JP6977347B2 - Evaluation base material manufacturing method and evaluation base material manufacturing equipment - Google Patents

Description

The present invention relates to a method for producing a base material for evaluation and an apparatus for producing a base material for evaluation.

In recent years, the demand for toxicity or drug efficacy evaluation tools in vitro has been increasing.

One of the reasons is to reduce the number of experimental animals by promoting 3Rs (“Replacement”, “Reduction”, and “Refinement”) of animal experiments, and to replace the experimental animals. Is required.

Reasons other than the promotion of 3Rs in animal experiments include that the toxicity or drug efficacy evaluation tool in vitro has many advantages such as reduction of cost for animal experiments and shortening of test time.

On the other hand, with the progress of tissue engineering, the development of a technique for artificially forming a tissue composed of a plurality of cells is being carried out. Among them, bioprinters are attracting attention in the construction of three-dimensional tissue models because cells can be rapidly fixed on the bottom surface of a culture vessel or on a gel depending on the type of fixing material. However, it is already known that it is difficult to completely reproduce the function of cells in a living body and the process of reaction to a drug in vitro, and many problems remain.

Therefore, for the purpose of providing a culture substrate having a cell-immobilized region, a container, and a manufacturing method, a method for forming a pattern of the cell-immobilized region, a step of arranging a different cell culture control substance, and a cell culture control substance are provided. A method for producing a substrate including a step of arranging the substrate has been proposed (see, for example, Patent Document 1).
Further, the compound comprising a step of culturing the established hepatocytes within 12 hours or more and 72 hours or less in a plurality of culture spaces formed in the culture vessel to prepare a plurality of spheroids having an equivalent diameter of 50 μm or more and less than 200 μm. A screening method has been proposed (see, for example, Patent Document 2).

An object of the present invention is to provide a method for producing an evaluation base material capable of adhering cells to a base material in a short time.

The method for producing the evaluation base material of the present invention as a means for solving the above-mentioned problems includes a fixing material A-containing solution applying step of applying a solution containing the fixing material A to the base material, and the fixing material A. The fixing material B and the cell-containing suspension-imparting step of imparting a suspension containing the fixing material B and the cells which can be mixed with the fixing material B are included, and at least each of the fixing material A and the fixing material B is provided. A portion is mixed to form a hydrogel, and the cells are fixed to the substrate via the hydrogel.

According to the present invention, it is possible to provide a method for producing an evaluation base material capable of adhering cells to a base material in a short time.

FIG. 1A is a schematic plan view showing an example of an evaluation base material produced by the method for producing an evaluation base material of the present invention. FIG. 1B is a cross-sectional view taken along the line AA'of the evaluation base material of FIG. 1A. FIG. 2A is a schematic view showing an example of a solenoid valve type discharge head. FIG. 2B is a schematic diagram showing an example of a piezo type discharge head. FIG. 2C is a schematic view of a modified example of the piezo type discharge head in FIG. 2B. FIG. 3A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. FIG. 3B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 4A is a schematic diagram showing an example of the state of the droplet. FIG. 4B is a schematic diagram showing an example of the state of the droplet. FIG. 4C is a schematic diagram showing an example of the state of the droplet. FIG. 5 is a schematic diagram showing an example of a droplet forming apparatus. FIG. 6 is a diagram illustrating a hardware block of the control means of FIG. FIG. 7 is a schematic diagram showing another modification of the droplet forming apparatus of FIG. FIG. 8A is a schematic view showing an example of a fixing material A-containing solution applying step. FIG. 8B is a schematic view showing an example of the fixing material B and the cell-containing suspension applying step. FIG. 8C is a schematic view showing an example of a step of applying a medium. FIG. 9A is a schematic view showing an example of the fixing material A-containing solution applying step. FIG. 9B is a schematic view showing an example of the fixing material B and the cell-containing suspension applying step. FIG. 9C is a schematic view showing an example of a step of applying a solution containing at least one of the fixing material A and the fixing material B. FIG. 9D is a schematic view showing an example of a step of applying a medium. FIG. 10 is a photograph showing the state of cells when the evaluation substrate prepared in Example 1 was observed with an optical microscope. FIG. 11A is a photograph showing the state of cells in Comparative Example 3 after a culture time of 0 hours. FIG. 11B is a photograph showing the state of cells after a culture time of 3 hours in Comparative Example 3. FIG. 11C is a photograph showing the state of cells after a culture time of 10 hours in Comparative Example 3. FIG. 12 is a graph showing the cell viability in Example 1 and Comparative Example 3. FIG. 13 is a graph showing the cell membrane damage rate in Example 1 and Comparative Example 3. FIG. 14 is a graph showing the amount of inflammatory substance secreted in Example 1 and Comparative Example 3.

(Manufacturing method of evaluation base material and equipment for manufacturing evaluation base material)
The evaluation base material manufacturing method of the present invention can be mixed (or reacted) with the fixing material A-containing solution applying step of applying the fixing material A-containing solution to the base material and the fixing material A. The fixing material B and the cell-containing suspension applying step of imparting the fixing material B and the suspension containing the cells are included, and at least a part of the fixing material A and the fixing material B are mixed (or at least a part of each). Reaction) to form a hydrogel, the cells being immobilized on the substrate via the hydrogel, and comprising a step of imparting a solution containing at least one of the fixing material A and the fixing material B. It is preferable, and further includes a step of applying a medium and other steps, if necessary.
In the method for producing a substrate for evaluation of the present invention, in the conventional method for producing a substrate and the method for screening a compound, cells used for the test are seeded in a culture vessel as a preparation for the test, and the cells are placed in the culture vessel. It is based on the finding that there is a problem that it takes about 24 hours for culturing to adhere by itself.
Further, in the method for producing the evaluation substrate of the present invention, in the conventional method for producing the substrate and the method for screening the compound, the number of cells adhering to the culture vessel is smaller than expected when seeded in the culture vessel with a small number of cells. Therefore, it is based on the finding that accurate test results may not be obtained, and there is a problem that the test time and the number of cells in the evaluation of drug efficacy or toxicity are considerably limited. Furthermore, in order to carry out one test, it is necessary to secure time for the cells to adhere to the culture vessel, and it is necessary to perform a drug efficacy or toxicity test on cells for which it is difficult to secure a certain number of cells or more. It is based on the finding that there is a problem that the test method and the cell type to be tested are limited.

The evaluation base material manufacturing apparatus of the present invention mixes (or mixes) with the base material, the fixing material A-containing solution applying means for imparting a solution containing the fixing material A to the base material, and the fixing material A. It has a fixing material B capable of (reaction), a fixing material B for imparting a suspension containing cells, and a cell-containing suspension applying means, and the cells have the fixing material A and the fixing material B. Means for imparting a solution containing at least one of the fixing material A and the fixing material B to the substrate via a hydrogel formed by mixing (or reacting with) at least a part of each of the fixing material A and the fixing material B. It is preferable to have, and if necessary, a means for applying a medium and other means are provided.

FIG. 1A is a schematic plan view showing an example of an evaluation base material produced by the method for producing an evaluation base material of the present invention. FIG. 1B is a cross-sectional view taken along the line AA'of the evaluation base material of FIG. 1A.
According to the method for producing an evaluation base material of the present invention, as shown in FIG. 1A, a hydrogel 3 formed by mixing at least a part of each of the fixing material A and the fixing material B on the base material 2 , The evaluation base material 1 in which the cells 4 are arranged at a desired position can be obtained. Further, as shown in FIG. 1B, the cells 4 can be adhered to the base material 2 via the hydrogel by being fixed to the hydrogel 3. As a result, the time for the cells to be cultured and adhered to the substrate can be shortened, and the evaluation substrate 1 can be obtained in a short time.
Further, according to the method for producing an evaluation base material of the present invention, it is possible to obtain an evaluation base material in which cells are arranged at a desired position.

<Fixing material A-containing solution applying step and fixing material A-containing solution applying means>
The fixing material A-containing solution applying step is a step of applying the fixing material A-containing solution to the base material.
The fixing material A-containing solution applying means is a means for applying the fixing material A-containing solution to the base material.
The fixing material A-containing solution applying step can be suitably carried out by the fixing material A-containing solution applying means.

<< Base material >>
The base material is not particularly limited in size, shape, structure, material and the like as long as it does not inhibit the activity and proliferation of cells, and can be appropriately selected according to the purpose.
The size of the base material is not particularly limited and may be appropriately selected depending on the intended purpose.
The shape of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a three-dimensional shape such as a dish, a multi-plate, a flask, or a cell insert; a flat plate such as a glass plate, a slide glass, or a cover glass. Shape; Examples include flat membrane shape.
The structure of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a porous structure, a mesh structure, an uneven structure, and a honeycomb structure.
Examples of the material of the base material include organic materials and inorganic materials. These may be used alone or in combination of two or more.
The organic material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI). , Nylon (Ny), low density polyethylene (LDPE), medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene terephthalate, polypropylene, urethane acrylate and other acrylic materials, cellulose, etc. Can be mentioned.
The inorganic material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include glass and ceramics.

-Solution containing fixing material A-
The solution containing the fixing material A contains the fixing material A and, if necessary, other components.

--Fixing material A--
The fixing material A is not particularly limited and may be appropriately selected depending on the intended purpose. For example, biological polymers such as collagen, elastin, gelatin and fibroin; coagulation factors such as fibrinogen; fibronectin, laminin, recombinant peptide and the like. Adhesion factors; polysaccharide compound metal salts such as alginic acid and gellan gum; synthetic polymers such as polylactic acid and the like. These may be used alone or in combination of two or more. Of these, coagulation factors are preferred, and fibrinogen is more preferred.

-Other ingredients-
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a medium, a cross-linking agent, a pH adjuster, a preservative, and an antioxidant.

The method for applying the fixing material A is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a pipette dispensing method using a micropipette or the like, a micromanipulator method, an aspirator method, or a droplet ejection method. (Inkjet method), gel extrusion method, transfer method such as screen printing method and the like can be mentioned.

<Fixing material B and cell-containing suspension applying step, and fixing material B and cell-containing suspension applying means>
The step of imparting the fixing material B and the cell-containing suspension is a step of imparting a suspension containing the fixing material B and cells that can be mixed with the fixing material A.
The fixing material B and the cell-containing suspension applying means are means for applying the fixing material B and the cell-containing suspension that can be mixed with the fixing material A.
The step of applying the fixing material B and the cell-containing suspension can be preferably carried out by the fixing material B and the cell-containing suspension applying means.
The inkjet method can be preferably used as the fixing material B and the cell-containing suspension applying step and the fixing material B and the cell-containing suspension applying means.

-Suspension containing fixing material B and cells that can be mixed with fixing material A-
The suspension containing the fixing material B and the cells that can be mixed with the fixing material A contains the fixing material B and the cells, and further contains other components as needed.

--Fixing material B--
The fixing material B is not particularly limited as long as it is a mixture of the fixing material A and the fixing material B to form a hydrogel, and can be appropriately selected depending on the intended purpose. The fixing material A and the fixing material B can be appropriately selected. However, it is preferable to thicken by mixing.

The fixing material B is not particularly limited as long as it is a mixture of the fixing material A and the fixing material B to form a hydrogel, and can be appropriately selected depending on the intended purpose. For example, polysaccharides and polyvalents. Examples include metal salts, fibrinogen, thrombin, fibronectin, laminin, recombinant peptides, chitosan, chitin and the like. These may be used alone or in combination of two or more. Among these, when the fixing material A is fibrinogen, thrombin is preferable as the fixing material B.

--Cell ---
The type of cell is not particularly limited and may be appropriately selected according to the purpose. Classificationally, for example, eukaryotic cell, prokaryotic cell, multicellular biological cell, single cell biological cell, etc. It can be used for all cells.
Examples of eukaryotic cells include animal cells, insect cells, plant cells, fungi and the like. These may be used alone or in combination of two or more. Among these, animal cells are preferable, and when the cells form a cell aggregate, the cells adhere to each other and have cell adhesion to such an extent that they cannot be isolated without physicochemical treatment. Sex cells are more preferred.

The adhesive cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include differentiated cells and undifferentiated cells.
The differentiated cells include, for example, hepatocytes, which are parenchymal cells of the liver; stellate cells; Kupper cells; vascular endothelial cells; endothelial cells such as analog path endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; bone crushed bones. Cells; root membrane-derived cells; epidermal cells such as epidermal keratinized cells; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; breast cells; pericite; smooth muscle cells, myocardium Muscle cells such as cells; renal cells; pancreatic Langerhans islet cells; peripheral nerve cells, nerve cells such as optic nerve cells; cartilage cells; bone cells and the like. The adhesive cells may be primary cells taken directly from a tissue or organ, or may be passaged from them for several generations.
The undifferentiated cells are not particularly limited and may be appropriately selected depending on the intended purpose. For example, embryonic stem cells which are undifferentiated cells, pluripotent stem cells such as pluripotent mesenchymal stem cells; Unipotent stem cells such as vascular endothelial precursor cells having monodifferentiation ability; iPS cells and the like can be mentioned.

Examples of prokaryotic cells include eubacteria and archaea.

The cells are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include normal human skin fibroblasts.
As the normal human skin fibroblast, a commercially available product can be used, and examples of the commercially available product include trade name: CC2507 (manufactured by Lonza).

-Other ingredients-
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a medium, a cross-linking agent, a pH adjuster, a preservative, and an antioxidant.

As the materials used in the fixing material A and the fixing material B, the materials listed in the fixing material A are used as the fixing material B in the fixing material B and the cell-containing suspension applying step, and the materials listed in the fixing material B are used. As the fixing material A, it may be used in the fixing material A-containing solution applying step, and can be appropriately replaced depending on the purpose.

As the means for imparting the fixing material B and the cell-containing suspension, the means for discharging the fixing material B and the cell-containing suspension as a suspension by a droplet ejection method (inkjet method) (hereinafter, also referred to as “discharging head”) may be used. Are) can be preferably used. However, the method of applying the fixing material B is not particularly limited, and any method may be used as long as it can be appropriately selected according to the purpose and the droplet (suspension) can be applied to a desired position. In addition to the inkjet method described in the above, examples thereof include a transfer method such as a micromanipulator method, a gel extrusion method, and a screen printing method. The inkjet method is a method in which droplets are continuously dropped from a nozzle.

Examples of the inkjet method include an on-demand method and a continuous method. Among these, in the case of the continuous method, empty discharge until a stable discharge state is achieved, the amount of droplets is adjusted, and droplets are continuously formed even when moving between each well of the well plate. For these reasons, the dead volume of the suspension used tends to increase. In the present invention, it is preferable to reduce the influence of dead volume from the viewpoint of adjusting the number of cells, and therefore, in the above two methods, the on-demand method is more preferable.

Examples of the on-demand method include a pressure application method in which a liquid is discharged by applying pressure to the liquid, a thermal method in which the liquid is discharged by boiling a film due to heating, and a droplet is formed by pulling a droplet by electrostatic attraction. A plurality of known methods such as an electrostatic method can be mentioned. Among these, the pressure application method is preferable for the following reasons.

In the electrostatic method, it is necessary to install an electrode facing the ejection portion that holds the suspension and forms a droplet. In the method for producing an evaluation base material of the present invention, the base materials for receiving the droplets are arranged so as to face each other, and it is preferable that the electrodes are not arranged in order to increase the degree of freedom in the composition of the base material.
Since the thermal method generates local heating, there is a concern about the influence on cells, which are biomaterials, and the scorching (cogation) on the heater part. Since the influence of heat depends on the content and the use of the base material, it is not necessary to exclude it unconditionally, but the pressure application method is preferable because there is no concern about burning to the heater portion as compared with the thermal method.

Examples of the pressure application method include a method of applying pressure to a liquid using a piezo element, a method of applying pressure by a valve such as a solenoid valve, and the like. 2A to 2C show configuration examples of a droplet generation device that can be used to eject a droplet of a suspension.
FIG. 2A is a schematic view showing an example of a solenoid valve type discharge head. The solenoid valve type discharge head includes an electric motor 13a, a solenoid valve 112, a liquid chamber wall 11a constituting a liquid chamber as a liquid accommodating portion, a suspension 300a, and a nozzle 111a.
As the solenoid valve type discharge head, for example, a dispenser manufactured by TechElan can be preferably used.
Further, FIG. 2B is a schematic diagram showing an example of a piezo type discharge head. The piezo type discharge head has a piezoelectric element 13b, a liquid chamber wall 11b constituting a liquid chamber as a liquid accommodating portion, a suspension 300b, and a nozzle 111b.
As the piezo type discharge head, a single cell printer manufactured by Cytena or the like can be preferably used.
Although any of these discharge heads can be used, the piezo method is used to increase the throughput of manufacturing the evaluation substrate because the pressure application method using a solenoid valve cannot form repeated droplets at high speed. It is preferable to use. Further, in the piezo type ejection head using the general piezoelectric element 13b, unevenness of cell concentration may occur due to sedimentation, or nozzle clogging may occur.
Therefore, as a more preferable configuration, the configuration shown in FIG. 2C and the like can be mentioned. FIG. 2C is a schematic view of a modified example of the piezo type discharge head using the piezoelectric element in FIG. 2B. The discharge head of FIG. 2C has a piezoelectric element 13c, a liquid chamber wall 11c constituting a liquid chamber as a liquid accommodating portion, a suspension 300c, and a nozzle 111c.
In the discharge head of FIG. 2C, by applying a voltage to the piezoelectric element 13c from a control device (not shown), compressive stress is applied in the lateral direction of the paper surface, and the membrane can be deformed in the vertical direction of the paper surface.

Examples of the method other than the on-demand method include a continuous method in which droplets are continuously formed. In the continuous method, when the droplet is pressurized and extruded from the nozzle, a piezoelectric element or a heater gives a periodic fluctuation, whereby minute droplets can be continuously produced. Further, by controlling the ejection direction of the flying droplet by applying a voltage, it is possible to select whether to land on the well or collect the droplet on the collecting unit. Such a method is used in a cell sorter or a flow cytometer, and for example, an apparatus name: Cell Sorter SH800 manufactured by Sony Corporation can be used.

FIG. 3A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. Further, FIG. 3B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 3A shows the drive voltage for forming a droplet. Voltage _{(V A, V B, V} C) by intensity, it is possible to form a droplet. FIG. 3B shows the voltage for stirring the suspension without ejecting the droplets.

It is possible to agitate the suspension in the liquid by inputting multiple pulses that are not strong enough to eject the droplets during the non-ejection period, and the concentration distribution due to cell sedimentation. The occurrence can be suppressed.

The droplet forming operation of the ejection head that can be used in the present invention will be described below.
The ejection head can eject droplets by applying a pulsed voltage to the upper and lower electrodes formed on the piezoelectric element. 4A to 4C are schematic views showing the state of the droplet at each timing. In FIG. 4A, first, when a voltage is applied to the piezoelectric element 13c, the membrane 12c is rapidly deformed, so that the liquid chamber wall 11c is held in the liquid chamber formed by the liquid chamber wall 11c and the membrane 12c on which the nozzle portion is formed. A high pressure is generated between the suspension and the membrane 12c, and this pressure pushes the droplets out of the nozzle. Next, as shown in FIG. 4B, the liquid is continuously extruded from the nozzle portion for a time until the pressure is relaxed upward, and the droplet grows. Finally, as shown in FIG. 4C, when the membrane 12c returns to its original state, the liquid pressure near the interface between the suspension and the membrane 12c decreases, and a droplet 310'is formed.

The droplet forming apparatus used in the present invention will be described with reference to FIGS. 5 to 7.
FIG. 5 is a schematic diagram showing an example of a droplet forming apparatus. As shown in FIG. 5, the droplet forming device 1A includes a ejection head (droplet ejection means) 10, a driving means 20, and a control means 70.

The discharge head 10 has a liquid chamber wall 11, a membrane 12, and a driving element 13, and can discharge the suspension 300 containing the fixing material B and the cells 350 as droplets.

The liquid chamber formed by the liquid chamber wall 11 and the membrane 12 on which the nozzle portion is formed is a liquid holding portion that holds the suspension 300 containing the fixing material B and the cells 350, and has a through hole on the lower surface side. Nozzle 111 is formed. The liquid chamber can be formed of, for example, metal, silicon, ceramics, or the like.

The membrane 12 is a film-like member fixed to the upper end of the liquid chamber wall 11. The planar shape of the membrane 12 may be, for example, circular, but may be elliptical, quadrangular, or the like.

The drive element 13 is provided on the upper surface side of the membrane 12. The shape of the drive element 13 can be designed according to the shape of the membrane 12. For example, when the planar shape of the membrane 12 is circular, it is preferable to provide a circular driving element 13.

The membrane 12 can be vibrated by supplying a drive signal from the drive means 20 to the drive element 13. The vibration of the membrane 12 allows the droplets containing the cells 350 to be ejected from the nozzle 111.

When a piezoelectric element is used as the driving element 13, for example, a structure may be provided in which electrodes for applying a voltage are provided on the upper surface and the lower surface of the piezoelectric material. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means 20, compressive stress is applied in the lateral direction of the paper surface, and the membrane 12 can be vibrated in the vertical direction of the paper surface. As the piezoelectric material, for example, lead zirconate titanate (PZT) can be used. In addition to this, various piezoelectric materials such as bismuth iron oxide, metal niobate, barium titanate, or these materials to which a metal or a different oxide is added can be used.

The control means 70 has a function of controlling the drive means 20. Hereinafter, the operation of the droplet forming apparatus 1A including the operation of the control means 70 will be described with reference to FIG.

FIG. 6 is a diagram illustrating a hardware block of the control means of FIG.

As shown in FIG. 6, the control means 70 includes a CPU 71, a ROM 72, a RAM 73, an I / F 74, and a bus line 75. The CPU 71, ROM 72, RAM 73, and I / F 74 are connected to each other via a bus line 75.

The CPU 71 controls each function of the control means 70. The ROM 72, which is a storage means, stores a program executed by the CPU 71 to control each function of the control means 70 and various information. The RAM 73, which is a storage means, is used as a work area or the like of the CPU 71. Further, the RAM 73 can temporarily store predetermined information. The I / F 74 is an interface for connecting the droplet forming device 1A to another device or the like. The droplet forming apparatus 1A may be connected to an external network or the like via the I / F 74.

FIG. 7 is a schematic view showing another modification of the discharge head of FIG. As shown in FIG. 7, the droplet forming apparatus 1B differs from the droplet forming apparatus 1A (see FIG. 5) in that the droplet ejection means 10 is replaced with the droplet ejection means 10B. It should be noted that the description of the same component as that of the embodiment already described may be omitted.
The droplet ejection means 10B has a liquid chamber wall 11B, a membrane 12B, and a driving element 13B. The liquid chamber composed of the liquid chamber wall 11B has an atmospheric opening portion 115 that opens the liquid chamber to the atmosphere at the upper part, and is configured so that air bubbles mixed in the suspension 300 can be discharged from the atmospheric opening portion 115. Has been done.

The membrane 12B is a film-like member fixed to the lower end of the liquid chamber composed of the liquid chamber wall 11B. A nozzle 121, which is a through hole, is formed at substantially the center of the membrane 12B, and the suspension 300 held in the liquid chamber is discharged as droplets from the nozzle 121 by the vibration of the membrane 12B. Since droplets are formed by the inertia of the vibration of the membrane 12B, even a suspension 300 having a high surface tension (high viscosity) can be discharged. The planar shape of the membrane 12B may be, for example, circular, but may be elliptical, quadrangular, or the like.

The material of the membrane 12B is not particularly limited, but if it is too soft, the membrane 12B easily vibrates and it is difficult to immediately suppress the vibration when it is not discharged. Therefore, it is preferable to use a material having a certain degree of hardness. .. As the material of the membrane 12B, for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.

In particular, when using cells, it is preferable that the material has low adhesion to cells and proteins. It is generally said that the cell adhesion depends on the contact angle of the material with water, and when the material has high hydrophilicity or hydrophobicity, the cell adhesion is low. Various metal materials and ceramics (metal oxides) can be used as the material having high hydrophilicity, and fluororesin or the like can be used as the material having high hydrophobicity.

Other examples of such materials include, for example, stainless steel, nickel, aluminum and the like, silicon dioxide, alumina, zirconia and the like. In addition to these, it is also conceivable to reduce cell adhesion by coating the surface of the material. For example, the surface of the material can be coated with the above-mentioned metal or metal oxide material, or with a synthetic phospholipid polymer imitating a cell membrane (for example, Lipidure manufactured by NOF CORPORATION).

It is preferable that the nozzle 121 is formed as a substantially circular through hole in the substantially center of the membrane 12B. In this case, the diameter of the nozzle 121 is not particularly limited, but it is preferably twice or more the size of the cell 350 in order to prevent the cells 350 from being clogged with the nozzle 121. When the cell 350 is, for example, an animal cell, particularly a human cell, the size of the human cell is generally about 5 μm to 50 μm, so that the diameter of the nozzle 121 should be 10 μm or more according to the cell to be used. It is preferably 100 μm or more, more preferably 100 μm or more.

On the other hand, the diameter of the nozzle 121 is preferably 200 μm or less. That is, in the droplet ejection means 10B, the diameter of the nozzle 121 is typically in the range of 10 μm to 200 μm.

The drive element 13B is formed on the lower surface side of the membrane 12B. The shape of the drive element 13B can be designed according to the shape of the membrane 12B. For example, when the planar shape of the membrane 12B is circular, it is preferable to form a driving element 13B having an annular (ring-shaped) planar shape around the nozzle 121. The drive system of the drive element 13B can be the same as that of the drive element 13.

The driving means 20 selectively (for example, alternately) imparts an ejection waveform that vibrates the membrane 12B to form a droplet and a stirring waveform that vibrates the membrane 12B within a range that does not form a droplet. can do.

For example, by making the discharge waveform and the stirring waveform both rectangular waves and lowering the driving voltage of the stirring waveform to be lower than the driving voltage of the discharging waveform, it is possible to prevent the formation of droplets by applying the stirring waveform. That is, the vibration state (degree of vibration) of the membrane 12B can be controlled by adjusting the driving voltage.

In the droplet ejection means 10B, since the drive element 13B is formed on the lower surface side of the membrane 12B, when the membrane 12 vibrates due to the drive element 13B, it is possible to generate a flow from the lower side to the upper side of the liquid chamber. Is.

At this time, the movement of the cells 350 becomes a movement from the bottom to the top, convection occurs in the liquid chamber, and the suspension 300 containing the fixing material B and the cells 350 is agitated. Due to the flow from the lower part to the upper part of the liquid chamber, the settled and aggregated cells 350 are uniformly dispersed inside the liquid chamber.

That is, the driving means 20 applies the discharge waveform to the driving element 13B and controls the vibration state of the membrane 12B to transfer the suspension 300 containing the fixing material B and the cells 350 held in the liquid chamber from the nozzle 121. It can be ejected as a droplet. Further, the driving means 20 agitates the suspension 300 containing the fixing material B and the cells 350 held in the liquid chamber by adding the stirring waveform to the driving element 13B and controlling the vibration state of the membrane 12B. Can be done. During stirring, no droplets are ejected from the nozzle 121.

In this way, by stirring the suspension 300 containing the fixing material B and the cells 350 while the droplets are not formed, the cells 350 are prevented from settling and aggregating on the membrane 12B, and the cells 350 are prevented from being settled and aggregated. Can be evenly dispersed in the suspension 300 containing the fixing material B. This makes it possible to suppress the clogging of the nozzle 121. As a result, the suspension 300 containing the fixing material B and the cells 350 can be continuously and stably discharged as droplets for a long period of time.

In addition, air bubbles may be mixed in the suspension 300 containing the fixing material B and the cells 350. Even in this case, since the discharge head 10B is provided with the air opening portion 115 at the upper part of the liquid chamber, the air bubbles mixed in the suspension 300 containing the fixing material B and the cells 350 can be removed from the air opening portion 115. It can be discharged to the outside air through. This makes it possible to continuously and stably form droplets without discarding a large amount of liquid for discharging bubbles.

That is, when bubbles are mixed in the vicinity of the nozzle 121 or when a large number of bubbles are mixed on the membrane 12B, the ejection state is affected. Therefore, in order to stably form the droplets for a long time, it is necessary to form the droplets stably. It is necessary to discharge the mixed air bubbles. Normally, the air bubbles mixed on the membrane 12B move upward naturally or by the vibration of the membrane 12B, but since the liquid chamber is provided with the air opening portion 115, the mixed air bubbles are discharged from the air opening portion 115. It will be possible. Therefore, it is possible to prevent non-ejection even if air bubbles are mixed in the liquid chamber, and it is possible to continuously and stably form droplets.

The membrane 12B may be vibrated at a timing when the droplets are not formed within a range where the droplets are not formed, and the bubbles may be positively moved above the liquid chamber.

-Hydrogel-
The hydrogel is formed by mixing (reacting) at least a part of each of the fixing material A and the fixing material B.
Around the cell, at least a part of the fixing material A and the fixing material B may be mixed to form a hydrogel, and the fixing material A and the fixing material B coexist in addition to the hydrogel. It may be in the state of being.
The hydrogel immobilizes the cells on the substrate on the substrate.
The hydrogel preferably covers at least a portion of the cell, more preferably the entire cell. The hydrogel can prevent the cells from drying out by covering at least a part of the cells.

<Step of applying a solution containing at least one of the fixing material A and the fixing material B, and means for applying the solution containing at least one of the fixing material A and the fixing material B>
The step of applying the solution containing at least one of the fixing material A and the fixing material B is the time when the cells partially coated with the solution containing at least one of the fixing material A and the fixing material B are dried. When it reaches, it is a step of applying a solution containing at least one of the fixing material A and the fixing material B.
The means for applying the solution containing at least one of the fixing material A and the fixing material B is at the time when the cells partially coated with the solution containing at least one of the fixing material A and the fixing material B are dried. When it reaches, it is a means for applying a solution containing at least one of the fixing material A and the fixing material B.
The step of applying the solution containing at least one of the fixing material A and the fixing material B can be preferably carried out by means for applying the solution containing at least one of the fixing material A and the fixing material B.
The step of applying the solution containing at least one of the fixing material A and the fixing material B is carried out when at least a part of the surface of the cells applied on the substrate is exposed (uncoated). It is preferable to do so. By applying a solution containing at least one of the fixing material A and the fixing material B, drying of the cells can be prevented.
As a solution containing at least one of the fixing material A and the fixing material B, it is preferable to apply the solution to the surface of cells at least partially exposed.
Examples of the time for the cells partially coated with the solution containing at least one of the fixing material A and the fixing material B to dry include, for example, obtaining the drying time of the cells in advance by an experiment.

As the material to be applied to the uncoated cells, at least one of the fixing material A and the fixing material B is preferable, and both the fixing material A and the fixing material B are applied to form at least a part of the hydrogel. Is more preferable, and it is particularly preferable to completely form a hydrogel.
In addition to the solution containing at least one of the fixing material A and the fixing material B, there is no particular limitation as long as the exposed cells can be coated from the viewpoint of preventing the cells from drying out, and an appropriate selection can be made according to the purpose. For example, a medium or the like can be used.

<Step of applying the medium and means for applying the medium>
The step of applying the medium is a step of applying the medium on the hydrogel after the cells are fixed to the substrate via the hydrogel.
The step of applying the medium can be preferably carried out by means for applying the medium.

The means for applying the medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a pipette dispensing method using a micropipette, a micromanipulator method, an aspirator method, an inkjet method, a screen printing method, etc. Etc., such as a transfer method.

-Medium-
The medium is a solution containing components necessary for the formation and maintenance of tissues, preventing drying, and adjusting the external environment such as osmotic pressure, and those known in the art can be appropriately selected. If it is not necessary to keep it in the medium solution at all times, it may be removed as appropriate.

The medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a medium classified according to the composition of a natural medium, a semi-synthetic medium, a synthetic medium, etc .; a semi-solid medium, a liquid medium, a powder medium ( Hereinafter, a medium classified according to the shape such as (sometimes referred to as “powder medium”) and the like can be mentioned. These may be used alone or in combination of two or more. When the cells are of animal origin, any medium used for culturing animal cells can be used.

The medium used for culturing animal cells is not particularly limited and may be appropriately selected depending on the intended purpose. For example, Dulvecco's Modified Eagles's Medium (D-MEM), Ham F12 Medium (Ham's Nutrition Mixture F12), D-MEM / F12 medium, McCoy's 5A medium, Eagle's Medium Essential Medium (EMEM), αMEM medium (EMEM), αMEM medium. Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI1640 medium, Iskov's Modified Dulvecco's Medium (IMDM) medium E StemPro34 (Invigen), X-VIVO 10 (Kembrex), X-VIVO 15 (Kembrex), HPGM (Kembrex), StemSpan H3000 (Stemcell Technology), StemSpanSMEM (Stemcell Technology) , StemlineII (manufactured by Sigma Aldrich), QBSF-60 (manufactured by Quality Biological), StemProhESCSFM (manufactured by Invitrogen), Essential8 (registered trademark) medium (manufactured by Gibco), mTeSR1 or 2 medium (manufactured by Stemcell Technology). , Repro FF or Repro FF2 (manufactured by Reprocell), PSGro hESC / iPSC medium (manufactured by System Bioscience), NutriStem (registered trademark) medium (manufactured by Bionic Industries), CSTI-7 medium (manufactured by Cell Science Laboratory). , MesenPRO RS medium (Gibco), MF-Media (registered trademark) Membranous stem cell growth medium (Toyo Spinning Co., Ltd.), Sf-900II (Invigen), Opti-Pro (Invigen) And so on. These may be used alone or in combination of two or more.

The carbon dioxide concentration in the medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2% or more and 5% or less, and more preferably 3% or more and 4% or less. When the carbon dioxide concentration is 2% or more and 5% or less, cells can be suitably cultured.

Here, the method for producing the evaluation base material of the present invention will be described with reference to FIGS. 8A to 8C. FIG. 8A is a schematic view showing an example of a fixing material A-containing solution applying step. FIG. 8B is a schematic view showing an example of the fixing material B and the cell-containing suspension applying step. FIG. 8C is a schematic view showing an example of a step of applying a medium.

As shown in FIG. 8A, the solution 21 containing the fixing material A is applied to the base material 20 by the micropipette 22. Next, as shown in FIG. 8B, the suspension containing the fixing material B and the cells 26 is discharged as droplets 24 by the inkjet method using the inkjet head 23, and the solution containing the fixing material A is discharged. It is applied to the base material 20 containing 21. By contacting the suspension containing the fixing material B and the cells 26 with the solution 21 containing the fixing material A, the fixing material A and the fixing material B react with each other to form the hydrogel 25. An evaluation base material in which cells 26 are adhered to the base material 20 can be produced via the formed hydrogel 25. Next, as shown in FIG. 8C, the medium 27 can be dropped onto the hydrogel 25 containing the cells 26 by a micropipette 22 to prevent the hydrogel 25 from drying out and to prepare for evaluation.

Next, a case will be described using FIGS. 9A to 9D to include a step of applying a solution containing at least one of the fixing material A and the fixing material B in the method for producing the evaluation base material of the present invention. FIG. 9A is a schematic view showing an example of the fixing material A-containing solution applying step. FIG. 9B is a schematic view showing an example of the fixing material B and the cell-containing suspension applying step. FIG. 9C is a schematic view showing an example of a step of applying a solution containing at least one of the fixing material A and the fixing material B. FIG. 9D is a schematic view showing an example of a step of applying a medium.

First, as shown in FIG. 9A, the solution 21 containing the fixing material A is applied to the base material 20 by the micropipette 22. Next, as shown in FIG. 9B, the suspension containing the fixing material B and the cells 26 is discharged as droplets 24 by the inkjet method using the inkjet head 23, and the solution containing the fixing material A is discharged. It is applied to the base material 20 containing 21. In this case, by contacting the suspension containing the fixing material B and the cells 26 with the solution 21 containing the fixing material A, at least a part of the fixing material A and the fixing material B is mixed, and the hydrogel 28 is mixed. Is formed, but a portion of the surface of the cell 26 is exposed from the hydrogel 28.
Therefore, as shown in FIG. 9C, a solution containing at least one of the fixing material A and the fixing material B is jetted from the inkjet head 23'with the droplet 24'on the cells (not coated with the hydrogel). It is discharged by the method and applied to the surface (not coated with hydrogel) of the cell 26 to form a fixing material A, a fixing material B, a hydrogel, or a coating film 24 ”in which these are mixed. Therefore, it is possible to prevent the surface of the cell 26 that is not covered with the hydrogel from drying. Regarding the prevention of the drying of the cell 26, for example, the drying time of the cell 26 is obtained in advance by an experiment, and the drying time thereof is obtained. Based on the drying time obtained from the experimental data, there is a method of preventing the cells from drying by discharging the fixing material B (or the fixing material A) to the corresponding cells at an appropriate time interval (timing). It can be considered as an example.
Next, as shown in FIG. 9D, the medium 27 can be dropped onto the hydrogel 28 containing the cells 26 by a micropipette 22 to prevent the hydrogel 28 from drying out and to prepare for evaluation.

As the evaluation base material of the present invention, it can be suitably used for evaluation of drug efficacy or toxicity.

When the base material for evaluation of the present invention is used for evaluation of drug efficacy or toxicity, the evaluation is performed by using a medium shown in FIGS. 8C and 9D to which a reagent for evaluating drug efficacy or toxicity is added in advance. It can be carried out.

The reagent for evaluating the medicinal efficacy is not particularly limited and may be appropriately selected depending on the intended purpose. For example, oxazolone, benzoquinone, 2,4-dinitrocorobenzine, 4-phenylenediamine, glutaraldehyde, benzoyl peroxide, etc. 4-Methylaminophenol sulfate, formaldehyde, cinnamaldehyde, ethylenediamine, 2-hydroxyethyl acrylate, isooigenol, nickel sulfate (II), benzylideneacetone, methyl 2-nonate, benzyl sartylate, diethylenetriamine, thioglycerol, 2 -Mercaptobenzothiazole, phenylacetaldehyde, hexylcinnamaldehyde, dihydroeugenol, benzoisothiazolione, citral, resorcinol, phenyl benzoate, eugenol, avietic acid, ethyl aminobenzoate, benzyl cinnamate, cinnamyl alcohol, hydroxycitronellal , Imidazolidinyl urea, butyl glycidyl ether, ethylene glycol dimethacrylate, glyoxal, 4-nitrobenzyl bromide and the like.
The reagent for evaluating toxicity is not particularly limited and may be appropriately selected depending on the intended purpose. For example, zinc chloride, 1-butanol, benzoic acid, ethyl vanillin, 4-hydroxybenzoic acid, sulfanilic acid, tartrate acid. , Methyl salicylate, salicylic acid, sodium lauryl sulfate, lactic acid, benzyl alcohol, dextran, diethylphthalate, glycerol, propylparaben, Tween80, dimethylisophthalate, phenol, chlorobenzene, sulfanilic acid, octanoic acid and the like.

When a flat plate-shaped base material such as a glass plate, a slide glass, or a cover glass is used as the evaluation base material of the present invention, the evaluation base material is immersed in another container to which a medium or the like is added, and hydrolyzed. It is also possible to culture cells immobilized via a gel.

Hereinafter, examples and comparative examples of the present invention will be described, but the present invention is not limited to these examples.

(Example 1)
<Preparation of solution containing fixing material A>
Fibrinogen (trade name: Fibrinogen from bovine plasma, manufactured by Sigma-Aldrich, Sigma-Aldrich, Sigma-Aldrich) is added to 1 mL of Dulpco's Solution Buffered Salone (trade name: DPBS (1 ×), manufactured by Life technologies). A / mL fibrinogen aqueous solution (solution containing fixing material A) was prepared.

<Preparation example of suspension containing fixing material B and cells>
Commercially available normal human skin fibroblasts (trade name: CC2507, manufactured by Lonza, hereinafter also referred to as "NHDF") 2 × 10 ⁴ cells / mL, Dulbecco's Modified Eagle Medium (trade name: DMEM (trade name: DMEM) 1 ×), manufactured by Life technologies), added to a 100 mm dish containing 10 mL, and cultured in an incubator (device name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment) for 72 hours. The culture medium containing the cultured normal human skin fibroblasts was removed, and 2 mL of Dulbeccoline acid buffered saline (hereinafter, also referred to as “DPBS”) was added and washed. Remove DPBS, add 2 mL of trypsin (trade name: 0.05% Trypsin-EDTA (1 ×), manufactured by Life technologies), _{and trypsin treatment in an incubator (37 ° C, 5% CO 2} environment) for 5 minutes. The cells were isolated.
The isolated cells were added to 20 U / mL of thrombin (trade name: Thrombin from bovine Plasma, manufactured by Sigma-Aldrich) so that the number of cells was 4 × 10 ⁶ cells / mL, and the fixing material B was added. And a suspension containing cells was obtained.

A 25 mg / mL fibrinogen aqueous solution (solution containing fixing material A) using a micropipette in each well of a 96-well plate (trade name: 96-well cell culture plate, flat-bottomed low-evaporation type, polystyrene with lid, manufactured by Corning). 10 μL was added (see FIG. 9A).

Next, the suspension containing the fixing material B and the cells was arranged using an inkjet bioprinter (device name: cell-compatible inkjet device, self-developed product) so that the cells did not overlap 96. Discharge onto the wells of the plate (see FIG. 9B). The fixing material A and the fixing material B were mixed to form a hydrogel, and the cells were allowed to stand in a _{CO 2 incubator at 37 ° C. until the cells were fixed.}

In a 96-well plate on which cells are immobilized, cells whose cells are not coated with hydrogel are coated with thrombin 20 U / mL by applying 20 U / mL to the cell surface using an inkjet bioprinter (see FIG. 9C). ), A substrate for evaluation was obtained. The obtained evaluation substrate was observed using an optical microscope (device name: CKX53, manufactured by Olympus Corporation). As a result, it was confirmed that the cells were arranged in one layer. The state of cells observed with an optical microscope is shown in FIG.

(Comparative Example 1)
In Example 1, a solution containing the fixing material A was obtained in the same manner as in Example 1 except that fibrinogen (fixing material A) was not added.
Next, in Example 1, an evaluation base material was obtained in the same manner as in Example 1 except that the solution containing the fixing material A was changed to the solution not containing the fixing material A.

(Comparative Example 2)
In Example 1, a suspension containing cells was obtained in the same manner as in Example 1 except that thrombin (fixing material B) was not added to the suspension containing the fixing material B and the cells. ..
Next, in Example 1, the evaluation was carried out in the same manner as in Example 1 except that the suspension containing the fixing material B and the cells was changed to the suspension containing the cells (not containing the fixing material B). A substrate for use was obtained.

Using the obtained evaluation substrate, the hydrogel formation time was measured as follows.
In the preparation of the base material for evaluation, the suspension was added to the solution and then allowed to stand in a _{CO 2 incubator at 37 ° C.} Then, the 96-well plate was shaken by hand, and the time until the hydrogel was formed was measured by visually confirming that the cells were fixed and did not move. The results are shown in Table 1 below.

As can be seen from Table 1, in Example 1, it was confirmed that the hydrogel was formed (thickened) in 9 minutes and the cells adhered to the 96-well plate (base material) via the hydrogel, and in a short time, It was found that a base material for evaluation can be produced.
On the other hand, in Comparative Examples 1 and 2, hydrogels were not formed and cells were not fixed.

(Comparative Example 3)
Dulvecco's Modified Eagle's Medium (trade name: DMEM (1X), manufactured by Life technologies) 10 mL container (trade name: Tissue Culture Dsch, manufactured by Asahi Glass Co., Ltd.), NHDF (trade name: CC2507, L) (Manufactured) was added so as to be 5 × 10 ⁴ cells / mL, and the cells were cultured in an incubator (device name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment) for 10 hours. After 0 hours, 3 hours, and 10 hours of culturing, cells were observed using an optical microscope (device name: CKX53, manufactured by Olympus Corporation). The results are shown in FIGS. 11A (after 0 hours), FIG. 11B (after 3 hours), and FIG. 11C (after 10 hours). From the results of FIGS. 11A to 11C, it can be seen that the cells did not adhere to the substrate after 0 hours and 3 hours of culture, but almost all the cells adhered to the substrate after 10 hours. I understand.

Using the evaluation base material of Example 1 and the evaluation base material of Comparative Example 3, the "cell survival rate", "cell membrane damage rate", and "secretion amount of inflammatory substance" were evaluated as follows. did.

(Cell survival rate)
Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd.) in which zinc chloride under four concentration conditions of 0 μM, 100 μM, 160 μM, and 220 μM was added to the evaluation base materials of Examples 1 and 3 as test substances, respectively. , Hereinafter referred to as “D-MEM”) was added by a micropipette in an amount of 100 μL, and the mixture was allowed to stand in a _{CO 2 incubator at 37 ° C. overnight (20 hours).}
Next, in order to evaluate the effect of zinc chloride on cell viability, 10 μL of WST-1 reagent (trade name: Premix WST-1 Cell Proliferation Assay System, manufactured by Takara Bio Inc.) was added per well for 1 hour. The reaction was allowed to stand in _{a CO 2} incubator at 37 ° C. Then, using a plate reader (device name: Cytation5, manufactured by BioTek), the absorbance at 450 nm and 570 nm as a reference value was measured to evaluate the “cell viability”. The results are shown in FIG.

(Cell membrane damage rate)
D-MEM supplemented with zinc chloride was added in the same manner as in the evaluation of cell viability, and the cells were allowed to stand in a _{CO 2 incubator at 37 ° C. overnight (20 hours).}
In order to evaluate the effect of zinc chloride on cell membrane damage, only the medium was collected using a micropipette, placed in a 96-well plate, and an LDH measurement reagent (trade name: Cytotoxicity Detection Kit, manufactured by Roche) was added. Then, the reaction was carried out at room temperature (23 ° C.) for 5 minutes. Then, the absorbance at 492 nm and 620 nm as a reference value was measured using a plate reader to evaluate the “cell membrane damage rate”. The results are shown in FIG.

(Amount of inflammatory substance secreted)
D-MEM supplemented with zinc chloride was added in the same manner as in the evaluation of cell viability, and the cells were allowed to stand in a _{CO 2 incubator at 37 ° C. overnight (20 hours).}
In order to evaluate the effect of zinc chloride on the amount of inflammatory substances secreted, only the medium was collected using a micropipette. Using a protein measurement kit (trade name: Human IL-8 ELISA Ready-SET-Go! (Registered trademark), manufactured by Affymetrix), the amount of IL-8 secreted by the ELISA method was 450 nm using a plate reader. Absorbance at 570 nm was measured as a reference value. The results are shown in FIG.

As can be seen from the results of FIGS. 12 to 14, in the evaluation base material of Example 1 and the evaluation base material of Comparative Example 3, the cell viability, the cell membrane damage rate, and the secretion of inflammatory substances with respect to zinc chloride. The amounts showed similar trends in toxic reactions. From these results, it can be seen that the evaluation base material of the present invention is effective for evaluating the toxic reaction.

Examples of aspects of the present invention are as follows.
<1> A fixing material A-containing solution applying step of applying a fixing material A-containing solution to the base material,
A fixing material B and a cell-containing suspension applying step of imparting a suspension containing a fixing material B and cells that can be mixed with the fixing material A are included.
At least a part of each of the fixing material A and the fixing material B is mixed to form a hydrogel.
A method for producing an evaluation base material, wherein the cells are fixed to the base material via the hydrogel.
<2> The method for producing an evaluation base material according to <1>, wherein at least a part of the cells is coated with the hydrogel.
<3> The method for producing an evaluation base material according to any one of <1> to <2>, wherein the fixing material A and the fixing material B are thickened by mixing.
<4> When the time is reached for the cells partially coated with the solution containing at least one of the fixing material A and the fixing material B to dry, at least the fixing material A and the fixing material B are at least. The method for producing an evaluation base material according to any one of <1> to <3>, further comprising a step of applying a solution containing any of the above.
<5> The method for producing an evaluation base material according to any one of <1> to <4>, wherein the evaluation base material is immersed in a separate container and cells fixed via the hydrogel are cultured. be.
<6> The above-mentioned <1> to <5>, wherein the fixing material A is at least one selected from a biological polymer, a coagulation factor, an adhesion factor, a polysaccharide compound metal salt, and a synthetic polymer. This is a method for manufacturing a base material for evaluation.
<7> The method for producing an evaluation base material according to <6>, wherein the biological polymer is at least one selected from collagen, elastin, gelatin, and fibroin.
<8> The method for producing an evaluation base material according to any one of <6> to <7>, wherein the coagulation factor is fibrinogen.
<9> The method for producing an evaluation base material according to any one of <6> to <8>, wherein the adhesion factor is at least one selected from fibronectin, laminin, and a recombinant peptide.
<10> The method for producing an evaluation base material according to any one of <6> to <9>, wherein the polysaccharide compound metal salt is at least one of alginic acid and gellan gum.
<11> Of the above <1> to <10>, wherein the fixing material B is at least one selected from polysaccharides, polyvalent metal salts, fibronectin, thrombin, fibronectin, laminin, recombinant peptides, chitosan, and chitin. It is the method for manufacturing the evaluation base material according to any one.
<12> The method for producing an evaluation base material according to <11>, wherein the fixing material B is thrombin.
<13> The method for producing an evaluation base material according to any one of <1> to <12>, which further comprises a step of applying a medium.
<14> Base material and
A fixing material A-containing solution applying means for applying a solution containing the fixing material A to the base material,
It has a fixing material B that can be mixed with the fixing material A and a fixing material B that imparts a suspension containing cells, and a cell-containing suspension applying means.
Production of an evaluation base material, wherein the cells are fixed to the base material via a hydrogel formed by mixing at least a part of each of the fixing material A and the fixing material B. It is a device.
<15> The apparatus for producing an evaluation base material according to <14>, wherein the fixing material A is at least one selected from a biological polymer, a coagulation factor, an adhesion factor, a polysaccharide compound metal salt, and a synthetic polymer. Is.
<16> The fixing material A is the evaluation base material manufacturing apparatus according to <15>, which is a coagulation factor.
<17> The device for producing an evaluation base material according to <16>, wherein the coagulation factor is fibrinogen.
<18> The evaluation base material manufacturing apparatus according to any one of <14> to <17>, wherein the fixing material B is thrombin.
<19> The device for producing an evaluation base material according to any one of <14> to <18>, further comprising a means for applying a medium.
<20> The device for producing an evaluation base material according to any one of <14> to <19>, wherein the cell is a normal human skin fibroblast.

The evaluation base material manufacturing method according to any one of <1> to <13> and the evaluation base material manufacturing apparatus according to any one of <14> to <20> are conventionally described above. The problem can be solved and the object of the present invention can be achieved.

Japanese Unexamined Patent Publication No. 2002-355026 Japanese Unexamined Patent Publication No. 2005-269902

2,20 Base material 3,25,28 Hydrogel 4,26 cells 22 Fixing material A-containing solution applying means 23 Fixing material B and cell-containing suspension applying means

Claims (6)

A fixing material A-containing solution applying step of applying a solution containing the fixing material A to the base material, and a step of applying the fixing material A-containing solution.
A fixing material B and a cell-containing suspension applying step of imparting a suspension containing a fixing material B and cells that can be mixed with the fixing material A are included.
At least a part of each of the fixing material A and the fixing material B is mixed to form a hydrogel.
The cells are fixed to the substrate via the hydrogel and
The suspension method of producing a base material for evaluating, wherein Rukoto granted as droplets to be placed so that the cells do not overlap. The method for producing an evaluation base material according to claim 1, wherein at least a part of the cells is coated with the hydrogel. The method for producing an evaluation base material according to any one of claims 1 to 2, wherein the fixing material A and the fixing material B are thickened by mixing. When the time is reached for the cells partially coated with the solution containing at least one of the fixing material A and the fixing material B to dry, at least one of the fixing material A and the fixing material B is applied. The method for producing an evaluation base material according to any one of claims 1 to 3, further comprising a step of applying the contained solution. The method for producing an evaluation base material according to any one of claims 1 to 4, wherein the evaluation base material is immersed in a separate container and cells fixed via the hydrogel are cultured. With the base material
A fixing material A-containing solution applying means for applying a solution containing the fixing material A to the base material,
It has a fixing material B that can be mixed with the fixing material A and a fixing material B that imparts a suspension containing cells, and a cell-containing suspension applying means.
The cells are fixed to the substrate via a hydrogel formed by mixing at least a part of the fixing material A and the fixing material B so that the cells do not overlap with the suspension. apparatus for manufacturing a base material for evaluating characterized that you grant as droplets to be placed.

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