Cell chip and three-dimensional tissue chip, and method for producing same

The present invention is to provide a cell chip and a three-dimensional tissue chip and a production method therefor such that even when a highly viscous cell-containing solution is a material, the highly safe and reproducible cell chip or three-dimensional tissue chip with a desired application volume can be produced within a short time and in a large quantity so as to have a desired cell density and exhibit high cell viability.

TECHNICAL FIELD

The present invention relates to an ex vivo cell chip or three-dimensional tissue chip, and a production method therefor.

BACKGROUND ART

Technology for ex vivo cellular three-dimensional tissue construction is critical in research on drug discovery and regenerative medicine. Human biological tissue-resembling cellular tissue construction, in particular, has been sought.

In the case of ex vivo cell manipulation, it is common to culture cells two-dimensionally by using cultureware, such as a plastic or glass dish. However, cells actually proliferate in vivo three-dimensionally to form a tissue or organ. Thus, in order to realize in vivo resembling culture conditions, it is critical for progression in drug discovery research and regenerative medicine research to construct a three-dimensional cell/tissue chip and use the three-dimensional tissue chip for evaluation and experiment.

Each three-dimensional tissue chip (cell assembly), on which cells assemble and aggregate to one another, is placed on, for instance, each well (recessed portion) aligned on a well plate to evaluate each cell assembly. This assay has been widely used in evaluation of cellular function and screening for a compound effective as a novel pharmaceutical. Such cell assemblies may be assayed to evaluate many items within a short time by using a small amount of sample. This is advantageous from the viewpoint of making the evaluation rapid, simple, safe, reproducible, and highly reliable.

Examples of technology for precisely patterning cells on a substrate include a method of processing a substrate using a photolithography technique for controlling cell adhesion and a method of directly printing cells for arrangement and immobilization. Recently, 3D printer technology for three-dimensionally (3D) arranging and layering cells has advanced and cell assembly construction using a 3D printer has been investigated. Here, it has considerably been developed to apply a tissue model obtained from cell assemblies to drug discovery research and regenerative medicine.

CITATION LIST

Patent Literature

SUMMARY OF INVENTION

Technical Problem

As the 3D printer for cell assembly construction, there are an inkjet printer (inkjet type: a thermal type, piezoelectric type), a microextrusion printer (of dispenser type), and a laser-assisted printer (of pulse laser type). Unfortunately, in these printers, a dischargeable material is limited and their printing rate (production rate) and resolution (discharge volume) have also been restricted. Further, when the material to be discharged is a cell-containing solution containing cells, conventional 3D printers have had problems with a cell assembly to be produced regarding, for instance, the cell viability and the cell density after discharged. In the case of producing a cell assembly from a highly viscous cell-containing solution, in particular, there is a risk of clogging of highly viscous solution in a printer nozzle. Thus, it is impossible to provide a high resolution, that is, to produce fine cell assemblies in a large quantity, which has been a problem.

The purpose of the present invention is to provide a cell chip and a three-dimensional tissue chip and a production method therefor such that the cell chip or three-dimensional tissue chip with a desired application volume can be produced within a short time (at a high speed) and in a large quantity even if a highly viscous cell-containing solution is a material and the produced cell chip or three-dimensional tissue chip has a desired cell density so as to exhibit high cell viability.

Solution to Problem

To achieve the goal, a method for producing a cell chip or three-dimensional tissue chip according to an aspect of the invention includes:an attachment step of attaching a cell-containing solution to a tip of an application needle;a transfer step of moving the cell-containing solution-attached tip of the application needle closer to an application target;an application step of bringing the cell-containing solution attached to the tip of the application needle into contact with the application target or into contact with the cell-containing solution having already been applied on the application target, thereby subjecting the cell-containing solution attached to the tip of the application needle to contact application; anda separation step of, after the cell-containing solution is subjected to the contact application, making the tip of the application needle apart from the application target.

In addition, to achieve the goal, a method for producing a cell chip or three-dimensional tissue chip according to an aspect of the invention by using a micro-applicator provided with an application unit including an application liquid container having an application liquid reservoir for storing a cell-containing solution in a prescribed amount and an application needle allowing for penetration through the application liquid reservoir having the cell-containing solution stored, the method including:a waiting step of making a tip of the application needle dipped into the cell-containing solution having been charged in the application liquid reservoir;a descending step of making the tip of the application needle penetrate through the application liquid reservoir to move downward the cell-containing solution-attached tip of the application needle;an application step of bringing the cell-containing solution-attached tip of the application needle into contact with an application target, thereby applying the cell-containing solution to the application target to form a liquid droplet spot; anda holding step of lifting the tip of the application needle and holding the tip of the application needle in the application liquid reservoir.

Further, to achieve the goal, a cell chip or three-dimensional tissue chip according to an aspect of the invention is produced by at least the cell chip or three-dimensional tissue chip production method.

Advantageous Effects of Invention

According to the invention, a cell chip and a three-dimensional tissue chip with a desired application volume can be produced within a short time and in a large quantity even if a highly viscous cell-containing solution is a material and the produced cell chip or three-dimensional tissue chip has a desired cell density so as to exhibit high cell viability.

DESCRIPTION OF EMBODIMENTS

First, various aspects in a cell chip or three-dimensional tissue chip production method according to the invention are described. Note that the cell chip in the invention refers to a chip in a state in which individual cells are substantially dispersed and adhered on, for instance, a substrate. The three-dimensional tissue chip refers to a cell assembly in a state in which cells are assembled, aggregated, and layered on a substrate to form a three-dimensional tissue and are functioning. Here, any shape of the cell assembly, such as a spherical shape or a flat shape, is acceptable.

A method for producing a cell chip or three-dimensional tissue chip according to a first aspect according to the invention is characterized by including:an attachment step of attaching a cell-containing solution to a tip of an application needle;a transfer step of moving the cell-containing solution-attached tip of the application needle closer to an application target;an application step of bringing the cell-containing solution attached to the tip of the application needle into contact with the application target or into contact with a cell-containing solution having already been applied on the application target, thereby subjecting the cell-containing solution attached to the tip of the application needle to contact application; anda separation step of, after the cell-containing solution is subjected to the contact application, making the tip of the application needle apart from the application target.

For a method for producing a cell chip or three-dimensional tissue chip according to a second aspect of the invention, multiple cycles of cell application operation may be repeated while one cycle of the cell application operation includes the attachment step, the transfer step, the application step, and the separation step in the first aspect.

A method for producing a cell chip or three-dimensional tissue chip according to a third aspect of the invention by using a micro-applicator provided with an application unit including an application liquid container having an application liquid reservoir for storing a cell-containing solution in a prescribed amount and an application needle allowing for penetration through the application liquid reservoir having the cell-containing solution stored includes:a waiting step of making a tip of the application needle dipped into the cell-containing solution having been charged in the application liquid reservoir;a descending step of making the tip of the application needle penetrate through the application liquid reservoir to move downward the cell-containing solution-attached tip of the application needle;an application step of bringing the cell-containing solution-attached tip of the application needle into contact with an application target, thereby applying the cell-containing solution to the application target to form a liquid droplet spot; anda holding step of lifting the tip of the application needle and holding the tip of the application needle in the application liquid reservoir.

For a method for producing a cell chip or three-dimensional tissue chip according to a fourth aspect of the invention, multiple cycles of cell application operation may be repeated while one cycle of the cell application operation includes the waiting step, the descending step, the application step, and the holding step performed with respect to certain positions relative to the application target in the third aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a fifth aspect of the invention, one cycle of the cell application operation may be conducted in 0.5 sec or shorter in the second or fourth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a sixth aspect of the invention, the cell application operation may be carried out using a material in which the viscosity of the cell-containing solution is 1×105mPa·s or less and preferably from 1 to 1×104mPa·s in any one of the second, fourth, or fifth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a seventh aspect of the invention, a liquid droplet spot obtained by the one cycle of the cell application operation may be formed with positional precision of ±15 μm or less with respect to the application target in any one of the second, fourth, fifth, or sixth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to an eighth aspect of the invention, a liquid droplet spot obtained by the one cycle of the cell application operation may be formed with positional precision of ±3 μm or less with respect to the application target in any one of the second, fourth, fifth, or sixth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a ninth aspect of the invention, multiple cycles of the cell application operation may be repeated while a stop position of the tip of the application needle in the application step is shifted upward with respect to a certain position relative to the application target by a given distance every cycle of the cell application operation in any one of the second, fourth, fifth, sixth, seventh, or eighth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a tenth aspect of the invention, the application unit may include a sliding mechanism part for slidably holding the application needle in the third or fourth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to an eleventh aspect of the invention, the sliding mechanism part may have a mechanism for absorbing a shock when the tip of the application needle comes into contact with the application target in the tenth aspect.

For a method for producing a cell chip or three-dimensional tissue chip according to a twelfth aspect of the invention, the tip of the application needle may be configured to move in a vertical direction during the application step of any one of the first to eleventh aspects.

For a method for producing a cell chip or three-dimensional tissue chip according to a thirteenth aspect of the invention, the tip of the application needle may include a flat surface perpendicular to a transfer direction of the application needle during the application step in any one of the first to twelfth aspects.

For a method for producing a cell chip or three-dimensional tissue chip according to a fourteenth aspect of the invention, the tip of the application needle may include a recessed surface in any one of the first to thirteenth aspects.

For a method for producing a cell chip or three-dimensional tissue chip according to a fifteenth aspect of the invention, it is possible to use, as a cell(s) in the cell-containing solution, a cell(s) having a cell surface coated with, for instance, an extracellular matrix protein, a sugar chain, and/or a natural or synthetic polymer so as to increase inter-cellular adhesion in any one of the first to fourteenth aspects.

A cell chip or three-dimensional tissue chip according to a sixteenth aspect of the invention is a cell chip or three-dimensional tissue chip produced by the method for producing a cell chip or three-dimensional tissue chip according to any one of the first to fifteenth aspects.

In the methods for producing a cell chip or three-dimensional tissue chip according to the invention, as described using specific examples in the below-described embodiments, a micro-applicator is used which can be utilized to highly precisely apply several pL (picoliter) of a tiny liquid droplet attached to the tip of an application needle to a predetermined position on a target within a very short time, such as 0.1 sec, per application. This allows for increased safety and reproducibility and automation. Thus, it is presented to produce a highly reliable cell chip or three-dimensional tissue chip (cell assembly) within a short time and in a large quantity.

The invention makes it possible to produce, with high precision, a plurality of three-dimensional tissue chips (cell assemblies) by using a high-speed micro-applicator to reliably apply, onto a predetermined position within a short time, a highly viscous (50 mPa·s or higher) material, such as a highly viscous solution containing cells and a gelatinizer, which has not been successfully handled using any conventional printer. As a result, the invention makes it possible to optionally control and arrange desired cells two-dimensionally and three-dimensionally. Thus, various three-dimensional tissue chips can be produced under an aseptic condition and in a large quantity while the production is automated. Hence, the methods for producing a cell chip or three-dimensional tissue chip according to the invention make it possible to produce, in a large quantity by automation, a cell chip or three-dimensional tissue chip having increased safety and reproducibility and reliability.

Next, the methods for producing a cell chip or three-dimensional tissue chip according to the invention will be described, using embodiments representing specific configuration examples, by referring to the Drawings attached. Note that the methods for producing a cell chip or three-dimensional tissue chip according to the invention are not limited to configurations using micro-applicators in the below-described embodiments. The methods can be implemented by cell application operation (cell application method) in the technical ideas comparable to those for the cell application operation (cell application method) carried out in micro-applicators.

First Embodiment

Hereinafter, the first embodiment will be specifically described with reference to the Drawings attached.FIG.1is a diagram illustrating the whole micro-applicator1used in the first embodiment. As shown inFIG.1, the micro-applicator1is provided with an applicator main body2and a display/control unit3configured to display, control, and set the applicator main body2. What is called a personal computer (PC) is a component of the display/control unit3of the micro-applicator1in the first embodiment.

The applicator main body2of the micro-applicator1includes: an XY table4movable over a main body base12in a horizontal direction; a Z table5movable in a top-to-bottom direction (vertical direction) with respect to the XY table4; an application unit6fixed to a driving mechanism movable, like the Z table5, in the top-to-bottom direction; and an optical detection unit (e.g., a CCD camera)7configured to observe an application target on the XY table4. For instance, a substrate, on which application liquid10, a cell-containing solution, is applied to form a plurality of cell chips or three-dimensional tissue chips, is placed on and fixed to the XY table4.

The application unit6in the micro-applicator1as so structured is configured to carry out cell application operation for aligning and forming a plurality of cell chips or three-dimensional tissue chips on, for instance, a substrate over the XY table4. Hereinbelow, the structure of the application unit6and the cell application operation by using the application unit6will be described.

[Structure of Application Unit]

FIG.2is a diagram illustrating an application needle holder part13mounted on the application unit6. An application needle9protrudes from the application needle holder part13.FIG.3is diagrams illustrating a tip portion of each application needle9. Regarding the application needle9in the first embodiment, a tip9aof cone-shaped leading edge portion has a flat surface (is flat) so as to face a horizontal surface of the XY table4(see (a) ofFIG.3). That is, the flat surface of the tip9ais a flat surface perpendicular to the vertical direction. The diameter d of the tip9asubstantially contributes to the shape of each cell chip or three-dimensional tissue chip to be produced, which will be described later. In the first embodiment, the tip9aof the application needle9has a diameter d of from 50 to 330 μm. In the first embodiment, as shown in (a) ofFIG.3, the leading edge portion of the application needle9is shaped like a cone. The tip9ahas a horizontal surface. Accordingly, the tip9amay be polished to a horizontal surface so as to easily adjust the diameter d of the tip9ato a desired value ranging from, for instance, 50 to 330 μm.

Note that, in the first embodiment, the tip9aof the application needle9is explained using an example of horizontal flat surface configuration (see (a) ofFIG.3). This tip9asurface shape may have a recessed surface (semi-spherical surface) with a prescribed diameter, such as a diameter of 30 μm or less, and cells may be retained on the recessed surface so as to carry out cell application operation (see (b) ofFIG.3). Such formation of the tip9aof the application needle9into a recessed surface makes it possible to produce a cell chip or three-dimensional tissue chip having a desired cell density and cell arrangement by the cell application operation using the application needle9. In addition, as shown in (c) ofFIG.3, the tip9aof the application needle9may have a stepped protrusion9b. This configuration with such a protrusion9bmakes it possible to produce a cell assembly extending upward relative to a substrate by performing continuous and repeated application over the substrate while a needle tip is shifted upward by a given distance, such as 0.5 μm, every cell application operation.

FIG.4is diagrams schematically illustrating cell application operation in the application unit6. As shown inFIG.4, the application unit6includes: an application liquid container8having an application liquid reservoir8ain which a prescribed amount of application liquid10, a cell-containing solution, is stored; and the application needle holder part13equipped with the application needle9that penetrates through the application liquid reservoir8a. The application needle holder part13is provided with a sliding mechanism part16that slidably holds the application needle9in the top-to-bottom direction (vertical direction). The application needle holder part13is detachably provided at a given position on a driving mechanism part17, and, for instance, may be detachable from the driving mechanism part17by using magnetic force of a magnet.

The application needle holder part13is so fixed to the driving mechanism part17in the applicator main body2and is configured to reciprocally move between prescribed distances at a high speed in the top-to-bottom direction (vertical direction). The display/control unit3executes, for instance, settings of driving control of such a driving mechanism part17and driving control of the XY table4and the Z table5. The sliding mechanism part16installed in the application needle holder part13can hold the vertically reciprocating application needle9and is configured to make the application needle9slide relative to the sliding mechanism part16in the vertical direction such that when coming into contact with a target, for instance, a substrate11, the tip9aof the application needle9stops at the contact position. That is, the sliding mechanism part16has a shock absorption mechanism when the application needle9comes into contact. Because of this, the tip9aof the application needle9stops at the position in contact with an application target and is made apart from the application target in response to the subsequent upward movement of the driving mechanism part17. Note that, the vertical reciprocating operation of the application needle9at that time is at an ultra-high speed. For instance, one reciprocating operation is set to preferably 0.5 sec or less and more preferably 0.1 sec or less.

As described above, the application needle holder part13in the application unit6is provided with the vertically slidable sliding mechanism part16while holding the application needle9and is detachably fixed to the vertically moving driving mechanism part17. In addition, the application needle holder part13is configured such that the application needle9can move in the top-to-bottom direction (vertical direction) and penetrate through the application liquid reservoir8athat stores the application liquid10, a cell-containing solution. An upper portion and a lower portion of the application liquid container8each have a hole (upper hole14aor lower hole14b) through which the application needle9penetrates.

Next, the cell application operation in the application unit6, as schematically shown inFIG.4, will be described. During the cell application operation illustrated inFIG.4, the application needle9passes through the application liquid reservoir8aof the application liquid container8and comes into contact with the substrate11, which is an application target; and the cell-containing application liquid10is applied on the substrate11to form a liquid droplet spot S. This cell application operation is repeated prescribed times and the application liquid10is applied multiple times to produce a desired cell chip or three-dimensional tissue chip on the substrate11. Note that in the first embodiment and other embodiments according to the invention, the substrate11is used, as an application target, for description. Here, the application target is not limited to a substrate and examples of the target include cultureware such as a plastic dish, a glass dish, or a well-plate commonly used for cell culture and/or various solutions on the cultureware.

(a) ofFIG.4shows a waiting state during the cell application operation. In this waiting state, the application needle9is inserted from the upper hole14aand the tip9aof the application needle9is dipped into the application liquid10in the application liquid reservoir8a. In such a waiting state (waiting step), the tip9aof the application needle9is dipped into the application liquid10, so that the application liquid10attached to the tip9ais not dried. At that time, because the diameter of the lower hole14bat the application liquid container8is very small (e.g., 1 mm or less), no leak of the application liquid10from the application liquid reservoir8aoccurs.

(b) ofFIG.4shows a state (descending step) in which the tip9aof the application needle9is projected from the lower hole14bat the application liquid container8and the tip9adescends toward the substrate11from the application liquid reservoir8a. That is, (b) ofFIG.4shows a descending state in which the tip9aof the application needle9penetrates through and protrudes from the application liquid reservoir8aof the application liquid container8. During this descending state, the application liquid10is attached to the application needle9and the surface tension of the application liquid10attached causes the tip9aof the application needle9to retain a certain volume of application liquid10.

(c) ofFIG.4shows a state (application step) in which the tip9aof the application needle9comes into contact with a surface of the substrate11and the application liquid10is applied on the surface of the substrate11. A liquid droplet spot S of the application liquid10applied at that time corresponds to a certain volume of application liquid10held on the tip9aof the application needle9. The first embodiment is configured such that an impact load at the moment when the tip9aof the application needle9is in contact with (comes into contact with) a surface of the substrate11is about 0.06 N or lower. In the first embodiment, as described above, because the application needle9is slidably held by the sliding mechanism part16so as to absorb a vertical shock, the impact load at the time of contact is a very small value.

(d) ofFIG.4shows a state immediately after the application needle9is used to apply the application liquid10on a surface of the substrate11and shows a state in which the application needle9is being lifted. This lifting state is followed by transition into a waiting state in which the tip9aof the application needle9is dipped into the application liquid10of the application liquid reservoir8a(holding step).

As described above, the one cycle of cell application operation includes, in sequence, (a), (b), (c), (d), and (a) operations as illustrated inFIG.4. In the first embodiment, the one cycle of cell application operation is conducted in 0.1 sec, and the cell application operation is executed in a ultra-short period. Note that in the first embodiment, the cell application operation is repeated prescribed times (e.g., 10 cycles) to produce a desired cell chip or three-dimensional tissue chip. Meanwhile, 10 cycles of the application operation require 1 sec so as to produce 1 spot of three-dimensional tissue.

During the cell application operation using the micro-applicator1in the first embodiment according to the invention, the application needle9having a tip attached to a very small volume of application liquid10is brought into contact with an application target (e.g., the substrate11); and a liquid droplet spot S in an application volume of several pL (picoliter) can be applied and formed with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. In addition, it is possible to apply a material with a viscosity of the application liquid10of 1×105mPa·s or lower and preferably from 1 to 1×104mPa·s. This allows for application of highly viscous cell dispersion. During the cell application operation using the micro-applicator1in the first embodiment, it is possible to use, as an application material, a material with a viscosity of from 10 mPa·s to 1×105mPa·s (inclusive), which material has not been successfully used because nozzle-type printers such as inkjet printers have a problem of clogging, etc. In addition, the application needle9having a tip attached to a very small volume of application liquid10is brought into contact with an application target for application. Accordingly, the application is not affected by a variation in the vertical position of the application needle9and can be repeated using a desired application volume of application liquid10in a stable fashion. As such, in the first embodiment according to the invention, a highly viscous cell dispersion can be precisely applied at a predetermined position on, for instance, the substrate11. This makes it possible to produce a cell chip with a given pattern or a three-dimensional tissue chip on which cells are shaped three-dimensionally. In view of the above, the method for producing a cell chip or three-dimensional tissue chip according to the invention exerts advantageous effects on progress in respective fields while the produced cell chip or three-dimensional tissue chip is utilized in the fields of regenerative medicine and drug discovery research such as drug efficacy or safety evaluation screening.

Experimental Example 1

The micro-applicator1, which has been described in the above first embodiment, was used to conduct application experiment 1 using application liquids10with different concentrations and viscosities. In this application experiment 1, 3 different application liquids10, including 5%, 10%, and 20% gelatin PBS (phosphate buffer solution) solutions, were used to examine the shape of each liquid droplet spot.

Three application liquid containers8in the application unit6were provided, and gelatin was dissolved at 5, 10, or 20% weight by volume (% w/v) in phosphate buffer solution (PBS) to prepare, as the application liquids10, three different gelatin PBS solutions. Note that each application liquid10used in this application experiment 1 is free of cells.

In application experiment 1, each application liquid container8was filled with 20 μL of 5%, 10%, or 20% gelatin PBS solution. A needle having a tip9a(flat surface shape) with a diameter d of 100 μm was used as the application needle9in the application unit6. In this application experiment 1, each application liquid10was subjected to point contact and was applied as 5×5 spots with a 150-μm interval on a slide glass fixed to the XY table4. The liquid droplet spots formed on the slide glass by the application were observed under a phase-contrast microscope.

FIG.5is photographs showing images of the liquid droplet spots observed under a phase-contrast microscope in application experiment 1. (a) ofFIG.5is an image showing liquid droplet spots formed by applying, as the application liquid10, 5% gelatin PBS solution (with a viscosity of 3 mPa·s) on a slide glass by using an application needle9having a tip9awith a diameter d (tip diameter) of 100 μm. (b) ofFIG.5is an image showing liquid droplet spots formed by applying, as the application liquid10, 10% gelatin PBS solution (with a viscosity of 30 mPa·s) on a slide glass by using the application needle9(with a tip diameter of 100 μm). In addition, (c) ofFIG.5is an image showing liquid droplet spots formed by applying, as the application liquid10, 20% gelatin PBS solution (with a viscosity of 220 mPa·s) on a slide glass by using the application needle9(with a tip diameter of 100 μm).

(a), (b), and (c) ofFIG.5clearly demonstrate that when the application unit6of the micro-applicator1was used, the liquid droplet spots formed using the application liquids10even with different concentrations and viscosities had substantially the same shape (the 5% gelatin liquid droplet spots had a diameter of 136±3 μm, the 10% gelatin liquid droplet spots had a diameter of 147±1 μm, and the 20% gelatin liquid droplet spots had a diameter of 151±1 μm). That is, application experiment 1 successfully verified constantly stable liquid droplet spots, which did not significantly depend on the gelatin concentration and viscosity. According to the experiment conducted by the present inventors, the diameter of each liquid droplet spot was within a size 1.3 to 1.6 times the tip diameter of the application needle9, and the liquid droplet was not at least twice as large as the tip diameter.

Experimental Example 2

The micro-applicator1, which has been described in the above first embodiment, was used to conduct application experiment 2 using application needles9with different tip9adiameters (tip diameters). In this application experiment 2, 5% gelatin PBS solution was used as the application liquid10to examine the shape of each liquid droplet spot formed. The application liquid10used in this application experiment 2 is free of cells.

In application experiment 2, three different application needles9having a tip diameter of 50 μm, 100 μm, or 150 μm were used. In application experiment 2, the application liquid10was subjected to point contact and was applied as 5×5 spots with a 150-μm interval on a slide glass fixed to the XY table4. The liquid droplet spots formed on the slide glass by the application were observed under a phase-contrast microscope.

FIG.6is photographs showing images of the liquid droplet spots observed under a phase-contrast microscope in application experiment 2. (a) ofFIG.6is an image showing liquid droplet spots formed by applying the application liquid10of 5% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 50 μm. (b) ofFIG.6is an image showing liquid droplet spots formed by applying the application liquid10of 5% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 100 μm. (c) ofFIG.6is an image showing liquid droplet spots formed by applying the application liquid10of 5% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 150 μm.

(a), (b), and (c) ofFIG.6successfully demonstrated that the liquid droplet spots formed had liquid droplet spot diameters approximately proportional to the diameters (50 μm, 100 μm, and 150 μm) of the application needles9(the 50-μm application needle: a liquid droplet spot diameter of 75±2 μm; the 100-μm application needle: a liquid droplet spot diameter of 137±3 μm; and the 150-μm application needle: a liquid droplet spot diameter of 219═5 μm).

Experimental Example 3

In application experiment 3, human dermal fibroblasts (NHDF) were dispersed at a concentration of 2×107cells/mL in 10% gelatin PBS solution to prepare an application liquid10. In application experiment 3, the micro-applicator1was used to apply the application liquid10on a slide glass by contact application using three different application needles9with a tip diameter of 100 μm, 150 μm, or 200 μm. The shapes of liquid droplet spots formed on the slide glass by the application were observed under a phase-contrast microscope.

FIG.7is photographs showing images of the liquid droplet spots observed under a phase-contrast microscope in application experiment 3. (a) ofFIG.7is an image showing liquid droplet spots formed by applying the NHDF-dispersed 10% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 100 μm. (b) ofFIG.7is an image showing liquid droplet spots formed by applying the NHDF-dispersed 10% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 150 μm. (c) ofFIG.7is an image showing liquid droplet spots formed by applying the NHDF-dispersed 10% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 200 μm.

According to application experiment 3, when the application needle9having a tip diameter of 100 μm was used, one liquid droplet spot was found to contain 0 to 3 cells applied. When the application needle9having a tip diameter of 150 μm was used, one liquid droplet spot was found to contain 2 to 6 cells applied. In addition, one liquid droplet spot formed using the application needle9having a tip diameter of 200 μm was found to contain up to about 10 cells applied. Hence, by selecting the tip diameter of the application needle9, it was found to be possible to control the number of cells applied in each liquid droplet spot to within about 1 to 10.

Experimental Example 4

In application experiment 4, instead of human dermal fibroblasts (NHDF) in the above application experiment 3, a liver cancer cell line (HepG2) was used to likewise conduct an experiment. In application experiment 4, HepG2 was dispersed at a concentration of 5×107cells/mL in 10% gelatin PBS solution to prepare an application liquid10.

FIG.8is an image showing liquid droplet spots formed by applying the HepG2-dispersed 10% gelatin PBS solution on a slide glass by using an application needle9having a tip diameter of 100 μm. Even application experiment 4 demonstrated that a given number of cells were present in each liquid droplet spot, so that stable application was possible regardless of the kind of cells. In the below-described application experiment 5, how the tip diameter of the application needle9correlated to the number of cells applied and contained in each liquid droplet spot formed was investigated.

Experimental Example 5

In application experiment 5, iPS-derived cardiomyocytes (iPS-CM) were dispersed at a concentration of 4×107cells/mL in PBS solution to prepare an application liquid10and respective application needles9with a tip diameter of 70 μm, 100 μm, 150 μm, 200 μm, or 330 μm were used to form liquid droplet spots. In application experiment 5, how the tip diameter of each application needle9correlated to the number of cells applied and contained in each liquid droplet spot formed was investigated.

In application experiment 5, the above application liquid10was applied once on a slide glass, and the number of cells after the application was calculated by fluorescence microscopy (using cells, the nuclei of which were stained with a fluorescent dye DAPI) and phase-contrast microscopy. In this application experiment 5, 20 or more liquid droplet spots formed by each of the application needles9with a tip diameter of 50 μm, 100 μm, 150 μm, 200 μm, or 330 μm were measured and averaged.

FIG.9is a graph showing the experimental results of application experiment 5. InFIG.9, the ordinate represents the number of cells applied [cells/spot], and the abscissa represents the tip diameter of application needle9[μm]. As shown inFIG.9, when iPS-CM was dispersed at a concentration of 4×107cells/mL in PBS solution, average 1.1 cells applied were present in each liquid droplet spot applied by using the application needle9with a tip diameter of 50 μm. Average 4.0 cells applied were present in each liquid droplet spot obtained by using the application needle9with a tip diameter of 100 μm; average 4.5 cells applied were present in each liquid droplet spot obtained by using the application needle9with a tip diameter of 150 μm; average 19.1 cells applied were present in each liquid droplet spot obtained by using the application needle9with a tip diameter of 200 μm; and average 85.3 cells applied were present in each liquid droplet spot obtained by using the application needle9with a tip diameter of 330 μm. Note that inFIG.9, the error bars indicate the standard deviations (in the positive direction) of the number of cells applied using the respective application needles9.

As described above, the tip diameter of each application needle9used was correlated with the number of cells applied and present in each liquid droplet spot formed, indicating that as the tip diameter of the application needle9became larger, the number of cells applied and present in each liquid droplet spot increased. Hence, by selecting the tip diameter of the application needle9, it was successfully verified that the number of cells applied in each liquid droplet spot was able to be controlled to within a certain range.

Experimental Example 6

In application experiment 6, the micro-applicator1was used to produce a cell assembly20. In application experiment 6, human dermal fibroblasts (NHDF) were dispersed at a concentration of 2×107cells/mL in 2.5% alginic acid PBS solution to prepare an application liquid10. This application liquid10was charged into the application liquid container8of the application unit6, and cell application operation was then executed by the micro-applicator1.

In application experiment 6, by using an application needle9(see (c) ofFIG.3) having a tip9awith a stepped protrusion9band a tip diameter of 100 μm, the alginic acid-containing cell dispersion was consecutively applied 1600 times on a slide glass. At that time, the needle tip was shifted upward by 0.5 μm every cell application operation to produce a cell assembly20. This resulted in production of the cell assembly20with a diameter of 50 μm and a height of 500 μm.FIG.10is an image picture showing a cell assembly20produced in application experiment 6.

As described above, the cell assembly20with a desired shape was found to be able to be produced by repeating multiple cycles of cell application operation while the stop position of the tip9aof the application needle9was shifted upward (e.g., by 0.5 μm), with respect to a certain application target position (certain point) on the substrate11, per cycle during the application step in the cell application operation.

Experimental Example 7

In application experiment 7, the viability of cells applied in liquid droplet spots formed by application using the micro-applicator1was examined. In application experiment 7, human dermal fibroblasts (NHDF) were dispersed at a concentration of 8×107cells/mL in PBS solution to prepare an application liquid10. In addition, in application experiment 7, this application liquid10was consecutively applied 40 times on a slide glass by using the application needle9with a tip diameter of 330 μm, and the viability of cells15after the application was evaluated by viable cell/dead cell (Live/Dead) fluorescent staining (dead cells were stained red).FIG.11is pictures showing viable cell/dead cell (Live/Dead) fluorescently stained images in application experiment 7.

InFIG.11, (a) shows a viable cell/dead cell (Live/Dead) fluorescently stained image of cells15in a pre-application application liquid10and (b) shows a viable cell/dead cell (Live/Dead) fluorescently stained image of cells15in a post-application liquid droplet spot. Note that in the viable cell/dead cell (Live/Dead) fluorescently stained images in application experiment 7, viable cells were stained green and dead cells were stained red. Here, in the pictures showing the viable cell/dead cell (Live/Dead) fluorescently stained images inFIG.11, viable cells were denoted by ∘ and dead cells were denoted by ●.

The results of examining the cell15viability in application experiment 7 demonstrated that the pre-application cell viability was 96% and the post-application cell viability was 91% and still high. This result clearly confirmed that during the cell application operation using the micro-applicator1, almost no damage was given directly to the cells15. Note that the pre-application cell viability was slightly dropped from 96% to the post-application cell viability of 91%. Here, this drop was a regular decrease occurring over time and was caused by another factor.

Experimental Example 8

In application experiment 8, the micro-applicator1was used to construct a three-dimensional tissue chip having iPS-derived cardiomyocytes (iPS-CM) on a cell disc. Then, pulsation behavior of a myocardial tissue body on the three-dimensional tissue chip was assessed.

In application experiment 8, iPS-CM was dispersed at a concentration of 4×107cells/mL in 20 mg/mL fibrinogen solution to prepare an application liquid10. In application experiment 8, application was consecutively carried out 10 times on a cell disc by using the application needle9with a tip diameter of 330 μm, and the cell disc was then soaked in 800 unit/mL (8.3 mg/mL) thrombin solution to immobilize a tissue by gelatination. Thrombin action caused fibrinogen to form fibrin (blood coagulation-related protein), and this gelatination reaction was utilized to immobilize a tissue on a substrate.

After that, during 6 days of culturing, pulsation behavior was recorded over time under a phase-contrast microscope. As a result, immediately after the application, myocardial tissue bodies with a diameter of about 300 μm were formed and even after 6 days of culturing, the structure of equally spaced myocardial tissue bodies was able to be observed.

FIG.12is a photograph showing states (at culture day 0) of myocardial tissue bodies, produced in application experiment 8, immediately after application.FIG.13is a photograph showing states of the myocardial tissue bodies shown inFIG.12after 6 days of culturing.FIG.14is an enlarged image of one of the myocardial tissue bodies after 5 days of culturing.FIGS.13and14demonstrated that the structure of each myocardial tissue body was examined and a three-dimensional tissue chip was found to be reliably constructed.

In the myocardial tissue bodies produced, cardiomyocytes started pulsation after culture day 2, and at culture day 6, average 82 pulsations per min were recorded in 6 samples. The standard deviation of the 6 samples was 15.

In addition, a pulsation video captured using a high-speed camera was analyzed to calculate a contraction-relaxation rate with a constant cycle.FIG.15is a graph showing a contraction-relaxation rate obtained by analyzing a pulsation video of the myocardial tissue body (three-dimensional tissue chip) cultured for 5 days as shown inFIG.14. InFIG.15, the ordinate represents a movement rate at which the myocardial tissue body contracts and relaxes, and the abscissa represents time [s].

As shown inFIG.15, the myocardial tissue body (three-dimensional tissue chip) exhibited constant pulsations and the contraction-relaxation rate with a constant cycle was found. At that time, the pulsation rate was 78 times/min, the average contraction rate was 8.7 μm/s, and the average relaxation rate was 4.6 μm/s.

The myocardial tissue bodies obtained in the above application experiment 8 may be produced in respective wells on, for instance, a 96-well plate. This makes it possible to produce a high-throughput cardio-toxicity evaluation kit allowing for automated robotic evaluation under aseptic conditions.

Second Embodiment

Next, a method for producing a cell chip or three-dimensional tissue chip according to a second embodiment of the invention will be described with reference to the Drawings attached. An apparatus having the same configuration and functions as of the micro-applicator1used in the above-described first embodiment is used as a micro-applicator in the second embodiment. The method for producing a cell chip or three-dimensional tissue chip according to the second embodiment is as described in, for instance, paragraph [0044] regarding the cell application operation in the above first embodiment. Here, an example of the cell chip or three-dimensional tissue chip production method is described in which the needle tip is shifted upward by a given distance every cycle of cell application operation. That is, in the method for producing a cell chip or three-dimensional tissue chip according to the second embodiment, a specific example of the cell chip or three-dimensional tissue chip production method described in the first embodiment is explained. Note that, in the description for the second embodiment, elements having the same action, structure, and function as of the above first embodiment may have the same reference numerals so as to omit and avoid redundant description.

FIG.16is diagrams schematically illustrating cell application operation in the method for producing a cell chip or three-dimensional tissue chip according to the second embodiment. The cell application operation shown inFIG.16represents one cycle of cell application operation. In the cell application operation shown inFIG.16, the tip9aof the application needle9is subjected to contact application on the substrate11, which is an application target, and a state is illustrated in which a liquid droplet spot S (cell assembly20) of the application liquid10, which is a cell-containing solution, has already been formed on the substrate11. That is, the cell application operation inFIG.16represents a cell application operation in at least the second cycle or later after the first cycle of cell application operation has been executed.

(a) ofFIG.16shows an attachment state (attachment step) in which the tip9aof the application needle9is dipped into the application liquid10in the application liquid reservoir8aand the application liquid10is attached to the tip9aof the application needle9, and this state corresponds to a waiting state during the cell application operation in the first embodiment.

(b) ofFIG.16shows a transfer state (transfer step) in which the tip9aof the application needle9is projected from the lower hole14bat the application liquid container8and the tip9adescends toward the substrate11from the application liquid reservoir8a. That is, (b) ofFIG.16corresponds to a descending state during the cell application operation in the first embodiment.

(c) ofFIG.16shows a contact application state (application step) in which the tip9aof the application needle9comes into contact with a liquid droplet spot S on the substrate11. The liquid droplet spot S of the application liquid10applied at that time corresponds to a certain volume of application liquid10attached to the tip9aof the application needle9.

(d) ofFIG.16shows a state after the application needle9is used to apply the application liquid10on a surface of the substrate11and shows a separation state (separation step) in which the application needle9has been separated and is being lifted from the substrate11. That is, (d) ofFIG.16corresponds to a holding state during the cell application operation in the first embodiment.

As described above, in the second embodiment, the one cycle of cell application operation includes, in sequence, (a), (b), (c), (d), and (a) operations as illustrated inFIG.16. In the second embodiment, the one cycle of cell application operation is conducted in 0.1 sec, and the cell application operation is executed in a ultra-short period. Regarding the cell application operation in the second embodiment, the tip9aof the application needle9is controlled such that the stop position at the time of contact application is gradually shifted upward after the cell application operation at the second cycle or later.

Diagrams (1) to (4) shown inFIG.17are diagrams schematically illustrating movements of the tip9aof the application needle9during contact application in the cell application operation.FIG.17schematically illustrates: (1) a state in which the application liquid10, which is a tiny volume of cell-containing solution, has been attached to the tip9aof the application needle9; (2) a state in which the application liquid10attached to the tip9aof the application needle9comes into contact with a liquid droplet spot S of the application liquid10that is an application target and has already been applied onto the substrate11; (3) a state immediately after the tip9aof the application needle9has come into contact with the application liquid10on the substrate11and is then lifted; and (4) a state in which the tip9aof the application needle9is lifted and separated from the substrate11and a new liquid droplet spot S is formed on the substrate11.

As described above, in the cell application operation at the second cycle or later according to the second embodiment, the tip9aof the application needle9does not come into contact with the substrate11, but comes into contact with the application liquid10on the substrate11and is thus subject to contact application to produce a desired cell chip or three-dimensional tissue chip.

FIG.18is an operation graph describing movements of the tip9aof the application needle9during the cell application operation. In the operation graph ofFIG.18, the ordinate represents an application needle tip position, and the abscissa represents time.

As shown inFIG.18, during the cell application operation of the application needle9, its tip9adescends at a first application velocity V1afrom an upper limit position P1aand descends at a low speed of second application velocity V2aslower that the first application velocity V1aafter having reached a predetermined velocity change position P2aimmediately before the application. The tip9aof the application needle9descends at the second application velocity V2a, and the application liquid10attached to the tip9ais subjected to contact application at a contact application position P3a. The contact application position P3amay be pre-set by calculating the position and height over the application target substrate11on the basis of data detected with, for instance, a position meter installed at a measuring device. Note that the micro-applicator1is provided with: a horizontally movable XY table4; a finely adjustable application unit6that moves in the top-to-bottom direction (vertical direction) relative to the XY table4; an observation optical unit (e.g., a CCD camera) such as an optical detection unit7configured to, for instance, visually examine an applied material (e.g., cells) over the XY table4; and a measuring device (e.g., a laser displacement meter, a white light interference meter) including a measuring instrument configured to, for instance, measure an applied material produced.

Regarding the contact application position P3a, an original starting position of application at the first cycle of the cell application operation may be detected and then pre-set. Based on the starting position, a contact application position P3aat the second time or later in the application operation may be set. That is, during a plurality of cycles of the cell application operation using the application needle9, the contact application position P3amay be gradually shifted upward every cell application operation as long as the contact application is permitted.

During the cell application operation, the contact application at the contact application position P3ais followed by return to the upper limit position P1a, which is the initial operation position, at a return velocity V3. Then, the next cell application operation is repeated. As shown inFIG.18, during one cycle of the cell application operation, the needle descends at the first application velocity V1afrom the upper limit position P1a(at time T1a) to the velocity change position P2a(at time T2a), descends at the second application velocity V2a(<V1a) from the velocity change position P2a(at time T2a) to the contact application position P3a(at time T3a), and is then subjected to contact application. After the contact application, the needle ascends toward the upper limit position P1a(at time T4a) at a prescribed return velocity V3.

Regarding the micro-applicator1in the second embodiment, the second application velocity V2aof the application needle9when reaching the contact application position P3ais lower than the first application velocity V1a. This enables the tip9aof the application needle9to be positioned, with high precision, on the pre-set contact application position P3a, thereby capable of executing finely tuned cell application operation. Note that even if the tip9aof the application needle9is brought into contact with the application target substrate surface, the resulting contact impact can be small and the cells may thus be affected little.

FIG.19is an operation chart describing multiple cycles of cell application operation for the micro-applicator1in the second embodiment. In the cell application operation shown inFIG.19, the cell contact application, in which the application liquid10is applied on the application target substrate11by using the application needle9, is repeated 10 times.

As illustrated inFIG.19, during 10 times of contact application in the cell application operation, the contact application position P3a, which is the lowest position of the application needle9, is set and gradually shifted upward every time a single contact application is completed. Regarding the cell application operation in the micro-applicator1in the second embodiment, the contact application position P3ais shifted upward by several μm every cycle.

For the micro-applicator1in the second embodiment, movements illustrated inFIG.20, as a modification embodiment of the movements shown inFIG.18, may be adopted as movements of the tip9aof the application needle9during the cell application operation.

FIG.20is an operation graph illustrating cell application operation of the tip9aof the application needle9while the application needle9is once paused for a predetermined period at the velocity change position P2ajust above the application target. As shown inFIG.20, the tip9aof the application needle9descends at the first application velocity V1aand reaches the velocity change position P2a. At that time (T2a), the tip stops for a predetermined short period (T2a→T2′a). After this predetermined period has passed, the tip descends again toward the application target at the second application velocity V2aand is then subjected to contact application. As such, the tip9aof the application needle9is stopped only for a predetermined short period at the position just above the application target, so that the volume of the application liquid10applied by the tip9acan be made equal. This makes it possible to stably apply, depending on characteristics of the application liquid, a constant application volume on the application target while the application needle9in the micro-applicator1is used to perform the cell application operation show inFIG.20.

As described above, during the cell application operation shown inFIG.20, the needle is once paused at the velocity change position P2aand descends, at the second application velocity V2aslower than the first application velocity V1abefore the pause, toward the contact application position P3aafter the predetermined period (X) has passed. The contact application at the contact application position P3ais followed by return to the upper limit position P1a, which is the initial operation position, at the return velocity V3. Then, the next cycle of cell application operation is repeated. As shown inFIG.20, during one cycle of the application operation, the needle descends at the first application velocity V1afrom the upper limit position P1a(at time T1a) to the velocity change position P2a(at time T2a), descends at the second application velocity V2a(<V1a) from the velocity change position P2a(at time T2′a) to the contact application position P3a(at time T3b) after the predetermined period (X) has passed, and is then subjected to contact application. After the contact application, the needle ascends toward the upper limit position P1a(at time T4b) at the prescribed return velocity V3.

FIG.21is an operation chart illustrating multiple cycles of cell application operation using the application needle9as shown inFIG.20. During the cell application operation using the application needle9as illustrated inFIG.21, the contact application, in which the application needle9is used to apply the application liquid10on the application target substrate11, is repeated 10 times.

During the cell application operation illustrated inFIG.21, like the cell application operation illustrated inFIG.19, the contact application position P3a, which is the lowest position of the application needle9, is set and gradually shifted upward every time a single application is completed.

[How to Determine Initial Set Position of Contact Application Position P3]

The following describes how to determine the initial set position, which is the contact application position P3aof the tip9aof the application needle9at the first cycle, during the cell application operation using the micro-applicator1in the second embodiment. During the cell application operation, the contact application position P3aof the tip9aof the application needle9is gradually shifted upward every cycle. Before the cell application operation is executed, the initial set position, which is the contact application position P3aat the first cycle during the cell application operation, is determined. Once the initial set position is determined, the contact application position P3ais set and shifted upward, based on the determined initial set position, by a pre-set given distance (e.g., several μm) every cycle.

FIG.22is a flowchart illustrating how to determine the initial set position with respect to the contact application position P3ain the micro-applicator1in the second embodiment. At step S101, an observation optical unit (e.g., a CCD camera) such as an optical detection unit7installed at the micro-applicator1is used to focus on an application point, which is on an application surface of an application target (e.g., the substrate11).

At step102, the micro-applicator1tries to carry out the cell application operation on the application point of the application target. In this trial, the application operation may be executed like actual cell application operation while a reference position based on the focused application point is set as a temporal contact application position P3a. Note that the temporal contact application position P3a, which is initially set in the trial, may be visually determined using the observation optical unit or may be determined based on the position focused using the observation optical unit as described above.

At step103, the observation optical unit is used to check whether or not a liquid droplet spot S has been formed on the application point of the application target. If formation of a liquid droplet spot S on the application point of the application target is detected, the initial set position is shifted upward to a pre-set, one-step upper position (a several-μm upper position) (step104). At step105, the cell application operation is re-tried after the liquid droplet spot S on the application point of the application target has been removed.

At step106, the observation optical unit is used to check whether or not a liquid droplet spot S is newly formed on the application point of the application target. If a liquid droplet spot S is newly formed on the application point, the process returns to step104and the initial set position is shifted upward to an additional one-step upper position. Then, the cell application operation is retried.

At step106, if any liquid droplet spot S is not formed on the application point, the process goes to step107. At step107, the initial set position is shifted downward to a pre-set one-step lower position. This lowered position is determined as and set to the initial set position, which is the contact application position P3aat the first cycle during the cell application operation (step111).

Meanwhile, if formation of any liquid droplet spot S on the application point of the application target is not detected by the observation optical unit at step103, the initial set position, which is the contact application position P3aat the first cycle during the cell application operation, is shifted downward to a pre-set, one-step lower position (e.g., a several-μm lower position) (step108). At step109, the cell application operation is retried on the application point of the application target.

At step110, if formation of any liquid droplet spot S on the application point of the application target is not detected by the observation optical unit, the process returns to step108and the initial set position is shifted downward to an additional one-step lower position. Then, the cell application operation is retried.

When formation of a liquid droplet spot on the application point is detected at step110, this position is determined as and set to the initial set position, which is the contact application position P3aat the first cycle during the cell application operation (step111).

Note that each step in the flowchart shown inFIG.22is carried out by executing control programs stored in a memory such as ROM of the display/control unit3included in the micro-applicator1.

By determining the contact application position P3aat the first cycle during the cell application operation as described above, the contact application positions P3aat the respective cycles during the cell application operation are determined. The contact application position P3aat each cycle is set by adding a pre-set prescribed distance (e.g. several μm) to the determined initial set position. The prescribed distance added at that time is set to a distance that secures the contact application.

As described above, the micro-applicator1in the second embodiment is set such that in the multiple cycles (e.g., 10 cycles) of contact application during the cell application operation, the contact application position P3a, which is the lowest position, of the tip9aof the application needle9is gradually shifted upward every time a single contact application is completed. For instance, the contact application position P3ais shifted upward by several μm every contact application.

In the method for producing a cell chip or three-dimensional tissue chip by cell application operation using the micro-applicator1according to the second embodiment, the contact application is repeated multiple times on the application target or the application liquid10on the application target while a tiny volume of application liquid10is attached to the tip9aof the application needle9. Liquid droplet spots S in an application volume of several pL (picoliter) can be applied and formed with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. In addition, in the method for producing a cell chip or three-dimensional tissue chip according to the second embodiment, it is possible to apply a material with a viscosity of the application liquid10of 1×105mPa·s or lower and preferably from 1 to 1×104mPa·s. This allows for application of highly viscous cell dispersion. During the cell application operation using the micro-applicator1in the second embodiment, a tiny volume of application liquid10attached to the tip9aof the application needle9is subjected to contact application, with high precision, on the application target or the application liquid on the application target. This makes it possible to stably and repeatedly apply the application liquid10. As such, according to the cell application operation using the micro-applicator1in the second embodiment, a highly viscous cell dispersion can be precisely applied at a predetermined position relative to the application target. This makes it possible to produce a cell chip with a given pattern or a three-dimensional tissue chip on which cells are shaped three-dimensionally. In view of the above, the method for producing a cell chip or three-dimensional tissue chip according to second embodiment of the invention exerts advantageous effects on progress in respective fields while the produced cell chip or three-dimensional tissue chip is utilized in the fields of regenerative medicine and drug discovery research such as drug efficacy or safety evaluation screening.

Third Embodiment

In the micro-applicator1in the first or second embodiment, it is configured such that the application needle9is made to penetrate through the application liquid reservoir8aof the application liquid container8and is subjected to contact application on the application target or the application liquid10on the application target. The present invention is not limited to such a configuration. For instance, it may be configured such that the application needle dipped in the application liquid of the application liquid reservoir is lifted, moved to an application position, and subjected to contact application. The micro-applicator as so configured can exert substantially the same effects as of the micro-applicator1in the above first or second embodiment. Even when a highly viscous cell-containing gelatinizer is a material, a cell chip or three-dimensional tissue chip formed using a desired application volume can be reliably produced within a short time (at a high speed).

FIG.23is diagrams schematically illustrating cell application operation of an application unit60in a micro-applicator in a third embodiment. As shown inFIG.23, the application unit60includes: an application liquid container80having an application liquid reservoir80ain which a prescribed amount of application liquid10, a cell-containing solution, is stored; and an application needle holder part130holding the application needle9having the tip9aattached to the application liquid10while the application needle9is dipped into the application liquid10in the application liquid reservoir80a. The application needle holder part130is provided with a sliding mechanism part160that slidably holds the application needle9in the top-to-bottom direction (vertical direction). The application needle holder part130is detachably provided at a given position on a driving mechanism part170, and, for instance, is detachable from the driving mechanism part170by using magnetic force of a magnet. The driving mechanism part170is configured such that the application needle9held by the mounted application needle holder part130is moved to an application position on an application target (e.g., the substrate11) and is then subjected to contact application.

The cell application operation in the application unit60, as schematically shown inFIG.23, will be described. During the cell application operation as shown in (a) and (b) ofFIG.23, the tip9aof the application needle9is dipped into the application liquid10, a cell-containing solution, and the application liquid10is attached to the tip9aof the application needle9. The tip9ahaving the application liquid10attached is moved and then comes into contact with an application target, namely the substrate11or the application liquid10on the substrate11to form a liquid droplet spot S on the substrate11(see (c) and (d) ofFIG.23). This cell application operation is repeated a predetermined number of times and the application liquid10is subjected to multiple cycles of contact application to produce a desired cell chip or three-dimensional tissue chip on the substrate11.

(a) ofFIG.23shows an attachment step during the cell application operation. In this attachment step, the tip9aof the application needle9is dipped into (held in) the application liquid10, which is a cell-containing solution stored in the application liquid reservoir80aof the application liquid container80.

(b) ofFIG.23shows a transfer step in which the tip9aof the application needle9is lifted and the tip9amoves toward the substrate11from the application liquid reservoir80a. During this transfer step, the application liquid10is attached to the tip9aof the application needle9and the surface tension of the application liquid10attached causes the tip9aof the application needle9to securely retain a certain volume of application liquid10. Note that the final stage of this transfer step includes at least an operation in which the application needle descends toward the application target.

(c) ofFIG.23shows an application step of bringing the tip9aof the application needle9into contact with the application target, namely the substrate11or the application liquid10on the substrate11to form a liquid droplet spot S on the substrate11. The liquid droplet spot S of the application liquid10contact-applied at that time corresponds to the volume of application liquid10attached to the tip9aof the application needle9.

(d) ofFIG.23shows a state immediately after the application needle9is used to apply the application liquid10on a surface of the substrate11and shows a separation step in which the application needle9is lifted from the substrate11and moves toward the application liquid reservoir80aof the application liquid container80. That is, the separation step is in a state in which after the application liquid10, a cell-containing solution, is subjected to contact application, the tip9aof the application needle9is separated from the application target. This separation step is followed by transition to the attachment step in which the tip9aof the application needle9is dipped into (held in) the application liquid10of the application liquid reservoir8a.

As described above, the one cycle of cell application operation includes, in sequence, (a), (b), (c), (d), and (a) operations as illustrated inFIG.23. In the third embodiment, the cell application operation is repeated a predetermined number of times (e.g., 10 cycles) to produce a desired cell chip or three-dimensional tissue chip. Note that a derivative form of the third embodiment may be configured such that the application unit60is only moved vertically during the steps (b) to (c), the application liquid container80and the substrate11are together moved horizontally, and the application needle is then brought into contact with the application target or the application liquid on the application target to form a liquid droplet spot S on the application target.

In the method for producing a cell chip or three-dimensional tissue chip according to the third embodiment like the above first or second embodiment, the contact application is performed on the application target or the application liquid10on the application target while a tiny volume of application liquid10is attached to the tip9aof the application needle9. A liquid droplet spot S in an application volume of several pL (picoliter) can be applied and formed with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. During the cell application operation using the micro-applicator1in the third embodiment, a tiny volume of the application liquid10attached to the tip9aof the application needle9can be contact-applied, with high precision, onto the application target or the application liquid on the application target. Thus, the application liquid10can be applied stably and repeatedly.

Fourth Embodiment

A micro-applicator in a fourth embodiment is not configured such that the application needle penetrates through the application liquid reservoir of the application liquid container, and is configured, like the configuration described in the above second or third embodiment, such that the application liquid, a cell-containing solution, is attached to the tip of the application needle. In the micro-applicator as so configured in the fourth embodiment, the tip of the application needle is subjected to contact application. Accordingly, even when a highly viscous cell-containing gelatinizer is a material, the tip of the application needle is free of clogging and a cell chip or three-dimensional tissue chip formed using a desired application volume can be reliably produced.

FIG.24is diagrams schematically illustrating cell application operation of an application unit61in the micro-applicator in the fourth embodiment. As shown inFIG.24, the application unit61includes: an application liquid container81having an application liquid reservoir81ain which a prescribed amount of application liquid10, a cell-containing solution, is stored; a nozzle82for jetting the application liquid10in the application liquid reservoir81atoward the tip9aof the application needle9; and the application needle holder part130holding the application needle9having the application liquid10attached. Specifically, the application unit61in the micro-applicator in the fourth embodiment has, as a component, an inkjet bio-printer and is configured such that a desired volume of the application liquid10, a cell-containing solution, is attached to the tip9aof the application needle9by jetting from the nozzle82.

The application needle holder part130is provided with the sliding mechanism part160that slidably holds the application needle9in the top-to-bottom direction (vertical direction). The application needle holder part130is detachably provided at a given position on the driving mechanism part170, and, for instance, is detachable from the driving mechanism part170by using magnetic force of a magnet. The driving mechanism part170is configured such that the application needle9held by the mounted application needle holder part130is moved to an application position on an application target (e.g., the substrate11) and is then subjected to contact application.

The cell application operation in the application unit61, as schematically shown inFIG.24, will be described. During the cell application operation illustrated inFIG.24, the application liquid10, a cell-containing solution, is jetted from the nozzle82and a prescribed volume of the application liquid10is then attached to the tip9aof the application needle9(see (a) and (b) ofFIG.24). The tip9aof the application needle9having the application liquid10attached is moved and then comes into contact with an application target, namely the substrate11or the application liquid10on the substrate11to form a liquid droplet spot S on the substrate11(see (c) and (d) ofFIG.24). This cell application operation is repeated a predetermined number of times and the application liquid10is subjected to multiple cycles of contact application to produce a desired cell chip or three-dimensional tissue chip on the substrate11.

(a) ofFIG.24shows an attachment step during the cell application operation. In this attachment step, the application liquid10, which is a cell-containing solution stored in the application liquid reservoir81aof the application liquid container81, is jetted from the nozzle82and then attached to the tip9aof the application needle9.

(b) ofFIG.24shows a transfer step in which the tip9aof the application needle9is moved toward the application target (e.g., the substrate11). During this transfer step, the application liquid10is attached to the tip9aof the application needle9and the surface tension of the application liquid10attached causes the tip9aof the application needle9to securely retain a certain volume of application liquid10.

(c) ofFIG.24shows an application step of bringing the tip9aof the application needle9into contact with the application target (substrate11) or the application liquid10on the application target to form a new liquid droplet spot S on the application target. The liquid droplet spot S of the application liquid10contact-applied at that time corresponds to the volume of application liquid10attached to the tip9aof the application needle9.

(d) ofFIG.24shows a state immediately after the application needle9is used to apply the application liquid10on a surface of the application target (substrate11) and shows a separation step in which the application needle9is lifted from the application target and moves toward a nozzle82-mediated jetting area. That is, the separation step is in a state in which after the application liquid10, a cell-containing solution, is subjected to contact application, the tip9aof the application needle9is separated from the application target. This separation step is followed by transition to the attachment step in which the application liquid10is jetted from the nozzle82toward the tip9aof the application needle9and the application liquid10is then attached to the tip9aof the application needle9.

As described above, the one cycle of cell application operation includes, in sequence, (a), (b), (c), (d), and (a) operations illustrated inFIG.24. In the fourth embodiment, the cell application operation is repeated a predetermined number of times (e.g., 10 cycles) to produce a desired cell chip or three-dimensional tissue chip.

In the method for producing a cell chip or three-dimensional tissue chip according to the fourth embodiment as described in the above first to third embodiments, the contact application is performed on the application target or the application liquid10on the application target while a tiny volume of application liquid10is attached to the tip9aof the application needle9. A liquid droplet spot S in an application volume of several pL (picoliter) can be applied and formed with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. During the cell application operation using the micro-applicator in the fourth embodiment, a tiny volume of the application liquid10attached to the tip9aof the application needle9can be contact-applied, with high precision, onto the application target or the application liquid on the application target. Thus, the application liquid10can be applied stably and repeatedly.

Fifth Embodiment

A micro-applicator in a fifth embodiment is not configured such that the application needle penetrates through the application liquid reservoir of the application liquid container, and is configured, like the configuration described in the above second to fourth embodiments, such that the application liquid, a cell-containing solution, is attached to the tip of the application needle. In the micro-applicator as so configured in the fifth embodiment, the tip of the application needle is subjected to contact application. Accordingly, even when a highly viscous cell-containing gelatinizer is a material, the tip of the application needle is free of clogging and a cell chip or three-dimensional tissue chip formed using a desired application volume can be reliably produced.

FIG.25is diagrams schematically illustrating cell application operation of an application unit62in the micro-applicator in the fifth embodiment. As shown inFIG.25, the application unit62includes: an application liquid container83having an application liquid reservoir83ain which a prescribed amount of application liquid10, a cell-containing solution, is stored; a nozzle84for attaching the application liquid10in the application liquid reservoir83ato the tip9aof the application needle9; and the application needle holder part130holding the application needle9having the application liquid10attached. Specifically, the application unit62in the micro-applicator in the fifth embodiment has, as a component, a dispenser-type bio-printer and is configured such that a desired volume of the application liquid10, a cell-containing solution, is attached to the tip9aof the application needle9by discharging a prescribed volume of the application liquid10from the nozzle84.

The application needle holder part130is provided with the sliding mechanism part160that slidably holds the application needle9in the top-to-bottom direction (vertical direction). The application needle holder part130is detachably provided at a given position on the driving mechanism part170, and, for instance, is detachable from the driving mechanism part170by using magnetic force of a magnet. The driving mechanism part170is configured such that the application needle9held by the mounted application needle holder part130is moved to an application position on an application target (e.g., the substrate11) and is then subjected to contact application.

The cell application operation in the application unit62, as schematically shown inFIG.25, will be described. During the cell application operation illustrated inFIG.25, the application liquid10, a cell-containing solution, is discharged from the nozzle84and a prescribed volume of the application liquid10is then attached to the tip9aof the application needle9(see (a) and (b) ofFIG.25). The tip9aof the application needle9having the application liquid10attached is moved and then comes into contact with an application target, namely the substrate11or the application liquid10on the substrate11to form a liquid droplet spot S on the substrate11(see (c) and (d) ofFIG.25). This cell application operation is repeated a predetermined number of times and the application liquid10is subjected to multiple cycles of contact application to produce a desired cell chip or three-dimensional tissue chip on the substrate11.

(a) ofFIG.25shows an attachment step during the cell application operation. In this attachment step, a prescribed volume of the application liquid10, which is a cell-containing solution stored in the application liquid reservoir83aof the application liquid container83, is discharged from the nozzle84and then attached to the tip9aof the application needle9.

(b) ofFIG.25shows a transfer step in which the tip9aof the application needle9is moved toward the application target (e.g., the substrate11). During this transfer step, the application liquid10is attached to the tip9aof the application needle9and the surface tension of the application liquid10attached causes the tip9aof the application needle9to securely retain a certain volume of application liquid10.

(c) ofFIG.25shows an application step of bringing the tip9aof the application needle9into contact with the application target or the application liquid10on the application target to form a new liquid droplet spot S on the application target. The liquid droplet spot S of the application liquid10contact-applied at that time corresponds to the volume of application liquid10attached to the tip9aof the application needle9.

(d) ofFIG.25shows a state immediately after the application needle9is used to apply the application liquid10on a surface of the application target and shows a separation step in which the application needle9is lifted from the application target and moves toward a nozzle84-mediated discharging area. That is, the separation step is in a state in which after the application liquid10, a cell-containing solution, is subjected to contact application, the tip9aof the application needle9is separated from the application target. This separation step is followed by transition to the attachment step in which the application liquid10is discharged from the nozzle84toward the tip9aof the application needle9and the application liquid10is then attached to the tip9aof the application needle9.

As described above, the one cycle of cell application operation includes, in sequence, (a), (b), (c), (d), and (a) operations illustrated inFIG.25. In the fifth embodiment, the cell application operation is repeated a predetermined number of times (e.g., 10 cycles) to produce a desired cell chip or three-dimensional tissue chip.

In the method for producing a cell chip or three-dimensional tissue chip according to the fifth embodiment, as described in the above first to fourth embodiments, the contact application is performed on the application target or the application liquid10on the application target while a tiny volume of application liquid10is attached to the tip9aof the application needle9. A liquid droplet spot S in an application volume of several pL (picoliter) can be applied and formed with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. During the cell application operation using the micro-applicator in the fifth embodiment, a tiny volume of the application liquid10attached to the tip9aof the application needle9can be contact-applied, with high precision, onto the application target or the application liquid on the application target. Thus, the application liquid10can be applied stably and repeatedly.

Examples of the cells that can be used in the invention include: but are not particularly limited to, various primary cells such as fibroblasts, vascular endothelial cells, epithelial cells, smooth muscle cells, cardiomyocytes, gastrointestinal cells, neurons, hepatocytes, renal cells, and/or pancreatic cells; iPS cell-derived differentiated cells; and various cancer cells. As the cells, it is possible to use: cells coated with, for instance, a protein, a sugar chain, nucleic acid, a natural polymer, and/or a synthetic polymer; or cells coated by a coating process(es) or with a known coating agent(s) such as fibrinogen, gelatin, collagen, laminin, elastin, vitronectin, fibrinogen, dextran sulfate, heparan sulfate, polyamino acid, and/or a peptide(s).

Note that to give included cells a stable adhesion/proliferation environment, the cell-containing solution may include: an extracellular matrix component(s) such as fibronectin, gelatin, collagen, laminin, elastin, and/or Matrigel; a cell growth factor(s) such as fibroblast growth factor and/or platelet-derived growth factor; or an additional additive agent(s) such as vascular endothelial cells, lymphatic endothelial cells, and/or various stem cells. In addition, as the gelatinizer, it is possible to include a protein, a sugar chain, a natural polymer, a synthetic polymer, and/or a peptide such as fibrinogen, alginic acid, polyamino acid, polyethylene glycol, and/or a thermally responsive polymer.

As described using the above embodiments and respective experimental examples, the invention provides a novel method for producing a cell chip or three-dimensional tissue chip. Compared to the case of production using a conventional printer with a nozzle, the invention is configured to use the application needle to apply a solution attached to its tip surface. Thus, the solution is not clogged and the resolution and the formation rate of cell assembly is improved, so that a less sample volume (sample) can be used to definitely produce a highly reliable cell chip or three-dimensional tissue chip. In addition, compared to the case of using conventional printers, the invention enables a cell chip or three-dimensional tissue chip to be produced by applying a highly viscous cell dispersion onto a target. This can suppress evaporation of the cell dispersion after the application, thereby capable of maintaining high cell viability.

Regarding the micro-applicators in the invention, the needle (application needle) having a tiny volume of the application liquid attached to the tip is brought into contact with a target (e.g., a substrate); and a liquid droplet in an application volume of several pL (picoliter) can be applied with high positional precision, such as positional precision of ±15 μm or less and preferably ±3 μm or less. In addition, it is possible to apply a material such as a material with a viscosity of the application liquid of 1×105mPa·s or lower and preferably from 1 to 1×104mPa·s. This allows for application of highly viscous cell dispersion. As such, according to the invention, a highly viscous cell dispersion can be precisely applied at a predetermined position relative to a target (e.g., a substrate). This makes it possible to produce a cell chip with a given pattern or a three-dimensional tissue chip on which cells are shaped three-dimensionally. As a result, the produced cell chip or three-dimensional tissue chip can be utilized in the fields of regenerative medicine and drug discovery research such as drug efficacy or safety evaluation screening.

The invention has been described in the embodiments in detail to some extent. However, these configurations are examples and the content disclosed in the embodiments may be modified with respect to specifics of the configurations. The elements in the embodiments of the invention may be replaced by other elements and the combinations and the order thereof may be changed, which can be realized without departing from the scope and the spirit of the invention claimed.

INDUSTRIAL APPLICABILITY

According to the present cell chip, three-dimensional tissue chip, and production method therefor, various highly reliable cell chips and three-dimensional tissue chips can be produced in a large quantity. The invention is therefore critical technology in research on drug discovery and regenerative medicine and is highly industrially applicable.

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