition search can be realized at low cost.
Cell functions are determined through complex intracellular signaling that depends on the external environment composed of scaffolding factors and humoral factors, making it difficult to centrally understand the effects of the environment on cells, Extensive testing is required for combinations of scaffolding factors and humoral factors.
In general, exhaustive testing of a large number of combinations is time-consuming and the reliability is likely to decrease due to the necessity of a plurality of tests. However, in the present invention, a large number of combinations can be tested at a time. Therefore, since a highly efficient and reliable test is possible, the technical significance in searching for the optimal culture conditions for cells is extremely high.

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 a top view which shows the modification site | part of a scaffold factor. It is sectional drawing which shows the principal part of the microchip modified with the scaffold factor. It is a top view which shows the microchip modified with the scaffold factor. It is a top view which shows the microchip modified with the scaffold factor. It is a top view which shows the principal part of the microchip of the 2nd example of this invention. It is sectional drawing 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 the microchip of the 3rd example of this invention. It is a top view which shows the principal part of the microchip of the 4th example of 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.

Hereinafter, a microchip 1 as a first example of the present invention will be described with reference to the drawings.
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 (left-right direction in FIG. 1) is sometimes referred to as a first direction, and the short direction (vertical direction in FIG. 1) of the microchip 1 is sometimes referred to as a second direction.

As shown in FIG. 2 and FIG. 3, the microchip 1 can be constituted by a flat main plate portion 2 and a flat substrate portion 3 superimposed on the main plate portion 2.
On the surface 2a (surface 2a) of the main plate portion 2 facing the substrate portion 3, a chamber recess 5 serving as a chamber portion 4 into which the sample liquid is introduced is formed.
The chamber recess 5 includes a main space recess 5a having a circular shape in plan view and an outer periphery space recess 5b formed on the outer periphery side of the main space recess 5a so as to surround it. The outer peripheral space recess 5b is formed shallower than the main space recess 5a.
As shown in FIG. 3, the chamber part 4 consists of the space (main space part 4a) in the main space recessed part 5a, and the space (outer periphery space part 4b) in the outer periphery space recessed part 5b.

As shown in FIGS. 1 and 2, the chamber portions 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 chamber portions 4 constituting the rows are arranged along the first direction (left-right direction in FIG. 1), and the chamber portions 4 constituting the rows are arranged along the second direction (up-down direction in FIG. 1). The chamber parts 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.
In the illustrated example, the row and column formation directions are substantially perpendicular to each other.

An inlet channel 7 and an outlet channel 15 communicating with the chamber recess 5 (chamber part 4) are formed on the surface 2a of the main plate part 2.
The inlet channel 7 includes a liquid supply channel 11 formed along the first direction (the left-right direction in FIG. 1), and a plurality of introduction channels that branch from the liquid supply channel 11 and communicate with the chamber portions 4. 12, and these introduction flow paths 12 are each connected to the chamber section 4, so that the sample liquid from the storage section 21 is introduced from the liquid supply flow path 11 into the chamber section 4 through the introduction flow path 12. be able to.

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 chamber portions 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 chamber portions 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 is the fifth row 9E. And the fourth introduction flow path 12D branched from the liquid supply flow path 11D of the inlet flow path 7D is connected to the chamber sections 4 of the seventh line 9G and the eighth line 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. The sample liquid 6B from the storage part 21B can be introduced into the chamber part 4 of the third row 9C and the fourth line 9D from the inlet channel 7B, and the sample liquid 6C from the storage part 21C can be introduced into the fifth line 9E. And the sample liquid 6D from the reservoir 21D can be introduced into the chamber portions 4 of the seventh row 9G and the eighth row 9H.
In the present invention, one introduction flow path is connected to a chamber portion belonging to some rows among a plurality of rows, and another introduction flow passage is connected to a chamber portion belonging to at least some rows among other rows. Need only be connected.

As shown in FIGS. 1 and 4, the outlet channel 15 has a plurality of outlet channels 13 communicating with each chamber portion 4 and a discharge channel 14 to which the plurality of outlet channels 13 are connected. The liquid in the chamber portion 4 can be discharged from the outlet flow channel 13 to the discharge portion 22 via the discharge flow channel 14. The discharge flow path 14 is formed along the first direction (left-right direction in FIG. 1).
As shown in FIG. 4, five outlet channels 15 are formed (referred to as outlet channels 15A to 15E), and the outlet channel 14A of the outlet channel 15A is outside the first row 9A (upward 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 between the fourth row 9D and the fifth row 9E. The outlet channel 15D 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 extends outside the eighth row 9H (FIG. 4). (Below).

  4, the outlet channel 13A of the outlet channel 15A is connected to the chamber portion 4 of the first row 9A, and the outlet channel 13B of the outlet channel 15B is a chamber of the second row 9B and the third row 9C. The outlet channel 15C of the outlet channel 15C is connected to the chamber part 4 of the fourth row 9D and the fifth row 9E, and the outlet channel 13D of the outlet channel 15D is connected to the sixth row 9F and the seventh column. The outlet channel 13E of the outlet channel 15E is connected to the chamber part 4 of the eighth row 9H.

If the outlet channel 13 has a larger cross-sectional area than the introduction channel 12 or is formed shorter than the introduction channel 12 to reduce the flow resistance compared to the introduction channel 12, the pressure in the chamber portion 4 is excessive. Can be prevented from rising.
In the illustrated example, the introduction channel 12 and the outlet channel 13 communicate with the outer peripheral space recess 5 b of the chamber 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.
A surface 3a (upper surface 3a) of the substrate portion 3 facing the main plate portion 2 may be flat.

  The main plate portion 2 can be formed by a replica molding method. Moreover, the chamber recessed part 5, the flow path 7, 8 etc. can also be formed by RIE (Reactive Ion Etching), laser processing, NC processing, optical modeling processing, injection molding processing, nanoimprint processing, etc.

As a material for the main plate portion 2 and the substrate portion 3, a resin material such as a silicone resin (for example, polydimethylsiloxane (PDMS)) or an acrylic resin (for example, polymethyl methacrylate), or glass (for example, quartz glass) is preferable. .
In the microchip 1, it is preferable that at least the upper surface 3a of the substrate portion 3 is made of PDMS or glass.
In the microchip of the present invention, it is preferable that at least a part of the inner surface of the chamber portion is made of PDMS. For example, both the main plate portion 2 and the substrate portion 3 may be made of PDMS, or the main plate portion 2 may be made of PDMS and the substrate portion 3 may be made of glass.

Next, a method for manufacturing and using the microchip 1 will be described.
As shown in FIG. 5, the upper surface 3 a of the substrate portion 3 corresponding to the inner surface of the chamber portion 4 (the portion facing the chamber recess 5) is modified with a plurality of types of scaffolding factors 8.
The scaffold factor 8 promotes cell adhesion to the substrate part 3, and for example, one or two or more can be selected from collagen, fibronectin, laminin, gelatin, and polylysine. The scaffold factor 8 is required not only to attach cells to the upper surface 3a but also to obtain a state in which physiological activity according to the purpose can be obtained.

In the illustrated example, the modification sites by the scaffold factor 8 are circular and are two-dimensionally arranged to form a matrix of 8 rows and 8 columns. The modification site may be the entire portion corresponding to the inner surface of the predetermined chamber portion 4 or a part thereof.
In this example, the portion corresponding to the chamber portion 4 of the first row 10A and the second row 10B is modified with the first scaffold factor 8A, and the portion corresponding to the third row 10C and the fourth row 10D is the second scaffold. The portion modified with the factor 8B, the portion corresponding to the fifth column 10E and the sixth column 10F is modified with the third scaffold factor 8C, and the portion corresponding to the seventh column 10G and the eighth column 10H is the fourth scaffold factor 8D. (See FIG. 1). The modified sites by the scaffolding factors 8A to 8D are denoted by reference numerals 8a to 8d, respectively.
The first to fourth scaffold factors 8A to 8D are different from each other.
In the present invention, the inner surface of the chamber portion belonging to some of the plurality of rows is modified with the first scaffold factor, and the inner surface of the chamber portion belonging to at least some of the other rows is the first. It only needs to be modified with a scaffold factor of 2.

As a method for modifying the upper surface 3a of the substrate part 3 with the scaffold factor 8, for example, a polymer material is grafted to the upper surface 3a of the substrate part 3, and the scaffold factor 8 is bonded to the upper surface 3a via this polymer material. Is possible.
Specifically, for example, a liquid containing acrylic acid is applied to the upper surface 3a, and acrylic acid at a site corresponding to the modified sites 8a to 8d is photopolymerized using a photolithographic technique to the upper surface 3a of this site. A method can be employed in which polyacrylic acid is grafted, and then the scaffolding factors 8A to 8D are brought into contact with this site to bind the amino groups of the scaffolding factors 8A to 8D and the carboxyl groups of the polyacrylic acid by dehydration condensation.

As shown in FIGS. 6 and 7, the microchip 1 is obtained by superimposing and joining the substrate part 3 modified with the first to fourth scaffolding factors 8 </ b> A to 8 </ b> D and the main plate part 2.
In the microchip 1, among the eight columns 10A to 10H, the first and second columns 10A and 10B are modified with the first scaffold factor 8A, and the third and fourth columns 10C and 10D are the second scaffold factor 8B. The fifth and sixth columns 10E, 10F are modified with the third scaffold factor 8C, and the seventh and eighth columns 10G, 10H are modified with the fourth scaffold factor 8D.

The cells are adhered and immobilized by contacting the modified sites 8a to 8d of the scaffolding factors 8A to 8D with a culture solution containing the target cells.
The target cells are not particularly limited, and animal-derived cells (for example, human cells), plant-derived cells, microorganism-derived cells, and the like can be used depending on the purpose.
Specific examples 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 Blood cells such as myeloblasts, monoblasts, megakaryoblasts and megakaryocytes, endothelial cells, epithelial cells, liver parenchymal cells, pancreatic islet cells and the like can be used.

To bring the cells into contact with the modified sites 8a to 8d, for example, a method of passing the culture solution from the discharge channel 14 of the outlet channel 15 to the chamber part 4 through the outlet channel 13 can be employed.
It can be presumed that the cells can be fixed by the scaffolding factors 8A to 8D because the membrane proteins and the like on the cell surface bind firmly to the ligands of the scaffolding factors 8A to 8D.

As shown in FIG. 8, sample liquids 6A to 6D are introduced into the four reservoirs 21A to 21D, respectively.
The sample liquids 6A to 6D include first to fourth liquid factors 6a to 6d, respectively. That is, the sample liquid 6A includes the first liquid factor 6a, the sample liquid 6B includes the second liquid factor 6b, the sample liquid 6C includes the third liquid factor 6c, and the sample liquid 6D includes the fourth liquid factor 6b. Of humoral factor 6d. These humoral factors 6a to 6d are different from each other.
As the humoral factors 6a to 6d, for example, one or two or more can be selected from an activator, a suppressor, a growth factor, a hormone, and a cytokine.
Specific examples of the humoral factor include phospholipids, steroids, arylacetic acid-based nonsteroidal compounds, and organic acids, and one or more of these can be selected and used.

  The sample liquid 6A from the reservoir 21A is introduced into the chamber portions 4 of the first row 9A and the second row 9B from the inlet channel 7A, and the sample solution 6B from the reservoir 21B is introduced from the inlet channel 7B to the third row 9C and The sample liquid 6C from the reservoir 21C is introduced into the chamber part 4 of the fifth row 9E and the sixth line 9F, and the sample liquid 6D from the reservoir 21D is introduced into the seventh line. 9G and 8th row 9H are introduced into the chamber portion 4.

Thus, the chamber part 4 is given any of the four conditions for the scaffolding factors 8A to 8D for each column, and any of the four conditions for the humoral factors 6a to 6d for each row.
That is, the chamber parts 4 in the first and second rows 9A, 9B and the first and second columns 10A, 10B are modified with the first scaffold factor 8A, and the first humoral factor 6a is introduced. The chamber portions 4 in the third and fourth rows 9C, 9D and the first and second columns 10A, 10B are modified with the first scaffold factor 8A and the second humoral factor 6b is introduced. The chamber portions 4 in the first and second rows 9A, 9B and the third and fourth columns 10C, 10D are modified with the second scaffold factor 8B, and the first humoral factor 6a is introduced. The chamber portions 4 in the third and fourth rows 9C, 9D and the third and fourth columns 10C, 10D are modified with the second scaffold factor 8B, and the second humoral factor 6b is introduced.
Similarly, one combination of scaffolding factor and humoral factor is obtained for each of the four chamber portions 4, and a total of 16 combinations of scaffolding factor and humoral factor are realized.
For this reason, by comparing the state of the cells in each chamber part 4, the effects of these 16 combinations of scaffolding factors and humoral factors on the target cells can be compared with each other.

Specifically, for example, it is possible to search for optimum environmental conditions for inducing differentiation of stem cells into specific cells.
That is, by comparing the influences of the 16 conditions on the target cell with each other, it is possible to know the combination of the scaffold factor and the humoral factor that is most suitable for inducing differentiation into a specific cell.

In the microchip 1, the chamber portions 4 arranged two-dimensionally so as to form a matrix are modified with a predetermined scaffold factor 8 for each column, and a predetermined liquid factor is introduced into these chamber portions 4 for each row. Since the introduction flow path 12 to be connected is connected, many combinations of the scaffold factor and the liquid factor can be tested at a time.
For this reason, it is possible to exhaustively test the culture conditions of the target cells. For example, it is possible to search for optimum environmental conditions for inducing differentiation of stem cells into specific cells.
Since a large number of combinations can be realized at a time, an efficient and reliable condition search can be realized at low cost.

Cell functions are determined through complex intracellular signaling that depends on the external environment composed of scaffolding factors and humoral factors.
For this reason, it is difficult to understand the effects of the environment on the cells in a unified manner. Examining the effects of scaffolding factors and humoral factors on the cells alone does not necessarily reveal the optimal combination. Extensive testing is required.
In general, exhaustive testing of a large number of combinations is time-consuming and the reliability is likely to decrease due to the necessity of a plurality of tests. However, in the present invention, a large number of combinations can be tested at a time. Therefore, since a highly efficient and reliable test is possible, the technical significance in searching for the optimal culture conditions for cells is extremely high.

Next, the microchip 31 which is the 2nd example of this invention is demonstrated.
FIG. 9 is a plan view showing the main part of the microchip 31, and FIG. 10 is a view showing the XX ′ cross section of the microchip 31 in FIG. 9. FIG. 11 is a view showing a YY ′ cross section of the microchip 31 in FIG. 9.
As shown in FIG. 9, the microchip 31 is formed with chamber portions 34 two-dimensionally arranged so as to form a matrix composed of a plurality of rows and a plurality of columns. In the illustrated example, the chamber portion 34 forms a matrix having 4 rows and 4 columns.
Reference numerals 32 </ b> A to 32 </ b> D respectively indicate four rows formed by the chamber part 34, and reference numerals 33 </ b> A to 33 </ b> D respectively indicate four columns formed by the chamber part 34.

The chamber portions 34 of the second and third rows 32B and 32C communicate with inlet channels 35A and 35B for liquid factors and outlet channels (not shown).
The first inlet channel 35A (first introduction channel) is connected to each chamber part 34 belonging to the second row 32B, and the second inlet channel 35B (second introduction channel) is each chamber belonging to the third row 32C. Connected to the unit 34.
For this reason, the sample liquid 36A from the storage part 37A can be introduced into the chamber part 34 of the second row 32B from the first inlet flow path 35A, and the sample liquid 36B from the storage part 37B is supplied from the second inlet flow path 35B to the third line. It can be introduced into the chamber portion 34 of 32C.
The sample liquid 36A includes a first liquid factor 36a, and the sample liquid 36B includes a second liquid factor 36b different from the first liquid factor 36a.

The chamber part 34 in the first row 32A is connected to a mixing channel 35C (mixed solution introduction channel) for a liquid factor.
The mixing channel 35C is connected to a first branch channel 38A branched from the first inlet channel 35A and a second branch channel 38B branched from the second inlet channel 35B.
For this reason, the liquid mixture of the sample liquid 36A and the sample liquid 36B can be introduced into the chamber portion 34 of the first row 32A.

As shown in FIGS. 9 to 11, the branch flow paths 38A and 38B are formed at different height positions from the mixing flow path 35C, and communicate with the mixing flow path 35C via the communication flow paths 39A and 39B, respectively. doing.
A blank channel 35D is connected to the chamber portion 34 of the fourth row 32D. The blank channel 35D is connected to a blank storage portion 37D.
In the present invention, the first and second branch flow paths 38A and 38B can be directly connected to the chamber portion 34 of the first row 32A.

Next, a method for using the microchip 31 will be described.
According to the above-described method, the inner surface of the chamber portion 34 is modified with a scaffold factor.
For example, the inner surface of the chamber portion 34 in the first row 33A is modified with the first and second scaffold factors, the inner surface of the chamber portion 34 in the second row 33B is modified only with the first scaffold factor, and the chambers in the third row 33C The inner surface of the portion 34 is modified only by the second scaffolding factor, and the scaffolding factor is not modified in the chamber portion 34 of the fourth row 33D.
As a result, there are four different ways of modifying the chamber portions 34 of the first to fourth rows 33A to 33D.

Next, a sample solution is introduced into the chamber portions 34 as follows.
The sample liquid 36A from the reservoir 37A is introduced into the chamber part 34 of the second row 32B through the inlet channel 35A, and the sample liquid 36B from the reservoir 37B is introduced into the chamber part of the third row 32C through the inlet channel 35B. 34.
Similarly, the mixed liquid of the sample liquid 36A and the sample liquid 36B is introduced into the chamber part 34 of the first row 32A through the mixing flow path 35C.
A sample solution not containing a liquid factor is introduced into the chamber portion 34 of the fourth row 32D to which the blank channel 35D is connected.
Since the scaffolding factor and the humoral factor are not introduced into the chamber part 34 in the fourth row 32D and the fourth column 33D, a blank test can be performed under conditions without the scaffolding factor and the humoral factor.

There are four ways in which liquid factors are introduced into the chamber portions 34 of the first to fourth rows 32A to 32D, which are different from each other for each row.
That is, two liquid factors 36a and 36b are introduced into the chamber portion 34 of the first row 32A, only the first liquid factor 36a is introduced into the chamber portion 34 of the second row 32B, and the third row 32C. The second liquid factor 36b is introduced into the chamber part 34, and no liquid factor is introduced into the chamber part 34 in the fourth row 32D.
In this manner, the chamber part 34 is given different conditions for the scaffolding factor for each column and different conditions for the humoral factor for each row, so there are a total of 16 combinations of the scaffolding factor and the humoral factor. Realized.

In the microchip 31, since the mixing flow path 35C into which a mixed liquid of a plurality of sample liquids can be introduced is formed, the number of liquid factors is more than that without complicating the flow path configuration. Can be tested.
Therefore, efficient and reliable condition search can be realized at low cost.

Next, the microchip 51 which is the 3rd example of this invention is demonstrated.
FIG. 12 is a plan view showing a main part of the microchip 51. The microchip 51 is different from the microchip 31 shown in FIG. 9 in that the scaffolding factor channels 45A to 45D, 48A, 48B and the reservoirs 47A, 47B, 47D are provided.

The chamber portions 34 of the second and third rows 33B and 33C are connected to inlet passages 45A and 45B (introduction passages) for scaffolding factors.
The first inlet channel 45A is connected to the chamber portions 34 in the second row 33B, and the second inlet channel 45B is connected to the chamber portions 34 in the third row 33C.
Therefore, the sample liquid 46A from the reservoir 47A can be introduced into the chamber part 34 of the second row 33B from the inlet channel 45A, and the sample solution 46B from the reservoir 47B can be introduced from the inlet channel 45B to the chamber part of the third row 33C. 34.
The sample solution 46A includes a first scaffold factor 46a, and the sample solution 46B includes a second scaffold factor 46b that is different from the first scaffold factor 46a.

A mixing channel 45C (mixed solution introduction channel) for a scaffolding factor is connected to the chamber portion 34 of the first row 33A.
The mixing channel 45C is connected to a first branch channel 48A branched from the first inlet channel 45A and a second branch channel 48B branched from the second inlet channel 45B.
For this reason, the liquid mixture of the sample liquid 46A and the sample liquid 46B can be introduced into the chamber portion 34 of the first row 33A.

The branch flow channels 48A and 48B are formed at different heights from the mixing flow channel 45C, and communicate with the mixing flow channel 45C via connecting flow channels 49A and 49B, respectively.
A blank channel 45D is connected to the chamber portion 34 of the fourth row 33D. The blank channel 45D is connected to the blank reservoir 47D.

Next, a method for using the microchip 51 will be described.
The sample liquid 46A from the reservoir 47A is introduced into the chambers 34 in the second row 33B through the inlet channel 45A, the inner surface thereof is modified with the first scaffold factor 46a, and the sample liquid 46B from the reservoir 47B is modified. It introduces into the chamber part 34 of the 3rd row | line | column 33C through the inlet flow path 45B, and modifies the inner surface with the 2nd scaffold factor 46b.
Similarly, the mixed solution of the sample solution 46A and the sample solution 46B is introduced into the chamber part 34 of the first row 33A through the mixing channel 45C, and the inner surface thereof is modified with two scaffold factors 46a and 46b.
Since the sample liquid not containing the scaffolding factor is introduced into the chamber part 34 of the fourth row 33D to which the blank channel 45D is connected, the modification with the scaffolding factor is not performed.
As described above, there are four different ways of modifying the chamber portions 34 of the first to fourth rows 33A to 33D.

Next, in the same manner as the microchip 31 of the second example, the sample liquid containing the liquid factor (or the liquid factor flow paths 35A to 35D, 38A, 38B and the reservoirs 37A, 37B, 37D) (or A sample solution containing no liquid factor is introduced into the chamber 34.
Since the chamber part 34 is given different conditions for the scaffolding factor for each column and different conditions for the humoral factor for each row, a total of 16 combinations of scaffolding factor and humoral factor are realized.

In the microchip 51, since the mixing channels 35C and 45C into which a mixture of a plurality of sample solutions can be introduced are formed, there are a plurality of liquid factors and scaffolding factors without complicating the configuration of the channels. More combinations, including cases, can be tested.
Therefore, efficient and reliable condition search can be realized at low cost.

FIG. 13 is a plan view showing a main part of the microchip 61 of the fourth example in which the number of flow paths is increased as compared with the microchip 51 of the third example in order to realize more combinations. Hereinafter, a method of using the microchip 61 will be described.
First, the inner surface of each chamber portion 34 is modified with scaffolding factors 66a to 66d as follows.
The sample solution 66A from the storage section 67A is introduced into the chamber section 34 of the eighth row 33H through the inlet channel 65A (first introduction channel), and the inner surface thereof is modified with the first scaffold factor 66a.
The sample solution 66B from the reservoir 67B is introduced into the chamber part 34 in the twelfth row 33L through the inlet channel 65B (second introduction channel), and the inner surface thereof is modified with the second scaffold factor 66b.
The sample liquid 66C from the storage part 67C is introduced into the chamber part 34 of the 14th row 33N through the inlet channel 65C (third introduction channel), and the inner surface thereof is modified with the third scaffold factor 66c.
The sample solution 66D from the storage part 67D is introduced into the chamber part 34 of the fifteenth row 33O through the inlet channel 65D (fourth introduction channel), and the inner surface thereof is modified with the fourth scaffold factor 66d.

The sample liquids 66A, 66B, 66C, 66D are introduced into the chamber part 34 of the first row 33A through the first to fourth branch flow paths 68A, 68B, 68C, 68D and the mixing flow path 69A (mixed liquid introduction flow path). The inner surface is modified with scaffolding factors 66a, 66b, 66c, 66d.
The sample liquids 66A, 66B, 66C are introduced into the chamber portions 34 of the second row 33B through the branch flow paths 68A, 68B, 68C and the mixing flow path 69B, and the inner surfaces thereof are modified with the scaffolding factors 66a, 66b, 66c.
The sample liquids 66A, 66B, 66D are introduced into the chamber portions 34 of the third row 33C through the branch flow paths 68A, 68B, 68D and the mixing flow path 69C, and the inner surfaces thereof are modified with the scaffolding factors 66a, 66b, 66d.
The sample liquids 66A and 66B are introduced into the chamber portions 34 of the fourth row 33D through the branch flow paths 68A and 68B and the mixing flow path 69D, and the inner surfaces thereof are modified with the scaffolding factors 66a and 66b.
The sample liquids 66A, 66C, 66D are introduced into the chamber portions 34 of the fifth row 33E through the branch flow paths 68A, 68C, 68D and the mixing flow path 69E, and the inner surfaces thereof are modified with the scaffolding factors 66a, 66c, 66d.
The sample liquids 66A and 66C are introduced into the chamber portions 34 of the sixth row 33F through the branch flow paths 68A and 68C and the mixing flow path 69F, and the inner surfaces thereof are modified with the scaffolding factors 66a and 66c.
The sample liquids 66A and 66D are introduced into the chamber part 34 of the seventh row 33G through the branch flow paths 68A and 68D and the mixing flow path 69G, and the inner surfaces thereof are modified with the scaffolding factors 66a and 66d.
The sample solutions 66B, 66C, 66D are introduced into the chamber portions 34 in the ninth row 33I through the branch flow paths 68B, 68C, 68D and the mixing flow path 69I, and the inner surfaces thereof are modified with the scaffolding factors 66b, 66c, 66d.
The sample solutions 66B and 66C are introduced into the chamber portions 34 of the tenth row 33J through the branch flow paths 68B and 68C and the mixing flow path 69J, and the inner surfaces thereof are modified with the scaffolding factors 66b and 66c.
The sample solutions 66B and 66D are introduced into the chamber portions 34 in the eleventh row 33K through the branch channels 68B and 68D and the mixing channel 69K, and the inner surfaces thereof are modified with the scaffolding factors 66b and 66d.
The sample solutions 66C and 66D are introduced into the chamber portion 34 of the 13th row 33M through the branch flow paths 68C and 68D and the mixing flow path 69M, and the inner surfaces thereof are modified with the scaffolding factors 66c and 66d.
Since the sample liquid not containing the scaffold factor is introduced into the chamber portion 34 of the 16th row 33P from the blank reservoir 67E through the blank flow path 65E, the chamber portion 34 is not modified with the scaffold factor.
There are 16 ways of modifying the chamber portions 34 of the first to sixteenth rows 33A to 33P, which are different from one another for each row.

Next, a sample solution containing a liquid factor (or a sample solution not containing a liquid factor) is introduced into the chamber 34 as follows.
The sample liquid 56A (liquid factor 56a) from the reservoir 57A is introduced into the chamber part 34 in the eighth row 32H through the inlet channel 55A (first introduction channel).
The sample liquid 56B (liquid factor 56b) from the storage part 57B is introduced into the chamber part 34 of the 12th row 32L through the inlet channel 55B (second introduction channel).
The sample liquid 56C (liquid factor 56c) from the storage part 57C is introduced into the chamber part 34 of the 14th row 32N through the inlet flow path 55C (third introduction flow path).
The sample liquid 56D (liquid factor 56d) from the reservoir 57D is introduced into the chamber portion 34 of the fifteenth row 32O through the inlet channel 55D (fourth introduction channel).

The sample liquids 56A, 56B, 56C, 56D are introduced into the chamber portion 34 of the first row 32A through the first to fourth branch flow paths 58A, 58B, 58C, 58D and the mixing flow path 59A (mixed liquid introduction flow path). .
The sample liquids 56A, 56B, 56C are introduced into the chamber part 34 of the second row 32B through the branch flow paths 58A, 58B, 58C and the mixing flow path 59B.
The sample liquids 56A, 56B, 56D are introduced into the chamber portion 34 of the third row 32C through the branch flow paths 58A, 58B, 58D and the mixing flow path 59C.
The sample liquids 56A and 56B are introduced into the chamber portion 34 in the fourth row 32D through the branch flow paths 58A and 58B and the mixing flow path 59D.
The sample liquids 56A, 56C, 56D are introduced into the chamber portion 34 of the fifth row 32E through the branch flow paths 58A, 58C, 58D and the mixing flow path 59E.
The sample liquids 56A and 56C are introduced into the chamber portion 34 of the sixth row 32F through the branch flow paths 58A and 58C and the mixing flow path 59F.
The sample liquids 56A and 56D are introduced into the chamber portion 34 of the seventh row 32G through the branch flow paths 58A and 58D and the mixing flow path 59G.
The sample liquids 56B, 56C, 56D are introduced into the chamber portion 34 of the ninth row 32I through the branch flow paths 58B, 58C, 58D and the mixing flow path 59I.
The sample liquids 56B and 56C are introduced into the chamber portion 34 of the 10th row 32J through the branch flow paths 58B and 58C and the mixing flow path 59J.
The sample liquids 56B and 56D are introduced into the chamber portion 34 in the 11th row 32K through the branch flow paths 58B and 58D and the mixing flow path 59K.
The sample liquids 56C and 56D are introduced into the chamber portion 34 of the 13th row 32M through the branch flow paths 58C and 58D and the mixing flow path 59M.
A sample liquid not containing a liquid factor is introduced into the chamber part 34 of the 16th row 32P from the blank storage part 57E through the blank flow path 55E.
There are 16 ways in which the liquid factor is introduced into the chamber portions 34 of the first to sixteenth rows 32A to 32P.
Since the scaffolding factor and the humoral factor are not introduced into the chamber part 34 in the 16th row 32P and the 16th column 33P, a blank test can be performed under the conditions without the scaffolding factor and the humoral factor.

  In this way, the chamber portion 34 is given 16 different conditions for the scaffolding factor for each column and 16 different conditions for the humoral factor for each row, so that there are 256 different scaffolding factors in total. A combination of humoral factors is realized.

In the microchip 61, more combinations including the case where there are a plurality of liquid factors and scaffold factors can be tested without complicating the configuration of the flow path.
Therefore, efficient and reliable condition search can be realized at low cost.

FIGS. 14 to 16 show the results of perfusion culture of CHO cells for 24 hours in the chamber using a microchip made of PDMS.
FIG. 14 is a photograph showing the chamber part when the inner surface of the chamber part is modified with collagen, and FIG. 15 is a photograph showing the chamber part when the inner surface of the chamber part is modified with fibronectin. FIG. 16 is a photograph showing the chamber part when CHO cells were perfused in the same manner except that the scaffold factor was not modified.
From these results, it can be seen that different results were obtained regarding the adhesion and proliferation of CHO cells depending on the scaffolding factor, and when fibronectin was used as the scaffolding factor, adhesion and proliferation of CHO cells were promoted.

According to the present invention, it is possible to individually and efficiently search for differentiation induction conditions for stem cells having different properties for each cell line by comprehensively analyzing the culture environment.
Therefore, the differentiation-inducing process when the patient-derived iPS cells are differentiated into specific tissues and the disease model cells are created 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, cell functions and the like are determined through complex intracellular signal transmission depending on the external environment composed of humoral factors and scaffolding factors. The present invention can contribute to the elucidation of the operating principle of such a complex life system.

  1, 31, 51, 61 ... microchip, 4 ... chamber part, 8A-8D, 46a, 46b, 66a-66d ... scaffolding factor, 6a-6d, 36a, 36b, 56a-56d Liquid factor, 4a: main space part, 4b: outer peripheral space part, 12A, 35A, 55A ... first introduction flow path, 12B, 35B, 55B ... second introduction flow path, 35C , 59A to 59G, 59I to 59K, 59M ... mixing channel (mixed liquid introducing channel), 35D, 55E ... blank channel, 38A, 58A ... first branch channel, 38B, 58B ... Second branch flow path.

Claims (8)

In a microchip formed with a plurality of chamber parts for cell culture,
The plurality of chamber portions are arranged to form a matrix composed of a plurality of rows and a plurality of columns,
The inner surface of the chamber portion belonging to some of the plurality of rows is modified with a first scaffolding factor that promotes cell adhesion,
The inner surface of the chamber part belonging to at least some of the other rows is modified with a second scaffolding factor that promotes cell adhesion,
A chamber portion belonging to some of the plurality of rows is connected to a first introduction flow channel for introducing a liquid containing a first liquid factor,
A chamber part belonging to at least some of the other rows is connected to a second introduction flow channel for introducing a liquid containing a second liquid factor ,
The main plate portion having a chamber recess serving as the chamber portion and the substrate portion having a flat facing surface in which a portion facing the chamber recess is modified by the scaffold factor is characterized in that it is configured. To microchip. A first branched flow path branched from the first introduction flow path is provided in a chamber portion belonging to at least a part of the rows other than the row including the chamber portion to which the first and second introduction flow paths are connected. If, according to claim 1, characterized in that it is possible to introduce the liquid containing the first and second humoral factors supplied by the second branch flow path branched from the second inlet flow path Microchip. The first and second liquid factors are applied to chamber portions belonging to at least some of the rows other than the row including the chamber portion into which the liquid containing the first and second liquid factors can be introduced. The microchip according to claim 2 , wherein a blank channel into which a liquid not contained can be introduced is connected. The microchip according to any one of claims 1 to 3 , wherein the scaffold factor is selected from collagen, fibronectin, laminin, gelatin, and polylysine. The microchip according to any one of claims 1 to 4 , wherein the humoral factor is selected from an activator, an inhibitory factor, a growth factor, a hormone, and a cytokine. At least microchip according to any one of claims 1-5, wherein the surface to be modified with the scaffold element is characterized by comprising a polydimethyl siloxane or glass. At least microchip according to any one of claims 1 to 6 in which a portion of the chamber inner surface is characterized by comprising a polydimethyl siloxane. A method for searching for culture conditions using the microchip according to any one of claims 1 to 7 ,
Adhering target cells to the chamber part belonging to the row modified with the first and second scaffold factors,
Introducing a liquid containing the first and second liquid factors into a chamber part belonging to a row to which the first and second introduction flow paths are connected, and comparing the state of cells in each chamber part; The culture condition search method characterized by these.

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