JP7024970B2 - Coating unit and fine coating equipment - Google Patents

Description

The present invention relates to a coating unit and a fine coating device including a coating unit for applying a liquid substance, and more particularly to a coating unit and a fine coating device capable of finely coating using a coating needle.

As a pattern formed by applying a liquid substance, for example, there is a circuit pattern used for electric / electronic parts and the like. In order to form such a circuit pattern, a coating device such as an inkjet method is generally used. In an inkjet coating device, if a highly viscous material is used as the liquid substance to be coated, the tip of the nozzle may be clogged, which causes a problem in drawing a fine circuit pattern or the like. It was a thing. Therefore, instead of the inkjet method, there is a demand for a fine coating device capable of drawing fine circuit patterns and the like easily and with high accuracy.

Further, in a fine coating device capable of drawing a fine pattern, in addition to the above-mentioned use in the field of industrial use such as electric / electronic parts, its use is also considered for applications in other fields. ing.

In recent years, it has been studied to construct a three-dimensional tissue of cells in vitro by a coating operation using a fine coating device using a solution in which cells are suspended as a liquid substance. Techniques for constructing three-dimensional tissues of cells in vitro are important in drug discovery research and regenerative medicine research, and construction of cell tissues close to human living tissues is particularly required.

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

An assay method in which a cell tissue chip (cell aggregate) in which cells are aggregated and aggregated is placed in each of a plurality of aligned wells (recesses) in a well plate, for example, and each cell aggregate is evaluated is a cell. It is widely used in functional evaluation and screening to select effective compounds as new drugs. By performing an assay method on such a cell aggregate, it is possible to evaluate multiple items in a short time with a small amount of sample, and it is possible to evaluate quickness, convenience, safety, reproducibility and high reliability. It is advantageous in terms of securing.

Techniques for precision patterning and placement of cells on a substrate include a method of processing a substrate by photolithography technology to control cell adhesion and a method of directly printing cells for placement and immobilization. In recent years, with the development of 3D printer technology for arranging and stacking cells in three dimensions (3D), construction of a cell aggregate using a 3D printer has been studied, and a tissue model obtained from the cell aggregate has been studied. Is being actively developed for drug discovery research and regenerative medicine.

Japanese Unexamined Patent Publication No. 2008-126459 Japanese Unexamined Patent Publication No. 2008-017798 Japanese Unexamined Patent Publication No. 2010-022251 JP-A-2015-229148 Japanese Unexamined Patent Publication No. 2016-087822 Japanese Unexamined Patent Publication No. 2017-163931 Japanese Unexamined Patent Publication No. 2017-169560 Japanese Unexamined Patent Publication No. 2017-131144 Japanese Patent Application Laid-Open No. 2015-112576

Inkjet printers (inkjet printers: inkjet thermal type, piezo type), microextrusion printers (dispenser type), laser-assisted printers (pulse lasers) are used as 3D printers for constructing cell aggregates. Method) exists. However, in these printers, the materials that can be ejected are limited, and the print speed (production speed) and the resolution (ejection amount) are also greatly restricted. Further, in the conventional 3D printer, when the material to be discharged is a cell-containing solution containing cells, there is a problem regarding the cell viability and cell density of the cell aggregate to be produced in the state after discharge. In particular, when cell aggregates are produced from a cell-containing solution having high viscosity, the highly viscous solution may be clogged at the nozzle of the printer, so high resolution, that is, fine cell aggregates are produced in large quantities. It was not possible to do so and had a problem.

In addition, in order to prevent damage caused by drying of 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 liquids are mixed is used. When an ultraviolet curable gelling agent or a thermosetting gelling agent is used, the use of such a curing agent is preferable because the ultraviolet rays and heat at the time of curing adversely affect the cells. It's not a thing.

When producing a cell aggregate using a two-component gelling agent, two printers are used, and one gelling agent containing cells is ejected from the nozzle of one printer to a predetermined position and the other. It is necessary to eject the gelling agent from the nozzle of the other printer to the one gelling agent with high accuracy to ensure that the gelling agents react with each other. As described above, it is not easy to produce a cell aggregate by ejecting two liquid gelling agents to the same position with high accuracy and gelling them using different printers. Further, since two printers are used, there is a problem that the entire device becomes large. In particular, when a cell aggregate is produced using an inkjet printer or a dispenser printer, if a highly viscous material is used as the gelling agent, the tip of the nozzle may be clogged, which is highly reliable. It was difficult to reliably discharge the desired amount of gelling agent from the nozzle while maintaining the properties and high accuracy.

The present invention is a coating unit capable of reliably drawing a large amount of fine patterns or droplet spots formed with a desired coating amount in a short time (high speed) even if it is a highly viscous liquid substance. It is an object of the present invention to provide a fine coating apparatus equipped with a coating unit.

In order to achieve the above object, the coating unit of one aspect according to the present invention is
The first coating liquid container for storing the first coating liquid and
A first coating needle whose tip can be immersed in the first coating liquid stored in the first coating liquid container and reciprocates.
A second coating liquid container for storing the second coating liquid, and
A second coating needle whose tip can be immersed in the second coating liquid stored in the second coating liquid container and which reciprocates is provided.
The second coating liquid held at the tip of the second coating needle is contact-applied to the position where the first coating liquid held at the tip of the first coating needle is contact-applied to the object to be coated. Has been done.

According to the coating unit and the fine coating apparatus of the present invention, even if it is a highly viscous liquid substance, a fine pattern or droplet spots formed with a desired coating amount can be reliably produced in a large amount in a short time with high reliability. can do.

It is a front view which shows the tabletop type fine coating apparatus used in Embodiment 1 which concerns on this invention. (A) is a figure showing a coating needle (first coating needle and second coating needle) attached to the coating unit in the fine coating apparatus of the first embodiment, and (b) is a diagram showing a tip portion of a needle portion in the coating needle. It is an enlarged view which shows. The figure which shows the coating operation of the coating unit in the fine coating apparatus of Embodiment 1. (A) is a diagram showing the first coating operation by the first coating needle, and (b) is a diagram showing the second coating operation by the second coating needle. (A) is an operation diagram showing the movement of the tip in the first coating operation by the first coating needle, and (b) is an operation diagram showing the movement of the tip in the second coating operation by the second coating needle. FIG. 5 is an operation diagram showing a continuous coating operation of a first coating operation by a first coating mechanism and a second coating operation by a second coating mechanism in the fine coating apparatus of the first embodiment. It is a graph which shows an example of the relationship between the number of coatings and the shape (coating diameter and coating height) of a droplet spot. It is an operation diagram which shows the 3rd coating operation of the 1st coating needle. It is an operation diagram which shows the continuous coating operation of the 3rd coating operation of the 1st coating needle, and the 2nd coating operation of a 2nd coating needle. It is an image which shows the state of the tissue immediately after the 2nd coating liquid was applied to the droplet spot of the 1st coating liquid. It is an image which shows the state when the tissue shown in FIG. 10 was cultured for 3 days. It is a graph which shows the experimental result of the tip diameter of a coating needle, and the number of coating cells contained in a droplet spot. It is a graph which shows the experimental result which measured the number of coated cells when the 3rd coating operation was performed using the 1st coating needle with a tip diameter of 330 μm.

The coating unit and the fine coating device in the present invention are devices used for coating operations in various industrial / industrial equipment, for example, for various industrial / industrial equipment using a highly viscous adhesive or the like. Used. Further, the coating unit and the fine coating device in the present invention can be used as an auxiliary device for medical use, for example, for a coating operation in cell culture. In the first embodiment of the present invention described later, the coating operation of the two-component gelling agent containing cells will be described as an example of the 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 / industrial equipment, for example, in a coating operation in various industrial / industrial equipment using a highly viscous adhesive or the like.

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

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

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

The coating unit according to the third aspect of the present invention has a coating speed at which the second coating liquid held at the tip of the second coating needle is contact-applied in the first aspect or the second aspect. The first coating liquid held at the tip of the first coating needle may be configured to be slower than the coating speed at which the first coating liquid is contact-applied to the object to be coated.

In the coating unit according to the fourth aspect of the present invention, in any one of the first to third aspects described above, the first coating liquid held at the tip of the first coating needle is applied. Immediately before coming into contact with the object, the coating speed of the first coating needle is slowed down, and the second coating liquid held at the tip of the second coating needle becomes the first coating liquid coated on the object to be coated. It may be configured to slow down the coating speed of the second coating needle immediately before contact.

In the coating unit according to the fifth aspect of the present invention, in any one of the first to fourth aspects described above, the first coating liquid held at the tip of the first coating needle is the object to be coated. Immediately before coming into contact with an object, the first coating needle may be configured to be slower than the coating speed before stopping after being stopped for a predetermined time.

In the coating unit according to the sixth aspect of the present invention, the second coating liquid held at the tip of the second coating needle in any one of the first to fifth aspects is the first. Immediately before contacting with the coating liquid , the second coating needle may be configured to be slower than the coating speed before the stop after temporarily stopping for a predetermined time.

In any of the first to sixth aspects, the coating unit according to the seventh aspect of the present invention has the height from the object to be coated when the second coating needle is applied. It may be set higher than the height from the object to be coated when the first coating needle is applied.

In the coating unit according to the eighth aspect of the present invention, in any one of the first to seventh aspects, the first coating liquid is applied to the object to be coated a plurality of times by the first coating needle. The height from the object to be coated at the time of application of the first coating needle may be gradually increased each time the coating object is applied.

In the coating unit according to the ninth aspect of the present invention, in any one of the first to eighth embodiments, the first coating needle and the tip of the second coating needle move during coating. It may be configured to include planes orthogonal to the direction.

In the coating unit according to the tenth aspect of the present invention, in any one of the first to ninth embodiments, the first coating needle penetrates the first coating liquid container and the first coating liquid container is used. The coating liquid may be configured to be applied to the object to be coated, and the second coating needle may be configured to penetrate the second coating liquid container and apply the second coating liquid to the object to be coated.

The coating unit according to the eleventh aspect of the present invention has a storage capacity of the second coating liquid stored in the second coating liquid container in any one of the first to tenth aspects. , It may be configured to be larger than the storage capacity of the first coating liquid stored in the first coating liquid container.

The fine coating apparatus according to the twelfth aspect of the present invention includes the coating unit according to any one of the first to eleventh aspects described above.
An XY table on which an object to be coated, which is contact-coated by the first coating needle and the second coating needle in the coating unit, is placed and fixed, and which can be moved in the horizontal direction.
A vertical movement mechanism that enables the coating unit to move in the vertical direction,
It is equipped with.

The fine coating apparatus according to the thirteenth aspect of the present invention is an observation optical system unit for observing a coated object contact-coated with an object to be coated placed on the XY table in the twelfth aspect. May be provided.

The fine coating apparatus according to the fourteenth aspect of the present invention is for evaluating the coated object contact-applied to the coated object placed on the XY table in the twelfth aspect or the thirteenth 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, and contains cells in addition to the coating liquids used for industrial and industrial purposes such as two-component adhesives. It is possible to produce cell chips and cell tissue chips using a liquid gelling agent as a coating liquid. In the following embodiment, a case where a two-component gelling agent containing cells is used as the coating liquid of the fine coating apparatus will be described as an example of the coating liquid used for industrial use.

The fine coating apparatus according to the present invention has a small droplet of several pL (picolitre) attached to the tip of the coating needle as an electrode per time, as described with reference to specific examples in the embodiments described later. It is a device that can be applied to a predetermined position of an object in a short time, for example, 0.1 seconds, with high safety and reproducibility in the production of cell chips and cell tissue chips, and can be automated in a short time. It is possible to produce a large amount of highly reliable cell chips and cell tissue chips (cell aggregates).

The cell chip used in the embodiment is, for example, a chip formed of substantially one cell in a state in which individual cells are dispersed and adhered to each other in a culture vessel. A cell tissue chip is a cell aggregate in a state in which cells are aggregated, aggregated, laminated, organized, and functioning on a substrate, and is a two-dimensional cell aggregate such as a cell sheet or a cell sheet. Includes superposed three-dimensional cell aggregates.

In the present invention, by using a two-needle type coating unit having a plurality of coating needles, for example, a two-needle type coating unit having two coating needles, and a fine coating apparatus having a coating unit, a conventional coating apparatus (printer) can be used. It becomes possible to stably produce gelled cell chips and cell tissue chips (cell aggregates), which have been difficult to deal with, at high speed. As a result, various cell tissue chips can be automated and manufactured in large quantities in an aseptic state. Therefore, the fine coating apparatus according to the present invention is desired not only for the production of cell chips and cell tissue chips, but also when a coating liquid used for industrial or industrial purposes such as a two-component adhesive is used. It is possible to reliably and reliably manufacture a large amount of fine drawing patterns and droplet spots in a short time.

Next, the coating unit and the fine coating apparatus according to the present invention will be described with reference to the accompanying drawings by using an embodiment showing a specific configuration example. The coating unit and the fine coating device according to the present invention are not limited to the configurations of the embodiments described below, but include configurations based on the same technical ideas as the following coating units and fine coating devices.

(Embodiment 1)
Hereinafter, the specific embodiment 1 will be described with reference to the attached drawings. FIG. 1 is a front view showing a tabletop type fine coating device 1 used in the first embodiment. The fine coating device 1 shown in FIG. 1 includes a display / control unit (not shown) for setting, controlling, and displaying in the fine coating device 1. The display / control unit in the fine coating device 1 of the first embodiment is composed of a so-called personal computer (PC).

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

In the first embodiment, as shown in FIG. 1, the configuration of the tabletop type fine coating device 1 will be described, but the present invention is not limited to such a tabletop type configuration and is substantially limited to such a tabletop type configuration. It includes a large-sized fine coating device 1 having the same configuration and being enlarged and installed in a culture room or the like.

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. There is. Hereinafter, the configuration of the coating unit 4 and the cell coating operation by the coating unit 4 will be described.

[Structure of coating unit]
The coating unit 4 in the fine coating apparatus 1 is provided with two coating mechanisms (first coating mechanism 4A and second coating mechanism 4B), and the respective coating mechanisms 4A and 4B are configured to independently perform coating operations. Has been done. The fine coating device 1 has a configuration in which a two-component gelling agent is used as a coating liquid in order to produce a cell chip or a cell tissue chip, and is provided with a first coating mechanism 4A and a second coating mechanism 4B. ..

FIG. 2A is a diagram showing a coating needle 7 mounted on the first coating mechanism 4A and the second coating mechanism 4B in the coating unit 4. The coating needle 7 has a needle holding portion 8 and a needle portion 9 projecting from the needle holding portion 8. FIG. 2B is an enlarged view showing a tip portion of the needle portion 9 of the coating needle 7. The tip portion of the coating needle 9 of the first embodiment is formed in a conical shape, and the tip portion 9a is formed in a flat surface so as to face the horizontal plane 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 device 1, the coating needle 7 (first coating needle 7A) mounted on the first coating mechanism 4A and the coating needle 7 (second coating needle 7B) mounted on the second coating mechanism 4B are The diameter of the tip 9a of the coating needle 9 is different, but the other configurations are substantially the same. The diameter d of the tip 9a of the coating needle 9 is largely related to the shape of the cell chip or the cell tissue chip to be produced. In the fine coating device 1 of the first embodiment, the diameter d of the tip 9a of the coating needle 9 is such that the first coating needle 7A attached to the first coating mechanism 4A is, for example, 330 μm, and is attached to the second coating mechanism 4B. The second coating needle 7B is, for example, 1000 μm, and fine cell chips or cell tissue chips are produced using the first coating needle 7A and the second coating needle 7B having different tip diameters. In the first embodiment, the needle holding portion 8, the needle portion 9, and the tip 9a in the first coating needle 7A and the second coating needle 7B will be described with reference to the same reference numerals.

As shown in FIG. 2B, since the tip portion of the coating needle 7 has a conical shape and the tip 9a is a horizontal plane, the diameter d of the tip 9a is desired by polishing the tip 9a to a horizontal plane. It is easy to form to the diameter of.

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

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

The first coating operation of the first coating needle 7A is achieved by a drive mechanism that converts the rotational operation of the drive source (for example, a motor) into a reciprocating operation via a link mechanism. The high-speed multiple reciprocating movements of the first coating needle 7A in the vertical direction (vertical direction) are performed by the through hole (upper hole 10b) formed at the upper end of the first coating liquid container 10A. The portion 9 is slidably held, and the reliability of high-speed operation of the first coating needle 7A is ensured. The first coating needle 7A is detachably provided at a predetermined position of the drive mechanism that operates the first coating needle 7A at high speed in the first coating mechanism 4A, and is provided, for example, by the magnetic force of a magnet with respect to the drive mechanism or by a screw. It is configured to be removable by stopping.

As described above, the first coating needle 7A fixed to the drive mechanism in the first coating 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 the drive control of the vertical movement mechanism for moving the entire coating unit 4 including the YX table 3 and the first coating mechanism 4A in the vertical direction (vertical direction), etc. are displayed and controlled by the display / control unit. It is done in. The vertical reciprocating motion of the first coating needle 7A is an ultra-high speed, and for example, one reciprocating motion can be set to 0.5 seconds or less, and further 0.1 seconds or less.

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

The first coating mechanism 4A in the coating unit 4 is configured as described above to drive the first coating needle 7A, but the second coating mechanism 4B in the coating unit 4 is similarly configured and the second coating needle. 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 high speed (one reciprocating operation) in the vertical direction (vertical direction). That is, the second coating operation of the second coating needle 7A is achieved by a drive mechanism that converts the rotational operation of the drive source (for example, a motor) into a reciprocating operation via the link mechanism. In the first coating operation of the first coating needle 7A and the second coating operation of the second coating needle 7A, the rotational operation of one drive source is transmitted via the first link mechanism and the second link mechanism, respectively. It is a configuration to switch to. The first link mechanism and the second link mechanism are configured to be capable of speed control during reciprocating operation.

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

As described above, the first coating operation by the first coating mechanism 4A and the second coating operation by the second coating mechanism 4B are substantially the same except for the number of coatings and the position of the lowest point of the tip at the time of coating. Is. Therefore, regarding the second coating operation by the second coating mechanism 4B, the first coating operation of the first coating mechanism 4A will be mainly described with reference to the schematic diagram of FIG.

[Applying operation]
In the first coating operation by the first coating mechanism 4A shown in FIG. 3, the first coating liquid 11A (first gelling agent) containing the cells stored in the coating liquid reservoir 10a of the first coating container 10A is the object to be coated. This is a cell coating operation applied on the bottom surface of the well of the culture vessel 6.

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 coating, a very small amount of the first coating liquid 11A held at the tip 9a of the first coating needle 7A comes into contact with the bottom surface of the well and is applied, and the tip 9a of the first coating needle 7A is applied. It is different from the application by contact between the well and the bottom surface of the well.

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

In FIG. 3B, the tip 9a of the first coating needle 7A protrudes from the lower hole 10c of the first coating container 10A, and the first coating liquid 11A held by the tip 9a contacts the bottom surface of the well of the culture container 6. It shows the state of application. That is, in a descending state in which the tip 9a of the first coating needle 7A penetrates the coating liquid reservoir 10a and protrudes, a certain amount of the first coating liquid 11A is held at the tip portion of the first coating needle 7A. Therefore, when the first coating liquid 11A held by the tip 9a of the first coating needle 7A comes into contact with 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. It is applied by contact. The fine droplet spot S that has moved to the bottom surface of the well of the culture vessel 6 is in a state of being raised in a substantially hemispherical shape on the bottom surface of the well due to its surface tension.

The cell coating operation by the first coating needle 7A is repeated a predetermined number of times, and the first coating liquid 11A is contact-coated on the bottom surface of the well. In the cell coating operation of the first coating needle 7A a predetermined number of times, in the second and subsequent contact coatings, the first coating liquid 11A held at the tip 9a of the first coating needle 7A and the first coated on the bottom surface of the well. The coating liquid 11A is applied by contact with the droplet spot S. In this way, the cell coating operation is repeated a predetermined number of times (for example, 10 times), 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 coating operation by the first coating needle 7A shown in FIGS. 3A and 3B 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. 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 coating operation, the second coating liquid 11B is contact-coated only once with respect to the formed droplet spot S of the first coating liquid 11A. The first coating liquid 11A is a first gelling agent in which cells are suspended, and the second coating liquid 11B is a second gelling agent that reacts and gels by mixing with the first gelling agent. When the second coating liquid 11B, which is the second gelling agent, is contact-coated so as to cover the first coating liquid 11A, the droplet spots S are reliably gelled by sufficiently reacting with each other.

The figures (a-1) to (a-4) shown in FIG. 4A schematically show the movement of one first coating operation (cell coating operation) by the first coating needle 7A. Is. The figures (b-1) to (b-3) shown in FIG. 4 (b) are diagrams schematically showing the second coating operation by the second coating needle 7B. In FIG. 4A, a state (a-1) in which a very small amount of the first coating liquid 11A is held at the tip 9a of the first coating needle 7A, and the bottom surface of the well of the culture container 6 to be coated. A state in which the first coating liquid 11A of the tip 9a of the first coating needle 7A is in contact with the top (a-2) and a state immediately after the tip 9a of the first coating needle 7A is in contact with the first coating liquid 11A (a-2). a-3) and a state (a-4) in which the tip 9a of the first coating 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 repeated a predetermined number of times, for example, 10 times, and the cells are suspended as the first gelling agent. A droplet spot S (height Z2) of the coating liquid 11A is formed. As shown in FIG. 4B, the second gelling agent held at the tip 9b of the second coating needle 7B with respect to the droplet spot S (height Z2) of the first coating liquid 11A. 2 The coating liquid 11B is contact-coated.

In the fine coating device 1 of 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 9b of the second coating needle 7B used for the second coating operation. The diameter of is, for example, 1000 μm. Therefore, the coating amount by the second coating needle 7B is larger than the coating amount by the first coating needle 7A, and fine droplets of the first coating liquid 11A formed in the first coating operation by the first coating needle 7A. The second coating operation by the second coating needle 7B ensures that the second coating liquid 11B covers the spot S. For example, according to our experiment, the coating amount of the first coating needle 7A (tip diameter 330 μm) by the first coating operation is about 15 pL, whereas the coating amount of the second coating needle 7B by the second coating operation is about 15 pL. Was about 80 pL.

As described above, the first coating operation ((a) in FIG. 4) is performed a predetermined number of times by the first coating needle 7A, and then the second coating operation ((b) in FIG. 4) by the second coating needle 7B is performed. By doing so, the second coating liquid 11B is applied so as to cover the first coating liquid 11A, and the first coating liquid 11A and the second coating liquid 11B react with each other to prepare a gelled cell chip or cell tissue chip. Will be done.

The coating amount by the first coating needle 7A and the coating amount by the second coating needle 7B are significantly different, and the consumption of the second coating liquid 11B is larger than the consumption of the first coating liquid 11A. 2 The capacity of the coating liquid container 10B may be larger than that of the first coating liquid container 10A. That is, the storage capacity of the second coating liquid 11B stored in the second coating liquid container 10B may be larger than the storage capacity of the second 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 coating operation by the first coating needle 7A, and FIG. 5B is an operation diagram showing the movement of the tip 9a in the second coating operation by the second coating needle 7B. It is an operation diagram which shows the movement. In the operation diagrams of FIGS. 5A and 5B, the vertical axis indicates the position of the tip of the coating needle, and the horizontal axis indicates time.

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

In the first coating operation, after the contact coating is performed at the contact coating position P3a, the process returns to the upper limit position P1a, which is the initial position, at the return speed V3, and the next coating operation is repeated. As shown in FIG. 5A, in one coating operation, the speed is lowered from the upper limit position P1a (time T1a) to the speed changing position P2a (time T2a) at the first coating speed V1a, and the speed changing position P2a ( The contact coating is performed by descending from the time T2a) to the contact coating position P3a (time T3a) at the second coating speed V2a (<V1a). After the contact coating, the speed increases to the upper limit position P1a (time T4a) at a predetermined return speed V3.

In the fine coating device 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, so that the first coating is performed. The tip 9a of the needle 7A can be arranged with high accuracy with respect to the set contact coating position P3a, and a fine coating operation can be performed. Even if the tip 9a of the first coating needle 7A comes into contact with the surface of the substrate to be coated, the contact impact is small and the influence on the cells can be small.

On the other hand, in the second coating operation by the second coating needle 7B, as shown in FIG. 5 (b), the speed is lower than that of the first coating operation of the first coating needle 7A (FIG. 5 (a)). It is set. In the second coating operation of the second coating needle 7B, the tip 9a of the second coating needle 7B descends from the upper limit position P1b at the first coating speed V1b (V1b <V1a), and the speed change position immediately before coating is set in advance. When P2b (P2b = P2a) is reached, the speed decreases at a second coating speed V2b, which 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 liquid 11B held at the tip 9a at the contact coating position P3b contacts the droplet spot S of the first coating liquid 11A. It is applied. Even if the contact coating position P3b is calculated and set by the position measuring device provided in the evaluation system device 5B based on the detection data indicating the position and height of the droplet spot S of the first coating liquid 11A. good.

In the second coating operation, after the contact coating is performed at the contact coating position P3b, the speed returns to the upper limit position P1b, which is the initial position, at the return speed V3. As shown in FIG. 5B, in the second coating operation, the speed is changed by descending from the upper limit position P1b (time T1b) to the speed changing position P2b (time T2b) at the first coating speed V1b (<V1a). The contact coating is performed by descending from the position P2b (time T2b) to the contact coating position P3b (time T3b) at the second coating speed V2b (<V1b). After the contact coating, the speed increases to the upper limit position P1b (time T4b) at a predetermined return speed V3.

FIG. 6 is an operation diagram showing a continuous coating operation of the first coating operation by the first coating mechanism 4A and the second coating operation by the second coating mechanism 4B in the fine coating device 1 of the first embodiment. In the first coating operation by the first coating mechanism 4A shown in FIG. 6, the first coating liquid 11A is contact-applied 10 times to the culture vessel 6 which is the object to be coated by the first coating needle 7A. ..

As shown in FIG. 6, in 10 contact coatings in the first coating operation, the contact coating position P3a, which is the lowest point position of the first coating needle 7A, gradually changes each time one contact coating is completed. It is set to move upwards. 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 for each contact coating.

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

As shown in FIG. 7, in the coating diameter of the droplet spot S, the number of coatings gradually increased up to the fifth coating, and the coating diameter did not change significantly after the sixth coating. Regarding the coating height of the droplet spot S, the droplet spot S having a height of about half of the final coating height is formed by the first coating, and then the final coating height is reached by the fifth coating. 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 was substantially determined by performing the coating times 5 times. However, since the relationship between the number of coatings and the shape of the droplet spot S changes greatly depending on the characteristics 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 10 coatings. Be done.

In the fine coating device 1 of the first embodiment, the coating liquid held at the tip of the coating needle comes into contact with the object to be coated (bottom surface of the well of the culture vessel, droplet spot, etc.) to apply the coating liquid by contact coating. A special coating operation is performed. Therefore, even if the droplet spot S formed on the object to be coated is contact-coated, it is not a coating operation in which all of the coating liquid held at the tip of the coating 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 retention amount of the coating liquid held at the tip of the coating needle, the characteristics of the coating liquid, and the surface texture (surface roughness) of the object to be coated. It is thought that it will be decided by such factors.

After the first coating operation is completed as described above, the coating object on the XY table 3 is moved from directly under the first coating needle 7A to directly under the second coating needle 7B within a predetermined time. The second coating operation by the second coating needle 7B is started. In the second coating operation, the second coating liquid 11B is contact-coated by the second coating needle 7B so as to cover the droplet spot S of the first coating liquid 11A.

After applying the first coating liquid 11A (first gelling agent) in which cells are suspended with the first coating needle 7A, the second coating liquid 11B (second gelling agent) is applied with the second coating needle 7B. Since it has 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 cells are suspended in the first coating liquid 11A. It is possible to prevent the outflow of turbid cells.

In the fine coating device 1 of the first embodiment, as shown in the operation diagram of FIG. 5, the proximity speed when the first coating needle 7A by the first coating mechanism 4A is closest to the coating object in the first coating operation ( It is a setting to slow down V2a) (V2a <V1a). Depending on the characteristics such as viscosity of the coating liquid, the coating amount held at the tip 9a of the first coating needle 7A may not be constant just by slowing down the proximity speed. When a constant coating amount cannot be stably applied only by slowing the proximity speed of the tip 9a in this way, the first coating needle 7A is temporarily stopped for a predetermined time at the speed changing position P2a which is the position immediately before the object to be coated. 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 coating needle 7A showing the third coating operation of temporarily stopping the first coating needle 7A at the speed change position P2a immediately before the object to be coated for a predetermined time. As shown in FIG. 8, when the tip 9a of the first coating needle 7A descends at the first coating speed V1a and reaches the speed change position P2a (T2), it stops for a short predetermined time (T2 → T2'). After the lapse of this predetermined time, the second coating speed V2a is lowered toward the object to be coated and contact coating is performed. By stopping the tip 9a of the first coating needle 7A at a position immediately before the object to be coated for a minute predetermined time in this way, the holding amount of the first coating liquid 11A by the tip 9a can be made uniform. Therefore, by performing the third coating operation of the first coating needle 7A in the first coating mechanism 4A, a constant coating amount is stably and highly reliablely coated on the object to be coated according to the characteristics of the coating liquid. It becomes possible to do.

As described above, in the third coating operation, the speed is temporarily stopped at the speed change position P2a, and after a predetermined time (X) elapses, it descends to the contact coating position P3a at a second coating speed V2a slower than the first coating speed V1a before the stop. do. After the contact coating is performed at the contact coating position P3a, the process returns to the upper limit position P1a, which is the initial position, at the return speed V3, and the next coating operation is repeated. As shown in FIG. 8, in one coating operation, the speed is lowered from the upper limit position P1a (time T1 ) to the speed change position P2a (time T2 ) at the first coating speed V1a, and the speed is changed after a predetermined time (X) has elapsed. The contact coating is performed by descending from the position P2a (time T2' ) to the contact coating position P3a (time T3c ) at the second coating speed V2a (<V1a). After the contact coating, the speed increases to the upper limit position P1a (time T4c) at a predetermined return speed V3.

Although FIG. 8 shows the third coating operation of the first coating needle 7A, the same coating operation can be performed on the second coating needle 7B as well. That is, just before the second coating liquid 11B held at the tip of the second coating needle 7B comes into contact with the object to be coated, the second coating needle 7B is once stopped for a predetermined time and then brought into contact at a slower speed than the coating speed before the stop. You may. By stopping the tip 9a of the second coating needle 7B at a position immediately before the object to be coated for a minute predetermined time in this way, the holding amount of the second coating liquid 11B by the tip 9a can be made uniform.

FIG. 9 is an operation diagram showing a continuous coating operation of the third coating operation of the first coating needle 7A shown in FIG. 8 and the second coating operation of the second coating needle 7B shown in FIG. 5 (b). be. In the third coating operation by the first coating needle 7A shown in FIG. 9, the first coating liquid 11A is contact-coated 10 times to the culture vessel 6 which is the object to be coated by the first coating needle 7A. ..

In the third coating operation shown in FIG. 9, as in the first coating operation shown in FIG. 6, every time the contact coating position P3a, which is the lowest point position of the first coating needle 7A, is completed once. It is set to gradually move upward. Further, after the switching time (Tc) of the predetermined time has elapsed after the third coating operation is completed, the second coating liquid 11B is contact-coated with the droplet spot S of the first coating liquid 11A by the second coating needle 7B. ..

In the coating operation of the coating unit 4 and the fine coating device 1 in the first embodiment, a very small amount of the coating liquid 11 (11A, 11B) is adhered to the tip 9a of the coating needle 7 (7A, 7B) and comes into contact with the object to be coated. By doing so, the droplet spot S having a coating amount of several pL (picolitre) can be applied and formed with high placement accuracy, for example, with a placement accuracy of ± 15 μm or less, preferably ± 3 μm or less. Further, as the viscosity of the coating liquid 11, it is possible to apply a material having a viscosity of 1 × 10 ⁵ mPa · s or less, and it is possible to apply a highly viscous cell dispersion liquid. In the coating operation of the fine coating device 1 in the first embodiment, a printer using a nozzle such as an inkjet printer cannot be used because of problems such as clogging. Viscosity of 10 mPa · s or more, 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 brought into contact with the object to be coated for coating, the coating liquid 11 is stable without being affected by the variation in the vertical position of the coating needle 7. Can be applied repeatedly. As described above, in the coating unit 4 and the fine coating device 1 of the first embodiment, since the highly viscous cell dispersion can be precisely coated at a predetermined position on the object to be coated, cells having an arbitrary patterning can be applied. It is possible to manufacture chips and cell tissue chips in which cells are three-dimensionally shaped. Therefore, by using the coating unit 4 and the fine coating device 1 of the first embodiment according to the present invention, it is possible to surely form a fine drawing pattern, and the coating unit and the fine coating device are manufactured. Cell chips and cell tissue chips are used in the fields of drug discovery research and regenerative medicine such as screening for evaluation of drug efficacy and safety, and are effective for progress in each field.

(Experimental example)
Hereinafter, specific experiments carried out 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 thereto.

In the following experiment, a coating experiment with a two-component gelling agent was performed using the fine coating device 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 × ¹⁰⁷ cells / mL. The solution (suspension) was used. On the other hand, as the second coating liquid 11B (second gelling agent), an 800 unit / mL thrombin solution was used.

In the coating experiment, as shown in FIG. 9, a third coating operation by the first coating needle 7A (see FIG. 8) and a second coating operation by the second coating needle 7B (see (b) in FIG. 5) are performed. rice field.

First, in the third coating operation by the first coating needle 7A, the first coating needle 7A descends at the first coating speed V1a, and in order to stabilize the coating amount by the first coating needle 7A, the first coating needle 7A is first at the speed changing position P2. The coating needle 7A is temporarily stopped for a predetermined time (period (T2 → T2') in FIG. 8: X). By allowing the first coating needle 7A to stand still for a predetermined time X, the first coating liquid 11A held at the tip 9a becomes stable. After the first coating needle 7A has been stationary for a predetermined time X, the first coating needle 7A descends to the contact coating position P3a at the lowermost end at the second coating speed V2a, which is slower than the first coating speed V1a, to perform contact coating. The contact coating position P3a, which is the lowermost end, is moved upward by several μm for each coating. The moving distance of the contact coating position P3a for each coating is preset based on an empirical rule, but the coating spot measured by a position measuring device or the like provided in the evaluation system device 5B of the fine coating device 1 It may be set according to the height of. In the coating experiment, the moving distance that the first coating needle 7A moves upward each time the first coating needle 7A applies a height lower than the height Z1 of the droplet spot S formed by one coating of the first coating needle 7A. It is supposed to be.

In the third coating operation by the first coating needle 7A in the coating experiment, the needle tip tip position is raised as described above, and the plastic coverslip which is the object to be coated is Celldesk LF (registered trademark) (Sumitomo Bakelite Co., Ltd.). The droplet spot S was formed by contact-applying 10 times continuously on the bottom surface of the well of the culture vessel 6 (manufactured by).

After the third coating operation by the first coating needle 7A is completed, the droplet spot S of the first coating 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 coating operation by the second coating needle 7B was started. In the second coating operation, the second coating liquid 11B held at the tip 9a of the second coating needle 7B can be reliably contacted with the droplet spot S of the first coating liquid 11A on the bottom surface of the well to be coated. The tip position of the second coating needle 7B is set. For example, the tip position of the second coating needle 7B may be set to the height Z2 (height after 10 times coating) of the droplet spot S of the first coating liquid 11A on the bottom surface of the well, and the second coating needle may be set. It may be set in consideration of the lowest point position of the second coating liquid 11B that hangs down from the tip 9a of 7B. By setting the tip position of the second coating needle 7B in this way, the second coating liquid 11B is reliably contact-coated on the upper surface of the droplet spot S of the first coating liquid 11A. As a result, thrombin, which is the second gelling agent of the second coating liquid 11B, is applied so as to cover the suspension of the fibrinogen solution containing human skin fibroblasts (NHDF), which is the first coating liquid 11A (Fig.). (See (b) of 4), the droplet spot S was tissue-fixed by gelation to prepare a cell tissue chip. The observation results of the prepared cell tissue chips are shown in FIGS. 10 and 11.

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 (cultured for 0 days). 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 (cell aggregate) was confirmed by culturing for 3 days, and it was confirmed that the cell tissue chip was surely constructed.

Further, the inventor conducted a coating experiment by changing the tip diameter of the coating needle 7, and measured the tip diameter of the coating needle 7 and the number of coated cells contained in the droplet spot S coated on the substrate. In this experiment, a suspension in which iPS-CM was dispersed in a PBS solution at a concentration of 4 × ¹⁰⁷ cells / mL was used as the first coating liquid 11A. As a result of the experiment, when the tip diameter of the coating needle 7 is 50 μm, an average of 1.1 coated cells are present in the droplet spot S, and the droplet spot S when the tip diameter is 100 μm is formed by one coating. Has an average of 4.0 coated cells and an average of 4.5 coated cells in the droplet spot S when the tip diameter is 150 μm, and an average of 4.5 coated cells in the droplet spot S when the tip diameter is 200 μm. There were an average of 19.1 coated cells. When the coating needle 7 having a tip diameter of 330 μm was applied once, an average of 85.3 coated cells were present in the droplet spot S. FIG. 12 is a graph showing the experimental results for considering the relationship between the tip diameter of the coating needle 7 and the number of coated 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 coated needle 7.

As shown in the graph of FIG. 12, the larger the tip diameter of the coating needle 7, the more coated cells were present in the droplet spot S. Therefore, in the fine coating device 1 of the first embodiment, by selecting the tip diameter of the first coating needle 7A, a droplet spot S having a desired number of coated cells is formed on the substrate or the like of the object to be coated. It can be understood that it is possible. That is, by selecting the tip diameter of the coating needle 7, the solution cell concentration, the solution viscosity, and the like, it is possible to produce 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 represents the number of coated cells [cells / spot], and the horizontal axis represents the number of coatings by the first coating 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 × ¹⁰⁷ cells / mL was used as the first coating solution 11A. As shown in FIG. 13, when the first coating needle 7A applied the first coating liquid 11A on the bottom surface of the well 10 times, an average of 162.4 coated cells were present (4 experiments). .. In the graphs of FIGS. 12 and 13, the standard deviation (positive direction) of the number of coated cells is indicated by an error bar.

As described above, in the fine coating device 1 of the first embodiment, the droplet spot S containing a desired number of cells can be formed and formed by the first coating mechanism 4A having the first coating needle 7A. The second coating liquid 11B is surely applied and reacted with the droplet spot S of the first coating liquid 11A by the second coating mechanism 4B having the second coating needle 7B, and gelled cell chips or cell tissue chips. Can be reliably manufactured.

The fine coating device 1 of the first embodiment has a configuration in which the coating needle 7 penetrates the coating liquid reservoir 10a of the coating liquid container 10 and is contact-coated with the object to be coated. Not limited. For example, the coating needle 7 immersed in the coating liquid of the coating liquid pool 10a may be lifted, moved to the coating position of the coating object, and contact-coated. Even in the fine coating apparatus configured as described above, even if it is a highly viscous liquid substance, for example, a two-component adhesive, a predetermined coating amount is predetermined as in the effect of the fine coating apparatus 1 of the first embodiment. A drawing pattern, a droplet spot, or the like can be reliably formed. Further, even if the highly viscous liquid substance is, for example, a cell-containing gelling agent as a material, cell chips formed in 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 produce cell tissue chips in a short time (high speed).

The cells that can be used in the present invention are not particularly limited, but are, for example, fibroblasts, vascular endothelial cells, epidermal cells, smooth muscle cells, myocardial cells, gastrointestinal cells, nerve cells, hepatocytes, renal cells, and the like. Various primary cells such as pancreatic cells, differentiated cells derived from iPS cells, various cancer cells and the like can be used. The cells include unmodified cells or cells modified with proteins, sugar chains, nucleic acids and the like, for example, cells coated with already known coating agents and coating methods such as fibronectin, gelatin, collagen, laminin and elastin. Can be used.

In addition, in order to provide an environment in which the contained cells can stably adhere and proliferate, the cell-containing solution contains extracellular matrix components such as fibronectin, gelatin, collagen, laminin, elastin, and matrigel, fibroblast growth factor, and platelets. It may contain an additive such as a cell growth factor such as a derived growth factor, vascular endothelial cells, lymphatic endothelial cells, and various stem cells. Further, fibrinogen, alginic acid, a heat-sensitive polymer and the like may be included as the gelling agent.

As described with reference to the above embodiments and experimental examples, the present invention provides a new manufacturing method for manufacturing cell chips and cell tissue chips using a new bioprinter. Compared to the case of manufacturing using a printer using a conventional nozzle, the structure is such that the solution adhering to the tip surface is applied using a coating needle, so the solution is not clogged and the cell aggregate is not clogged. The resolution and formation rate of the cells are improved, and highly reliable cell chips and cell tissue chips can be reliably produced with a smaller sample amount (sample). Further, since the cell chip and the cell tissue chip are produced by applying a high-viscosity cell dispersion to the object as compared with the case of using a conventional printer, the cell dispersion after application is used. Evaporation can be suppressed and high cell viability can be maintained.

In the coating unit and the fine coating apparatus of the present invention, a needle (coating needle) having a very small amount of coating liquid attached to the tip thereof is brought into contact with an object to be coated (culture container, etc.) to obtain several pL (picolitre). The droplets to be applied can be coated with high placement accuracy, for example, ± 15 μm or less, preferably ± 3 μm or less. Further, as the viscosity of the coating liquid, it is possible to apply a material up to 1 × 10 ⁵ mPa · s, and for example, it is possible to apply a highly viscous cell dispersion liquid.

Further, by using the coating unit and the fine coating apparatus of the present invention, for example, it becomes easy to produce a cell aggregate by a two-component gelling agent, and a large amount of cell aggregate can be produced with high accuracy. Can be done. Further, according to the present invention, even if a highly viscous cell-containing gelling agent is used as a material, desired cell chips and cell tissue chips can be produced in large quantities in a short time (high speed). It is possible to maintain a high cell viability with a desired cell density in a cell chip and a cell tissue chip.

As a result, according to the present invention, a highly viscous cell dispersion can be precisely applied to a predetermined object (culture container, etc.) at a predetermined position, and cell chips and cells having arbitrary patterning can be sterically applied. It is possible to produce a cell tissue chip that has been specifically shaped. As a result, the produced cell chips and cell tissue chips can be used in the fields of drug discovery research and regenerative medicine such as screening for evaluation of drug efficacy and safety.

Further, the coating unit and the fine coating apparatus of the present invention are not limited to the coating of the two-component gelling agent containing cells described in the first embodiment, and 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, and silicone adhesives. It can be used for bonding metal parts, bonding electrical and electronic parts, bonding building materials, and the like.

Although the present invention has been described in embodiments with some detail, this configuration is exemplary and the disclosures of this embodiment should vary in detail. In the present invention, replacement, combination, and change of order of elements in an embodiment with other elements can be realized without departing from the claimed scope and ideas of the present invention.

Since the coating unit and the fine coating apparatus according to the present invention can reliably produce various fine patterns or droplet spots with high reliability in a large amount, in addition to industrial and industrial applications, and It is an important technology for researching drug discovery and regenerative medicine, and is an invention with high industrial applicability.

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 needles Holding part 9 Needle part 9a Tip 10 Coating liquid container 10A First coating liquid container 10B Second coating liquid container 10a Coating liquid pool 10b Upper hole 10c Lower hole 11 Coating liquid 11A First coating liquid (cell-containing solution: first gelling) Agent)
11B 2nd coating liquid (2nd gelling agent)

Claims (14)

The first coating liquid container for storing the first coating liquid and
A first coating needle whose tip can be immersed in the first coating liquid stored in the first coating liquid container and reciprocates.
A second coating liquid container for storing the second coating liquid, and
A second coating needle whose tip can be immersed in the second coating liquid stored in the second coating liquid container and which reciprocates is provided.
The second coating liquid held at the tip of the second coating needle is contact-applied to the position where the first coating liquid held at the tip of the first coating needle is contact-applied to the object to be coated. Applying unit. The coating unit according to claim 1, wherein the tip diameter of the second coating needle is larger than the tip diameter of the first coating needle. The coating speed at which the second coating liquid held at the tip of the second coating needle is contact-applied, and the first coating liquid held at the tip of the first coating needle is contact-applied to the object to be coated. The coating unit according to claim 1 or 2, which is configured to be slower than the coating speed. Immediately before the first coating liquid held at the tip of the first coating needle comes into contact with the object to be coated, the coating speed of the first coating needle is slowed down, and the first coating liquid is held at the tip of the second coating needle. The coating unit according to claim 1 or 2, wherein the coating unit is configured to slow down the coating speed of the second coating needle immediately before the second coating liquid comes into contact with the first coating liquid coated on the object to be coated. .. Immediately before the first coating liquid held at the tip of the first coating needle comes into contact with the object to be coated, the first coating needle is once stopped for a predetermined time and then slowed down from the coating speed before the stop. , The coating unit according to any one of claims 1 to 4. Immediately before the second coating liquid held at the tip of the second coating needle comes into contact with the first coating liquid , the second coating needle is temporarily stopped for a predetermined time and then slowed down from the coating speed before the stop. The coating unit according to any one of claims 1 to 5. The item according to any one of claims 1 to 6, wherein the height from the object to be coated at the time of application of the second coating needle is set higher than the height from the object to be coated at the time of application of the first coating needle. The coating unit described. The first coating needle is configured to apply the first coating liquid to the object to be coated a plurality of times, and the height from the object to be coated at the time of application of the first coating needle is gradually increased each time. The coating unit according to any one of claims 1 to 7, which is configured to cause the coating. The coating unit according to any one of claims 1 to 8, wherein the tips of the first coating needle and the second coating needle are configured to include a plane orthogonal to the moving direction at the time of coating. The first coating needle is configured to penetrate the first coating liquid container to apply the first coating liquid to the object to be coated, and the second coating needle penetrates the second coating liquid container. The coating unit according to any one of claims 1 to 9, which is configured to apply the second coating liquid to the object to be coated. Claims 1 to 10, wherein the storage capacity of the second coating liquid stored in the second coating liquid container is larger than the storage capacity of the first coating liquid stored in the first coating liquid container. The coating unit according to any one item. The coating unit according to any one of claims 1 to 11.
An XY table on which an object to be coated, which is contact-coated by the first coating needle and the second coating needle in the coating unit, is placed and fixed, and which can be moved in the horizontal direction.
A vertical movement mechanism that enables the coating unit to move in the vertical direction,
A fine coating device equipped with. The fine coating apparatus according to claim 12, further comprising an observation optical system unit for observing a coating material contact-coated with a coating material placed on the XY table. The fine coating apparatus according to claim 12 or 13, further comprising an evaluation system apparatus for evaluating an coating material contact-applied to the coating object placed on the XY table.

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