Method for manufacturing substrates on which nerve cells are arranged

Abstract

To provide technologies for precisely arranging nerve cells on a substrate while suppressing the migration of nerve cells.SOLUTION: Provided is a method of manufacturing a substrate on which nerve cells are arranged, comprising: arranging a plurality of droplets containing nerve cells on a substrate having a region in which a cell-adhesive material is placed and a region in which a non-cell-adhesive material is placed, by an inkjet method to form one or more liquid pools; and allowing it to stand still until the nerve cells in the liquid pools settle and temporarily adhere to the substrate to form cell aggregates, the diameter of each of the liquid pools being 500 μm or less, and the density of nerve cells per the liquid pool being 105 cells / cm2 or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a substrate on which nerve cells are arranged. [Background technology]

[0002] Neurons form networks in the body and function in a functionally connected state. In elucidating brain function, evaluating toxicity in nervous system diseases, and developing new drugs, it is considered important to reproduce the activity of neurons in the body as accurately as possible in vitro in order to increase the extrapolation of test results (correlation with clinical data).

[0003] To achieve this, it would be effective to develop a neural circuit model in which arbitrary neurons are placed in predetermined positions and functionally connected to each other by extending axons. However, it is difficult to precisely place neurons on a substrate by hand. Summary of the Invention [Problem to be solved by the invention]

[0004] Patent Document 1 (JP 2019-162097 A) attempts cell placement using a pattern of cell-adhesive and non-cell-adhesive materials. However, the inventors found that when cells are placed using a pattern of cell-adhesive and non-cell-adhesive materials, neurons, in particular, tend to migrate at the cell adhesion sites, making it difficult to keep them in a desired position. Therefore, the present invention aims to provide a technology for precisely placing neurons on a substrate while suppressing neuronal migration. [Means for solving the problem]

[0005] The method of the present invention for producing a substrate on which neurons are arranged includes the steps of: arranging a plurality of droplets containing neurons by an inkjet method on a substrate having an area where a cell adhesive material is arranged and an area where a cell non-adhesive material is arranged, thereby forming one or more liquid pools; and leaving the substrate to stand until the neurons in the liquid pools settle and temporarily adhere to the substrate to form a cell aggregate, wherein the diameter of each liquid pool is 500 μm or less, and the density of neurons per liquid pool is 10 5 pieces / cm 2 That's all. [Effects of the Invention]

[0006] According to the present invention, it is possible to provide a technique for precisely arranging neurons on a substrate while suppressing migration of the neurons. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram showing an example of an inkjet head. [Figure 2] FIG. 2 is a schematic diagram showing an example of a waveform input to an inkjet head. [Figure 3] FIG. 3 is a schematic diagram showing an example of a waveform input to the inkjet head. [Figure 4] FIG. 4 is a schematic diagram showing an example of a droplet placement device. [Figure 5] FIG. 5 is a schematic diagram showing an example of a droplet placement device. [Figure 6] FIG. 6 is a schematic diagram showing an example of a droplet placement device. [Figure 7] FIG. 7 is a schematic diagram showing an example of a droplet placement device. [Figure 8] FIG. 8 is a flow chart showing an example of a method for manufacturing a neural circuit model. [Figure 9] FIG. 9 is a schematic cross-sectional view illustrating a method for manufacturing a neural circuit model. [Figure 10] 10(a) to 10(c) are micrographs taken in Experimental Example 1. FIG. [Figure 11]FIG. 11 is a graph showing the results of Experimental Example 2. [Figure 12] FIG. 12 is a schematic diagram illustrating a procedure for arranging a pattern of a non-cell-adhesive material and a pattern of a cell-adhesive material on a substrate and then arranging cells. [Figure 13] 13(a) to 13(d) are micrographs taken in Experimental Example 3. FIG. [Figure 14] FIG. 14 is a fluorescence microscope photograph taken in Experimental Example 4. [Figure 15] 15(a) to 15(e) are schematic diagrams illustrating the procedure of Experimental Example 5. FIG. [Figure 16] 16(a) to 16(d) are schematic diagrams showing the arrangement of cells in Experimental Example 6. FIG. [Figure 17] Fig. 17(a) is a schematic diagram showing the arrangement pattern of the non-cell-adhesive material and neurons in the neural circuit model prepared in Experimental Example 7. Fig. 17(b) is a representative micrograph of the neural circuit model immediately after ejection of the cell ink in Experimental Example 7. [Figure 18] FIG. 18 is a fluorescent micrograph of the neural circuit model prepared in Experimental Example 7. [Figure 19] 19(a) and (b) are diagrams showing the pattern of the non-cell-adhesive material placed on the substrate in Experimental Example 8. FIG. [Figure 20] 20(a) and (b) are fluorescence microscope photographs taken in Experimental Example 8. [Figure 21] 21(a) and (b) are micrographs taken in Experimental Example 9. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and redundant explanations will be omitted. The dimensional ratios in the drawings may be exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0009] [Method for manufacturing substrates with neurons arranged on them] In one embodiment, the present invention provides a method for producing a cell aggregate comprising the steps of: disposing, by inkjet printing, a plurality of droplets containing neurons on a substrate having an area where a cell adhesive material is disposed and an area where a non-cell adhesive material is disposed, to form one or more liquid pools; and allowing the neurons in the liquid pools to settle and temporarily adhere to the substrate to form a cell aggregate. 5 pieces / cm 2 As described above, a method for producing a substrate on which nerve cells are arranged is provided.

[0010] As will be described later in the Examples, the inventors have demonstrated that the diameter of each liquid pool arranged on the substrate is 500 μm or less, and the density of neurons in the liquid pool is 10 5 pieces / cm 2 The researchers found that this suppressed neuronal migration and enabled precise placement of neurons on a substrate. Furthermore, by using an inkjet method to eject droplets containing cells onto a substrate, they were able to stably place a few cells at a time in a microscopic area on the order of micrometers.

[0011] The diameter of each reservoir may be 400 μm or less, 300 μm or less, or 200 μm or less, which is preferable because a smaller diameter reservoir makes it easier to reduce the amount of expensive cells used.

[0012] Here, droplets refer to droplets ejected from an inkjet head by the inkjet method. A puddle refers to a droplet formed when multiple droplets ejected from an inkjet head land on a substrate. The diameter of one puddle refers to the diameter of the area where one puddle contacts the substrate. If the area where the puddle contacts the substrate is not circular, the diameter refers to a circle with the same area as the area where the puddle contacts the substrate. The density of neurons per puddle refers to the area where one puddle contacts the substrate. The area where the cell adhesive material is placed This refers to the number of cells per area in contact with the

[0013] The droplets preferably contain 1 to 50 neurons per droplet. Furthermore, the number of neurons contained in each liquid pool is preferably approximately 7 to 10,000. The number of neurons contained in each liquid pool may be 7 or more, 30 or more, or 70 or more. Furthermore, the number of neurons contained in each liquid pool may be 100 or less, 70 or less, or 30 or less. These upper and lower limits can be combined in any manner.

[0014] The substrate is not particularly limited as long as it can be used for cell culture, and examples of the substrate material include the organic and inorganic materials described below. These may be used alone or in combination of two or more.

[0015] The organic material is not particularly limited and can be appropriately selected depending on the purpose. Examples include acrylic materials such as polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, and urethane acrylate; silicone materials such as cellulose and polydimethylsiloxane (PDMS); polyvinyl alcohol (PVA); metal alginates such as calcium alginate; polyacrylamide; methylcellulose; and gel-like materials such as agarose.

[0016] The inorganic material is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include glass and ceramics.

[0017] The structure of the substrate is not particularly limited as long as it can be used for cell culture, and examples thereof include a porous structure and a non-porous structure. The substrate may have a porous structure, or may be a non-porous flat plate member on which a porous member is laminated.

[0018] There are no limitations on the size or shape of the pores in the porous structure, and the porous structure may be, for example, a mesh structure, a concave-convex structure, a honeycomb structure, etc. A porous structure is preferred as a substrate structure because it increases the surface area on which the non-cell-adhesive material or the cell-adhesive material can be fixed, and it can hold a large amount of solution, preventing drying.

[0019] If a substrate having a porous structure is used as the substrate and a liquid such as a culture medium is held in the substrate beforehand, and then a liquid pool is formed, the liquid is held in place by the porous structure, thereby preventing the liquid pool from drying out.

[0020] Furthermore, when droplets are ejected onto a dried substrate to form a liquid pool, a process for suppressing evaporation of the liquid in the liquid pool (drying suppression process) is carried out, which makes it possible to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate.

[0021] Examples of the drying suppression process include (i) a process of increasing the humidity around the liquid pool, (ii) a process of forming a liquid pool after placing a fluid that suppresses evaporation of the liquid on the substrate (here, examples of the fluid that suppresses evaporation of the liquid include oil, culture medium, buffer solution, etc.), and (iii) a process of forming a liquid pool on the substrate and then covering the liquid pool with a fluid that suppresses evaporation of the liquid (e.g., oil, etc.).

[0022] When suppressing evaporation by increasing humidity, it is preferable to control humidity locally to minimize the impact on the surrounding area. Furthermore, when using oil to suppress drying, it is preferable to use a biocompatible oil to minimize the impact on cells.

[0023] The manufacturing method of this embodiment may further include a step of supplying a culture medium to the substrate on which the cell aggregates have been formed. The culture medium can be appropriately selected and used from those suitable for the cells to be used. Specific media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, D-MEM / F12, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Minimum Essential Medium, RPMI1640 (Roswell Park Memorial Institute-1640) medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, IPL41 medium, Fischer's medium, M199 medium, High Performance Medium 199, StemPro34 (Thermo Fisher Scientific), and X-VIVO 10 (Chembrex), X-VIVO 15 (Chembrex), HPGM (Chembrex), StemSpan H3000 (Stem Cell Technologies), StemSpanSFEM (Stem Cell Technologies), Stemline II (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Thermo Fisher Scientific), Essential8 (registered trademark) medium (Thermo Fisher Scientific), mTeSR1 or mTeSR2 medium (Stem Cell Technologies), ReproFF or ReproFF2 (Repro Cell), PSGro hESC / iPSC medium (SystemExamples of suitable medium include Thermo Fisher Scientific), NutriStem (registered trademark) medium (Biological Industries), CSTI-7 medium (Cell Science Institute), MesenPRO RS medium (Thermo Fisher Scientific), MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Sf-900II (Thermo Fisher Scientific), and Opti-Pro (Thermo Fisher Scientific).

[0024] These may be used alone or in combination of two or more. In particular, in the case of a DMEM / F12 medium, it is preferable to mix the DMEM medium and the F12 medium in a mass ratio ranging from 6:4 to 4:6.

[0025] Additives may also be added to the medium. Examples of additives include those commonly used in neuronal culture, such as SM1 Supplement (Stem Cell Technologies), N2 Supplement A (Stem Cell Technologies), rat astrocyte culture supernatant (Fujifilm Wako Pure Chemical Industries, Ltd.), human astrocyte culture supernatant (Sciencell Research), Component N (Elixirgen Scientific), Component G2 (Elixirgen Scientific), Component P (Elixirgen Scientific), N2 Supplement (Thermo Fisher Scientific), iCell Neural Supplement B (CDI), iCell Neuvous System Supplement, B-27 plus (Thermo Fisher Scientific), etc.

[0026] In addition, the manufacturing method of this embodiment may further include a step of forming multiple liquid pools in the liquid pool forming step, forming multiple cell aggregates in the standing step, and incubating the substrate supplied with culture medium to functionally bond at least two of the cell aggregates.

[0027] When multiple liquid pools are formed on the substrate, cell aggregates are formed in each liquid pool. When these cell aggregates are then supplied with culture medium and incubated, at least two of these cell aggregates become functionally connected, allowing the production of a neural circuit model. Here, functional connection between cell aggregates refers to the extension of a process called an axon by a neuron, forming a synaptic connection with the dendrites of another neuron. This results in the formation of a neural circuit.

[0028] Neurons can be broadly divided into, for example, peripheral nerves and central nerves. Peripheral nerves include, for example, sensory nerves, motor nerves, and autonomic nerve cells. Central nerves include, for example, interneurons and projection neurons. Projection neurons include, for example, cortical neurons, hippocampal neurons, and amygdala neurons. Central nerve cells can also be broadly divided into excitatory neurons and inhibitory neurons. Examples include glutamatergic neurons, which are primarily responsible for excitatory transmission in the central nervous system, and GABAergic (γ-aminobutyric acid) neurons, which are primarily responsible for inhibitory transmission.

[0029] Other neurons that release neuromodulators include cholinergic neurons, dopaminergic neurons, noradrenergic neurons, serotonergic neurons, and histaminergic neurons.

[0030] In the manufacturing method of this embodiment, a cell aggregate containing cells other than neurons may be further arranged on the substrate on which neurons are arranged. The cell aggregate other than the cell aggregate containing neurons may contain cells capable of receiving a transmission signal from neurons. Examples of cells capable of receiving a transmission signal from neurons include neurons and muscle cells. Examples of muscle cells include cardiac muscle cells, skeletal muscle cells, smooth muscle cells, etc. One type of these cells may be used alone, or two or more types may be used in combination.

[0031] In the production method of this embodiment, the nerve cells may be primary cultured cells, passaged cells, established cell lines, immortalized cells, or nerve cells that have been subjected to various gene editing processes. Furthermore, from the viewpoint of facilitating the production of a cell population containing a large number of desired nerve cells, nerve cells may be those induced to differentiate from stem cells.

[0032] Examples of stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells, mesenchymal stem cells, umbilical cord blood-derived stem cells, neural stem cells, etc. Examples of induced pluripotent stem cells include nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), etc. Examples of mesenchymal stem cells include bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, etc. Among these, iPS cells are preferred as stem cells.

[0033] iPS cells may be derived from healthy individuals or from patients with various nervous system disorders. They may also be gene-edited, for example, cells that have been gene-edited to contain genes that are causative or risk factors for various nervous system disorders.

[0034] iPS cells derived from patients with various nervous system disorders can be used to construct models of those disorders. Examples of nervous system disorders include, but are not limited to, neurodegenerative disorders, autism, epilepsy, attention-deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder. Examples of neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

[0035] The animal species from which the nerve cells are derived is not particularly limited, and examples include humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, etc. Among these, humans are preferred.

[0036] In the manufacturing method of this embodiment, the nerve cells may be those collected from a living body, may be established and cultured, or may be those induced to differentiate from stem cells.

[0037] When producing a neural circuit model using multiple types of neurons, it is preferable that one cell assembly contains one type of neuron, and each of the multiple cell assemblies may contain different types of neuron depending on the purpose.

[0038] The following describes the process of placing droplets containing neurons on a substrate to form one or more pools. This process is carried out by ejecting droplets of a cell suspension (cell ink) containing at least neurons and a cell drying inhibitor using an inkjet system.

[0039] Examples of droplet ejection means using the inkjet method include the so-called piezo method (see, for example, Japanese Patent Publication No. 2-51734), which uses a piezoelectric element as a pressure generating means to pressurize the cell ink and change the volume of the cell suspension to eject droplets; the so-called thermal method (see, for example, Japanese Patent Publication No. 61-59911), which uses a heating resistor to heat the cell ink and generate bubbles; and the electrostatic method (see, for example, Japanese Patent Publication No. 6-71882), which arranges a vibrating plate and an electrode opposite each other and deforms the vibrating plate using electrostatic force generated between the vibrating plate and the electrode to change the volume of the cell ink and eject droplets.

[0040] (inkjet head) Specific embodiments of an inkjet head used to eject droplets of cell ink containing nerve cells and a cell drying inhibitor will be described below. Fig. 1 is a schematic diagram showing an example of an inkjet head. In Fig. 1, a piezoelectric element is used as a pressure generating means. Figs. 2 and 3 are schematic diagrams showing examples of input waveforms to the inkjet head.

[0041] The droplet ejection head 10 has a liquid chamber 12 that holds cell ink 11, a nozzle 15, a membrane 13 that is a film-like member, a vibration unit 16 that imparts vibrations to the membrane 13, and a drive unit 14 that imparts voltage to the vibration unit 16 as a specific drive signal to vibrate the vibration unit 16.

[0042] The liquid chamber 12 is provided with an air vent 17 for opening the liquid chamber to the atmosphere. The droplet ejection head 10 applies vibrations to the cell-based ink, thereby ejecting droplets of the cell-based ink from the nozzles.

[0043] The driver 14 can apply an ejection waveform Pj to the vibration unit 16 as a drive signal, thereby controlling the vibration state of the membrane 13 and ejecting the cell-cell ink 11 held in the liquid chamber 12 in the form of droplets. The ejection waveform Pj can be set to a drive signal that includes the membrane 13's natural vibration period To in order to resonate the membrane 13 and eject the cell-cell ink 11 with less voltage. The ejection waveform Pj can be not only a triangular wave or a sine wave, but also a triangular wave with gentler edges obtained by applying a low-pass filter. Furthermore, the driver 14 can apply a vibration-isolating waveform Ps to the vibration unit 16 as a drive signal to suppress residual membrane vibration after droplet ejection. This quickly suppresses residual membrane vibration after droplet formation, enabling higher-frequency continuous ejection. Furthermore, the reduction in satellites and mist allows for more precise control of droplet volume. The vibration isolation waveform Ps can be not only a triangular wave or a sine wave, but also a triangular wave whose edges have been softened by passing it through a low-pass filter.

[0044] There is no particular limit to the amount of cell ink 11 that can be held in the liquid chamber 12, and it is possible to hold, for example, about 1 μL to 1 mL of liquid. In particular, when using an expensive liquid such as cell ink with dispersed cells, it is preferable that droplets can be formed with a small amount of liquid, and a configuration that can hold about 1 μL to 50 μL of liquid is desirable.

[0045] The shape of the membrane 13 may be circular, elliptical, or rectangular. The material of the membrane 13 is not particularly limited, but if the membrane is too soft, it will vibrate easily, making it difficult to immediately stop the vibration when droplets are not being ejected. Therefore, it is preferable that the material has a certain degree of hardness. Examples of materials that can be used for the membrane 13 include metal materials, ceramic materials, and polymer materials with a certain degree of hardness.

[0046] The nozzle 15 is preferably formed as a substantially circular through-hole in the center of the membrane 13. The vibrator 16 may be, for example, a piezoelectric element. By applying a voltage, compressive stress is applied laterally to the plane of the page, causing the membrane 13 to deform. The piezoelectric element may be made of, for example, the commonly used lead zirconate titanate. Other piezoelectric materials that may be used include bismuth iron oxide, metal niobate, barium titanate, or any of these materials to which a metal or a different oxide has been added.

[0047] The means for applying vibrations to membrane 13 to deform it is not limited to a piezoelectric element, and it is also possible, for example, to attach a material with a different linear expansion coefficient from that of the membrane 13 to the membrane 13, and heat it to utilize the difference in linear expansion coefficient to deform membrane 13. In this case, for example, a heater is formed on the material with the different linear expansion coefficient, and the heater heats up when current is applied, allowing membrane 13 to deform.

[0048] (Cell Ink) Next, we will explain the cell ink. The cell ink contains at least neurons and a cell drying inhibitor. Furthermore, the cell suspension (cell ink) contains a dispersion medium to disperse the cells, and may contain other additives such as dispersants and pH adjusters as necessary. The neurons are the same as those described above.

[0049] The cell drying inhibitor covers the surface of cells and has the function of inhibiting the cells from drying out, and examples thereof include polyhydric alcohols, gel-like polysaccharides, and proteins selected from extracellular matrices.

[0050] The polyhydric alcohol is not particularly limited as long as it does not damage cells, and examples thereof include glycerin, diglycerin, diethylene glycol, 1,3-butanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, triethylene glycol, tetraethylene glycol, propylene glycol, and polyethylene glycol. These may be used alone or in combination of two or more. Among these, glycerin is preferred. Glycerin has low toxicity to cells and can be expected to have the effect of suppressing drying even when added in small amounts.

[0051] Gel-like polysaccharides refer to polysaccharides in a gel state. Gel-like polysaccharides are not particularly limited and can be selected appropriately depending on the purpose. Examples include calcium alginate, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, diutan gum, and carboxymethylcellulose. These may be used alone or in combination. Among these, calcium alginate is preferred. Calcium alginate is a salt in which calcium ions are bound to the carboxyl groups of alginic acid. Because the calcium ions are divalent, they bind across the two carboxyl groups (ionic cross-linking), thickening the ink and preventing it from drying. The calcium ions contained in the dispersion medium are thought to bind to excess calcium ions that become concentrated during drying, potentially regulating osmotic pressure.

[0052] The protein selected from the extracellular matrix is not particularly limited and can be appropriately selected depending on the purpose, and examples include collagen, laminin, fibronectin, elastin, fibrin, etc. These may be used alone or in combination of two or more. Among these, collagen is preferred. There are many types of collagen, but it is known to thicken depending on the concentration and temperature. By including it in the cell ink, the viscosity can be increased as the concentration increases.

[0053] As the dispersion medium, a cell culture medium or a buffer solution is preferable. The medium is the same as that described above. The buffer solution is used to adjust the pH, and known buffer solutions can be appropriately selected and used.

[0054] Because the liquid pools placed on the substrate are very small, evaporation of the liquid in the pools occurs within a few tens of seconds to a few minutes, causing a rapid rise in the concentration of components in the pools. This rapid change in concentration, such as osmotic pressure, can damage the cells in the pools and, in the worst case, can lead to cell death. Therefore, it is preferable to suppress evaporation of the liquid in the pools using the following methods.

[0055] For example, if a substrate having a porous structure is used as the substrate and a liquid such as a culture medium is held in the substrate beforehand, the liquid is held in the substrate by the porous structure, and the drying of the liquid pool can be prevented.

[0056] Furthermore, when droplets are ejected onto a dried substrate to form a liquid pool, a step of suppressing evaporation of the liquid in the liquid pool (a drying suppression step) can be carried out to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate. Examples of the drying suppression step include the steps similar to those described above.

[0057] (Step of placing droplets) Next, we will explain the process of placing one or more droplets containing cells on a substrate to form one or more puddles. In the droplet placement process, droplets of cell ink are ejected at target positions on the substrate. Droplets containing nerve cells may be placed at one location on the substrate, or at multiple locations on the substrate. Multiple droplets of cell ink ejected from an inkjet head are placed at one location on the substrate, forming one puddle.

[0058] By adjusting the timing of droplet ejection, it is possible to adjust the positions of cells adhering to the area where the cell adhesive material is arranged (cell adhesion area).Furthermore, by adjusting the amount of cell ink ejected (number of droplets and droplet volume) and cell concentration, it is possible to adjust the number of cells arranged on the substrate.

[0059] The cells in the liquid pool placed on the substrate settle and temporarily adhere to the substrate, forming a cell aggregate. With the inkjet method, the droplets of cell ink are much smaller than those obtained by manual techniques, so the time until the cells temporarily adhere to the substrate is very short. Furthermore, by placing droplets using the inkjet method, neurons are placed at predetermined positions, resulting in high accuracy in the shape of the cell pattern. There is a high probability that a predetermined number of neurons can be placed at a predetermined position, or that the number of placed cells is the desired number, and the accuracy in placing the desired number of neurons is high. Furthermore, the survival rate of the placed neurons after a predetermined time has passed is also high.

[0060] (droplet placement device) Specific embodiments of an apparatus for disposing droplets on a substrate (hereinafter, sometimes referred to as a "droplet disposing apparatus") will be described below, but the present invention is not limited to these embodiments.

[0061] The droplet placement device has a stage unit and an inkjet head, which is a droplet ejection means for ejecting droplets of cell-based ink. The stage unit holds the substrate. The droplet ejection head has the same configuration as the inkjet head described above.

[0062] 4 to 7 show an example of a droplet placement device equipped with an inkjet head. The droplet placement device 400 shown in Fig. 4 has a stage unit 31 and an inkjet head 21. As described above, the inkjet head 21 has a liquid chamber unit 25, a vibration unit 27, a drive unit 26, and a membrane 28. The inkjet head 21 also has an atmosphere opening unit 24 formed therein.

[0063] The droplet placement device may be configured to eject droplets of not only cell ink but also a solution containing a non-cell-adhesive material or a cell-adhesive material. The droplet placement device 500 shown in FIG. 5 includes an inkjet head 21 that ejects droplets of cell ink, as well as an inkjet head 23 that ejects droplets of a solution containing a non-cell-adhesive material or a cell-adhesive material. The basic configuration of the inkjet head 23 is the same as that of the inkjet head 21. In FIG. 5, reference numeral 29 denotes a drive unit, and reference numeral 30 denotes a liquid chamber that holds the solution of the non-cell-adhesive material or the cell-adhesive material. Like the inkjet head 21, the inkjet head 23 has a vibration unit and a membrane (not shown).

[0064] The droplet placement device can also place droplets containing two or more types of cells. The droplet placement device 600 shown in FIG. 6 can be equipped with multiple inkjet heads 21 that eject droplets of cell ink. FIG. 6 shows an example equipped with inkjet head 21 and inkjet head 22 having the same configuration as inkjet head 21. FIG. 7 shows a droplet placement device 700 equipped with inkjet head 23 in addition to inkjet heads 21 and 22. In addition to the configuration described above, the droplet placement device may also be equipped with a holding unit that holds the inkjet heads, a mechanism that controls the relative position of the stage and inkjet heads, etc.

[0065] The substrate on which neurons are arranged has an area where a cell adhesive material is arranged and an area where a non-cell adhesive material is arranged. In this specification, the area where the cell adhesive material is arranged may be referred to as a cell adhesive portion. Also, the area where the non-cell adhesive material is arranged may be referred to as a non-cell adhesive portion. As will be described later in the examples, by arranging a pattern of a non-cell adhesive material on the surface, it becomes possible to more flexibly control axonal outgrowth when neurons are arranged.

[0066] By disposing a non-cell-adhesive material on the substrate, the growth direction of cells can be regulated. Furthermore, by disposing a non-cell-adhesive material on the substrate, multiple types of cells can be disposed without intermixing with each other. Here, when disposing multiple types of cells by the inkjet method, it is preferable to prepare one inkjet head that ejects droplets of cell ink for each type of cell in order to avoid contamination between the cells.

[0067] The pattern of the non-cell-adhesive material may have regions where the non-cell-adhesive material is arranged and regions where the non-cell-adhesive material is not arranged, and the regions where the non-cell-adhesive material is not arranged may have a linear shape (line shape). In this case, it is preferable that the width of the linear shape is 100 μm or less.

[0068] When the width of the linear shape is 100 μm or less, axons can extend along the linear shape where the non-cell-adhesive material is not arranged, i.e., the area sandwiched between the two linear patterns of the non-cell-adhesive material can be used as a path for axon extension.

[0069] The substrate on which the cells are arranged not only has a pattern of a non-cell-adhesive material arranged on its surface, but also has a pattern of a cell-adhesive material arranged on its surface. The cell-ink droplets may be arranged in contact with the cell-adhesive material. When the cell-ink droplets are arranged in contact with the cell-adhesive material and the area of the cell-adhesive material pattern is smaller than the area of the cell-ink puddle arranged on the substrate in contact with the substrate, the cells contained in the droplets tend to settle and temporarily adhere to the substrate, and then migrate and aggregate on the cell-adhesive material pattern. Therefore, by arranging a pattern of a cell-adhesive material on the substrate, the arrangement of cells can be controlled.

[0070] (Cell non-adhesive material) Examples of non-cell adhesive materials include polydimethylsiloxane (PDMS), gels of metal alginates (such as calcium alginate), polyhydroxyethyl methacrylate (pHEMA), polyethylene glycol (PEG), and derivatives thereof.

[0071] Here, the case where the non-cell-adhesive material is polyethylene glycol will be described. In this case, for example, by contacting a first solution containing a hyperbranched polymer having a polyethylene glycol backbone and one or more nucleophilic or electrophilic functional groups at its side chains and / or terminals with a second solution containing a hyperbranched polymer having a polyethylene glycol backbone and one or more nucleophilic or electrophilic functional groups at its side chains and / or terminals, the polymers are crosslinked to form a pattern of the non-cell-adhesive material.

[0072] "Multi-armed polymers with a polyethylene glycol backbone" (hereinafter simply referred to as "multi-armed polymers" or "multi-arm PEGs") are polymers used as gelling materials. Two types of multi-armed PEGs, each with a nucleophilic functional group and an electrophilic functional group at the end of multiple polyethylene glycol (PEG) branches, can crosslink with each other to form a gel with a meshwork network (multi-armed PEG gel).

[0073] For example, in the case of two types of four-branched polymers (hereinafter sometimes referred to as "Tetra-PEG") each having a nucleophilic functional group and an electrophilic functional group at the end of each of the four PEG branches, a gel called "Tetra-PEG gel" having a uniform network structure can be formed.

[0074] The number of branches in a multi-arm polymer is not particularly limited. Generally, any PEG with three or more branches, including electrophilic and nucleophilic ends, can be used, and can be selected appropriately as needed. As long as each PEG has a nucleophilic functional group and an electrophilic functional group, the two or more PEGs constituting the multi-arm PEG may have different numbers of branches. Among the multi-arm PEGs, Tetra-PEG gel is known to have an ideal uniform network structure.

[0075] Furthermore, Tetra-PEG gel can be easily and quickly formed on-site by simply mixing the two types of Tetra-PEG contained in the first and second solutions, and the gelation time can be controlled by adjusting the pH and concentration of each Tetra-PEG. Furthermore, since PEG is the main component, it also has excellent biocompatibility. By ejecting a solution containing cells and Tetra-PEG using a droplet ejection device and allowing the two types of Tetra-PEG to react to form a Tetra-PEG gel, it becomes possible to position cells three-dimensionally.

[0076] In one embodiment, Tetra-PEG may be a compound having a structure represented by the following general formula (1):

[0077] [ka]

[0078] The m's in the above formula (1) may be the same or different. The closer the m's values are, the more uniform the three-dimensional structure can be, and the stronger the gel will be. Therefore, to obtain a gel with high strength, it is preferable that they are the same. If the m's value is too high, the gel strength will be weak, and if each m's value is too low, the gel will be difficult to form due to the steric hindrance of the compound. Therefore, each m's value can be an integer value of 25 to 250, preferably 35 to 180, more preferably 50 to 115, and particularly preferably 50 to 60. The molecular weight of the compound is 5×10 3 ~5×10 4 Da is 7.5 x 10 3 ~3×10 4 Da is preferred, 1 x 10 4 ~2×10 4 Da is more preferred.

[0079] In the above formula (1), X 1 is a linker moiety connecting the functional group and the core moiety. 1 may be the same or different, but are preferably the same in order to produce a high-strength gel with a uniform three-dimensional structure. 1 represents a C1-C7 alkylene group, a C2-C7 alkenylene group, -NH-Ra-, -CO-Ra-, -Rb-O-Rc-, -Rb-NH-Rc-, -Rb-CO2-Rc-, -Rb-CO2-NH-Rc-, -Rb-CO-Rc-, or -Rb-CO-NH-Rc-, where Ra represents a C1-C7 alkylene group, Rb represents a C1-C3 alkylene group, and Rc represents a C1-C5 alkylene group.

[0080] The term "C1-C7 alkylene group" refers to an alkylene group having from 1 to 7 carbon atoms, which may have a branch, and includes a linear C1-C7 alkylene group and a C2-C7 alkylene group having one or more branches (having from 2 to 7 carbon atoms, including the branches). Examples of C1-C7 alkylene groups include methylene, ethylene, propylene, and butylene. Examples of C1-C7 alkylene groups include -CH2-, -(CH2)2-, -(CH2)3-, -CH(CH3)-, -(CH(CH3))2-, -(CH2)2-CH(CH3)-, -(CH2)3-CH(CH3)-, -(CH2)2-CH(C2H5)-, -(CH2)6-, -(CH2)2-C(C2H5)2-, and -(CH2)3C(CH3)2CH2-.

[0081] The term "C2-C7 alkenylene group" refers to a straight-chain or branched alkenylene group having 2 to 7 carbon atoms and one or more double bonds in the chain. For example, it may be a divalent group having a double bond formed by removing 2 to 5 hydrogen atoms from adjacent carbon atoms of an alkylene group.

[0082] In the above formula (1), Y 1 As described above, is a functional group for forming a meshwork network by a cross-end coupling reaction, which is a crosslinking reaction by a covalent bond, and is selected from nucleophilic functional groups or electrophilic functional groups.

[0083] The "nucleophilic functional group" is not limited, but is preferably a thiol group, for example, from the viewpoint of shortening the gelation time. The functional groups may be the same or different, but are preferably the same. When the functional groups are the same, the reactivity with the nucleophilic functional group that is the target of the crosslinking reaction becomes uniform, making it easier to obtain a high-strength gel with a uniform three-dimensional structure. Hereinafter, Tetra-PEG having a nucleophilic functional group may be referred to as "nucleophilic Tetra-PEG."

[0084] The "electrophilic functional group" is not limited, but for example, a maleimidyl group is preferred from the viewpoint of shortening the gelation time. The functional groups may be the same or different, but are preferably the same. When the functional groups are the same, the reactivity with the nucleophilic functional group that is the target of the crosslinking reaction becomes uniform, making it easier to obtain a high-strength gel with a uniform three-dimensional structure. Hereinafter, Tetra-PEG having an electrophilic functional group may be referred to as "electrophilic Tetra-PEG."

[0085] Nucleophilic Tetra-PEG and electrophilic Tetra-PEG can be mixed so that the molar ratio of nucleophilic functional groups to electrophilic functional groups is 0.5:1 to 1.5:1. Because these functional groups can react and crosslink at a 1:1 ratio, a mixing molar ratio closer to 1:1 is preferable, but to form a high-strength hydrogel, a ratio of 0.8:1 to 1.2:1 is preferred. Furthermore, when the pH of the dispersion medium in the first or second solution is 5 to 10, the concentration of Tetra-PEG contained in each solution should be in the range of 0.3 to 20%, and when the pH is 6 to 10, a range of 1.7 to 20% is preferred.

[0086] The first solution may be composed of either nucleophilic or electrophilic Tetra-PEG, and the second solution may be composed of the other Tetra-PEG.

[0087] First Solution The "first solution" is an aqueous solution containing, as components, a multi-branched polymer having a skeleton of either polyethylene glycol having one or more nucleophilic functional groups at least on the side chains and / or ends or polyethylene glycol having one or more electrophilic functional groups, and a dispersion medium, and may contain cells or cell-active additives as necessary.

[0088] There may be multiple types of first solutions. In this case, the hyperbranched polymer, dispersion medium, cells, and cell-interacting additives, each of which has a skeleton of either polyethylene glycol having one or more nucleophilic functional groups at its side chains and / or terminals or polyethylene glycol having one or more electrophilic functional groups, contained in each first solution, may be entirely or partially different. For example, the dispersion medium contained in first solution a and the dispersion medium contained in first solution b may be the same or different. When multiple types of first solutions are present, for example, when forming a layered hydrogel, the dispersion medium can be changed as desired in each layer.

[0089] Second Solution The "second solution" is an aqueous solution containing a multi-branched polymer with a polyethylene glycol backbone and one or more functional groups (nucleophilic functional groups or electrophilic functional groups) at its side chains and / or ends that are different from those in the first solution, and a dispersion medium as essential components, and may contain cells or cell-active additives as needed.

[0090] There may be multiple types of second solutions. In this case, the hyperbranched polymer having a different functional group from that of the first solution, which has a polyethylene glycol skeleton having one or more nucleophilic or electrophilic functional groups at its side chains and / or ends, the dispersion medium, the cells, and the cell-interacting additives contained in each second solution may be different in whole or in part. For example, the cells contained in second solution a and the cells contained in second solution b may be the same or different.

[0091] <Gel formation process> The "gel formation process" is a process in which the first and second solutions are mixed, causing a reaction between the hyperbranched polymers with polyethylene glycol as a skeleton contained in each solution, thereby forming a hydrogel. Either the first or second solution may be ejected using a droplet ejection device, or both the first and second solutions may be ejected using a droplet ejection device.

[0092] A "droplet ejection device" is a means of ejecting droplets of a solution stored in a liquid chamber, causing them to land on a target site. The ejection method of an ejection device includes the inkjet method and the gel extrusion dispenser method. In the inkjet method, the solution is ejected from an ejection hole (nozzle). The ejection method is capable of ejecting minute amounts of solution (sometimes called droplets) from the ejection hole, making it possible to create highly accurate three-dimensional structures.

[0093] The amount of droplets to be ejected can be any amount, preferably 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, and 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, or 300 pL or less.

[0094] "Depositing" refers to bringing a solution into contact with a target site. This is achieved by ejecting droplets onto the target site using an ejection method. The position at which the solution is ejected is not particularly limited, as long as the solution is ejected so that it lands on the desired target site. Furthermore, the respective landing sites may be separated, or may be partially in contact or overlapping.

[0095] Gel formation occurs through a reaction between a hyperbranched polymer having a polyethylene glycol skeleton with nucleophilic and electrophilic functional groups, and thus a hydrogel is formed when the second solution lands on the first solution. When the second solution is ejected using a droplet ejection device, a single impact typically results in the formation of a dot-shaped hydrogel. In this specification, the term "dot (shape)" refers to a point-like shape. Therefore, the shape is not limited to a perfect circle or a hemisphere, and may be any shape, such as a roughly circular or roughly hemispherical shape, a polygonal shape, a roughly polygonal shape, an irregular shape, or a combination thereof. Furthermore, the dot (shape) may have a predetermined length and thickness in terms of its three-dimensional structure. When the second solution is ejected multiple times, a variety of hydrogel shapes, with dot-shaped hydrogels being the smallest unit, are formed.

[0096] The volume of the hydrogel formed by a crosslinking reaction resulting from a single impact, i.e., the dot-shaped hydrogel, depends on the number of times the second solution is ejected onto the same location. Furthermore, the larger the ejection hole diameter and the more times the solution is ejected onto the same location, the larger the volume of the dot-shaped hydrogel. Therefore, the volume of the dot-shaped hydrogel can be adjusted by changing the number of times the solution is ejected onto the same location and the ejection hole diameter. Although not limited thereto, the volume of the dot-shaped hydrogel herein is preferably 9 pL or more, 15 pL or more, 20 pL or more, 30 pL or more, 40 pL or more, 50 pL or more, 60 pL or more, 70 pL or more, 80 pL or more, 90 pL or more, or 100 pL or more, and preferably 900 pL or less, 800 pL or less, 700 pL or less, 600 pL or less, 500 pL or less, 400 pL or less, or 300 pL or less. The diameter of the dot-shaped hydrogel is not limited, but may be in the range of 10 μm to 300 μm, and the thickness may be in the range of 5 μm to 150 μm.

[0097] Since the second solution is ejected any number of times, multiple dot-shaped hydrogels may be formed. The dot-shaped hydrogels may be in contact with each other in whole or in part. By arranging multiple dot-shaped hydrogels in a series, it is possible to form not only dot-shaped but also any shaped hydrogel. The shape can be appropriately selected depending on the purpose. For example, by arranging dots in one axial direction, it is possible to form a linear hydrogel. Furthermore, by arranging linear hydrogels in the same plane without any gaps, it is also possible to form a membrane-shaped (planar) hydrogel.

[0098] 《Removal process》 The "removal step" is a step of removing the unreacted, i.e., uncrosslinked, hyperbranched polymer present on the substrate or on the hydrogel after the gel formation step. This step is an optional step and may be performed as needed.

[0099] The removal method is not particularly limited, and any known removal method can be used as long as it does not have a physical, biological, or chemical effect on the formed hydrogel. A commonly used and simple removal method is to wash the support or hydrogel with an appropriate washing solution.

[0100] The washing solution used in the washing method is not particularly limited as long as it does not affect the hydrogel or cells. It may be appropriately selected taking into consideration pH, osmotic pressure, etc. Preferred examples include a buffer solution or a culture medium.

[0101] The washing method may involve pouring a washing solution over the support or hydrogel, or immersing the support or hydrogel in the washing solution to reduce physical damage to the hydrogel. Washing may be performed multiple times in one step.

[0102] (Cell adhesive material) Examples of the cell adhesive material include proteins selected from the extracellular matrix. Proteins selected from the extracellular matrix are the same as those described above. The cell adhesive material may be discharged using a droplet discharge device and placed on the substrate. The droplet discharge device is the same as that described above.

[0103] [Neural circuit model] In one embodiment, the present invention provides a neural circuit model comprising a substrate and a plurality of cell assemblies discretely arranged on the substrate, each of the plurality of cell assemblies including a neuron, and at least two of the cell assemblies being functionally connected. In the neural circuit model of this embodiment, each of the cell assemblies preferably includes 7 to 10,000 cells.

[0104] The neural circuit model of this embodiment can accurately reproduce the activity state of nerve cells in a living body in vitro, and can be used to elucidate brain function, evaluate toxicity to nervous system diseases, develop new drugs, and so on.

[0105] In the neural circuit model of this embodiment, the substrate and neurons are the same as those described above. Furthermore, functional connection between cell assemblies is the same as that described above, and refers to a neuron extending a process called an axon and forming a synaptic connection with the dendrites of another neuron. As a result, a neural circuit is formed.

[0106] In the neural circuit model of this embodiment, the substrate may have a pattern of a non-cell-adhesive material arranged on its surface, or the substrate may have a pattern of a cell-adhesive material arranged on its surface.

[0107] The arrangement pattern and number of cell aggregates on the substrate are not particularly limited, and may be, for example, a circle, a rectangle, a lattice, etc. Furthermore, the functional bonds between the cell aggregates arranged on the substrate may be random, or may be bonded so that each cell aggregate is circulated.

[0108] [Method of manufacturing neural circuit models] In one embodiment, the present invention provides a method for producing a neural circuit model, comprising: a cell culture carrier formation step of arranging a cell-adhesive material and a cell-non-adhesive material on a substrate to form a microstructure for cell arrangement; a cell arrangement step of arranging neurons on the substrate; a step of leaving the arranged neurons to settle and temporarily adhere to the substrate to form a cell aggregate; a medium addition step of supplying medium to the substrate on which the cell aggregate has formed; and a culture step of culturing the cell aggregate to form a neural circuit model.

[0109] In the manufacturing method of this embodiment, the substrate may be one of those described above, or may be a non-porous flat plate member on which a porous member is laminated. Examples of the non-porous flat plate member include those formed from the materials of the substrate described above, and more specifically, examples thereof include culture dishes, glass, etc.

[0110] The porous member may be, for example, a porous membrane. The pore size of the porous member is preferably such that cells are not buried therein, for example, a pore size of 1 μm or less.

[0111] Fig. 8 is a flow diagram of the manufacturing method of this embodiment. As shown in Fig. 8, first, in the cell culture carrier formation step, a cell adhesive material and a cell non-adhesive material are arranged on a substrate to form a microstructure for cell arrangement. The cell adhesive material and the cell non-adhesive material are the same as those described above.

[0112] Next, in the cell arrangement step, cells are arranged in the cell arrangement region of the cell arrangement microstructure. The cell arrangement is preferably performed by an inkjet method. Furthermore, here, when multiple types of cells are arranged in the cell arrangement microstructure, all types of cells are arranged.

[0113] In the manufacturing method of this embodiment, it is preferable to perform a drying prevention treatment. For example, if a substrate having a porous structure is used as the substrate and a liquid such as a culture medium is held in the substrate beforehand, the liquid is held in the porous structure and the drying of the liquid pool can be prevented.

[0114] Furthermore, when droplets are ejected onto a dried substrate to form a liquid pool, a step of suppressing evaporation of the liquid in the liquid pool (a drying suppression step) can be carried out to maintain the shape of the liquid pool and stably achieve adhesion of cells to the substrate. Examples of the drying suppression step include the steps similar to those described above. The drying suppression treatment may be carried out after the cell placement step or before the cell culture carrier formation step.

[0115] Next, in the step of leaving the cells to stand, the cells are left to stand until all the cells have adhered. Next, in the step of adding a medium, a medium is added. The medium is the same as that described above. Next, in the step of culturing, the cell aggregate is cultured to form a neural circuit model.

[0116] 9 is a schematic cross-sectional view illustrating a method for producing a neural circuit model. As shown in FIG. 9, a cell culture support 930 is formed by laminating a porous member 920 on a flat plate member 910.

[0117] There is no particular limitation on the method for forming a microstructure for cell placement on a porous member, but in the example of Figure 9, the porous member 920 is immersed in the above-mentioned first solution 940, then placed on a flat member 910, and the second solution 950 is ejected from an inkjet head.

[0118] When the first solution 940 and the second solution 950 react with each other, a non-cell adhesive material, hydrogel (Tetra-PEG gel) 960, is formed. The reaction between the first solution 940 and the second solution 950 can be carried out by leaving the mixture to stand for 30 minutes in a high humidity environment. Here, the high humidity environment is preferably a relative humidity of 90% or higher.

[0119] Next, the cells are placed. In the example of FIG. 9, the cells 980 are placed by ejecting the above-mentioned cell ink 970 using an inkjet method. At this time, the culture medium is held in the porous member 920, which reduces the impact when the ejected microdroplets of cell ink 970 land, thereby reducing damage to the cells. Furthermore, the culture medium is held in the porous member 920, which prevents the microdroplets from drying out. [Example]

[0120] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0121] [Experimental Example 1] (Evaluation of cell migration 1) Cells were seeded on the substrate and migration was evaluated. The substrate used was a glass slide coated with Matrigel (registered trademark, Corning Incorporated). PC12 cells, a cell line derived from rat pheochromocytoma, were used. Cell seeding was performed using the inkjet method.

[0122] Preparation of cell ink First, the cells were stained by dissolving a green fluorescent dye (trade name: Cell Tracker Green, manufactured by Thermo Fisher Scientific) in dimethyl sulfoxide (DMSO) at a concentration of 10 mmol / L (mM) and mixing it with the medium to prepare a medium containing the green fluorescent dye at a concentration of 10 μmol / L (μM).

[0123] Next, 5 mL of serum-free medium containing green fluorescent dye was added to the dish containing the cultured PC12 cells, and the cells were cultured for 30 minutes in an incubator (KM-CC17RU2, Panasonic Corporation, 37°C, 5% CO2 by volume). The cells were then detached from the dish by trypsinization to obtain a cell suspension. A portion of the cell suspension was then placed on a PMMA plastic slide, and the cell count was measured using a Countess Automated Cell Counter (Thermo Fisher Scientific).

[0124] The dispersion medium for the cell ink was PBS(-) supplemented with 0.5% by mass of glycerin (molecular biology grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a cell drying inhibitor. PC12 cells were cultured at 6 × 10 6 The cells were dispersed in a dispersion medium at a concentration of 1000 cells / mL to obtain a cell ink.

[0125] <Cell ejection> The liquid chamber of the cell ejection head of the device shown in Figure 4 was filled with cell ink. Next, droplets of the cell ink were ejected onto the substrate, forming pools. The diameter of each pool was approximately 200 μm. Each pool on the substrate contained approximately 100 cells, giving a cell density of 3 × 10 5 pieces / cm 2 The cell density in the liquid pool refers to the number of cells per unit area where the liquid pool placed on the substrate is in contact with the substrate.

[0126] Figure 10(a) is a micrograph taken immediately after the cell ink was ejected. After the droplets of cell ink had landed, they were left in a high humidity environment of 95% or higher for approximately 10 minutes, during which time the cells in the droplets settled and temporarily adhered to the substrate, forming cell aggregates. Next, culture medium was gently added. Figure 10(b) is a micrograph taken immediately after the culture medium was added.

[0127] Cell culture The cells were then cultured for one day in an incubator at 37°C with 5% CO2 by volume. Figure 10(c) is a fluorescence microscope image of the Cell Tracker Green fluorescence observed in the cells one day after the start of culture. The results revealed that the cells had migrated and moved from their position immediately after ejection. When this type of migration occurs, it becomes difficult to keep the cells in place.

[0128] [Experimental Example 2] (Evaluation of cell migration 2) The number of cells in the pool was varied and cells were seeded onto the substrate to evaluate the occurrence of migration. A slide glass with a surface coated with Matrigel (registered trademark) was used as the substrate. PC12 cells were used. The cells were seeded using the inkjet method.

[0129] The preparation of cell ink and the ejection of cells were carried out in the same manner as in Experimental Example 1. However, when ejecting cells, the number of cells in the liquid pool was changed. Specifically, the number of cells was changed from 1 to less than 3 (cell density 2 × 10) per liquid pool arranged on the substrate. 4 pieces / cm 2 ), 3 to 6 cells (cell density 6 × 10 4 pieces / cm 2 ), 7~11 cells (cell density 1 × 10 5 pieces / cm 2 ) cells were placed on the substrate. Here, cell density refers to the number of cells per unit area where the liquid pools placed on the substrate are in contact with the substrate. The diameter of each liquid pool was 100 μm.

[0130] The cells were then cultured for one day in an incubator at 37°C and 5% CO2 by volume. Individual cells were observed over time using an inverted microscope (model "CKX41," manufactured by Olympus Corporation), and the migration distance of each cell was measured. Figure 11 is a graph showing the results of measuring the migration distance of cells one day after the start of culture. The vertical axis of the graph represents the migration distance (μm).

[0131] As a result, the number of cells per reservoir was 7 or more, and the cell density in the reservoir was 10 5 pieces / cm 2 As a result, it was revealed that cell migration was suppressed.

[0132] [Experimental Example 3] (neuronal pattern control) A pattern of a non-cell-adhesive material and a pattern of a cell-adhesive material were placed on a substrate. The substrate was a 13 mm diameter polyester porous culture membrane (trade name: ipCELLCULTURE Track Etched Membrane, pore size: 0.45 μm, pore density: 4 × 10) placed on a glass slide. 6 pieces / cm 2 A laminate of 12 μm thick film (manufactured by it4ip) was used.

[0133] Next, neurons were seeded onto this substrate and axonal outgrowth was observed. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as neurons. Figure 12 is a schematic diagram illustrating the procedure for placing patterns of non-cell-adhesive material and cell-adhesive material on the substrate and then placing cells.

[0134] Preparation of First Solution Tetra-PEG-SH (trade name "SUNBRIGHT PTE-100SH", manufactured by Yuka Sangyo Co., Ltd.) was dissolved in PBS(-) and then filtered through a filter with an average pore size of 0.2 μm (trade name "Minisart Syringe Filter 175497K", manufactured by Sartorius) to obtain a first solution containing 2% Tetra-PEG-SH.

[0135] Preparation of the second solution Tetra-PEG-maleimidyl (trade name "SUNBRIGHT PTE-100MA", manufactured by Yuka Sangyo Co., Ltd.) and Matrigel (registered trademark, manufactured by Corning Incorporated) were dissolved in PBS(-) and filtered through a filter with an average pore size of 0.2 μm to prepare a second solution containing 2% Tetra-PEG-maleimidyl and 1% Matrigel.

[0136] Pattern Formation The substrate was immersed in the first solution and removed, forming a liquid phase of the first solution on the substrate. The inkjet head's liquid chamber was then filled with the second solution, and the second solution was dropped onto the substrate to form a linear pattern approximately 200 μm wide. As a result, Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution crosslinked to form a hydrogel pattern. This hydrogel is a non-cell-adhesive material. Furthermore, the portion coated with Matrigel (registered trademark) formed a pattern of cell-adhesive material. The substrate was then immersed in phosphate-buffered saline (Life Technologies, hereinafter also referred to as PBS(-)), and excess first and second solutions were removed.

[0137] Preparation of cell ink Cell ink was prepared in the same manner as in Experimental Example 1, except that human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used.

[0138] <Cell ejection> Cell ink was filled into the liquid chamber of the cell ejection head of the device shown in Figure 1. Subsequently, droplets of the cell ink were ejected onto the pattern of cell adhesive material on the substrate, and a liquid pool was formed.

[0139] After the droplets of cell ink were left to stand for approximately 30 minutes, the cells in the droplets settled and temporarily adhered to the substrate, forming cell aggregates. Then, culture medium was gently added.

[0140] Cell culture The cells were then cultured in an incubator at 37°C with 5% CO2 by volume. Figure 13(a) is a bright-field micrograph of the cells four days after the start of culture. The scale bar is 100 μm. Figures 13(b) and 13(c) are fluorescence micrographs of the Cell Tracker Green fluorescence on the substrate. The scale bar is 100 μm. Figure 13(d) is a fluorescence micrograph of the Cell Tracker Green fluorescence in an area with low cell density. The scale bar is 50 μm.

[0141] As a result, it was revealed that cells did not migrate to the area where the pattern of the non-cell-adhesive material was placed. Furthermore, it was revealed that axons were formed only in the area excluding the pattern of the non-cell-adhesive material. These results demonstrate that it is possible to control the axonal elongation of nerve cells by placing a pattern of the non-cell-adhesive material on a substrate.

[0142] [Experimental Example 4] (Non-pattern control of neurons) A substrate containing only the cell adhesive material was prepared using the same method as in Experimental Example 3. Next, droplets of cell ink were ejected onto the pattern of cell adhesive material on the substrate, and neurons were seeded using the same method as in Experimental Example 3. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons.

[0143] The cells were then cultured in an incubator at 37°C with 5% CO2 by volume, and neuronal axon outgrowth was confirmed 7 days after the start of culture. Specifically, Calcein-AM (Thermo Fisher Scientific) diluted with DMSO was added to the medium to a final concentration of 10 μM, and the cells were incubated at 37°C with 5% CO2 for 30 minutes. The neurons were then observed under a fluorescence microscope.

[0144] Figure 14 shows a fluorescence micrograph of a neuron. The scale bar is 200 μm. The results confirmed that when a pattern of non-cell-adhesive material was not placed on the substrate, the direction of neuronal axon outgrowth was not controlled.

[0145] [Experimental Example 5] (Creating a neural circuit model 1) Neural circuit models were fabricated and evaluated by varying the diameter of the reservoirs on the substrate, the cell density in the reservoirs, the number of cells per reservoir, the pattern size of the non-cell-adhesive material, and the pattern size of the cell-adhesive material in various combinations as shown in Table 1 below. A 10 cm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons.

[0146] Figures 15(a) to (e) are schematic diagrams illustrating the procedure of this experimental example. Figures 15(a) to (d) are side views, and Figure 15(e) is a top view of Figure 15(b). As shown in Figures 15(a) to (e), the cell adhesive material was arranged in a circular shape to form a cell adhesive region. In addition, a non-cell adhesive material was arranged to surround the cell adhesive region to form a non-cell adhesive region. The shape of the non-cell adhesive region was a roughly donut shape concentric with the adhesive material.

[0147] Figure 15(a) is a schematic diagram illustrating the process of placing a droplet containing neurons on a substrate. Figure 15(b) is a schematic diagram illustrating the state in which multiple droplets land on a substrate to form a liquid pool. The edge of the liquid pool may be located on the non-cell-adhesive area, but it is preferable that it does not extend beyond the non-cell-adhesive area. Figure 15(c) is a schematic diagram illustrating the state in which neurons in the liquid pool have settled and temporarily adhered to the substrate to form a cell aggregate. Figure 15(d) is a schematic diagram illustrating the process of supplying a culture medium to the substrate on which the cell aggregate has formed.

[0148] In Table 1, "liquid pool diameter" indicates the diameter of the liquid pool placed on the substrate, "cell density" indicates the cell density in the liquid pool, "cell number" indicates the number of cells per liquid pool, "inner diameter / outer diameter of non-adhesive portion" indicates the inner and outer diameters of the donut-shaped portion of the cell non-adhesive portion, "diameter of adhesive portion" indicates the diameter of the cell adhesive portion, and "cell placement method" indicates whether the cells were placed by inkjet (IJ) or manual technique. "Cell density" is the number of cells per area where the liquid pool placed on the substrate comes into contact with the cell adhesive portion. "ND" indicates that measurement was not possible.

[0149] The pattern of the non-cell-adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution in the same manner as in Experimental Example 3. When forming a pattern of the cell-adhesive material, Matrigel (registered trademark, Corning Inc.) was filled into the liquid chamber of the inkjet head and ejected in a pattern.

[0150] The neural circuit model was evaluated based on the diameter of the pool, the initial cell placement, and the fixation of the cells. Table 1 shows the results of these evaluations.

[0151] The goal was to control the diameter of the pool to 500 μm or less, which was difficult to achieve by manual techniques. However, when cells were placed using the inkjet method, it was possible to control the diameter of the pool to either more than 500 μm or less.

[0152] The evaluation criteria for the initial placement of cells were as follows: -: Cells were randomly arranged. +: A discrete pattern of cells was formed.

[0153] The evaluation criteria for cell fixation were as follows, and cell migration was evaluated on days 1 to 7 of culture. -: Cells migrated outside the droplets placed on the substrate or were randomly placed. +: Cells remained inside the droplet placed on the substrate.

[0154] [Table 1]

[0155] As a result, good evaluation results were obtained in the evaluation items of the diameter of the liquid pool, the initial cell positioning, and the fixation of the cells in the neural circuit models of Examples 1 to 6. On the other hand, in the neural circuit models of Comparative Examples 1 to 6, the diameter of the liquid pool exceeded 500 μm, or the evaluation results of one of the evaluation items were poor.

[0156] From the above results, it was found that the droplets placed on the substrate contained 7 or more cells per droplet, and the cell density in the droplet was 10 5 pieces / cm 2 From the above, it became clear that cells can be stably arranged on the substrate even when the diameter of each of the liquid pools is set to 500 μm or less.

[0157] [Experimental Example 6] (Creating a neural circuit model 2) Figures 16(a) to (d) are schematic diagrams showing the arrangement of cells in a neural circuit model of an experimental example, in which both a non-cell-adhesive material and a cell-adhesive material were arranged. Figures 16(a) and (b) are side views, and Figures 16(c) and (d) are top views. Figures 16(a) and (c) show the state immediately after the cells were arranged, and Figures 16(b) and (d) show the state after cell culture, in which axons have extended between the cell aggregates and synapses have been formed.

[0158] In this experimental example, the neural circuit model shown in Figures 16(a) to 16(d) was fabricated. The pattern of the non-cell-adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution, as in Experimental Example 3. To form the pattern of the cell-adhesive material, Matrigel (registered trademark, Corning Incorporated) was filled into the liquid chamber of an inkjet head and ejected in a pattern. A 10 cm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons. As a result, a neural circuit model was obtained.

[0159] [Experimental Example 7] (Creating a neural circuit model 3) As shown in Figure 17(a), a neural circuit model was created by arranging neurons in the pattern shown in Figure 17(a) on a substrate with two concentric circular non-cell adhesive materials. The pattern of the non-cell adhesive material was formed by crosslinking Tetra-PEG-SH contained in the first solution and Tetra-PEG-maleimidyl contained in the second solution, as in Experimental Example 3. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons.

[0160] Figure 17(b) is a representative micrograph of the neural circuit model immediately after ejection of the cell ink. In Figure 17(b), "Position: +" indicates that the initial cell positioning was good, and "Position: -" indicates that the initial cell positioning was poor.

[0161] After arranging the cells under conditions that allowed for good initial arrangement, they were cultured for four weeks, and axons were allowed to extend to obtain a neural circuit model. Figure 18 shows a fluorescence micrograph of the neural circuit model, showing the fluorescence of Cell Tracker Green.

[0162] [Experimental Example 8] (Creating a neural circuit model 4) As shown in Figures 19(a) and (b), a cell non-adhesive material (Tetra-PEG gel) was placed on the substrate.

[0163] The substrate was a polyester porous culture membrane (trade name: ipCELLCULTURE Track Etched Membrane, pore size: 0.45 μm, pore density: 4 × 10) with a diameter of 13 mm, impregnated with the same first solution as in Experimental Example 3, on a slide glass. 6 pieces / cm 2 A laminate of 12 μm thick film (manufactured by IT4IP) was used.

[0164] Next, the liquid chamber of the inkjet head was filled with the same second solution as in Experimental Example 3, and the second solution was dropped onto the substrate to form patterns with a width of approximately 200 μm as shown in Figures 19(a) and (b).

[0165] In the pattern shown in Figure 19(a), six linear patterns were formed. The distance between these lines was approximately 0.5 mm. The length of each line was approximately 5 mm. In the pattern shown in Figure 19(b), three concentric circular patterns were formed. The diameters of each pattern, from the center to the outside, were approximately 1 mm, 2 mm, and 3 mm, respectively.

[0166] Next, we created a neural circuit model by arranging two types of neurons in the patterns shown in Figure 19(a) and (b), respectively. The neurons used were human iPS cell-derived GABAergic neurons (Elixirgen Scientific) and PC12 cells.

[0167] After arranging the cells under conditions that allowed for good initial arrangement, they were cultured for one week to allow neurite outgrowth and obtain neural circuit models, which were then analyzed by immunohistochemistry.

[0168] First, each neural circuit model was washed with PBS and fixed for 30 minutes in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C. After fixation, the model was washed once with PBS and blocked for 20 minutes at room temperature using 1% bovine serum albumin (Thermo Fisher Scientific). After blocking, the model was washed once with PBS and incubated overnight at 4°C with a mixture of primary antibodies: anti-βIII tubulin antibody (Sigma-Aldrich) and anti-tyrosine hydroxylase antibody (Abcam), each diluted 200-fold with PBS.

[0169] Subsequently, the sections were washed three times with PBS, and a mixture of APC-labeled goat anti-mouse IgG (H+L) antibody (Thermo Fisher Scientific) and Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) antibody (Thermo Fisher Scientific), each diluted 500-fold with PBS, was added as secondary antibodies, and the sections were incubated at room temperature for 1 hour, after which they were washed twice with PBS.

[0170] Next, a cover glass was placed over the sample using a sealing liquid (product name "ProLong Diamond Antifade Mountain", Thermo Fisher Scientific) to seal it, and the sample was observed under a fluorescence microscope.

[0171] Figures 20(a) and (b) are fluorescence micrographs showing the fluorescence of βIII tubulin, the neuroskeleton of the obtained neural circuit model, and tyrosine hydroxylase, which is expressed only in PC12. Figure 20(a) is a fluorescence micrograph of a neural circuit model in which neurons are arranged in the pattern shown in Figure 19(a), and Figure 20(b) is a fluorescence micrograph of a neural circuit model in which neurons are arranged in the pattern shown in Figure 19(b).

[0172] As a result, in both neural circuit models with neuronal cell arrangement patterns, βIII tubulin fluorescence was observed throughout the entire cell arrangement area, and neurite growth was confirmed. Furthermore, tyrosine hydroxylase fluorescence was observed only in the area where PC12 cells were arranged, confirming that the cell arrangement pattern was maintained.

[0173] [Experimental Example 9] (Improvement of the drying suppression process) Cells were seeded on the dry substrate, and the suppression of drying of the liquid pool and the occurrence of cell adhesion were evaluated. A glass slide was used as the substrate. PC12 cells, a cell line derived from rat pheochromocytoma, were used. Cells were seeded using the inkjet method.

[0174] Preparation of cell ink Five mL of serum-free medium containing green fluorescent dye was added to the cultured PC12 cell dish and cultured for 30 minutes in an incubator (KM-CC17RU2, Panasonic Corporation, 37°C, 5% CO2). The cells were then detached from the dish by trypsinization to obtain a cell suspension. A portion of the cell suspension was then used to count the number of cells using a NucleoCounter NC-3000 (ChemoMetec).

[0175] The dispersion medium for the cell ink was PBS(-) supplemented with 0.5% by mass of glycerin (molecular biology grade, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a cell drying inhibitor. PC12 cells were cultured at 3 × 10 6 The cells were dispersed in a dispersion medium at a concentration of 1000 cells / mL to obtain a cell ink.

[0176] <Cell ejection> The liquid chamber of the cell ejection head of the device shown in Figure 4 was filled with cell ink. Next, droplets of the cell ink were ejected onto the substrate, forming pools. The diameter of each pool was approximately 400 μm. Each pool on the substrate contained approximately 100 cells, giving a cell density of 8 × 10 4pieces / cm 2 It was.

[0177] Figure 21(a) is a micrograph of a liquid pool that was covered with biocompatible oil (Oil for Embryo Culture, Fujifilm Wako Pure Chemical Industries, Ltd.) immediately after ejection of the cell ink to prevent drying. By covering the liquid pool with oil, the tiny liquid pool did not dry out even after being left at 37°C for 60 minutes, and the cells in the droplet settled and temporarily adhered to the substrate, forming a cell aggregate. This method can be applied to many cell types, not just PC12 cells.

[0178] The oil was then gently removed and the medium was gently added. Figure 21(b) shows a micrograph taken immediately after the medium was added. Because the liquid pool was only a small amount, it would dry out in a few minutes in a typical laboratory environment, reducing the volume of the pool by more than 90%.

[0179] In addition to the method of leaving the substrate in a high humidity environment as in Experimental Examples 1 and 2, and the method of creating a liquid pool on a wet substrate as in Experimental Examples 3 to 8, it was confirmed that by covering the liquid pool with oil or the like, it is possible to stably perform cell sedimentation and temporary adhesion even when a dry substrate is used.

[0180] Next, the number of cells per pool and the presence or absence of drying prevention treatment for the pools on the dry substrate were varied in various combinations as shown in Table 2 below, and neural circuit models of Reference Examples 1 to 4 were fabricated and evaluated. A 35 mm dish was used as the substrate. Human iPS cell-derived GABAergic neurons (Elixirgen Scientific) were used as the neurons.

[0181] In Table 2 below, "prevention of drying of puddle" indicates whether or not a drying prevention process was performed on the puddle placed on the substrate and the type of process, and "retention of shape of puddle" indicates whether or not the shape of the puddle was maintained after being left to stand at 37°C for 60 minutes. The other items are the same as those shown in Table 1 above. The evaluation criteria for "retention of shape of puddle" were as follows: +: The shape of the liquid pool was maintained. -: The liquid pool was more than 80% dry and cell death was observed.

[0182] [Table 2]

[0183] The present invention includes the following aspects. [1] A method for producing a neuronal aggregate comprising the steps of: disposing, by an inkjet method, a plurality of droplets containing neurons on a substrate having an area where a cell adhesive material is disposed and an area where a cell non-adhesive material is disposed, to form one or more liquid pools; and allowing the neurons in the liquid pools to settle and temporarily adhere to the substrate to form a cell aggregate; wherein the diameter of each liquid pool is 500 μm or less, and the density of neurons in each liquid pool is 10 5 pieces / cm 2 This completes the method for manufacturing a substrate on which nerve cells are arranged. [2] The manufacturing method described in [1], wherein each of the liquid reservoirs contains 7 to 10,000 of the nerve cells. [3] The method according to [1] or [2], wherein each droplet contains 1 to 50 of the nerve cells. [4] The method according to any one of [1] to [3], wherein the droplet is placed in contact with the cell adhesive material. [5] The manufacturing method according to any one of [1] to [4], further comprising a step of suppressing evaporation of the liquid in the liquid pool. [6] The method of any one of [1] to [5], further comprising the step of supplying a culture medium to the substrate on which the cell aggregates are formed. [7] The manufacturing method described in [6], further comprising the steps of forming multiple liquid pools in the liquid pool forming step, forming multiple cell aggregates in the leaving step, and incubating the substrate supplied with the culture medium to functionally bond at least two of the cell aggregates. [8] A manufacturing method described in any one of [1] to [7], wherein the substrate has an area where the non-cell-adhesive material is placed and an area where the non-cell-adhesive material is not placed, and the area where the non-cell-adhesive material is not placed has a linear shape, and the width of the linear shape is 100 μm or less. [9] The method according to any one of [1] to [8], wherein the substrate has a porous structure. [Explanation of symbols]

[0184] 10...droplet ejection head, 11,970...cell ink, 12,25...liquid chamber, 13,28...membrane, 14,26...drive unit, 15...nozzle, 16,27...vibration unit, 17,24...atmospheric opening unit, 21,22,23...inkjet head, 31...stage unit, droplet placement device...400,500,600,700, 910...flat plate member, 920...porous member, 930...cell culture carrier, 940...first solution, 950...second solution, 960...non-cell adhesive material, 980...cell, Pj...ejection waveform, Ps...vibration isolation waveform. [Prior art documents] [Patent documents]

[0185] [Patent Document 1] Japanese Patent Application Publication No. 2019-162097

Claims

1. a step of disposing, by an inkjet method, a plurality of droplets containing nerve cells on a substrate having an area where a cell adhesive material is disposed and an area where a cell non-adhesive material is disposed, to form one or a plurality of liquid pools; and leaving the solution pool to stand until the nerve cells in the solution pool settle and temporarily adhere to the substrate to form a cell aggregate, The diameter of each of the liquid pools is 100 μm or more and 500 μm or less, The density of neurons per pool is 10 ５ ~10 ６ pieces / cm ２ and the droplet is placed so as to be in contact with the region where the cell adhesive material is placed, and the density of the nerve cells is the number of cells per area where the droplet is in contact with the region where the cell adhesive material is placed. A method for manufacturing substrates on which nerve cells are arranged.

2. The method according to claim 1, wherein each of the reservoirs contains 7 to 315 of the nerve cells.

3. The method according to claim 1 or 2, wherein each droplet contains 1 to 50 of the nerve cells.

4. The method according to any one of claims 1 to 3, wherein the droplet is placed in contact with the cell adhesive material.

5. The manufacturing method according to any one of claims 1 to 4, further comprising the step of suppressing evaporation of the liquid in the liquid pool.

6. The manufacturing method according to any one of claims 1 to 5, further comprising the step of supplying a culture medium to the substrate on which the cell aggregates are formed.

7. forming a plurality of the liquid reservoirs in the step of forming the liquid reservoirs; forming a plurality of cell aggregates in the standing step; The method of claim 6 , further comprising the step of incubating the substrate supplied with the culture medium to functionally bond at least two of the cell aggregates.

8. the substrate has an area where the non-cell-adhesive material is disposed and an area where the non-cell-adhesive material is not disposed, the region where the non-cell-adhesive material is not disposed has a linear shape; The method according to any one of claims 1 to 7, wherein the width of the linear shape is 100 µm or less.

9. The method according to any one of claims 1 to 8, wherein the substrate has a porous structure.

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