JP4364732B2 - Manufacturing method of cell observation unit and cell observation unit manufactured by the manufacturing method - Google Patents

Description

  The present invention relates to a cell observation chip for observing cells, and particularly to a manufacturing method for manufacturing the cell observation chip with a resin.

  Various techniques for mixing and analyzing liquid specimens such as medicines and biological substances have been proposed and put into practical use. For example, blood viscosity measurement and cell state observation are evaluated, and cell chemotaxis is observed. Chemotaxis is the property that cells move in response to a concentration gradient of chemical substances called chemotaxis factors. There is a movement to apply this cell chemotaxis to the development of therapeutic drugs. In particular, it is thought that by using this chemotaxis, a new approach can be found in the development of therapeutic agents for various inflammations, allergies, and cancers, and the importance of chemotaxis research is increasing. As a technique for mixing and analyzing such a liquid specimen, a flow-type microchip has attracted attention (see, for example, Patent Document 1 and Non-Patent Document 1).

  Here, a chip for observing chemotaxis will be described. A cell observation chip for observing chemotaxis has a well filled with a chemotactic factor and a well filled with a chemotactic cell or the like. These wells communicate with each other through a flow path, and a number of fine passages separated by barriers are provided in the flow path. A cell moves through the separated minute passage.

  FIG. 6 is a cross-sectional view for explaining the overall configuration of a cell observation unit 100 including a conventional cell observation chip 1B. In the figure, the cell observation chip 1B is provided with wells 31 and 32 which are regions filled with cells or chemotactic factors. The wells 31 and 32 are provided with through holes 21 ′ and 22 ′, respectively. The through holes 21 ′ and 22 ′ are connected to the through holes 21 and 22 of the block 5 provided on the upper surface of the cell observation chip 1 B and communicate with the upper part of the cell observation unit 100. In the figure, the cell observation chip 1B and the block 5 are separately molded and combined, but may be integrally molded.

  The through holes 21 and 22 are fine holes for injecting and collecting a sample such as a cell or a chemotactic factor. Further, between the wells 31 and 32, a flow path portion 4 that communicates between them is provided. The channel portion 4 is formed with fine passages separated by a barrier. The size of such a passage is made slightly smaller than the normal size of the cell, and if a concentration gradient of the chemotactic factor is created, the cell moves to the higher concentration through the passage, so that the observation of chemotaxis is possible. it can.

  This cell observation chip 1B is used in close contact with a hard substrate 10 having transparency. The cells passing through the fine passages of the flow path portion 4 from the substrate 10 side are observed. The conventional cell observation chip 1B mainly uses a method of processing a silicon wafer (single crystal silicon) using a micromachining technique such as anisotropic dry etching, or PDMS (Polydimethylsiloxane: polydimethylsiloxane) which is a kind of silicon rubber. ).

  However, in the method of processing a silicon wafer using a micromachining technique, the processing cost of the cell observation chip 1B becomes expensive. Further, since silicon rubber is a thermosetting resin that is cured by vulcanization or cross-linking, it has a long tact time, and the number of steps required for processing is large. Therefore, attempts have been made to increase the productivity while reducing the manufacturing cost by forming the resin.

FIG. 7 shows a state in which the cell observation chip 1B is manufactured from a resin. In this case, a thermoplastic resin is injected into a space formed by abutting the pins 13 and 14 in a region surrounded by the first mold 11 and the second mold 12, and is cooled and cured. Thus, the cell observation chip 1B can be manufactured. The 1st metal mold | die 11 and the 2nd metal mold | die 12 can be produced, for example by electroforming.
JP 2002-159287 A Nikkei Biobusiness November 2001, p. 48-50

  As shown in FIG. 8A, the cell observation chip 1B as described above is placed on the substrate 10 with the cell observation chip 1B formed of resin, and in some cases with the block 5, and then the pressing jig 50. Then, the cell observation chip 1B and the substrate 10 were directly joined by tightening from the outside to secure a sealing property.

  However, in practice, the cell observation chip made of resin is almost always distorted like the cell observation chip 1 shown in FIG. For example, if the entire width of the cell observation chip 1 is 14 mm, this distortion appears on the order of about 100 μm. If tightening is performed with the jig in this state, the jig moves around the cell observation chip 1 and the pressing force is biased.

  Then, as shown in FIG. 8B, when the substrate 10 and the cell observation chip 1 are directly joined, an excessively adhered portion such as a P portion and an insufficiently adhered portion such as a Q portion are formed. As a result, the direct bonding between the two does not work and the sealing performance cannot be secured. Moreover, since the unevenness forming the flow path in the flow path section 4 has a very fine structure of 10 μm or less, the structure is crushed by an unnecessarily large force caused by excessive adhesion, and the cell observation chip is used. Sometimes it becomes impossible to use.

  The present invention has been made to solve such problems, and directly bonds a cell observation chip formed of a resin material having distortion and a transparent substrate with a sufficient sealing property. An object of the present invention is to provide a cell observation unit that is bonded by the above-described method and that does not cause collapse of the fine structure.

  The method for manufacturing a cell observation unit according to the present invention is for cell observation in which a substrate having at least transparency and a cell observation chip having a fine structure formed of a resin material softer than the substrate are fixed to each other. A method for manufacturing a unit, wherein the cell observation chip and the substrate are pressed by pressing the cell observation chip against the substrate using a pressing block that is softer than the cell observation chip molded from a resin material. Is fixed. With such a configuration, a cell observation chip formed of a resin material having distortion and a transparent substrate are bonded by direct bonding with sufficient sealing properties, and the microstructure is crushed. No cell observation unit can be provided.

  Here, the pressing block has a through hole connected to a hole provided in the cell observation chip, and the cell observation chip is connected so that the hole of the cell observation chip and the hole of the pressing block are connected. And the pressing block may be fixed, and the pressing block may be part of the cell observation unit.

  The cell observation chip and the substrate are preferably fixed by direct bonding by pressing the cell observation chip against the substrate with the pressing block. As a result, an adhesive member for adhering the cell observation chip 1 and the substrate becomes unnecessary, and the cost can be reduced. In addition, since at least the step of applying the adhesive member and the step of waiting for the effect of the adhesive member are unnecessary, the mass productivity can be improved.

  The cell observation unit according to the present invention is a cell observation unit in which a substrate having at least transparency and a cell observation chip having a fine structure formed of a resin material softer than the substrate are fixed, The cell observation chip and the substrate are fixed by pressing the cell observation chip against the substrate with a softer pressing block than the cell observation chip. With such a configuration, a cell observation chip formed of a resin material having distortion and a transparent substrate are bonded by direct bonding with sufficient sealing properties, and the microstructure is crushed. No cell observation unit can be provided.

  On the other hand, a cell observation chip according to the present invention includes at least a transparent substrate, a cell observation chip having a fine structure fixed on the upper surface of the substrate and molded from a resin material, and the cell observation chip. The cell observation unit further comprises an elastic block fixed on the upper surface, wherein the cell observation unit is softer than the substrate, and the elastic block is softer than the cell observation unit. With such a configuration, a cell observation chip formed of a resin material having distortion and a transparent substrate are bonded by direct bonding with sufficient sealing properties, and the microstructure is crushed. No cell observation unit can be provided.

  Here, the elastic block has a through hole connected to a hole provided in the cell observation chip, and the cell observation chip is connected so that the hole of the cell observation chip and the hole of the pressing block are connected. It is preferable to fix the elastic block.

  According to the present invention, a cell observation chip formed of a resin material having strain and a transparent substrate are joined by direct joining while ensuring sufficient sealing properties, and the microstructure does not collapse. An observation unit can be provided.

  Hereinafter, embodiments to which the present invention can be applied will be described. The following description is to describe the embodiment of the present invention, and the present invention is not limited to the following embodiment. For the sake of clarity, the following description is omitted and simplified as appropriate. Moreover, those skilled in the art can easily change, add, and convert each element of the following embodiments within the scope of the present invention. Further, in each of the following embodiments, a case where a chip is used for observing the chemotaxis of cells will be described for concreteness, but the liquid specimen is not limited to this, for example, protein, nucleic acid, DNA, It is a liquid containing a measurement object such as RNA, peptide, hormone, antigen, antibody, ligand, receptor, enzyme, substrate, low molecular organic compound, cell, ion, etc., and also includes a dispersion system such as a suspension or a colloid. That is, the microchip according to the present invention can also be used for mixing and analysis of liquid specimens as described above.

Embodiment 1
First, the cell observation unit 100 will be described with reference to FIG. FIG. 1 is a cross-sectional view of the cell observation unit 100. The cell observation chip 1 is provided with wells 31 and 32. In this example, the wells 31 and 32 are formed as depressions on the observation surface of the cell observation chip 1. The wells 31 and 32 form spaces when the cell observation chip 1 and the transparent substrate 10 are in close contact with each other, and the spaces are filled with cells or chemotactic factors. The wells 31 and 32 according to this example are formed to be recessed by about 100 μm from one surface of the cell observation chip 1. In addition, there is no special restriction | limiting in the volume of the wells 31 and 32, What is necessary is just to have the minimum volume required at the time of filling a sample. For example, the depth may be about 100 μm, and the width and depth may be about 1 to 2.5 mm.

  The wells 31 and 32 are provided with through holes 21 ′ and 22 ′, respectively. The through holes 21 ′ and 22 ′ are connected to the through holes 23 and 24 of the pressing block 6 provided on the upper surface of the cell observation chip 1. Thus, the cell observation unit 100 is connected to the upper part. The through holes 23 and 24 are fine holes for injecting and collecting a sample. The cross-sectional shapes and sizes of the through holes 23 and 24 and the through holes 21 ′ and 22 ′ are not particularly limited. For example, it is only necessary that a sample can be injected using a microsyringe having a diameter of 0.5 mm. . For this reason, if the cross-sectional shape of the through holes 21 and 22 is circular, the diameter is about 1 mm, and if it is square, the length of one side is about 1 mm.

  Between the well 31 and the well 32, the flow path part 4 which connects both is provided. FIG. 2 is an enlarged perspective view of the flow path portion 4 of FIG. The direction indicated by the arrow in FIG. 1 coincides with the direction indicated by the arrow in FIG. As shown in FIG. 2, the channel portion 4 is formed with a fine passage 42 separated by a barrier 41, and when a concentration gradient of the chemotactic factor is formed, cells pass through the passage 42 and have a higher concentration. Since it moves to, chemotaxis can be observed. The cell observation chip 1 in which such a fine passage 42 is formed can be used not only for chemotaxis observation but also for evaluation of cell state observation and blood viscosity measurement.

  The passage 42 is formed so as to be recessed by about 5 μm from one surface of the cell observation chip 1. That is, the height of the barrier 41 that is a convex portion is about 5 μm. Moreover, the width | variety of the fine channel | path 42 is about 2-10 micrometers.

  The cell observation chip 1 is formed of a thermoplastic elastomer, for example, an olefin elastomer. If manufactured using an olefin-based elastomer, fine processing such as the barrier 41 and the passage 42 of the flow path portion 4 can also be manufactured by injection molding. The resin injection molding method can be mass-produced at low cost if a mold is manufactured. Therefore, it is less expensive and less time-consuming than conventional silicon wafer processing methods using micromachining technology or PDMS molding methods. It is suitable for mass production, but the finished molding has distortion.

  Next, a method of joining the cell observation chip 1 and the substrate 10 will be described with reference to FIG. FIG. 3A shows a state where the cell observation unit 100 shown in FIG. 1 is housed in the pressing jig 50 before the respective members are joined, and description of similar symbols is omitted. To do. The pressing block 6 is provided between the clamping portion 51 of the pressing jig 50 and the cell observation chip 1 in order to press the cell observation chip against the substrate 10. The pressing block 6 is made of a softer material than the cell observation chip 1. The pressing block 6 is provided with through holes 23 and 24 that are connected to the through holes 21 ′ and 22 ′ of the cell observation chip 1 and penetrate to the upper surface of the pressing block 6.

  The pressing block 6 is preferably sufficiently thicker than the cell observation chip 1. The pressing block 6 is made of a material softer than the cell observation chip 1, but as described above, the through holes 23 and 24 connected to the through holes 21 'and 22' provided in the cell observation chip 1 are provided. If it is too soft, the through holes 23 and 24 are blocked by the collapse of the pressing block itself. Therefore, the minimum hardness is required so that the through holes 23 and 24 are not blocked by the collapse of the pressing block 6 itself.

  With such a configuration, the cell observation chip 1 is pressed against the substrate 10 by tightening with the tightening portion 51 as in the conventional case. The arrow in FIG. 3B shows the actual tightening state. Since the pressing block 6 is made of a softer material than the cell observation chip 1, the pressure block 6 is deformed according to the strain of the cell observation chip 1 as shown in FIG. When the pressure is further pushed, the force of the distorted portion escapes as shown by the arrow in the portion where the pressing force is biased due to the distortion of the cell observation chip 1, and the force is uniformly applied as a whole. Due to this effect, the cell observation chip 1 is uniformly pressed against the substrate 10. Since the direct bonding surface is pressed with an equal pressure, the sealing performance is ensured. Further, the excessive pressing force escapes to the portion where the pressing force is insufficient as shown by the arrow in the figure, so that the fine structure applied to the chip material is not crushed.

  Naturally, it is essential to adjust the pressing force to such an extent that the fine structure of the flow path portion 4 is not crushed while ensuring the sealing property between the cell observation chip 1 and the substrate 10. Since it can be adjusted not by adjusting the pressure but by the hardness and thickness of the material of the pressing block 6, more precise adjustment is possible than by adjusting the pressing force of the pressing jig 50. Is valid.

  Furthermore, since the pressing block 6 has the through holes 23 and 24 connected to the through holes 21 ′ and 22 ′ provided in the cell observation chip 1 as described above, it is joined to the cell observation chip 1. Thus, a flow path connected to the wells 31 and 32 is formed. This flow path becomes a microhole for injecting and collecting samples such as cells or chemotactic factors. If the pressing block 6 and the cell observation chip 1 are joined by direct joining, an adhesive medium such as an adhesive becomes unnecessary, so that the manufacturing cost can be reduced. However, the present invention is not limited to this. In addition, if the cell observation chip 1 and the pressing block 6 are bonded directly, it is preferable to simplify the manufacturing process that is performed simultaneously when the cell observation chip 1 and the substrate 10 are directly bonded. However, it may be a separate step.

  In the present embodiment, it is assumed that the pressing jig 50 is removed after the pressing is completed. However, the pressing jig 50 may be part of the cell observation unit without being removed. FIG. 4 is a cross-sectional view of an example of the cell observation unit 101 when the pressing jig 70 is also a part of the cell observation unit. As shown in FIG. 4A, the pressing jig 70 includes at least a tightening portion 71 and an observation portion 72. The tightening portion 71 and the pressing jig 70 are not particularly limited, but as an example, the tightening portion 71 and the pressing jig 70 are screw tightening in the present embodiment, and are tightened by rotating the tightening portion 71.

  The fastening portion 71 has through holes 25 and 26 connected to the through holes 23 and 24 provided in the pressing block 6. The through holes 25 and 26 serve to inject and collect a sample such as a cell or a chemotactic factor. The tightening portion 71 is position-adjusted and tightened so that the through holes 23 and 24 and the through holes 25 and 26 are connected.

  The observation of chemotaxis is performed from the observation unit 72. The observation part 72 should just be able to observe the state of the flow-path part 4 through the board | substrate 10 which has transparency. Although not particularly limited, a notch may be provided, or it may be formed of a transparent material. When the observation part 72 is formed of a material having transparency, not only the observation part 72 but the entire pressing jig 70 may be formed of a material having transparency.

  The pressing jig 70 as described above is used in place of the pressing jig 50 and tightened as described above. FIG. 4B is a cross-sectional view in a tightened state. The pressing block 6 is deformed by pressing, and the effect of eliminating one side due to the distortion of the cell observation chip 1 is the same as in FIG. If such a configuration is used, it is possible to omit the step of taking out the cell observation unit after being clamped by the pressing block 70, which is in accordance with the purpose of improving mass productivity. Since the cell observation unit according to the present invention is a fine 14 mm square, for example, the material cost of the pressing jig 70 is lower than the cost that can be reduced by omitting the step of removing the cell observation unit 100 from the pressing jig. The production cost can also be reduced.

  As another method of directly joining the cell observation chip 1 and the substrate 10 without causing one side, a method by vacuum suction as shown in FIG. 9 can be considered. In FIG. 9A, the cell observation chip 1 and the substrate 10 are enclosed in a bag-like suction bag 60 such as a plastic bag. The suction bag 60 is completely sealed except for the suction port 61. When air in the suction bag 60 is sucked from the suction port 61 in this state, the suction bag 60 is in close contact with the cell observation chip 1 and the substrate 10 as shown in FIG. 9B, and the cell observation chip 1 is pressed. Since the pressure is evenly pressed against the substrate 10 without being biased, the sealing performance by direct bonding is ensured and the fine structure does not collapse. However, in this method, at least the cell observation chip 1 and the substrate 10 are enclosed in the suction bag 60, vacuum suctioned, and taken out from the suction bag, which is necessary for cell observation in order to reduce costs and increase mass productivity. The meaning of using a resin material as the material of the chip 1 is weakened. Therefore, the method according to the present invention is more suitable for achieving the object than the method using vacuum suction.

Embodiment 2
Next, a method for manufacturing the cell observation unit according to the second embodiment of the present invention will be described with reference to FIG. The parts denoted by the same symbols are the same as those in the first embodiment, and the description thereof is omitted. In FIG. 5A, the pressing block 7 is provided between the tightening portion 51 of the jig 50 and the cell observation chip 1 in order to press the cell observation chip against the substrate 10. The pressing block 7 is preferably sufficiently thicker than the cell observation chip 1. The pressing block 7 is made of a softer material than the cell observation chip 1. Here, in the first embodiment, the pressing block 6 is provided with the through holes 23 and 24, and it is necessary to have such a hardness that the through holes 23 and 24 are not crushed by the collapse of itself. In this embodiment, the pressing block 7 is not provided with a through hole, and may be removed as soon as the direct joining is completed. Therefore, the pressing block 7 does not need to have a certain degree of hardness and is softer than the cell observation chip 1. You just have to.

  With such a configuration, the cell observation chip 1 is pressed against the substrate 10 by tightening with the tightening portion 51 as in the conventional case. FIG. 5B shows the actual tightening state. Since the pressing block 7 is made of a softer material than the cell observation chip 1, it is deformed according to the strain of the cell observation chip 1 as shown in FIG. When the pressure is further pushed, the force of the distorted portion escapes as shown by the arrow in the portion where the pressing force is biased due to the distortion of the cell observation chip 1, and the force is uniformly applied as a whole. Due to this effect, the cell observation chip 1 is uniformly pressed against the substrate 10. Since the direct bonding surface is pressed with an equal pressure, the sealing performance is ensured. Further, the excessive pressing force escapes to the portion where the pressing force is insufficient as shown by the arrow in the figure, so that the fine structure applied to the chip material is not crushed.

  The pressing block 7 is not necessarily required if the direct bonding between the cell observation chip 1 and the substrate 10 is completed and the bonding strength sufficiently satisfies the requirements of the application, and may be removed.

It is sectional drawing showing the unit for cell observation concerning this invention. It is an expansion perspective view showing the flow-path part of the chip | tip for cell observation concerning this invention. It is sectional drawing showing the manufacturing method of the unit for cell observation concerning Embodiment 1 of this invention. It is sectional drawing showing the manufacturing method of the unit for cell observation concerning Embodiment 1 of this invention. It is sectional drawing showing the manufacturing method of the unit for cell observation concerning Embodiment 2 of this invention. It is sectional drawing showing the unit for cell observation concerning a prior art. It is sectional drawing showing the manufacturing method of the chip | tip for cell observation concerning a prior art. It is sectional drawing showing the manufacturing method of the unit for cell observation concerning a prior art. It is sectional drawing showing the manufacturing method of the unit for cell observation concerning the method compared with embodiment of this invention.

Explanation of symbols

1 Chip for cell observation, 21, 22, 21 ′, 22 ′, 23, 24 Through hole,
31, 32 wells, 4 channels, 41 barriers, 42 passages, 5 blocks,
6,7 Press block, 10 substrate, 11,12 mold, 13,14 pin,
50 pressing jig, 51 clamping part, 60 suction bag, 61 suction port,
70 Pressing jig, 71 Tightening part, 72 Observation part 100, 101 Cell observation unit, P excessive adhesion part, Q insufficient adhesion part,

Claims (5)

A method for producing a cell observation unit in which a substrate having at least transparency and a cell observation chip having a fine structure formed of a resin material softer than the substrate are fixed to each other,
A cell observation unit in which the cell observation chip and the substrate are fixed by pressing the cell observation chip against the substrate using a softer pressure block than the cell observation chip molded from a resin material. Manufacturing method.   The pressing block has a through-hole connected to a hole provided in the cell observation chip, and the cell observation chip and the pressing so that the hole of the cell observation chip and the hole of the pressing block are connected to each other. The method for producing a cell observation unit according to claim 1, wherein the block is fixed and the pressing block is also a part of the cell observation unit.   3. The cell observation chip and the substrate are fixed to each other by direct bonding by pressing the cell observation chip against the substrate with the pressing block. A method for producing a cell observation unit. A substrate having at least transparency, a cell observation chip having a fine structure fixed to the upper surface of the substrate and molded from a resin material, and an elastic block fixed to the upper surface of the cell observation chip are further provided. A cell observation unit, wherein the cell observation chip is softer than the substrate, and the elastic block is softer than the cell observation chip . The elastic block has a through hole connected to a hole provided in the cell observation chip, and the cell observation chip and the elasticity are connected so that the hole of the cell observation chip and the hole of the pressing block are connected. The cell observation unit according to claim 4, wherein the block is fixed .

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