WO2019235513A1

WO2019235513A1 - Coating unit, microcoating device, and method for producing cell chips and cell tissue chips

Info

Publication number: WO2019235513A1
Application number: PCT/JP2019/022299
Authority: WO
Inventors: 明石　満; 隆美赤木; 江上　正樹; 山中　昭浩; 陽香中村
Original assignee: 国立大学法人大阪大学; Ntn株式会社
Priority date: 2018-06-08
Filing date: 2019-06-05
Publication date: 2019-12-12

Abstract

A method for producing cell chips and cell tissue chips using the coating unit and microcoating device according to the present invention, said method comprising: a step for immersing the tip of a first coating needle in a first coating liquid pooled in a first coating liquid container, thus allowing the first coating needle to hold the first coating liquid on the tip thereof, and then contact-coating an object to be coated with the first coating liquid which is held on the tip of the first coating needle; and a step for immersing the tip of a second coating needle in a second coating liquid pooled in a second coating liquid container, thus allowing the second coating needle to hold the second coating liquid on the tip thereof, and then contact-coating the object to be coated with the second coating liquid which is held on the tip of the second coating needle. Thus, a large quantity of cell chips and cell tissue chips having a desired coating amount can be produced within a short period of time, even in the case of using a highly viscous cell-containing solution as a starting material.

Description

Coating unit, fine coating apparatus, and cell chip and cell tissue chip manufacturing method

The present invention relates to a coating unit for coating a liquid substance and a fine coating apparatus including the coating unit, and in particular, a coating unit and a fine coating apparatus capable of performing fine coating using a coating needle, and a coating unit and a fine coating apparatus. The present invention relates to a method for producing a cell chip and a cell tissue chip in vitro using the above.

Examples of forming a pattern by applying a liquid material include circuit patterns used for electric / electronic parts. In order to form such a circuit pattern, a coating apparatus such as an ink jet method is generally used. Inkjet coating devices have problems in drawing fine circuit patterns and the like because they may clog at the tip of the nozzle when a highly viscous material is used as the liquid material to be coated. It was a thing. Therefore, there is a demand for a fine coating apparatus that can easily and accurately draw a fine circuit pattern or the like in place of the ink jet method.

In addition, in the fine coating apparatus capable of drawing a fine pattern, in addition to the use in the industrial field such as the electric and electronic parts as described above, its use is also examined in other fields. ing.

Recently, it has been studied to use a solution in which cells are suspended as a liquid substance to construct a three-dimensional cell structure in vitro by a coating operation using a fine coating apparatus. A technique for constructing a three-dimensional tissue of a cell in vitro is important in drug discovery research and regenerative medicine research, and in particular, construction of a cellular tissue similar to a human biological tissue is required.

When handling cells in vitro, it is common to perform two-dimensional cell culture using a culture vessel such as a plastic dish or glass petri dish. However, since cells grow three-dimensionally in an actual living body to form tissues and organs, a three-dimensional cell tissue chip is constructed in order to realize a culture environment close to the living body. Performing evaluation experiments on tissue chips is important for the progress of drug discovery research and regenerative medicine research.

An assay method for evaluating each cell aggregate by arranging a cell tissue chip (cell aggregate) in which cells are aggregated and aggregated in each of a plurality of wells (recesses) arranged in a well plate, for example, It is widely used in function evaluation and screening for selecting effective compounds as new drugs. By performing an assay on such cell aggregates, it is possible to evaluate many items in a short time with a small amount of sample, and the rapidity, simplicity, safety, reproducibility, and high reliability are achieved. It is advantageous in terms of securing.

There are two types of techniques for precisely patterning and arranging cells on a substrate: a method of controlling a cell adhesion by processing a substrate by a photolithography technique and a method of arranging and immobilizing cells by directly printing them. In recent years, with the development of 3D printer technology for arranging and stacking cells in three dimensions (3D), construction of cell aggregates using 3D printers has been studied, and tissue models obtained from cell aggregates have been studied. Application to drug discovery research and regenerative medicine is actively developed.

JP 2008-126459 JP 2008-017798 JP 2010-022251 JP 2015-229148 JP 2016-087822 JP-A-2017-163931 JP 2017-169560 JP-A-2017-131144 JP-A-2015-112576

3D printers for cell assembly include ink jet printers (inkjet thermal type, piezo type), micro extrusion printers (microextrusion printer), laser-assisted printers (pulse laser) Method). However, in these printers, materials that can be ejected are limited, and printing speed (production speed) and resolution (ejection amount) are greatly limited. Furthermore, in the conventional 3D printer, when the material to be discharged is a cell-containing solution containing cells, there are problems regarding the cell viability and cell density of the cell aggregate to be manufactured in the state after discharge. In particular, when producing cell aggregates from a highly viscous cell-containing solution, high-viscosity solutions may be clogged at the printer nozzle, so high resolution, that is, production of a large number of fine cell aggregates. It could not be done and had a problem.

Also, in order to prevent damage due to drying of the cells in the cell aggregate, it is necessary to embed the cells with a gelling agent. When embedding cells with a gelling agent, a two-component gelling agent that gels when the two components are mixed is used. In addition, when an ultraviolet curable gelling agent or a thermosetting gelling agent is used, the use of such a curing agent is preferable because ultraviolet rays and heat at the time of curing adversely affect the cells. It is not a thing.

When a cell aggregate is produced using a two-component gelling agent, two printers are used, and one gelling agent containing cells is discharged from a nozzle of one printer to a predetermined position, and the other The gelling agent must be discharged from the nozzle of the other printer to the one gelling agent with high accuracy so that the gelling agents react with each other reliably. As described above, it is not easy to produce a cell aggregate by using two different printers so that the two liquid gelling agents are discharged to the same position with high accuracy to be gelled. Further, since two printers are used, there is a problem that the entire apparatus becomes large. In particular, when a cell aggregate is produced using an ink jet method or a dispenser type printer, if a highly viscous material is used as a gelling agent, there is a risk of clogging at the tip of the nozzle, which is highly reliable. It was difficult to reliably discharge a desired amount of the gelling agent from the nozzle while maintaining the properties and high accuracy.

The present invention provides a production method capable of reliably producing a large amount of cell chips and cell tissue chips formed with a desired coating amount in a short time (high speed) even if it is a highly viscous cell-containing material. Even if it is a high-viscosity liquid material, it can provide a fine pattern or droplet spot formed with a desired coating amount in a short time (high speed). A fine coating apparatus provided with a unit and a coating unit is provided.

A method for producing a cell chip and a cell tissue chip according to an aspect of the present invention includes:
A first coating solution container for storing a first coating solution containing cells;
A first application needle that can be immersed in the first application liquid stored in the first application liquid container;
A second coating solution container for storing a second coating solution;
A method of manufacturing a cell chip and a cell tissue chip using a fine coating apparatus comprising: a second coating needle that can be immersed in the second coating liquid stored in the second coating liquid container;
Immersing at least the tip of the first application needle in the first application liquid stored in the first application liquid container to hold the first application liquid at the tip of the first application needle;
A step of bringing the first application liquid held at the tip of the first application needle into contact with an application object and applying the first application liquid to the application object;
Immersing at least the tip of the second application needle in the second application liquid stored in the second application liquid container to hold the second application liquid at the tip of the second application needle; and A step of bringing the second application liquid held at the tip of the application needle into contact with the first application liquid applied to the object to be applied.

Another aspect of the coating unit according to the present invention is:
A first coating solution container for storing a first coating solution;
A first application needle whose tip can be immersed in the first application liquid stored in the first application liquid container and reciprocates;
A second coating solution container for storing a second coating solution;
A tip that can be immersed in the second coating solution stored in the second coating solution container, and a second coating needle that reciprocates,
The second coating liquid held at the tip of the second coating needle is contact-coated at the position where the first coating liquid held at the tip of the first coating needle is applied to the object to be coated. Has been.

According to the present invention, even when a highly viscous cell-containing solution is a material, cell chips and cell tissue chips formed with a desired coating amount can be reliably produced in a large amount in a short time. According to the coating unit and the fine coating apparatus of the present invention, even a highly viscous liquid material can reliably and reliably produce a large number of fine patterns or droplet spots formed in a desired coating amount in a short time. can do.

It is a front view which shows the desktop type fine coating device used in Embodiment 1 which concerns on this invention. (A) is a figure which shows the application needle (1st application needle and 2nd application needle) with which the application | coating unit in the fine application apparatus of Embodiment 1 is equipped, (b) is a tip part of the needle part in an application needle. It is an enlarged view shown. The figure which shows the application | coating operation | movement of the coating unit in the fine coating apparatus of Embodiment 1. (A) is a figure shown in the 1st application operation by the 1st application needle, and (b) is a figure showing the 2nd application operation by the 2nd application needle. (A) is an operation | movement diagram which shows the motion of the front-end | tip in the 1st application | coating operation | movement by a 1st application needle, (b) is an operation | movement diagram which shows the movement of the front-end | tip in the 2nd application | coating operation by a 2nd application needle. FIG. 5 is an operation diagram showing a continuous application operation of a first application operation by a first application mechanism and a second application operation by a second application mechanism in the fine application apparatus of the first embodiment. It is a graph which shows an example of the relationship between the frequency | count of application | coating and the shape (application | coating diameter and application | coating height) of a droplet spot. It is an operation | movement figure which shows the 3rd application | coating operation | movement of a 1st application needle | hook. It is an operation | movement figure which shows the continuous application | coating operation | movement with the 3rd application | coating operation | movement of a 1st application needle, and the 2nd application | coating operation | movement of a 2nd application needle. It is an image which shows the state of the structure | tissue immediately after the 2nd coating liquid was apply | coated with respect to the droplet spot of a 1st coating liquid. It is an image which shows a state when the tissue body shown in FIG. 10 is cultured for 3 days. It is a graph which shows the experimental result of the tip diameter of an application needle, and the number of application cells contained in a droplet spot. It is a graph which shows the experimental result which measured the number of application cells at the time of performing the 3rd application operation using the 1st applicator needle whose tip diameter is 330 micrometers.

First, various aspects in the method for producing a cell chip and a cell tissue chip according to the present invention will be described. The cell chip in the present invention is a chip formed of substantially one cell in a state where, for example, individual cells are substantially dispersed and adhered in a culture container. A cell tissue chip refers to a cell assembly in which cells are assembled, aggregated, laminated and organized on a substrate to function, and a two-dimensional cell aggregate such as a cell sheet or cell sheet Includes superimposed 3D cell aggregates.

The method for producing a cell chip and a cell tissue chip according to the first aspect of the present invention includes:
A first coating solution container for storing a first coating solution containing cells;
A first application needle that can be immersed in the first application liquid stored in the first application liquid container;
A second coating solution container for storing a second coating solution;
A method of manufacturing a cell chip and a cell tissue chip using a fine coating apparatus comprising: a second coating needle that can be immersed in the second coating liquid stored in the second coating liquid container;
Immersing at least the tip of the first application needle in the first application liquid stored in the first application liquid container to hold the first application liquid at the tip of the first application needle;
Applying the first application liquid to the application object by bringing the first application liquid held at the tip of the first application needle into contact with the application object;
Immersing at least the tip of the second application needle in the second application liquid stored in the second application liquid container to hold the second application liquid at the tip of the second application needle; and A step of bringing the second application liquid held at the tip of the application needle into contact with the first application liquid applied to the object to be applied;
including.

In the method for manufacturing a cell chip and a cell tissue chip according to the second aspect of the present invention, the first coating liquid and the second coating liquid according to the first aspect come into contact with each other to react and gel. It is good also as what includes a process.

In the method for manufacturing a cell chip and a cell tissue chip according to the third aspect of the present invention, the tip diameter of the second application needle of the first aspect or the second aspect is set to the tip of the first application needle. You may comprise larger than a diameter.

The method for producing a cell chip and a cell tissue chip according to a fourth aspect of the present invention is the method according to any one of the first to third aspects, wherein the second application liquid held at the tip of the second application needle is The application speed for contact application may be set to be slower than the application speed for contact application of the first application liquid held at the tip of the first application needle to the application object.

The method for producing a cell chip and a cell tissue chip according to a fifth aspect of the present invention is the method of manufacturing the cell chip and the cell tissue chip according to any one of the first to fourth aspects. Immediately before the first coating liquid comes into contact with the object to be coated, the coating speed of the first coating needle is decreased and brought into contact,
Immediately before the second application liquid held at the tip of the second application needle comes into contact with the first application liquid applied to the object to be applied, the application speed of the second application needle is decreased and brought into contact. Also good.

The method for producing a cell chip and a cell tissue chip according to the sixth aspect of the present invention is the method of producing a cell chip and cell tissue chip according to any one of the first to fifth aspects, wherein the tip is held at the tip of the first application needle. Immediately before the first coating liquid comes into contact with the object to be coated, the first coating needle may be brought into contact with the coating speed after stopping for a predetermined time and later than the coating speed before stopping.

The cell chip and cell tissue chip manufacturing method according to the seventh aspect of the present invention is the method of producing a cell chip and cell tissue chip according to any one of the first to sixth aspects, wherein the tip is held at the tip of the second application needle. Immediately before the second coating liquid comes into contact with the object to be coated, the second coating needle may be brought into contact after being stopped for a predetermined time and later than the coating speed before the stop.

The cell chip and cell tissue chip manufacturing method according to the eighth aspect of the present invention is the method of manufacturing the cell chip and cell tissue chip according to any one of the first to seventh aspects, wherein the tip is held at the tip of the first application needle. The first application liquid is brought into contact with the application object, and the second application liquid held at the tip of the second application needle is applied after a predetermined time has elapsed since the first application liquid is brought into contact with the application object. You may make it contact-apply to the said 1st coating liquid apply | coated to the target object.

The method for producing a cell chip and a cell tissue chip according to a ninth aspect of the present invention is the method according to any one of the first aspect to the eighth aspect, wherein the first application needle is applied to the tip of the first application needle. The process of holding the liquid and applying the first application liquid held at the tip of the first application needle to the object to be applied is repeated a plurality of times, and then the first of the second application needle held at the tip of the second application needle. Two coating solutions may be brought into contact with the first coating solution to be coated.

The method for producing a cell chip and a cell tissue chip according to a tenth aspect of the present invention is the method according to any one of the first aspect to the ninth aspect, wherein the first coating liquid and the second coating liquid are It may be a two-component mixed gelling agent.

The manufacturing method of the cell chip and the cell tissue chip according to the eleventh aspect of the present invention is the application object when applying the second application needle in any one of the first to tenth aspects. May be set higher than the height from the application object at the time of application of the first application needle.

The cell chip and cell tissue chip manufacturing method according to the twelfth aspect of the present invention is the ninth aspect, wherein the step of applying the first application liquid to the application object with the first application needle a plurality of times is performed. When it repeats, you may set so that the height from the application target object at the time of application | coating of the said 1st application needle | hook may be raised gradually.

The method for producing a cell chip and a cell tissue chip according to a thirteenth aspect of the present invention is the method according to any one of the first aspect to the twelfth aspect, wherein the first application needle and the second application needle are You may comprise so that a front-end | tip may include the plane orthogonal to the moving direction at the time of application | coating.

According to a fourteenth aspect of the present invention, there is provided the cell chip and cell tissue chip manufacturing method according to any one of the first to thirteenth aspects, wherein the first application needle is the first application liquid container. The first coating liquid may be applied to the coating object through the second coating needle, and the second coating needle may penetrate the second coating liquid container to apply the second coating liquid to the coating object. Good.

The method for producing a cell chip and a cell tissue chip according to the fifteenth aspect of the present invention is the method according to any one of the first to fourteenth aspects, wherein the cells in the first coating solution are unmodified. Or modified cells may be used.

The coating unit and the fine coating device in the present invention are devices used for coating operations in various industrial and industrial facilities. For example, in various industrial and industrial devices using a highly viscous adhesive or the like. Used. In addition, the coating unit and the fine coating apparatus according to the present invention can be used as a medical auxiliary device, for example, for a coating operation in cell culture. In Embodiment 1 of the present invention to be described later, a coating operation of a two-component gelling agent containing cells will be described as an example of a coating target, but the coating unit and the fine coating device in the present invention are generally industrial. It is used in a coating operation in various industrial and industrial facilities, for example, a coating operation in various industrial and industrial devices using a highly viscous adhesive or the like.

Next, various aspects of the coating unit and the fine coating apparatus according to the present invention will be described.

The coating unit according to the sixteenth aspect of the present invention is
A first coating solution container for storing a first coating solution;
A first application needle whose tip can be immersed in the first application liquid stored in the first application liquid container and reciprocates;
A second coating solution container for storing a second coating solution;
A tip that can be immersed in the second coating solution stored in the second coating solution container, and a second coating needle that reciprocates,
The second coating liquid held at the tip of the second coating needle is contact-coated at the position where the first coating liquid held at the tip of the first coating needle is applied to the object to be coated. Has been.

In the application unit of the seventeenth aspect according to the present invention, the tip diameter of the second application needle of the sixteenth aspect may be configured to be larger than the tip diameter of the first application needle.

The coating unit according to an eighteenth aspect of the present invention is the coating unit according to the sixteenth aspect or the seventeenth aspect, wherein the coating speed at which the second coating liquid held at the tip of the second coating needle is contact-coated is set. The first application liquid held at the tip of the first application needle may be configured to be slower than the application speed at which the first application liquid is applied in contact with the object to be applied.

The coating unit according to a nineteenth aspect of the present invention is the coating unit according to any one of the sixteenth to eighteenth aspects, wherein the first coating liquid held at the tip of the first coating needle is coated. Immediately before contact with the object, the application speed of the first application needle is decreased, and the second application liquid held at the tip of the second application needle is applied to the first application liquid applied to the application object. The application speed of the second application needle may be decreased immediately before contact.

A coating unit according to a twentieth aspect of the present invention is the coating unit according to any one of the sixteenth to nineteenth aspects, wherein the first coating liquid held at the tip of the first coating needle is a coating target. Immediately before contact with an object, the first application needle may be configured so as to be slower than the application speed before stopping after temporarily stopping for a predetermined time.

According to a twenty-first aspect of the present invention, there is provided the application unit in which the second application liquid held at the tip of the second application needle in any one of the sixteenth to twentieth aspects is an object to be applied. The second application needle may be configured so as to be slower than the application speed before the stop once after stopping for a predetermined time immediately before touching.

A coating unit according to a twenty-second aspect of the present invention is the coating unit according to any one of the sixteenth to twenty-first aspects, wherein the height from the coating object when the second coating needle is applied is You may set higher than the height from the application target object at the time of application | coating of a 1st application needle.

The application unit according to a twenty-third aspect of the present invention is the application unit according to any one of the sixteenth to twenty-second aspects, wherein the first application liquid is applied to an application object a plurality of times by the first application needle. The height from the application object at the time of application | coating of the said 1st application needle | hook may be comprised so that it may raise gradually.

The application unit according to a twenty-fourth aspect of the present invention is the application unit according to any one of the sixteenth to twenty-third aspects, wherein the tips of the first application needle and the second application needle move during application. It may be configured to include a plane orthogonal to the direction.

A coating unit according to a 25th aspect of the present invention is the coating unit according to any one of the 16th to 24th aspects, wherein the first coating needle penetrates the first coating liquid container and the first The application liquid may be applied to the application object, and the second application needle may be configured to pass through the second application liquid container and apply the second application liquid to the application object.

A coating unit according to a twenty-sixth aspect of the present invention is the coating unit according to any one of the sixteenth to twenty-fifth aspects, wherein the second coating liquid storage capacity is stored in the second coating liquid container. The storage capacity of the first coating liquid stored in the first coating liquid container may be larger than the storage capacity of the first coating liquid.

A fine coating apparatus according to a twenty-seventh aspect of the present invention is a coating unit according to any one of the sixteenth to twenty-sixth aspects,
An XY table in which a coating object to be contacted and coated by the first coating needle and the second coating needle in the coating unit is placed and fixed, and is movable in the horizontal direction;
A vertical movement mechanism that allows the application unit to move in the vertical direction;
May be provided.

A fine coating apparatus according to a twenty-eighth aspect of the present invention is the observation optical system unit for observing a coated material that is applied in contact with the coating object placed on the XY table in the twenty-seventh aspect. May be provided.

According to a twenty-ninth aspect of the present invention, there is provided a fine coating apparatus for evaluating a coating applied in contact with a coating object placed on the XY table in the twenty-seventh or twenty-eighth aspect. An evaluation system device may be provided.

The fine coating apparatus according to the present invention can use various coating liquids as liquid substances. For example, in addition to a coating liquid used for industrial and industrial purposes such as a two-component adhesive, 2 Cell chips and cell tissue chips can be produced using a liquid gelling agent as a coating solution. In the following embodiments, a case where a two-component gelling agent containing cells is used as a coating solution for a fine coating apparatus will be described as an example of a coating solution used for industrial and industrial purposes.

The fine coating apparatus according to the present invention, as will be described using specific examples in the embodiments to be described later, allows several pL (picoliters) of fine droplets adhered to the tip of the coating needle to be used as a pole per time. A device that can be applied to a predetermined position of an object with high accuracy in a short time, for example, in 0.1 seconds, and is highly safe and reproducible in the production of cell chips and cell tissue chips, and can be automated. A large amount of highly reliable cell chips and cell tissue chips (cell aggregates) can be manufactured.

The cell chip used in the embodiment is a chip formed of substantially one cell in a state in which individual cells are substantially dispersed and adhered in a culture container, for example, as described above. A cell tissue chip refers to a cell assembly in which cells are assembled, aggregated, laminated and organized on a substrate to function, and a two-dimensional cell aggregate such as a cell sheet or cell sheet It includes three-dimensional cell aggregates that are superimposed.

In the present invention, a conventional coating apparatus (printer) includes a plurality of coating needles, for example, a two-needle type coating unit including two coating needles and a fine coating apparatus including a coating unit. Gelled cell chips and cell tissue chips (cell aggregates) that have been difficult to deal with can be stably produced at high speed. As a result, various cell tissue chips can be automated and manufactured in large quantities in a sterile state. Therefore, the fine coating apparatus according to the present invention can be used not only in the production of cell chips and cell tissue chips, but also in the case of using a coating solution used for industrial and industrial purposes such as a two-component adhesive. It becomes possible to reliably produce a large amount of fine drawing patterns and droplet spots in a short time with high reliability.

Next, a method for manufacturing a cell chip and a cell tissue chip, a coating unit, and a fine coating apparatus according to the present invention will be described with reference to the accompanying drawings using embodiments showing specific configuration examples. The cell chip and cell tissue chip manufacturing method, the coating unit, and the fine coating apparatus according to the present invention are not limited to the configurations of the embodiments described below. The manufacturing method, and the structure based on the technical idea equivalent to the coating unit and the fine coating apparatus are included.

(Embodiment 1)
A specific embodiment 1 will be described below with reference to the accompanying drawings. FIG. 1 is a front view showing a desktop type fine coating apparatus 1 used in the first embodiment. A fine coating apparatus 1 shown in FIG. 1 includes a display / control unit (not shown) for performing setting, control, and display in the fine coating apparatus 1. The display / control unit in the fine coating apparatus 1 according to the first embodiment includes a so-called personal computer (PC).

The fine coating apparatus 1 can be finely adjusted in a main body base 2 arranged horizontally on a table so that the XY table 3 can be moved in the horizontal direction and can be moved in the vertical direction (vertical direction) with respect to the XY table 3. A coating unit 4, an observation optical system unit (for example, a CCD camera, etc.) 5A for visually observing a coated material (cells etc.) on the XY table 3, and a measuring instrument for measuring the produced coated material And an evaluation system device (for example, a laser displacement meter, a white interferometer, etc.) 5B. On the XY table 3, for example, an instrument such as a well plate having a large number of dents (wells) is placed and fixed as a coating object, for example, as a culture container 6 that is a cell culture container. For example, a coating solution that is a cell-containing solution is applied to each well of the well plate to produce a plurality of cell chips or cell tissue chips. Note that the fine coating apparatus of the present invention includes a configuration that does not include the observation optical system unit 5A and the evaluation system device 5B, or a configuration that includes either one.

In addition, in Embodiment 1, as shown in FIG. 1, it demonstrates by the structure of the desktop type fine coating apparatus 1, However, This invention is not limited to such a desktop type structure, but substantially. A large-sized fine coating apparatus 1 that is enlarged in size and installed in a culture room or the like is included.

The coating unit 4 in the fine coating apparatus 1 configured as described above is configured to perform a cell coating operation for producing a cell chip or a cell tissue chip in each well of the culture vessel 6 on the XY table 3. Yes. Hereinafter, the configuration of the coating unit 4 and the cell coating operation by the coating unit 4 will be described.

[Configuration of coating unit]
The coating unit 4 in the fine coating apparatus 1 includes two coating mechanisms (a first coating mechanism 4A and a second coating mechanism 4B), and each

coating mechanism

4A, 4B is configured to perform a coating operation independently. Has been. The fine coating apparatus 1 is configured to use a two-liquid gelling agent as a coating liquid in order to produce a cell chip or a cell tissue chip, and includes a first coating mechanism 4A and a second coating mechanism 4B. .

(A) of FIG. 2 is a view showing the application needle 7 attached to the first application mechanism 4A and the second application mechanism 4B in the application unit 4. The application needle 7 has a needle holding portion 8 and a needle portion 9 protruding from the needle holding portion 8. FIG. 2B is an enlarged view showing the distal end portion of the needle portion 9 in the application needle 7. The tip portion of the application needle 9 of Embodiment 1 is formed in a conical shape, and the tip 9 a is formed in a flat surface so as to face the horizontal surface of the XY table 4. That is, the plane of the tip 9a is a horizontal plane orthogonal to the vertical direction.

In the fine coating apparatus 1, the application needle 7 (first application needle 7A) attached to the first application mechanism 4A and the application needle 7 (second application needle 7B) attached to the second application mechanism 4B are: Although the diameter of the tip 9a of the application needle 9 is different, other configurations are substantially the same. The diameter d of the tip 9a of the application needle 9 is largely related to the shape of the cell chip or cell tissue chip to be produced. In the fine coating apparatus 1 of Embodiment 1, the diameter d of the tip 9a of the coating needle 9 is, for example, 330 μm for the first coating needle 7A mounted on the first coating mechanism 4A, and is mounted on the second coating mechanism 4B. The second application needle 7B is, for example, 1000 μm. Thus, a fine cell chip or cell tissue chip was produced using the first application needle 7A and the second application needle 7B having different tip diameters. In the first embodiment, the needle holding portion 8, the needle portion 9, and the tip 9a of the first application needle 7A and the second application needle 7B will be described using the same reference numerals.

As shown in FIG. 2 (b), the tip end portion of the application needle 7 has a conical shape, and the tip end 9a is a horizontal plane. By polishing the tip end 9a to a horizontal plane, the diameter d of the tip end 9a is desired. It is easy to form in the diameter.

FIG. 3 is a diagram schematically showing a coating operation in the coating unit 4. In the application operation of the application unit 4, after the first application operation by the first application mechanism 4A is performed, the second application operation by the second application mechanism 4B is performed. The first application liquid 11A applied by the first application operation by the first application mechanism 4A is a first gelling agent in which cells are suspended, and the second application liquid 11B applied by the second application mechanism 4B is the first. It is a 2nd gelatinizer for gelatinizing the coating liquid 11A.

FIG. 3 schematically shows the first application operation in the first application mechanism 4A. As shown in FIG. 3, the first coating mechanism 4A is provided with a first coating solution container 10A in which a coating solution reservoir 10a that stores a predetermined amount of the first coating solution 11A that is a cell-containing solution is formed. The first application mechanism 4A is equipped with a first application needle 7A that penetrates the application liquid reservoir 10a and moves (reciprocates) at high speed in the vertical direction (vertical direction).

The first application operation of the first application needle 7A is achieved by a drive mechanism that converts a rotation operation of a drive source (for example, a motor) into a reciprocating operation via a link mechanism. A plurality of high-speed reciprocations of the first application needle 7A in the vertical direction (vertical direction) are performed by the needle of the first application needle 7A through a through hole (upper hole 10b) formed in the upper end of the first application liquid container 10A. The portion 9 is slidably held, and the high-speed operation reliability of the first application needle 7A is ensured. The first application needle 7A is detachably provided at a predetermined position of a drive mechanism that causes the first application needle 4A to operate at a high speed in the first application mechanism 4A. It is configured to be detachable with a stopper or the like.

As described above, the first application needle 7A fixed to the drive mechanism in the first application mechanism 4A reciprocates at a high speed between predetermined intervals in the vertical direction (vertical direction). The drive control of such a drive mechanism, the setting of drive control of the vertical movement mechanism that moves the entire coating unit 4 including the YX table 3 and the first coating mechanism 4A in the vertical direction (vertical direction), etc. Done in The reciprocating motion of the first application needle 7A in the vertical direction is an extremely high speed. For example, one reciprocation can be set to 0.5 seconds or less and further to 0.1 seconds or less.

As described above, the first application mechanism 4A in the application unit 4 is configured to hold the first application needle 7A and reciprocate in the vertical direction at a high speed, and the first application needle 7A is a cell-containing solution. The coating liquid reservoir 10a for storing the first coating liquid 11A is configured to move in the vertical direction (vertical direction) and penetrate therethrough. Holes (upper hole 10b and lower hole 10c) that are penetrated by the first application needle 7A are formed in the upper and lower parts of the first application liquid container 10A having the application liquid reservoir 10a.

The first application mechanism 4A in the application unit 4 is configured as described above and drives the first application needle 7A. The second application mechanism 4B in the application unit 4 is also configured in the same manner, and the second application needle 4A. 7B penetrates the coating liquid reservoir (10a) of the second coating liquid container 10B (see the coating liquid container 10A in FIG. 3) and moves at a high speed in the vertical direction (one reciprocating operation). That is, the second application operation of the second application needle 7A is achieved by a drive mechanism that converts the rotation operation of a drive source (for example, a motor) into a reciprocating operation via a link mechanism. In the first application operation of the first application needle 7A and the second application operation of the second application needle 7A, the rotation operation of one drive source is transmitted via the first link mechanism and the second link mechanism, respectively. It is the structure switched to. The first link mechanism and the second link mechanism are configured to be able to perform speed control during a reciprocating operation.

In the first application mechanism 4A and the second application mechanism 4B, the diameters of the application needle tips of the first application needle 7A and the second application needle 7B are different as described above. Further, the difference in the coating operation between the first coating mechanism 4A and the second coating mechanism 4B is that the number of times of coating in the first coating operation is a plurality of times, whereas the number of times of coating in the second coating operation is one. And the lowest point position of the tip of the application needle during the reciprocation of the application operation.

As described above, the first application operation by the first application mechanism 4A and the second application operation by the second application mechanism 4B are substantially the same except for the number of applications and the position of the lowest point of the tip during application. It is. For this reason, the second coating operation by the second coating mechanism 4B will be described mainly with reference to the schematic diagram of FIG.

[Coating operation]
In the first application operation by the first application mechanism 4A shown in FIG. 3, the first application liquid 11A (first gelling agent) containing cells stored in the application liquid reservoir 10a of the first application container 10A is applied. It is the cell application | coating operation | movement apply | coated on the bottom face of the well of the culture container 6 which is.

In the cell coating operation, the first coating needle 7A passes through the coating liquid reservoir 10a of the first coating container 10A, and the first coating liquid 11A containing cells is contact-coated on the bottom surface of the well of the culture container 6. In this contact application, a very small amount of the first application liquid 11A held on the tip 9a of the first application needle 7A is applied in contact with the bottom surface of the well, and the tip 9a of the first application needle 7A is applied. And the application by contact with the bottom surface of the well is different.

(A) of FIG. 3 shows the coating solution immersion state of the first coating needle 7A in the cell coating operation. In this coating solution immersion state, the first coating needle 7A is inserted into the coating solution reservoir 10a from the upper hole 10b, and the tip 9a of the first coating needle 7A is immersed in the first coating solution 11A of the coating solution reservoir 10a. ing. In this coating liquid immersion state, since the tip 9a of the first coating needle 7A is immersed in the first coating liquid 11A, the first coating liquid 11A attached to the tip 9a is not dried. At this time, since the diameter of the lower hole 10c of the first application container 10A is fine (for example, 1 mm or less), the first application liquid 11A does not leak from the application liquid reservoir 10a.

3B, the tip 9a of the first application needle 7A protrudes from the lower hole 10c of the first application container 10A, and the first application liquid 11A held by the tip 9a contacts the bottom surface of the well of the culture container 6. The application state is shown. That is, in a lowered state in which the tip 9a of the first application needle 7A protrudes through the coating liquid reservoir 10a, a fixed amount of the first application liquid 11A is held at the tip of the first application needle 7A. For this reason, when the first coating liquid 11A held at the tip 9a of the first coating needle 7A contacts the bottom surface of the well, the first coating liquid 11A moves from the tip 9a of the first coating needle 7A to the bottom surface of the well. And applied by contact. The fine droplet spot S that has moved to the bottom surface of the well of the culture vessel 6 is raised in a substantially hemispherical shape on the bottom surface of the well due to its surface tension.

The cell application operation by the first application needle 7A is repeated a predetermined number of times, and the first application liquid 11A is applied on the bottom surface of the well. In the cell coating operation a predetermined number of times by the first coating needle 7A, in the second and subsequent contact coatings, the first coating liquid 11A held on the tip 9a of the first coating needle 7A and the first coated on the bottom surface of the well. It is applied by contact with the droplet spot S of the coating liquid 11A. As described above, the cell coating operation is repeated a predetermined number of times (for example, 10 times), and the first coating liquid 11A is contact-coated, and a fine droplet spot S is formed on the bottom surface of the well. For example, by performing the cell coating operation 10 times, when the diameter of the tip 9a of the first coating needle 7A is 330 μm, a droplet spot S having a diameter of about 250 to 300 μm is formed.

As described above, the cell application operation by the first application needle 7A shown in FIGS. 3 (a) and 3 (b) is repeated a predetermined number of times (for example, 10 times), and fine particles are formed on the bottom surface of the well of the culture vessel 6. A droplet spot S is formed. After the droplet spot S of the first coating liquid 11A is formed, the second coating operation by the second coating needle 7B of the second coating mechanism 4B is executed. In the second application operation, the second application liquid 11B is applied in contact with the droplet spot S of the formed first application liquid 11A only once. The first coating solution 11A is a first gelling agent in which cells are suspended, and the second coating solution 11B is a second gelling agent that reacts and gels by mixing with the first gelling agent. The second coating liquid 11B, which is the second gelling agent, is contact-coated so as to cover the first coating liquid 11A, thereby sufficiently reacting with each other to reliably gel the droplet spot S.

The diagrams (a-1) to (a-4) shown in (a) of FIG. 4 schematically show the movement of one first application operation (cell application operation) by the first application needle 7A. It is. The diagrams (b-1) to (b-3) shown in (b) of FIG. 4 are diagrams schematically showing the second application operation by the second application needle 7B. 4 (a), a state (a-1) in which a very small amount of the first application liquid 11A is held at the tip 9a of the first application needle 7A, and the bottom surface of the well of the culture vessel 6 that is the application target. The state (a-2) in which the first coating liquid 11A at the tip 9a of the first coating needle 7A is in contact with the top and the state immediately after the first coating liquid 11A is in contact with the tip 9a of the first coating needle 7A ( a-3), a state (a-4) in which the tip 9a of the first application needle 7A rises from the bottom surface of the well and a fine droplet spot S (height Z1) is formed on the bottom surface of the well; Is schematically shown.

In the fine coating apparatus 1 of the first embodiment, the first coating operation shown in FIG. 4A is a first gelling agent in which cells are suspended by repeating a predetermined number of times, for example, 10 times. A droplet spot S (height Z2) of the coating liquid 11A is formed. As shown in FIG. 4B, a second gelling agent which is a second gelling agent held on the tip 9a of the second application needle 7B with respect to the droplet spot S (height Z2) of the first application liquid 11A. 2 coating liquid 11B is contact-coated.

In the fine coating apparatus 1 according to the first embodiment, the diameter of the tip 9a of the first coating needle 7A used for the first coating operation is, for example, 330 μm, and the tip 9a of the second coating needle 7B used for the second coating operation. Is, for example, 1000 μm. For this reason, the application amount by the second application needle 7B is larger than the application amount by the first application needle 7A. For this reason, the second coating liquid 11B is applied to the fine droplet spot S of the first coating liquid 11A formed in the first coating operation by the first coating needle 7A by the second coating operation by the second coating needle 7B. Will be in a state of covering reliably. For example, according to our experiment, the application amount by the first application operation of the first application needle 7A (tip diameter 330 μm) is about 15 pL, whereas the application amount by the second application operation of the second application needle 7B. Was about 80 pL.

As described above, the first application operation by the first application needle 7A is performed a predetermined number of times (FIG. 4A), and then the second application operation by the second application needle 7B (FIG. 4B) is performed. By doing so, the second coating solution 11B is applied so as to cover the first coating solution 11A, and the first coating solution 11A and the second coating solution 11B react to produce a gelled cell chip or cell tissue chip. Is done.

The application amount by the first application needle 7A and the application amount by the second application needle 7B are greatly different, and the consumption amount of the second application liquid 11B is larger than the consumption amount of the first application liquid 11A. The capacity of the two coating solution container 10B may be larger than that of the first coating solution container 10A. That is, the storage capacity of the second coating liquid 11B stored in the second coating liquid container 10B may be configured to be larger than the storage capacity of the first coating liquid 11A stored in the first coating liquid container 10A.

FIG. 5A is an operation diagram showing the movement of the tip 9a in the first application operation by the first application needle 7A, and FIG. 5B is the operation of the tip 9a in the second application operation by the second application needle 7B. It is an operation | movement figure which shows a motion. In the operation diagrams of FIGS. 5A and 5B, the vertical axis indicates the tip position of the application needle, and the horizontal axis indicates time.

As shown in FIG. 5A, in the first application operation (reciprocating operation) of the first application needle 7A, the tip 9a descends from the upper limit position P1a at the first application speed V1a, and a preset application is performed. When the immediately preceding speed change position P2a is reached, the speed is lowered at a second coating speed V2a that is slower than the first coating speed V1a. The tip 9a of the first application needle 7A descends at the second application speed V2a, and the first application liquid 11A held on the tip 9a is contact-applied at the contact application position P3a. Regarding the contact application position P3a, the position and height of the well bottom surface of the culture vessel 6 that is the application object are calculated based on detection data from a position measuring device or the like provided in the evaluation system device 5B and set in advance. May be. As for the contact application position P3a, the first application origin position in the first application operation may be detected and set in advance, and the second and subsequent contact application positions P3a in the application operation may be set based on the origin position. . That is, in a plurality of application operations by the first application needle 7A, the contact application position P3a may be gradually increased within a range where contact application is possible every time the application operation is performed.

In the first application operation, after contact application is performed at the contact application position P3a, the initial application position returns to the upper limit position P1a at the return speed V3, and the next application operation is repeated. As shown in FIG. 5A, in one application operation, the upper limit position P1a (time T1a) to the speed change position P2a (time T2a) are lowered at the first application speed V1a, and the speed change position P2a ( From the time T2a) to the contact application position P3a (time T3a), the second application speed V2a (<V1a) is lowered to perform contact application. After the contact application, it rises at a predetermined return speed V3 up to the upper limit position P1a (time T4a).

In the fine coating apparatus 1 of the first embodiment, the second coating speed V2a of the first coating needle 7A when reaching the contact coating position P3a is lower than the first coating speed V1a. The tip 9a of the needle 7A can be placed with high accuracy with respect to the set contact application position P3a, and a fine application operation can be performed. Even if the tip 9a of the first application needle 7A comes into contact with the surface of the substrate that is the object to be applied, the contact impact is small and the influence on the cells can be small.

On the other hand, in the second application operation by the second application needle 7B, as shown in FIG. 5B, the first application operation of the first application needle 7A is slower than the first application operation (FIG. 5A). Is set. In the second application operation of the second application needle 7B, the tip 9a of the second application needle 7B descends from the upper limit position P1b at the first application speed V1b (V1b <V1a), and a speed change position immediately before application set in advance. When P2b (P2b = P2a) is reached, the lowering speed is lowered at a second coating speed V2b that is slower than the first coating speed V1b. The tip 9a of the second coating needle 7B descends at the second coating speed V2b, and the second coating solution 11B held on the tip 9a contacts the droplet spot S of the first coating solution 11A at the contact coating position P3b. Applied. Even if the contact application position P3b is calculated and set based on the detection data indicating the position and height of the droplet spot S of the first application liquid 11A by the position measurement device provided in the evaluation system apparatus 5B. Good.

In the second application operation, after the contact application at the contact application position P3b, the return speed V3 returns to the upper limit position P1b which is the initial position. As shown in FIG. 5B, in the second application operation, the speed changes from the upper limit position P1b (time T1b) to the speed change position P2b (time T2b) at the first application speed V1b (<V1a). From the position P2b (time T2b) to the contact application position P3b (time T3b), the coating is performed at the second application speed V2b (<V1b). After the contact application, it rises at a predetermined return speed V3 up to the upper limit position P1b (time T4b).

FIG. 6 is an operation diagram showing a continuous coating operation of the first coating mechanism 4A by the first coating mechanism 4A and the second coating mechanism 4B by the second coating mechanism 4B in the fine coating apparatus 1 of the first embodiment. In the first application operation by the first application mechanism 4A shown in FIG. 6, the first application needle 7A performs the contact application of the first application liquid 11A 10 times on the culture container 6 as the application object. .

As shown in FIG. 6, in 10 contact applications in the first application operation, the contact application position P3a, which is the lowest point position of the first application needle 7A, gradually increases every time one contact application is completed. It is set to move upward. In the first coating operation in the fine coating apparatus 1 of the first embodiment, for example, the contact coating position P3a is moved upward by several μm each time contact coating is performed.

The inventor conducted a measurement experiment on the shape (application diameter and application height) of the droplet spot S formed by the first application operation by the first application mechanism 4A. FIG. 7 is a graph showing an example of the relationship between the number of times of application and the shape of the droplet spot S (application diameter and application height).

As shown in FIG. 7, the application diameter of the droplet spot S gradually increased until the fifth application, and the application diameter did not change greatly after the sixth application. As for the coating height of the droplet spot S, the droplet spot S having a height that is about half of the final coating height is formed by the first coating, and thereafter the final coating height is reached by the fifth. A droplet spot S substantially equal to the coating height is formed. In the case of the example shown in FIG. 7, the shape of the droplet spot S is substantially determined by performing the number of times of coating five times. However, since the relationship between the number of times of application and the shape of the droplet spot S varies greatly depending on the properties such as the viscosity of the coating liquid, it is considered that the shape of the droplet spot S is stabilized by performing at least ten times of application. It is done.

In the fine coating apparatus 1 according to the first embodiment, the contact liquid is applied by applying the coating liquid by bringing the coating liquid held at the tip of the coating needle into contact with an object to be coated (such as the bottom surface of a well of a culture vessel or a droplet spot). A special application operation is performed. For this reason, even if contact application is performed on the droplet spot S formed on the application target, the application operation is not such that all of the coating liquid held at the tip of the application needle is transferred to the droplet spot S on the substrate. . In contact coating, the coating amount and shape of the droplet spot S to be coated are the amount of coating liquid held at the tip of the coating needle and the characteristics of the coating solution, and the surface properties (surface roughness) of the coating object. Etc.

After the first application operation is completed as described above, the object to be applied on the XY table 3 is moved from immediately below the first application needle 7A to just below the second application needle 7B during a predetermined time, The second application operation by the second application needle 7B is started. In the second application operation, the second application liquid 11B is applied by contact with the second application needle 7B so as to cover the droplet spot S of the first application liquid 11A.

The first application liquid 11A (first gelling agent) in which the cells are suspended is applied with the first application needle 7A, and then the second application liquid 11B (second gelling agent) is applied with the second application needle 7B. Since there is a short time (switching time Tc in FIG. 6), the cells are appropriately buffered in the droplet spot S of the first coating liquid 11A to stabilize the cells, and the first coating liquid 11A is suspended. It is possible to prevent turbid cells from flowing out.

In the fine coating apparatus 1 according to the first embodiment, as shown in the operation diagram of FIG. 5, the proximity speed when the first application mechanism 4A is closest to the application target in the first application operation of the first application needle 7A ( V2a) is set to be delayed (V2a <V1a). Depending on characteristics such as viscosity in the coating liquid, there is a case where the coating amount held at the tip 9a of the first coating needle 7A is not constant only by slowing the proximity speed. In this way, when the constant application amount cannot be stably applied only by slowing the proximity speed of the tip 9a, the first application needle 7A is temporarily stopped for a predetermined time at the speed change position P2a that is the position immediately before the application target. By doing so, it becomes possible to make the amount of the first coating liquid 11A held at the tip 9a of the first coating needle 7A uniform.

FIG. 8 is an operation diagram of the tip 9a of the first application needle 7A showing a third application operation for temporarily stopping the first application needle 7A at a speed change position P2a immediately before the application target for a predetermined time. As shown in FIG. 8, when the tip 9a of the first application needle 7A descends at the first application speed V1a and reaches the speed change position P2a (T2), it stops only for a minute predetermined time (T2 → T2 ′). Then, after the predetermined time has passed, the coating is again lowered toward the application object at the second application speed V2a and is applied in contact. In this way, by stopping the tip 9a of the first application needle 7A at a position just before the application target for a minute predetermined time, the holding amount of the first application liquid 11A by the tip 9a of the first application needle 7A is made uniform. be able to. Therefore, by performing the third application operation of the first application needle 7A in the first application mechanism 4A, a constant application amount is stably and reliably applied to the application object according to the characteristics of the application liquid. It becomes possible to do.

As described above, in the third application operation, the operation is temporarily stopped at the speed change position P2a, and after a predetermined time (X) has elapsed, it is lowered to the contact application position P3a at the second application speed V2a that is slower than the first application speed V1a before the stop. To do. After the contact application at the contact application position P3a, the process returns to the upper limit position P1a which is the initial position at the return speed V3, and the next application operation is repeated. As shown in FIG. 8, in one application operation, the first application speed V1a is lowered from the upper limit position P1a (time T1) to the speed change position P2a (time T2), and the speed is changed after a predetermined time (X) has elapsed. From the position P2a (time T2 ′) to the contact application position P3a (time T3c), the second application speed V2a (<V1a) is lowered to perform contact application. After the contact application, it rises at a predetermined return speed V3 up to the upper limit position P1a (time T4c).

Although FIG. 8 shows the third application operation of the first application needle 7A, this third application operation can also be performed on the second application needle 7B. That is, immediately before the second coating liquid 11B held at the tip 9a of the second coating needle 7B comes into contact with the object to be coated, the second coating needle 7B is temporarily stopped after a predetermined time and then brought into contact with the coating speed before stopping. You may let them. In this way, the tip 9a of the second application needle 7B is stopped at a position just before the object to be applied for a minute predetermined time so that the amount of the second application liquid 11B held by the tip 9a of the second application needle 7B is made uniform. be able to.

FIG. 9 is an operation diagram showing a continuous application operation of the third application operation of the first application needle 7A shown in FIG. 8 and the second application operation of the second application needle 7B shown in FIG. is there. In the third application operation by the first application needle 7A shown in FIG. 9, the first application needle 7A is applied to the culture container 6 that is the application object by the first application liquid 11A 10 times. .

In the third application operation shown in FIG. 9, as in the first application operation shown in FIG. 6, the contact application position P3a, which is the lowest point position of the first application needle 7A, is finished once. It is set to move upward gradually. In addition, after the elapse of a predetermined switching time (Tc) after the end of the third coating operation, the second coating liquid 11B is applied in contact with the droplet spot S of the first coating liquid 11A by the second coating needle 7B. . Note that the third application operation shown in FIG. 8 may be performed as the contact application operation by the second application needle 7B at this time.

In the coating operation of the coating unit 4 and the fine coating apparatus 1 in the first embodiment, a very small amount of the coating liquid 11 (11A, 11B) is attached to the tip 9a of the coating needle 7 (7A, 7B) to contact the coating target. By doing so, a droplet spot S having a coating amount of several pL (picoliter) can be applied and formed with high placement accuracy, for example, ± 15 μm or less, preferably ± 3 μm or less. Moreover, as a viscosity of the coating liquid 11, it is possible to apply | coat the material of 1 * 10 < ⁵ > mPa * s or less, and application | coating of a highly viscous cell dispersion liquid is attained. In the coating operation of the fine coating apparatus 1 according to the first embodiment, a viscosity of 10 mPa · s or more, which cannot be used due to a problem such as clogging in a printer using a nozzle such as an inkjet printer, is 1 × 10 ⁵ mPa · s. The following materials can be used as coating materials. Further, since a very small amount of the coating liquid 11 held at the tip 9a of the coating needle 7 is applied in contact with the object to be coated, the coating liquid 11 is stabilized without being affected by variations in the vertical position of the coating needle 7. And can be applied repeatedly. As described above, in the coating unit 4 and the fine coating apparatus 1 according to the first embodiment, a highly viscous cell dispersion can be precisely applied to a coating object at a predetermined position. A cell tissue chip in which chips and cells are three-dimensionally shaped can be manufactured. For this reason, by using the coating unit 4 and the fine coating apparatus 1 according to the first embodiment of the present invention, it is possible to reliably form a fine drawing pattern, and the coating unit and the fine coating apparatus are manufactured. Cell chips and tissue chips are used in the fields of drug discovery research and regenerative medicine, such as screening for drug efficacy and safety evaluation, and are effective in progress in each field.

(Experimental example)
Hereinafter, specific experiments performed by the inventors using the fine coating apparatus 1 described in the first embodiment will be described. However, the substance names and specific coating conditions described in the following description are shown as examples, and the present invention is not limited to these.

In the following experiment, a coating experiment using a two-component gelling agent was performed using the fine coating apparatus 1 of the first embodiment.

In this coating experiment, a tip diameter of 330 μm was used as the first coating needle 7A of the first coating mechanism 4A, and a tip diameter of 1000 μm was used as the second coating needle 7B of the second coating mechanism 4B. As the first coating solution 11A (first gelling agent) in the two-component gelling agent, human skin fibroblasts (NHDF) are dispersed in a 20 mg / mL fibrinogen solution at a concentration of 4 × 10 ⁷ cells / mL. Solution (suspension) was used. On the other hand, as the second coating solution 11B (second gelling agent), an 800 unit / mL thrombin solution was used.

In the application experiment, as shown in FIG. 9, the third application operation by the first application needle 7A (see FIG. 8) and the second application operation by the second application needle 7B (see FIG. 5B) are performed. It was.

First, in the third application operation by the first application needle 7A, the first application needle 7A descends at the first application speed V1a, and the first application needle 7A is stabilized at the first change rate P2 in order to stabilize the application amount. The application needle 7A is temporarily stopped for a predetermined time (period (T2 → T2 ′): X in FIG. 8). When the first application needle 7A is stationary for a predetermined time X, the first application liquid 11A held at the tip 9a is stabilized. After the first application needle 7A is stationary for a predetermined time X, the first application needle 7A descends to the contact application position P3a which is the lowest end at the second application speed V2a which is slower than the first application speed V1a, and performs contact application. The contact application position P3a, which is the lowest end, is moved upward several μm each time application is performed. The moving distance upward of the contact application position P3a for each application is set in advance based on empirical rules, but is measured by a position measuring device or the like provided in the evaluation system device 5B of the fine application device 1. It may be set according to the height of the applied spot. In the application experiment, a moving distance of moving upward each time the first application needle 7A applies a height lower than the height Z1 of the droplet spot S formed by one application of the first application needle 7A. It is said.

In the third application operation by the first application needle 7A in the application experiment, the position of the tip of the needle tip is raised as described above, and Cell Desk LF (registered trademark) (Sumitomo Bakelite Co., Ltd.), which is a plastic cover slip that is the application object. The droplet spot S was formed by continuous contact application 10 times on the bottom of the well of the culture vessel 6 (made).

After the third application operation by the first application needle 7A is completed, the droplet spot S of the first application liquid 11A on the bottom surface of the well is left for several seconds (switching time Tc in FIG. 9) to stabilize the droplet spot S. After that, the second application operation by the second application needle 7B was started. In the second application operation, the second application liquid 11B held at the tip 9a of the second application needle 7B can be applied while reliably contacting the droplet spot S of the first application liquid 11A on the bottom of the well. The tip position of the second application needle 7B is set. For example, the tip position of the second application needle 7B may be set to the height Z2 (the height after the tenth application) of the droplet spot S of the first application liquid 11A on the bottom of the well. The lowermost point position of the second coating liquid 11B that hangs down from the tip 9a of 7B may be set in consideration. By setting the tip position of the second application needle 7B in this way, the second application liquid 11B is reliably applied to the upper surface of the droplet spot S of the first application liquid 11A. As a result, thrombin, which is the second gelling agent of the second coating solution 11B, is applied so as to cover the suspension of the fibrinogen solution containing human skin fibroblasts (NHDF), which is the first coating solution 11A (FIG. 4 (b)), the droplet spot S was fixed to the tissue by gelation to prepare a cell tissue chip. The observation result of the produced cell tissue chip is shown in FIG. 10 and FIG.

FIG. 10 is an image showing the state of the tissue immediately after the second coating liquid 11B is applied to the droplet spot S of the first coating liquid 11A (0 day culture). FIG. 11 is an image showing a state when the tissue shown in FIG. 10 is cultured for 3 days. As shown in FIG. 11, the structure of the cell tissue body (cell aggregate) was confirmed by culturing for 3 days, and it was confirmed that the cell tissue chip was reliably constructed.

In addition, the inventor conducted an application experiment by changing the tip diameter of the application needle 7 and measured the tip diameter of the application needle 7 and the number of applied cells contained in the droplet spot S applied on the substrate. In this experiment, a suspension in which iPS-CM was dispersed in a PBS solution at a concentration of 4 × 10 ⁷ cells / mL was used as the first coating solution 11A. As a result of the experiment, when the tip diameter of the application needle 7 is 50 μm in one application, there is an average of 1.1 coated cells in the droplet spot S, and in the droplet spot S when the tip diameter is 100 μm. Is an average of 4.0 coated cells, the droplet spot S when the tip diameter is 150 μm has an average of 4.5 coated cells, and the droplet spot S when the tip diameter is 200 μm. There was an average of 19.1 coated cells. Then, when the coating needle 7 having a tip diameter of 330 μm was applied once, the droplet spot S had an average of 85.3 coated cells. FIG. 12 is a graph showing experimental results for considering the relationship between the tip diameter of the application needle 7 and the number of applied cells contained in the droplet spot S. In FIG. 12, the vertical axis represents the number of coated cells [cells / spot], and the horizontal axis represents the tip diameter [μm] of the coating needle 7.

As shown in the graph of FIG. 12, the larger the tip diameter of the application needle 7, the more applied cells in the droplet spot S. Therefore, in the fine coating apparatus 1 of the first embodiment, the droplet spot S having a desired number of coated cells is formed on the substrate or the like to be coated by selecting the tip diameter of the first coating needle 7A. It can be understood that this is possible. That is, by selecting the tip diameter, solution cell concentration, solution viscosity, and the like of the application needle 7, it becomes possible to manufacture a desired cell chip and cell tissue chip.

FIG. 13 is a graph showing the experimental results of measuring the number of coated cells when the third coating operation (10 contact coatings) was performed using the first coating needle 7A having a tip diameter of 330 μm. In FIG. 13, the vertical axis indicates the number of applied cells [cells / spot], and the horizontal axis indicates the number of times of application by the first application needle 7A. In this coating experiment, a solution in which iPS-CM was dispersed in a 20 mg / mL fibrinogen solution at a concentration of 4 × 10 ⁷ cells / mL was used as the first coating liquid 11A. As shown in FIG. 13, when the first application needle 7A applied the first application liquid 11A on the well bottom surface 10 times, an average of 162.4 applied cells were present (four experiments). . In the graphs of FIGS. 12 and 13, the standard deviation (positive direction) of the number of applied cells is indicated by an error bar.

As described above, in the fine coating apparatus 1 according to the first embodiment, the droplet spot S including a desired number of cells can be formed by the first coating mechanism 4A having the first coating needle 7A and formed. A cell chip or cell tissue chip that has been gelled and reacted by reliably applying the second coating liquid 11B to the droplet spot S of the first coating liquid 11A by the second coating mechanism 4B having the second coating needle 7B. Can be reliably manufactured.

In the fine coating apparatus 1 according to the first embodiment, the application needle 7 is configured to pass through the coating liquid reservoir 10a of the coating liquid container 10 and apply to the object to be applied, but the present invention has such a configuration. It is not limited. For example, the application needle 7 immersed in the application liquid in the application liquid reservoir 10a may be lifted and moved to the application position of the object to be applied for contact application. Even in the fine coating apparatus configured as described above, a high-viscosity liquid substance, for example, a two-component adhesive, has a predetermined coating amount in a predetermined amount, similarly to the effect of the fine coating apparatus 1 of the first embodiment. A drawing pattern, a droplet spot, etc. can be formed reliably. In addition, even when the highly viscous liquid substance is, for example, a cell-containing gelling agent, a cell chip formed with a desired coating amount by using the coating unit and the fine coating device 1 of the first embodiment. In addition, it becomes possible to reliably manufacture the cell tissue chip in a short time (high speed).

The cells that can be used in the present invention are not particularly limited. For example, fibroblasts, vascular endothelial cells, epidermal cells, smooth muscle cells, cardiomyocytes, gastrointestinal cells, nerve cells, hepatocytes, kidney cells, Various primary cells such as pancreatic cells, differentiated cells derived from iPS cells, various cancer cells, and the like can be used. Examples of cells include unmodified cells or cells modified with proteins, sugar chains, nucleic acids, etc., for example, cells coated with a known coating agent or coating method such as fibronectin, gelatin, collagen, laminin, and elastin. Can be used.

In addition, in order to give the cell-containing solution an environment in which the encapsulated cells can stably adhere and grow, extracellular matrix components such as fibronectin, gelatin, collagen, laminin, elastin, matrigel, fibroblast growth factor and platelets In addition to cell growth factors such as derived growth factors, additives such as vascular endothelial cells, lymphatic endothelial cells, and various stem cells may be included. Further, fibrinogen, alginic acid, a thermosensitive polymer or the like may be included as a gelling agent.

As described with reference to the above-described embodiments and experimental examples, the present invention provides a new manufacturing method for manufacturing a cell chip and a cell tissue chip using a new bioprinter. Compared to the case of manufacturing using a printer using a conventional nozzle, the solution adheres to the tip surface using an application needle, so that the solution is not clogged and the cell aggregate The resolution and the formation speed are improved, and a reliable cell chip and cell tissue chip can be reliably manufactured with a smaller sample amount (sample). In addition, since a cell chip and a tissue tissue chip are manufactured by applying a high-viscosity cell dispersion to an object as compared with the case of using a conventional printer, Evaporation can be suppressed and high cell viability can be maintained.

In the coating unit and the fine coating apparatus according to the present invention, a few pL (picoliter) is obtained by bringing a needle (coating needle) having a very small amount of coating solution attached to the tip thereof into contact with an object to be coated (culture vessel or the like). A droplet having a coating amount can be applied with high placement accuracy, for example, ± 15 μm or less, preferably ± 3 μm or less. Moreover, as a viscosity of a coating liquid, it is possible to apply | coat the material to 1 * 10 < ⁵ > mPa * s, for example, application | coating of a highly viscous cell dispersion liquid is attained.

In addition, by using the coating unit and the fine coating apparatus of the present invention, for example, it becomes easy to produce cell aggregates with a two-component gelling agent, and a large amount of cell aggregates can be manufactured with high accuracy. Can do. Furthermore, according to the present invention, even if a highly viscous cell-containing gelling agent is a material, a desired cell chip and cell tissue chip can be produced in a large amount in a short time (high speed). High cell viability can be maintained with a desired cell density in the cell chip and cell tissue chip.

As a result, according to the present invention, it becomes possible to precisely apply a high-viscosity cell dispersion liquid to an application target (such as a culture vessel) at a predetermined position, and to form a three-dimensional cell chip or cell having arbitrary patterning. A cell tissue chip that is shaped automatically can be manufactured. As a result, the produced cell chip and cell tissue chip can be used in the fields of drug discovery research and regenerative medicine such as screening for drug efficacy and safety evaluation.

Further, the application unit and the fine application apparatus in the present invention are not limited to the application of the two-component gelling agent containing cells described in the first embodiment. For example, a highly viscous adhesive or the like is used. It can also be used in coating operations in various industrial and industrial equipment. As a specific adhesive used in the present invention, it can be used for various two-component adhesives such as acrylic adhesives, epoxy adhesives, urethane adhesives, silicone adhesives, It can be used for adhesion of metal parts, adhesion of electrical / electronic parts, adhesion of building materials, and the like.

Although the present invention has been described in the embodiment with a certain degree of detail, this configuration is an exemplification, and the disclosure content of this embodiment should be changed in the details of the configuration. In the present invention, replacement of elements in the embodiments with other elements, combinations, and changes in order can be realized without departing from the scope and spirit of the claimed invention.

INDUSTRIAL APPLICABILITY The coating unit, the fine coating device, and the manufacturing method of the cell chip and the cell tissue chip according to the present invention can reliably manufacture a large amount of highly reliable various fine patterns or droplet spots. -It is an important technology for researching drug discovery research and regenerative medicine in addition to industrial uses, and is an invention with high industrial applicability.

DESCRIPTION OF SYMBOLS 1 Fine coating device 2 Main body base 3 XY table 4 Coating unit 4A 1st coating mechanism 4B 2nd coating mechanism 5A Observation optical system device 5B Evaluation system device 6 Culture container 7 Coating needle 7A 1st coating needle 7B 2nd coating needle 8 needle Holding part 9 Needle part 9a Tip 10 Application liquid container 10A First application liquid container 10B Second application liquid container 10a Application liquid reservoir

10b Upper hole

10c Lower hole 11 Application liquid 11A First application liquid (cell-containing solution: first gelation) Agent)
11B Second coating liquid (second gelling agent)

Claims

A first coating solution container for storing a first coating solution containing cells;
A first application needle that can be immersed in the first application liquid stored in the first application liquid container;
A second coating solution container for storing a second coating solution;
A method of manufacturing a cell chip and a cell tissue chip using a fine coating apparatus comprising: a second coating needle that can be immersed in the second coating liquid stored in the second coating liquid container;
Immersing at least the tip of the first application needle in the first application liquid stored in the first application liquid container to hold the first application liquid at the tip of the first application needle;
A step of bringing the first application liquid held at the tip of the first application needle into contact with an application object and applying the first application liquid to the application object;
Immersing at least the tip of the second application needle in the second application liquid stored in the second application liquid container to hold the second application liquid at the tip of the second application needle; and A step of bringing the second coating liquid held at the tip of the two coating needles into contact with the first coating liquid coated on the object to be coated;
A method for producing a cell chip and a cell tissue chip, comprising:
The method for producing a cell chip and a cell tissue chip according to claim 1, comprising a step of bringing the first coating solution and the second coating solution into contact with each other to react and gel.
The method for producing a cell chip and a cell tissue chip according to claim 1 or 2, wherein the tip diameter of the second application needle is larger than the tip diameter of the first application needle.
The coating speed at which the second coating liquid held at the tip of the second coating needle is contact-coated is such that the first coating liquid held at the tip of the first coating needle is contact-coated on the coating object. The method for producing a cell chip and a cell tissue chip according to any one of claims 1 to 3, which is set to be slower than a coating speed.
The step of bringing the first application liquid held at the tip of the first application needle into contact with the application speed of the first application needle immediately before contacting the object to be applied; and
Immediately before the second application liquid held at the tip of the second application needle comes into contact with the first application liquid applied to the object to be applied, the application speed of the second application needle is decreased and brought into contact. The method for producing a cell chip and a cell tissue chip according to any one of claims 1 to 4, comprising:
Immediately before the first application liquid held at the tip of the first application needle comes into contact with the object to be applied, the first application needle is brought into contact at a slower rate than the application speed before stopping after being stopped for a predetermined time. The manufacturing method of the cell chip and cell tissue chip | tip of any one of Claim 1 to 5 containing these.
Immediately before the second application liquid held at the tip of the second application needle comes into contact with the object to be applied, the second application needle is brought into contact at a slower rate than the application speed before stopping after being stopped for a predetermined time, The manufacturing method of the cell chip and cell tissue chip | tip of any one of Claim 1 to 6 containing these.
The first application liquid held at the tip of the first application needle is brought into contact with the application object, and after a predetermined time has elapsed since the first application liquid is brought into contact with the application object, the second application needle The cell chip and the cell tissue according to any one of claims 1 to 7, comprising a step of contacting and applying the second coating liquid held at the tip to the first coating liquid applied to a coating object. Chip manufacturing method.
The step of holding the first application liquid at the tip of the first application needle and the step of applying the first application liquid held at the tip of the first application needle to the application object were repeated a plurality of times. The method of any one of Claim 1 to 8 including the process after which the said 2nd coating liquid hold | maintained at the front-end | tip of the said 2nd coating needle is contacted with the said 1st coating liquid of a coating target object. Cell chip and cell tissue chip manufacturing method.
The method for producing a cell chip and a cell tissue chip according to any one of claims 1 to 9, wherein the first coating liquid and the second coating liquid are a gelling agent mixed with two liquids.
The height from the application target at the time of application of the second application needle is set to be higher than the height from the application target at the time of application of the first application needle. The manufacturing method of the cell chip and cell tissue chip | tip of description.
When repeating the step of applying the first application liquid to the application object with the first application needle a plurality of times, the height from the application object at the time of application of the first application needle is set to gradually increase. A method for producing a cell chip and a cell tissue chip according to claim 9.
The cell chip and the cell tissue chip according to any one of claims 1 to 12, wherein tips of the first application needle and the second application needle are configured to include a plane orthogonal to a moving direction at the time of application. Manufacturing method.
The first application needle penetrates the first application liquid container to apply the first application liquid to the object to be applied, and the second application needle penetrates the second application liquid container to the second application liquid. The method for manufacturing a cell chip and a cell tissue chip according to any one of claims 1 to 13, wherein the cell chip and the cell tissue chip are applied to an object to be applied.
The method for producing a cell chip and a cell tissue chip according to any one of claims 1 to 14, wherein unmodified cells or modified cells are used as the cells in the first coating solution.
A first coating solution container for storing a first coating solution;
A first application needle whose tip can be immersed in the first application liquid stored in the first application liquid container and reciprocates;
A second coating solution container for storing a second coating solution;
A tip that can be immersed in the second coating solution stored in the second coating solution container, and a second coating needle that reciprocates,
The second coating liquid held at the tip of the second coating needle is contact-coated at the position where the first coating liquid held at the tip of the first coating needle is applied to the object to be coated. Application unit.
The coating unit according to claim 16, wherein the tip diameter of the second coating needle is configured to be larger than the tip diameter of the first coating needle.
The application speed at which the second application liquid held at the tip of the second application needle is contact-applied is set so that the first application liquid held at the end of the first application needle is applied to the object to be applied. The coating unit according to claim 16 or 17, wherein the coating unit is configured to be slower than a coating speed.
The first application liquid held at the tip of the first application needle slows down the application speed of the first application needle immediately before coming into contact with the object to be applied, and is held at the tip of the second application needle. 18. The coating unit according to claim 16, wherein the second coating liquid is configured to slow down a coating speed of the second coating needle immediately before contacting the first coating liquid coated on a coating object. .
Immediately before the first application liquid held at the tip of the first application needle comes into contact with the object to be applied, the first application needle is temporarily stopped for a predetermined time and then made slower than the application speed before stopping. The coating unit according to any one of claims 16 to 19.
Immediately before the second application liquid held at the tip of the second application needle comes into contact with the object to be applied, the second application needle is temporarily stopped for a predetermined time and then made slower than the application speed before stopping. The coating unit according to any one of claims 16 to 20.
The height from the application object at the time of application of the second application needle is set higher than the height from the application object at the time of application of the first application needle. The coating unit described.
The first application needle is configured to apply the first application liquid to the application object a plurality of times by the first application needle, and gradually increases each time the height from the application object at the time of application of the first application needle is applied. 23. A coating unit according to any one of claims 16 to 22 configured to cause
The coating unit according to any one of claims 16 to 23, wherein tips of the first coating needle and the second coating needle are configured to include a plane orthogonal to a moving direction during coating.
The first application needle is configured to pass through the first application liquid container and apply the first application liquid to an object to be applied, and the second application needle passes through the second application liquid container and is The coating unit according to any one of claims 16 to 24, wherein the coating unit is configured to apply the second coating liquid to an application target.
26. The storage capacity of the second coating liquid stored in the second coating liquid container is configured to be larger than the storage capacity of the first coating liquid stored in the first coating liquid container. An application unit given in any 1 paragraph.
An application unit according to any one of claims 16 to 26;
An XY table in which a coating object to be contacted and coated by the first coating needle and the second coating needle in the coating unit is placed and fixed, and is movable in the horizontal direction;
A vertical movement mechanism that allows the application unit to move in the vertical direction;
A fine coating apparatus.
28. The fine coating apparatus according to claim 27, further comprising an observation optical system unit for observing a coating applied in contact with an application target placed on the XY table.
29. The fine coating apparatus according to claim 27 or 28, further comprising an evaluation system device for evaluating a coating applied in contact with a coating object placed on the XY table.