WO2023145781A1 - Cell culture method, screening method using cell culture method, culture device and culture kit - Google Patents

Cell culture method, screening method using cell culture method, culture device and culture kit Download PDF

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Publication number
WO2023145781A1
WO2023145781A1 PCT/JP2023/002304 JP2023002304W WO2023145781A1 WO 2023145781 A1 WO2023145781 A1 WO 2023145781A1 JP 2023002304 W JP2023002304 W JP 2023002304W WO 2023145781 A1 WO2023145781 A1 WO 2023145781A1
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Prior art keywords
culture
substrate
base material
container
cells
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PCT/JP2023/002304
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French (fr)
Japanese (ja)
Inventor
誠一郎 石原
永 芳賀
すみれ 石原
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国立大学法人北海道大学
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Publication of WO2023145781A1 publication Critical patent/WO2023145781A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a cell culture method, a screening method, a culture apparatus and a culture kit using the cell culture method.
  • the perfusion culture device described in Patent Document 1 is one example.
  • This device is configured as follows. Cells and a substrate are mixed and housed in a container whose bottom is made of a membrane, and a culture solution is housed on the substrate.
  • the container is placed in another container containing a medium, such that the membrane is in contact with the medium in the other container. Then, by sealing the contained inner container and pressurizing the inside of the inner container by supplying high-pressure gas, the culture solution in the inner container permeates into the substrate.
  • the culture solution in the inner container permeates into the substrate.
  • the culture solution can be supplied to the inside of the substrate. Therefore, it becomes easier to perform three-dimensional cell culture.
  • Patent Document 1 has the following problems.
  • the substrate inside the inner container is deformed by the pressure. If the substrate is deformed, the cells may not be properly retained in the substrate, and the culture may not be stably continued.
  • the substrate even if an attempt is made to suppress the deformation of the base material by adjusting the amount of pressure applied to the inside of the inner container, there is a limit to lowering the pressure inside the container due to the structure of the device that pressurizes the inside of the container using high-pressure gas. There is Therefore, it is difficult to suppress the deformation of the base material. If the substrate is deformed, the three-dimensional structure in the thickness direction cannot be maintained, and there is a possibility that the environment inside the tissue cannot be reproduced.
  • An object of the present invention is to provide a cell culture method that enables three-dimensional culture by suppressing deformation of the substrate, a screening method using the same, a cell culture apparatus, and a culture kit.
  • the culturing method of the present invention is a method for culturing cells in a substrate, comprising: a first housing compartment for housing a first culture medium so as to contact the substrate from above; and a second containing compartment containing a second culture medium so as to be in contact with each other, wherein the first and second At least one of the two culture solutions is permeated into the substrate.
  • a culture apparatus includes a substrate in which cells are embedded, a first storage compartment that stores a first culture medium so as to contact the substrate from above, and and a second storage compartment that stores a second culture medium so as to be in contact with each other, and by creating a water level difference between the first culture medium and the second culture medium, the first and At least one of the second culture fluids is permeated into the substrate.
  • a culture kit comprises a substrate in which cells are embedded, a first accommodation compartment that accommodates a first culture medium so as to contact the substrate from above, and and a second storage compartment that stores a second culture medium so as to be in contact with each other, and by creating a water level difference between the first culture medium and the second culture medium, the first and At least one of the second culture fluids is permeated into the substrate.
  • a pressure difference is generated between the first culture solution and the second culture solution due to the water level difference, thereby permeating the culture solution into the substrate. Since the pressure difference can be adjusted by adjusting the water level difference, it is easy to adjust the pressure applied to the base material, and to easily maintain a size that can suppress the deformation of the base material. Therefore, three-dimensional culture is possible.
  • the apparatus comprises a container having side walls and a bottom wall defining the first containing compartment, and a through hole in the bottom wall for communicating the first containing compartment and the second containing compartment. It is preferred that holes are formed.
  • a first containing compartment is defined by a container having side walls and a bottom wall. Therefore, the substrate can be supported by the bottom wall.
  • the water level difference is within a range in which the substrate is not deformed. According to this, since the substrate is not deformed, stable culture is ensured. It should be noted that the length of the period during which the base material is not deformed is assumed to be several hours to several days, for example.
  • the thickness of the substrate in the vertical direction exceeds 0.2 mm.
  • the thickness of the substrate for culturing cells is at most about 0.1 to 0.2 mm.
  • the amount of cells that can be cultured per device can be increased by increasing the thickness of the base material, it becomes easier to detect the expression of cell functions with high sensitivity.
  • the thickness of the substrate is preferably over 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more. Also, the thickness of the substrate is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less.
  • the thickness of the base material may be set within a range obtained by appropriately combining the above lower limit and upper limit depending on the culture conditions, for example, the thickness of the cells to be cultured and the desired viability.
  • the base material is a gel. According to this, it is easy to reproduce the in vivo situation by using the gel.
  • the colloid concentration in the substrate it is preferable to adjust the colloid concentration in the substrate within the range in which cell functions are expressed. According to this method, the concentration of the colloid is changed within the range in which the functions of the cells are expressed to change the properties such as the hardness of the base material. Therefore, it is possible to test cell functions while reproducing various conditions in vivo.
  • the colloid concentration in the base material within a range in which the survival rate of cells in the base material is 80% or more. According to this method, properties such as hardness of the base material are changed by changing the concentration of the colloid within the range in which the survival rate of the cells is 80% or more. Therefore, it is possible to test cells while reproducing various conditions in vivo.
  • the colloid concentration in the base material within the range of 1.0 to 2.5 mg/ml.
  • the properties of the base material can be adjusted by adjusting the concentration of the colloid within the range of 1.0-2.5 mg/ml.
  • the base material is a gel
  • the colloid concentration in the base material is adjusted within a range of 1.0 mg/ml or more. According to this, by adjusting the concentration of the colloid within the range of 1.0 mg/ml, deformation of the substrate can be easily suppressed.
  • the base material is a gel containing collagen.
  • collagen gel can be used as a base material.
  • the apparatus includes a first container defining the first containing compartment and a second container defining the second containing compartment, wherein the first container includes the first container.
  • the first culture solution is accommodated so that the liquid surface of the culture solution is arranged above the upper end of the second container. According to this, the liquid level of the first culture solution can be set relatively high. Therefore, it is easy to set a large initial value of the water level difference, and the number of times of replenishing the culture solution can be suppressed even when the culture is continued for a long time.
  • cells are cultured using a plurality of the devices, a compound is administered to cells of at least one of the plurality of devices, and at least any other or one cell is administered another compound than said compound or none of said compounds.
  • the present invention is a culture method that is particularly suitable for screening methods that employ multiple devices (eg, a large number of devices exceeding tens of devices).
  • FIG. 4 is a schematic configuration diagram of a culture device according to another embodiment of the present invention.
  • 2 is a graph showing the relative expression level of the detoxification enzyme according to Example 1, based on the expression level of the detoxification enzyme obtained by two-dimensional culture.
  • FIG. 1 is a schematic diagram of a conventional culture apparatus in which a culture solution is perfused by high-pressure gas;
  • FIG. (a) Image of a pre-perfusion substrate used in a conventional culture device.
  • (b) is an image of the substrate including a schematic diagram showing the perfusion of the culture solution in the culture using the substrate of (a).
  • (c) is an image of the substrate after continuing the culture of (b) for 3 days.
  • (a) is an image showing the distribution of cells in the center of the substrate by culturing without water level difference.
  • (b) is an image showing the distribution of cells in the central part of the base material by culturing in Example 2; 4 is a graph showing cell viability in the culture without water level difference and the culture in Example 2.
  • FIG. 10 is a graph showing the relative expression level of albumin when culturing was performed by changing the collagen concentration of the base material in Example 5.
  • FIG. 10 is a graph showing the relative expression levels of detoxification enzymes when culturing was carried out by changing the collagen concentration of the base material in Example 6.
  • FIG. 10 is a graph showing cell viability in culture without water level difference and culture in Example 7.
  • the culture apparatus 1 used in the method for culturing cells according to the present embodiment includes an inner container 10, an outer container 20, a substrate 50, and an upper culture solution 60 (the first 1 culture solution) and a lower culture solution 70 (the second culture solution referred to in the present invention).
  • the culturing device 1 may be used alone, or may be used in combination.
  • the horizontal direction of the culture apparatus 1 is called the left-right direction
  • the direction along the plane of the paper and orthogonal to the left-right direction is called the up-down direction.
  • a direction perpendicular to both the up-down direction and the left-right direction (direction perpendicular to the plane of the paper) is referred to as the front-rear direction.
  • the inner container 10 is a cup-shaped container that opens upward. Inside the inner container 10, a compartment (first storage compartment referred to in the present invention) for containing the base material 50 and the upper culture solution 60 is formed.
  • the inner container 10 has side walls 11 and a bottom wall 15 that define the interior space of the container.
  • the side wall 11 has a cylindrical structure that narrows slightly downward.
  • a hook portion 12a projecting leftward is formed at the left end portion of the upper end of the side wall 11 .
  • a right end portion of the upper end of the side wall 11 is formed with a hook portion 12b projecting rightward.
  • the hooks 12a and 12b are sized and shaped to be hooked on the upper end of the outer container 20 from above.
  • the bottom wall 15 is composed of a membrane having a plurality of through holes 16 .
  • a bottom wall 15 is arranged at the lower end of the side wall 11 .
  • the bottom wall 15 may be arranged somewhere in the middle from the upper end to the lower end of the side wall 11 .
  • the through hole 16 penetrates the bottom wall 15 in the vertical direction. Thereby, the through hole 16 allows the space inside the inner container 10 and the space inside the outer container 20 to communicate with each other.
  • the diameter of the through-hole 16 is adjusted so that cells cannot pass through it.
  • the hole diameter of the through holes 16 is preferably 0.1 ⁇ m or more, more preferably 0.4 to 8.0 ⁇ m.
  • the through-holes 16 are formed in the bottom wall 15 at a density of about 1 ⁇ 10 5 to 1 ⁇ 10 8 holes/cm 2 .
  • the size and number of the through-holes 16 may be appropriately adjusted according to the required flow rate of culture medium.
  • one or more through-holes 16 may be formed according to their size.
  • the size of the inner container 10 is appropriately selected according to the size of the outer container 20, the number of cells to be cultured, screening conditions, and the like.
  • a commercially available insert for cell culture may be used for the inner container 10 .
  • the outer container 20 is a cup-shaped container that opens upward. Inside the outer container 20, a compartment (second storage compartment referred to in the present invention) for containing the lower culture solution 70 is formed.
  • the outer container 20 is larger than the side walls 11 in the vertical, horizontal, and front-to-rear directions.
  • the size of the outer container 20 is appropriately selected depending on the number of cells to be cultured, screening conditions, and the like.
  • commercially available plates for cell culture such as 6-, 12-, 24-, 48-, and 96-well plates may be used. In this case, one well corresponds to one outer container 20 .
  • the side wall 11 of the inner container 10 is inserted into the outer container 20 from above.
  • the inner container 10 is supported by the outer container 20 by hooking the hooks 12 a and 12 b on the upper end of the outer container 20 .
  • One inner container 10 may be inserted into one outer container 20, or a plurality of inner containers 10 may be inserted.
  • the structure for supporting the inner container 10 on the outer container 20 may be of any type other than the structure according to the present embodiment.
  • the materials used for the inner container 10 and the outer container 20 preferably do not degrade the base material 50, the upper culture solution 60 and the lower culture solution 70, or adversely affect the cells.
  • the sidewall 11 of the inner container 10 and the outer container 20 are made of plastic such as polystyrene, polypropylene, polyethylene, etc., or metal such as glass or stainless steel.
  • the bottom wall 15 is made of, for example, polyester such as polyethylene terephthalate, synthetic resin such as polycarbonate, or metal such as stainless steel.
  • the materials of the inner container 10 and the outer container 20 may be the same or different.
  • the base material 50 is a scaffold material in which cells to be cultured are embedded.
  • the base material 50 is arranged at the bottom of the inner container 10 and supported by the bottom wall 15 .
  • the base material 50 has a thickness in the vertical direction, and has a thickness exceeding 0.2 mm, for example.
  • the thickness of the base material 50 preferably exceeds 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more, from the viewpoint of ensuring the thickness as much as possible.
  • the thickness of the substrate 50 is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less from the viewpoint of ensuring cell viability as much as possible.
  • the thickness of the base material 50 is 0.2 to 5 mm, 0.2 to 8 mm, 0.2 to 10 mm, 0.2 to 15 mm, 0.2 to 20 mm, 1 to 5 mm, 1 to 8 mm, 1 to 10 mm. , 1-15mm, 1-20mm, 2-5mm, 2-8mm, 2-10mm, 2-15mm, 2-20mm, 4-5mm, 4-8mm, 4-10mm, 4-15mm, 4-20mm, 7 ⁇ 8 mm, 7-10 mm, 7-15 mm, 7-20 mm, 10-15 mm and 10-20 mm.
  • the thickness of the base material 50 is set to a range obtained by appropriately combining the above lower limit and upper limit according to the culture conditions, for example, the thickness of the cells to be cultured and the desired viability.
  • the substrate 50 is preferably made of a gel containing collagen, fibronectin, laminin, etc., which constitute an extracellular matrix, in order to ensure tissue reproducibility.
  • the substance used for the base material 50 one type of substance may be selected, or several types may be mixed.
  • the component ratio of the substances forming the base material 50 may be appropriately adjusted according to the application. For example, when collagen is used, it may be derived from bone, cartilage, tendon, skin, fish scale, or the like.
  • the concentration of the colloid in the base material 50 is in a range in which the viability of the cells is sufficiently high (for example, 50% or more, preferably 60% or more, more preferably 60% or more, more preferably is 70% or more, more preferably 80% or more, and most preferably 90% or more). Moreover, it is preferable that the concentration of the colloid in the base material 50 is adjusted within a range in which the functions of the cells to be cultured are expressed.
  • the function of a cell is, for example, a function to be analyzed in various tests such as screening, and refers to a function of producing a specific substance, a function of exhibiting a specific reaction to a specific compound, and the like.
  • the concentration of the colloid in the base material 50 By changing the concentration of the colloid in the base material 50, it is possible to adjust properties such as hardness of the gel and ease of permeation and diffusion of substances. This makes it possible to test cells while reproducing various in vivo conditions such as normal organs such as the liver and pathological tissues such as cancer.
  • the concentration of collagen in the base material 50 is adjusted in the range of 0.5-3.0 mg/ml, preferably 1.0-2.5 mg/ml. Note that the upper limit of the concentration may be set to a possible value in terms of preparation of the base material 50 .
  • a culture medium is added to the base material 50 .
  • a medium commonly used for cells to be cultured is appropriately selected.
  • substances required for cell culture such as antibiotics such as streptomycin and penicillin, and serum such as fetal bovine serum, may be added as appropriate.
  • the cells to be cultured are embedded in the substrate 50.
  • the base material 50 has the thickness as described above and contains an extracellular matrix. Therefore, the cells embedded in the base material 50 and the entire base material 50 reproduce an actual in-vivo thick tissue. By culturing cells using such a substrate 50, it is possible to culture cells in a state in which the three-dimensional structure is reproduced.
  • Cells to be used may be derived from normal or pathological liver, pancreas, nerve, kidney, blood vessel, etc., but are not particularly limited.
  • the biological species of the cells is not particularly limited, and cells derived from animals such as humans, monkeys, mice, rats, dogs, cats, pigs, and cows can be used.
  • the seeding number of cells is appropriately determined according to the type of cells to be cultured and the purpose of culture, and is preferably 1 ⁇ 10 2 to 1 ⁇ 10 7 cells per culture apparatus.
  • the term "cell" in the present invention includes isolated cells as well as cells that constitute tissue pieces. The thickness of the piece of tissue may be within a range within the substrate 50 .
  • the upper culture solution 60 is housed in the inner container 10 and retained on the substrate 50 .
  • a medium commonly used for cells to be cultured is appropriately selected.
  • the medium may be the same as or different than the medium used for substrate 50 .
  • Substances necessary for cell culture, such as antibiotics, serum, and pH adjusters, may be added to the upper culture solution 60 as appropriate.
  • Other compounds such as screening candidate substances may also be added to the upper culture solution 60 .
  • a medium normally used for cells to be cultured is appropriately selected for the lower culture solution 70 .
  • the medium may be the same as or different than the medium used for substrate 50 or top culture 60 .
  • Substances necessary for cell culture such as antibiotics, serum, and pH adjusters, may be added to the lower culture solution 70 as appropriate.
  • other compounds such as screening candidate substances may be added to the lower culture solution 70 .
  • the installation method of the culture apparatus 1 is as follows. Cells are further seeded and mixed in a mixed solution containing an extracellular matrix, a 5-fold or 10-fold concentrated medium, a buffer solution, and the like. The cell suspension thus obtained is dispensed into the inner container 10, and the inner container 10 is allowed to stand still to gel the cell suspension, thereby producing the substrate 50.
  • the lower culture solution 70 is accommodated in the outer container 20 .
  • the lower culture medium 70 is accommodated in the outer container 20 until the liquid surface is positioned at or above the height at which the bottom wall 15 is arranged so as to come into contact with the base material 50 from below.
  • the upper culture medium 60 is accommodated in the inner container 10 .
  • the upper culture solution 60 comes into contact with the substrate 50 in the inner container 10 from above.
  • the liquid level of the upper culture solution 60 is made higher than the liquid level of the lower culture solution 70 . This causes a water level difference between the upper culture solution 60 and the lower culture solution 70 .
  • a pressure difference is generated between the upper culture solution 60 and the lower culture solution 70 , so that the upper culture solution 60 permeates the substrate 50 .
  • the magnitude of the pressure is preferably 0.01 to 1.0 kPa, more preferably 0.01 to 0.6 kPa, even more preferably 0.01 to 0.2 kPa.
  • the water level difference is such that the pressure applied to the substrate 50 does not deform the substrate 50 during the culture period, for example, the period during which the test on the cells is performed.
  • the length of the period during which the base material 50 is not deformed varies depending on the cells to be cultured and the content of the test, but is assumed to be several hours to several days, for example.
  • the term "deformation" as used herein indicates that it is evident from visual observation of the substrate 50 that the shape has changed due to pressure. The order of the steps in the installation method described above may be changed as long as there is no problem in culturing the cells.
  • the permeation of the upper culture solution 60 into the substrate 50 progresses over time.
  • the mixed solution contained in the base material 50 and the upper culture medium 60 permeating the base material 50 gradually pass through the base material 50 and flow into the lower culture medium 70 through the through holes 16 of the bottom wall 15 .
  • the inner container 10 is replenished with new culture medium at an appropriate timing as the upper culture medium 60 decreases.
  • fresh medium flows from the upper culture medium 60 into the substrate 50 and old medium flows out into the lower culture medium 70 . Therefore, cell culture can be appropriately continued.
  • the liquid level of the upper culture solution 60 is higher than the liquid level of the lower culture solution 70 and a water level difference is generated, the upper culture solution 60 permeates the base material 50 and fresh medium flows into the base material 50. becomes.
  • the base material 50 a three-dimensional structure by using a gel for the base material 50 .
  • the thickness of the base material 50 exceeds 0.2 mm, preferably exceeds 1 mm, and more preferably is 2 mm or more.
  • the thickness of the base material 50 preferably exceeds 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more.
  • the thickness of the base material 50 is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less.
  • the thickness of the substrate 50 may be set to a range obtained by appropriately combining the above lower limit and upper limit depending on the culture conditions, for example, the thickness of the cells to be cultured and the desired viability.
  • the thickness of the base material is at most about 0.1 to 0.2 mm because it is difficult for the culture medium to permeate into the base material.
  • Patent Document 1 which uses pressurization to supply the culture medium, there is a description that the thickness of the tissue can be reduced to about 1 mm at most.
  • the base material 50 according to the present embodiment as described above, it is possible to realize a thickness exceeding these conventional techniques. Therefore, it becomes easier to reproduce the three-dimensional structure in the tissue.
  • the amount of cells that can be cultured per culture apparatus 1 can be increased by increasing the thickness of the substrate 50, it becomes easier to detect the expression of cell functions with high sensitivity.
  • a pressure difference is generated between the upper culture solution 60 and the lower culture solution 70 due to the water level difference, and thus the upper culture solution 60 permeates into the substrate 50 .
  • Patent Literature 1 it is difficult to keep the pressure within a range that does not deform the base material due to the structure in which the inside of the container is pressurized using a high-pressure gas. If the base material is deformed, the state in which the three-dimensional structure is reproduced cannot be maintained. Therefore, even if the substrate has a thickness of about 1 mm, it may not be possible to maintain three-dimensional culture using this substrate.
  • the pressure difference can be adjusted by adjusting the water level difference, so the pressure applied to the base material 50 can be easily adjusted. Therefore, it is easy to keep the pressure applied to the base material 50 at a level that can suppress the deformation of the base material 50 . Therefore, three-dimensional culture can be continued while the base material 50 has a certain thickness.
  • the culture device 1 may be provided in the form of a kit including the inner container 10, the outer container 20, the substrate 50, the upper culture solution 60 and the lower culture solution 70 as a set.
  • the culture device 1 is produced by assembling the kit.
  • FIG. 2 A second embodiment in which the culture method according to the above embodiment is applied to a screening method will be described below.
  • a commercially available culture plate 1000 having a plurality of wells (one well corresponds to one outer container 20) is used as the outer container 20.
  • FIG. 1 a culture plate having a number of wells such as 6 wells, 12 wells, 24 wells, 48 wells, 72 wells, 96 wells, etc. is selected according to the intended use.
  • an upper culture solution 60 to which compounds A, B, C, .
  • a control upper culture solution 60 is prepared and dispensed into each culture device 1 corresponding to each well. After the cells are cultured in each culture apparatus 1, the functions and viability of the cells are evaluated.
  • the pressure applied to the substrate can be easily adjusted, for example, the pressure can be reduced, so the amount of compounds such as reagents used in the upper culture solution 60 can be reduced. Therefore, the culturing device 1 is a culturing method particularly suitable for screening methods that require a plurality of devices.
  • a high-pressure gas or pump-type pressurization machine it is necessary to provide a machine for each well or add a mechanism for adjusting the pressure individually for each well. , and the scale of each well or the entire device may become too large.
  • the culture apparatus 1 can suppress the size of each well and pressurize each apparatus without complicating the overall configuration or increasing the scale. Therefore, from this point of view as well, it is suitable for screening methods.
  • a third embodiment which is still another embodiment of the present invention, will be described with reference to FIG. Apparatus configurations similar to those of the above-described embodiments will be described using the same reference numerals, or description thereof will be omitted as appropriate.
  • the culture device 2 has an inner container 30, an outer container 40, a substrate 50, an upper culture solution 60, and a lower culture solution 70.
  • the culture device 2 may be used singly or in combination.
  • the lateral direction of the culture apparatus 2 is referred to as the left-right direction
  • the direction along the plane of the paper and orthogonal to the left-right direction is referred to as the up-down direction.
  • the inner container 30 has an upper container 80 , a lower container 90 and a support 93 . Inside the inner container 30, a compartment (first storage compartment referred to in the present invention) for containing the substrate 50 and the upper culture medium 60 is formed.
  • the material of the inner container 30 is the same as the material of the inner container 10 .
  • the upper container 80 has a syringe shape.
  • the upper container 80 has a cylindrical portion 81 , a convex portion 83 at the tip of the syringe, and a tapered portion 82 connected to the convex portion 83 .
  • the size of the tubular portion 81 in the vertical direction is determined by the amount of the upper culture solution 60 to be used.
  • the diameter of the upper end portion 82a of the tapered portion 82 is substantially the same as the diameter of the opening of the lower container 90 in the left-right direction.
  • the upper container 80 is inserted into the lower container 90 from above with the convex portion 83 arranged on the lower side.
  • the upper container 80 contains the upper culture solution 60 . This upper culture medium 60 flows down into the lower container 90 through the lower end opening of the projection 83 .
  • the lower container 90 is a cup-shaped container that opens upward.
  • the lower container 90 contains the upper culture medium 60 and the substrate 50 .
  • Lower container 90 has side walls 91 and bottom wall 95 .
  • the side wall 91 has a cylindrical structure that narrows slightly downward.
  • a hook portion 92a projecting leftward is formed at the left end portion of the upper end of the side wall 91 .
  • a right end portion of the upper end of the side wall 91 is formed with a hook portion 92b projecting rightward.
  • the bottom wall 95 is composed of a membrane having through holes 96 .
  • a bottom wall 95 is arranged at the lower end of the side wall 91 .
  • the bottom wall 95 may be arranged somewhere in the middle from the upper end to the lower end of the side wall 91 .
  • the through hole 96 vertically penetrates the bottom wall 95 .
  • the diameter of the through-hole 96 is adjusted so that cells cannot pass through it.
  • the hole diameter of the through holes 96 is preferably 0.1 ⁇ m or more, more preferably 0.4 to 8.0 ⁇ m.
  • the through holes 96 are formed in the bottom wall 95 at a density of about 1 ⁇ 10 5 to 1 ⁇ 10 8 holes/cm 2 .
  • the size and number of the through-holes 96 may be appropriately adjusted according to the required medium flow rate.
  • one or more through-holes 96 may be formed according to their size.
  • the tapered portion 82 and the convex portion 83 of the upper container 80 are inserted into the lower container 90 from above.
  • the lower container 90 supports the upper container 80 by contacting the inner surface of the upper end of the side wall 91 of the lower container 90 with the upper end 82 a of the tapered portion 82 .
  • the support 93 has a tubular portion 93a and a spacer 93b.
  • the support 93 supports the upper container 80 and the lower container 90 on the bottom surface of the outer container 40, and serves to adjust the position of the lower container 90 in the vertical direction.
  • the tubular portion 93a has a tubular shape extending in the vertical direction.
  • the vertical length of the support 93 is greater than the vertical length of the side wall 91 of the lower container 90 .
  • the tubular portion 93 a is placed on a plate-like spacer 93 b placed on the bottom surface of the outer container 40 .
  • the inside of the cylindrical portion 93a is large enough to allow the side wall 91 of the lower container 90 to be inserted therein.
  • the diameter of the cylindrical portion 93a is large enough to hook the hooks 92a and 92b on the upper end of the cylindrical portion 93a.
  • the lower container 90 is supported by the support 93 by inserting the side wall 91 of the lower container 90 into the cylindrical portion 93a and hooking the hooks 92a and 92b on the upper end of the cylindrical portion 93a.
  • the outer container 40 is a cup-shaped container that opens upward. Inside the outer container 40, a compartment (second storage compartment referred to in the present invention) for containing the lower culture solution 70 is formed.
  • the inner container 30 is supported on the bottom surface of the outer container 40 by the supports 93 as described above.
  • the inner container 30 is supported by supports 93 while the side walls 91 of the lower container 90 are inserted into the outer container 40 .
  • the entire inner container 30 protrudes greatly upward from the upper end of the outer container 40 .
  • the material of the outer container 40 is the same as the material of the outer container 20 .
  • the installation method of the culture device 2 is as follows. A cell suspension in which cells are seeded in the same manner as in the culture apparatus 1 is dispensed into the lower container 90, and the lower container 90 is allowed to stand still to gel the cell suspension, thereby producing the substrate 50. FIG. Next, a support 93 is placed on the bottom surface of the outer container 40 . Next, the lower container 90 is supported by the support 93 by hooking the hooks 92a and 92b on the upper end of the cylindrical portion 93a while inserting the side wall 91 into the cylindrical portion 93a. Next, the lower culture solution 70 is accommodated in the outer container 40 .
  • the lower culture medium 70 is accommodated in the outer container 40 until the liquid surface is positioned at a height equal to or higher than the bottom wall 95 so as to come into contact with the base material 50 from below.
  • the upper container 90 is filled with the culture solution by filling the lower container 90 with the upper culture solution 60 so as to come into contact with the base material 50 in the lower container 90 from above, and the upper container 80 is inserted into the lower container 90 from above. to support the upper container 80 .
  • a sealing material made of vacuum grease or the like is applied in advance to the upper container 80 and the lower container 90, and the upper end portion 82a of the upper container 80 and the hook portions 92a and 92b of the lower container 90 are sealed. do.
  • the culture apparatus 2 can be used for a compound screening method, like the culture apparatus 1 .
  • the liquid level of the upper culture solution 60 can be set relatively high. Therefore, it is easy to set a large initial value of the water level difference, and the number of times of replenishing the upper culture solution 60 can be suppressed even when the culture is continued for a long time.
  • the pressure may be in the range of 0.01 to 2.5 kPa, preferably in the range of 0.01 to 1.0 kPa, more preferably in the range of 0.1 to 0.6 kPa.
  • Example 1 As shown in FIG. 4, the expression level of the hepatocyte detoxification enzyme by the two-dimensional culture (comparative example) and the expression level of the hepatocyte detoxification enzyme by the culture apparatus 1 (Example 1) were evaluated.
  • the inner container 10 used a 24-well insert (manufactured by CORNING).
  • the hole diameter of the through hole 16 of the bottom wall 15 was 0.4 ⁇ m.
  • a 24-well cell culture plate (manufactured by Nippon Genetics or CORNING) was used as the outer container 20 .
  • HepG2 cells human liver cancer-derived cell line
  • the base material 50 is a 3.0 mg/ml collagen solution (Cellmatrix Type IP, manufactured by Nitta Gelatin), a 10-fold concentrated DMEM medium (Dulbecco's Modified Eagle Medium, manufactured by Thermo), and a buffer solution (reconstitution buffer, Nitta Gelatin Co., Ltd.) was used.
  • a DMEM medium (manufactured by Sigma) was used for the upper culture medium 60 and the lower culture medium 70 .
  • a culture apparatus for two-dimensional culture was produced as follows. About 500 ⁇ l of the mixed solution similar to the above was placed in a plastic dish (manufactured by Falcon) having an inner diameter of 35 mm, and gelled to prepare a substrate. 2.0 ⁇ 10 6 cells were seeded on the upper surface of the substrate, and about 2000 ⁇ l of the same culture solution as the upper culture solution 60 was placed in a container from above the cells.
  • the culture device 1 and the culture device for two-dimensional culture were placed in an incubator at 37°C, and cell culture was performed for 3 days. During this period, the upper culture medium 60 of the culture apparatus 1 was replenished every 12 hours, and the increased culture medium in the outer container 40 was removed. In the two-dimensional culture, the culture medium was replaced with fresh one every other day.
  • the expression level of the detoxification enzyme CYP1A1 was quantified by RT-qPCR (reverse transcription quantitative PCR, quantitative reverse transcription PCR method) using KAPA SYBR Fast qPCR Kit (manufactured by Nippon Genetics).
  • RNA was extracted from the cells in each culture apparatus, CYP1A1 mRNA was reverse transcribed into cDNA using reverse transcriptase, and the cDNA was amplified to quantify the expression level of CYP1A1.
  • the expression level of CYP1A1 was corrected with an endogenous control (Beta-actin). Based on the expression level of CYP1A1 in the two-dimensional culture, the relative expression level of CYP1A1 in the culture apparatus 1 was evaluated.
  • the relative expression level in culture apparatus 1 was about four times the expression level in two-dimensional culture. It is considered that the culture apparatus 1 enables cell culture while reproducing the internal environment of a thick tissue in vivo.
  • the culture device 300 has a pressure device 310, a culture solution bottle 320, a pressure device 330, and a waste solution bottle 340, as shown in FIG.
  • a substrate 350 is housed within the pressure device 330 .
  • the base material 350 contains a mixed solution with a collagen concentration of 2.4 mg/ml and 2.0 ⁇ 10 6 HepG2 cells, which are the same components as the base material 50 of Example 1.
  • a base material 350 is a gel-like base material produced in the same manner as the base material 50 of the first embodiment.
  • the culture device 300 was used as follows. A high-pressure gas of 3 kPa was supplied from the pressure device 310 to the culture solution bottle 320 . Next, the culture solution contained in the culture solution bottle 320 was supplied from the culture solution bottle 320 to the pressurizing device 330 by high-pressure gas. As a result, as shown in FIG. 6(b), the culture solution was perfused from the left to the right of the substrate 350. As shown in FIG. Next, the culture solution passed through the substrate 350 by perfusion was discharged from the pressure device 330 to the waste solution bottle 340 . In this manner, cell culture was performed for 3 days in an incubator at 37° C. while perfusing the substrate 350 with a fresh culture medium.
  • the substrate 350 Before perfusion, the substrate 350 had a rectangular planar shape as indicated by the solid line in FIG. 6(a). A dent was formed. In contrast, in Examples 1 to 7 according to the present invention, such deformation did not occur by visual observation.
  • Example 2 As shown in FIGS. 7 and 8, cell viability was evaluated for culture with no water level difference (comparative example) and culture with water level difference by the culture apparatus 1 (example 2).
  • the culture method of Example 1 was implemented assuming that there was a water level difference.
  • the culture method was performed under the following conditions when there was no water level difference. 1000 ⁇ l of the lower culture solution 70 was placed in the outer container 20, and the culture apparatus was prepared so that the water level difference between the upper culture solution 60 and the lower culture solution 70 was not generated. Other configurations and conditions were the same as when there was a water level difference.
  • the substrate 50 of each culture apparatus is immersed in a 4% paraformaldehyde solution (the solvent is a phosphate buffer), allowed to stand overnight at 4° C., and then treated with a phosphate buffer. After washing three times with , the gel in the center in the vertical and horizontal directions was collected. Next, the gel was immersed in a 0.5% Triton X100 solution (solvent: phosphate buffer) and shaken at room temperature for 15 minutes.
  • solvent is a phosphate buffer
  • FIG. 7(a) shows the case without the water level difference
  • FIG. 7(b) shows the image with the water level difference (here all cells are reflection interference images).
  • FIG. 8 shows, when there is a water level difference (when the culture apparatus 1 is used), more viable cells were observed than when there is no water level difference.
  • the survival rate was about 20% when there was no water level difference, and in the case of the culture apparatus 1, the survival rate was 90% or more.
  • Example 3 Cell viability was evaluated when the thickness of the substrate 50 in the vertical direction was 4.9 mm, 7.8 mm, and 10.0 mm.
  • Example 2 When the thickness of the substrate 50 in the vertical direction was 4.9 mm, the culture method of Example 2 was carried out under the same configuration and conditions as in Example 2. When the thickness of the substrate 50 in the vertical direction was 7.8 mm, 400 ⁇ l of the mixed solution was used, and the culturing method of Example 2 was performed with the other configurations and conditions being the same. When the thickness of the substrate 50 in the vertical direction was 10.0 mm, the culture method of Example 2 was performed using 600 ⁇ l of the mixed solution, and the other configurations and conditions were the same. In addition, the survival rate was evaluated in each case in the same manner as in Example 2.
  • the survival rate was 99% for the substrate 50 with a thickness of 4.9 mm, the survival rate was 84% for the substrate 50 with a thickness of 7.8 mm, and the survival rate was 50% for the thickness of 10.0 mm. Although the survival rate of cells decreased as the thickness of the substrate 50 in the vertical direction increased, 50% of the cells survived even at a thickness of 10.0 mm.
  • Example 4 Cell viability was evaluated when the base material 50 had a collagen concentration of 2.4 mg/ml, 1.6 mg/ml, and 1.2 mg/ml.
  • a base material 50 was produced in the same manner as in Example 2 when the collagen concentration was 2.4 mg/ml.
  • the culture method of Example 2 was carried out in the culture apparatus 1 having the same configuration and other conditions as the base material 50 with each of the collagen concentrations described above.
  • the survival rate was evaluated in each case in the same manner as in Example 2.
  • the survival rate was 99% for the substrate 50 with a collagen concentration of 2.4 mg/ml, 83% for 1.6 mg/ml, and 94% for 1.2 mg/ml. 80% or more of the cells survived in the culture device 1 with any collagen concentration.
  • Example 5 As shown in FIG. 9, the relative expression level of albumin in the substrate 50 with each collagen concentration in Example 4 was evaluated.
  • the culture method of Example 1 was performed with only the number of cells set to 1.5 ⁇ 10 5 and the other configurations and conditions being the same.
  • the albumin evaluation method is also RT-qPCR (using S18 as an endogenous control). Based on the expression level of albumin in the base material 50 with a collagen concentration of 2.4 mg/ml, the relative expression level of albumin in the base material 50 with each collagen concentration was evaluated. As shown in FIG. 9, there was no difference in the expression level of albumin between the concentrations.
  • Example 6 As shown in FIG. 10, the relative expression levels of detoxification enzymes in the substrate 50 of each collagen concentration in Example 4 were evaluated.
  • Example 2 Cells were cultured in the same manner as in Example 1 except that the number of cells was set to 1.5 ⁇ 10 5 in the culture apparatus 1 using the substrate 50 with each collagen concentration.
  • the expression level of the detoxification enzyme CYP1A1 was quantified in the same manner as in Example 1 (S18 was used as an endogenous control). Based on the expression level of CYP1A1 when using the base material 50 with a collagen concentration of 2.4 mg/ml, the relative expression level of CYP1A1 in the base material 50 with each collagen concentration was evaluated. As shown in FIG. 10, the expression level of the detoxification enzyme tended to decrease when the collagen concentration was low.
  • Example 7 As shown in FIG. 11, cell viability was evaluated for culture with no water level difference (comparative example) and culture with water level difference by culture apparatus 2 (example 7).
  • Example 7 Changing the initial amount of the upper culture medium 60 by using the culture apparatus 2 provided with the upper container 80 having a syringe shape instead of the culture apparatus 1, replenishing the upper culture medium 60, and increasing the culture medium in the outer container 40
  • the survival rate of cells was obtained for each of the case where there was a water level difference (Example 7) and the case where there was no water level difference (Comparative Example).
  • the syringe-shaped upper container 80 was cylindrical, and the initial volume of the upper culture solution 60 was about 5 ml. As shown in FIG. 11, the survival rate was less than 10% when there was no water level difference, and the survival rate was 90% or more in the case of the culture apparatus 2.
  • independent containers are used as the inner container 10 and the outer container 20, respectively, but these containers formed integrally may also be used. .
  • the inner container 30 is composed of the upper container 80 and the lower container 90, but the inner container 30 may be integrally formed.
  • a supply device may be provided that supplies the upper culture medium 60 to the inner container in an appropriate amount per unit time.
  • a discharge device may also be provided for discharging the lower culture solution 70 from the outer container to the outside.
  • a drip device may be used as these supply device and discharge device.
  • the culture apparatuses according to the first to third embodiments described above may be connected in multiple stages. For example, by causing the lower culture solution 70 in the outer container 20 of the culture device 1 in the preceding stage to flow into the inner container 10 of the culture device 1 in the latter stage, A plurality of stages of the culture apparatus 1 may be installed so as to be used as the upper culture solution 60 .
  • the support 93 may have a structure other than a tubular shape.
  • each of the inner containers 10 and 30 and the outer containers 20 and 40 according to the first to third embodiments described above may be configured in an appropriate shape.
  • it may be configured in a prismatic shape, a truncated cone shape, a hemispherical shape, or the like.
  • the water level difference is generated so that the liquid level of the upper culture solution 60 is higher than the liquid level of the lower culture solution 70 .
  • the water level difference may be generated so that the liquid level of the lower culture solution 70 is higher than the liquid level of the upper culture solution 60 .
  • Reference Signs List 1 2 culture apparatus 10, 30 inner container 20, 40 outer container 11, 91 side wall 15, 95 bottom wall 16, 96 through hole 50 substrate 60 upper culture solution 70 lower culture solution

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Abstract

The present invention enables three-dimensional culture by suppressing deformation of a base material.　A base material 50 formed of a collagen-containing gel is used in this culture device. The culture device 1 comprises: an inner container 10 holding the base material 50 and an upper culture medium 60 in such a manner that the upper culture medium 60 comes into contact with the base material 50 from above; and an outer container 20 holding a lower culture medium 70 in such a manner that the lower culture medium 70 comes into contact with the base material 50 from below. In the culture device 1, the upper culture medium 60 is allowed to penetrate into the base material 50 by making a difference in liquid level between the upper culture medium 60 and the lower culture medium 70.

Description

細胞の培養方法、細胞の培養方法を用いたスクリーニング方法、培養装置及び培養キットCell culture method, screening method using cell culture method, culture apparatus and culture kit
　本発明は、細胞の培養方法、その細胞の培養方法を用いたスクリーニング方法、培養装置及び培養キットに関する。 The present invention relates to a cell culture method, a screening method, a culture apparatus and a culture kit using the cell culture method.
　細胞を培養する技術において、実際の生体内の厚みがある組織を再現することが望まれている。一方、シャーレを用いた基本的な細胞の培養方法では、シャーレの底面のみに細胞が接着する。このため、単層の状態でしか細胞を培養できず、厚みを持たせた３次元的な培養が実現できない。これに関し、ある程度の厚さを持つゲル等の基材に培養液を浸透させることで基材内の細胞を培養する従来技術がある。 In the technology of culturing cells, it is desired to reproduce the actual thick tissue in vivo. On the other hand, in the basic cell culture method using a petri dish, cells adhere only to the bottom surface of the petri dish. For this reason, cells can only be cultured in a monolayer state, and three-dimensional culture with a thickness cannot be realized. In relation to this, there is a conventional technique for culturing cells in a base material such as a gel having a certain thickness by infiltrating a culture solution into the base material.
　特許文献１に記載の灌流培養装置はその一例である。この装置は以下のように構成されている。底部が膜で構成された容器に細胞と基材を混合して収容すると共に、基材上に培養液を収容する。また、当該容器を、培養液を収容した別の容器に収容し、膜が上記別の容器内の培養液と接触するようにする。そして、収容された内側の容器を密閉すると共に、高圧ガスの供給により内側の容器内を加圧することで、内側の容器内の培養液を基材内に浸透させる。これにより、基材中の厚みの方向に複数の細胞を存在させても基材内部まで培養液を供給できる。よって、３次元的な細胞培養を実行しやすくなる。 The perfusion culture device described in Patent Document 1 is one example. This device is configured as follows. Cells and a substrate are mixed and housed in a container whose bottom is made of a membrane, and a culture solution is housed on the substrate. Alternatively, the container is placed in another container containing a medium, such that the membrane is in contact with the medium in the other container. Then, by sealing the contained inner container and pressurizing the inside of the inner container by supplying high-pressure gas, the culture solution in the inner container permeates into the substrate. As a result, even if a plurality of cells exist in the thickness direction of the substrate, the culture solution can be supplied to the inside of the substrate. Therefore, it becomes easier to perform three-dimensional cell culture.
特許第６８１３７６５号Patent No. 6813765
　しかしながら、本発明者らは、特許文献１に記載の装置に以下の課題があることに気づいた。当該装置を用いて培養を続けると、内側の容器内の基材が加圧により変形していく。基材が変形すると、細胞を基材内に適切に保つことができず培養を安定に継続できないおそれがある。一方、内側の容器内を加圧する大きさを調整することで基材の変形を抑制しようとしても、高圧ガスを用いて容器内を加圧する装置の構成上、容器内の圧力を下げるのは限界がある。よって、基材の変形を抑制するのが困難である。基材が変形すると、厚みの方向に関する３次元的な構造が保てず、組織内部の環境を再現できないおそれがある。 However, the inventors have found that the device described in Patent Document 1 has the following problems. When culturing is continued using the device, the substrate inside the inner container is deformed by the pressure. If the substrate is deformed, the cells may not be properly retained in the substrate, and the culture may not be stably continued. On the other hand, even if an attempt is made to suppress the deformation of the base material by adjusting the amount of pressure applied to the inside of the inner container, there is a limit to lowering the pressure inside the container due to the structure of the device that pressurizes the inside of the container using high-pressure gas. There is Therefore, it is difficult to suppress the deformation of the base material. If the substrate is deformed, the three-dimensional structure in the thickness direction cannot be maintained, and there is a possibility that the environment inside the tissue cannot be reproduced.
　本発明の目的は、基材の変形を抑制することで３次元培養を可能とする細胞の培養方法、これを用いたスクリーニング方法、細胞の培養装置及び培養キットを提供することにある。 An object of the present invention is to provide a cell culture method that enables three-dimensional culture by suppressing deformation of the substrate, a screening method using the same, a cell culture apparatus, and a culture kit.
　本発明の培養方法は、基材中の細胞を培養する方法であって、前記基材に上方から接触するように第１の培養液を収容する第１収容区画と、前記基材に下方から接触するように第２の培養液を収容する第２収容区画とを有する装置において、前記第１の培養液と前記第２の培養液の間に水位差を生じさせることで前記第１及び第２の培養液の少なくともいずれかを前記基材中に浸透させる。本発明の別の観点に係る培養装置は、細胞が埋め込まれた基材と、前記基材に上方から接触するように第１の培養液を収容する第１収容区画と、前記基材に下方から接触するように第２の培養液を収容する第２収容区画とを備えており、前記第１の培養液と前記第２の培養液の間に水位差を生じさせることで前記第１及び第２の培養液の少なくともいずれかを前記基材中に浸透させる。本発明の別の観点に係る培養キットは、細胞が埋め込まれた基材と、前記基材に上方から接触するように第１の培養液を収容する第１収容区画と、前記基材に下方から接触するように第２の培養液を収容する第２収容区画とを備えており、前記第１の培養液と前記第２の培養液の間に水位差を生じさせることで前記第１及び第２の培養液の少なくともいずれかを前記基材中に浸透させる。 The culturing method of the present invention is a method for culturing cells in a substrate, comprising: a first housing compartment for housing a first culture medium so as to contact the substrate from above; and a second containing compartment containing a second culture medium so as to be in contact with each other, wherein the first and second At least one of the two culture solutions is permeated into the substrate. A culture apparatus according to another aspect of the present invention includes a substrate in which cells are embedded, a first storage compartment that stores a first culture medium so as to contact the substrate from above, and and a second storage compartment that stores a second culture medium so as to be in contact with each other, and by creating a water level difference between the first culture medium and the second culture medium, the first and At least one of the second culture fluids is permeated into the substrate. A culture kit according to another aspect of the present invention comprises a substrate in which cells are embedded, a first accommodation compartment that accommodates a first culture medium so as to contact the substrate from above, and and a second storage compartment that stores a second culture medium so as to be in contact with each other, and by creating a water level difference between the first culture medium and the second culture medium, the first and At least one of the second culture fluids is permeated into the substrate.
　本発明によると、水位差によって第１の培養液と第２の培養液の間に圧力差を生じさせ、もって、基材中に培養液を浸透させる。水位差の調整によって圧力差を調整できるので、基材に加わる圧力を調整しやすく、基材の変形を抑制できる大きさに保ちやすい。このため、３次元的な培養が可能となる。 According to the present invention, a pressure difference is generated between the first culture solution and the second culture solution due to the water level difference, thereby permeating the culture solution into the substrate. Since the pressure difference can be adjusted by adjusting the water level difference, it is easy to adjust the pressure applied to the base material, and to easily maintain a size that can suppress the deformation of the base material. Therefore, three-dimensional culture is possible.
　また、本発明においては、前記装置が、前記第１収容区画を規定する側壁及び底壁を有する容器を備えており、前記底壁に前記第１収容区画と前記第２収容区画を連通させる貫通孔が形成されていることが好ましい。これによると、側壁及び底壁を有する容器によって第１収容区画が規定されている。このため、底壁によって基材を支持できる。 Also in the present invention, the apparatus comprises a container having side walls and a bottom wall defining the first containing compartment, and a through hole in the bottom wall for communicating the first containing compartment and the second containing compartment. It is preferred that holes are formed. According to this, a first containing compartment is defined by a container having side walls and a bottom wall. Therefore, the substrate can be supported by the bottom wall.
　また、本発明においては、前記水位差を前記基材が変形しない範囲とすることが好ましい。これによると、基材が変形しないため、安定な培養が確保される。なお、基材が変形しない期間の長さは、例えば、数時間から数日間程度が想定される。 Further, in the present invention, it is preferable that the water level difference is within a range in which the substrate is not deformed. According to this, since the substrate is not deformed, stable culture is ensured. It should be noted that the length of the period during which the base material is not deformed is assumed to be several hours to several days, for example.
　また、本発明においては、前記基材の上下方向に関する厚さが０．２ｍｍを超えることが好ましい。従来技術によると、細胞を培養する基材に厚みを持たせるのが困難である。例えば、培養液の供給に加圧を用いない方法によると、細胞を培養する基材の厚みはせいぜい０．１から０．２ｍｍ程度までである。本発明によると、条件に応じ、厚さが０．２ｍｍを超える基材を使用することが可能である。これにより、厚みのある立体的な組織内の３次元的な構造を再現しやすくなる。また、基材の厚みが増すことにより、装置１つあたりに培養できる細胞量が増やせるため、細胞機能の発現を高感度に検出しやすくなる。なお、基材の厚みは、好ましくは１ｍｍを超え、より好ましくは２ｍｍ以上、４ｍｍ以上、７ｍｍ以上又は１０ｍｍ以上である。また、基材の厚みは、２０ｍｍ以下、好ましくは１５ｍｍ以下、より好ましくは１０ｍｍ以下、８ｍｍ以下又は５ｍｍ以下である。基材の厚みは、培養の条件、例えば、培養する細胞の厚みや所望する生存率の条件に応じて、上記下限値及び上限値を適宜組み合わせた範囲に設定されてよい。 Further, in the present invention, it is preferable that the thickness of the substrate in the vertical direction exceeds 0.2 mm. According to the prior art, it is difficult to provide a thick substrate for culturing cells. For example, according to a method that does not use pressurization to supply the culture solution, the thickness of the substrate for culturing cells is at most about 0.1 to 0.2 mm. According to the invention, it is possible to use substrates with a thickness greater than 0.2 mm, depending on the conditions. This makes it easier to reproduce a three-dimensional structure in a thick three-dimensional tissue. In addition, since the amount of cells that can be cultured per device can be increased by increasing the thickness of the base material, it becomes easier to detect the expression of cell functions with high sensitivity. The thickness of the substrate is preferably over 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more. Also, the thickness of the substrate is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less. The thickness of the base material may be set within a range obtained by appropriately combining the above lower limit and upper limit depending on the culture conditions, for example, the thickness of the cells to be cultured and the desired viability.
　また、本発明においては、基材がゲルであることが好ましい。これによると、ゲルを用いることで生体内の状況を再現しやすい。 In addition, in the present invention, it is preferable that the base material is a gel. According to this, it is easy to reproduce the in vivo situation by using the gel.
　また、本発明においては、前記基材におけるコロイドの濃度を、細胞の機能が発現する範囲内で調整することを好ましい。これによると、細胞の機能が発現する範囲内でコロイドの濃度を変えて基材の硬さ等の特性を変更する。このため、生体内の様々な状況を再現しつつ細胞の機能の試験が可能である。 In addition, in the present invention, it is preferable to adjust the colloid concentration in the substrate within the range in which cell functions are expressed. According to this method, the concentration of the colloid is changed within the range in which the functions of the cells are expressed to change the properties such as the hardness of the base material. Therefore, it is possible to test cell functions while reproducing various conditions in vivo.
　また、本発明においては、前記基材におけるコロイドの濃度を、前記基材中の細胞の生存率が８０％以上となる範囲内で調整することが好ましい。これによると、細胞の生存率が８０％以上となる範囲内でコロイドの濃度を変えて基材の硬さ等の特性を変更する。このため、生体内の様々な状況を再現しつつ細胞の試験が可能である。 In addition, in the present invention, it is preferable to adjust the colloid concentration in the base material within a range in which the survival rate of cells in the base material is 80% or more. According to this method, properties such as hardness of the base material are changed by changing the concentration of the colloid within the range in which the survival rate of the cells is 80% or more. Therefore, it is possible to test cells while reproducing various conditions in vivo.
　また、本発明においては、前記基材におけるコロイドの濃度を１．０～２．５ｍｇ／ｍｌの範囲内で調整することが好ましい。これによると、コロイドの濃度を１．０～２．５ｍｇ／ｍｌの範囲内で調整することで基材の特性を調整できる。 Further, in the present invention, it is preferable to adjust the colloid concentration in the base material within the range of 1.0 to 2.5 mg/ml. According to this, the properties of the base material can be adjusted by adjusting the concentration of the colloid within the range of 1.0-2.5 mg/ml.
　また、本発明においては、前記基材がゲルであり、前記基材におけるコロイドの濃度を１．０ｍｇ／ｍｌ以上となる範囲内で調整することが好ましい。これによると、コロイドの濃度を１．０ｍｇ／ｍｌの範囲内で調整することで基材の変形を抑制しやすい。 Further, in the present invention, it is preferable that the base material is a gel, and the colloid concentration in the base material is adjusted within a range of 1.0 mg/ml or more. According to this, by adjusting the concentration of the colloid within the range of 1.0 mg/ml, deformation of the substrate can be easily suppressed.
　また、本発明においては、前記基材がコラーゲンを含有したゲルであることが好ましい。これによると、コラーゲンのゲルを基材として利用可能である。 Further, in the present invention, it is preferable that the base material is a gel containing collagen. According to this, collagen gel can be used as a base material.
　また、本発明においては、前記装置が、前記第１収容区画を規定する第１容器と、前記第２収容区画を規定する第２容器とを備えており、前記第１容器に、前記第１の培養液の液面が前記第２容器の上端より上方に配置されるように前記第１の培養液を収容することが好ましい。これによると、第１の培養液の液面を比較的高く設定できる。このため、水位差の初期値を大きく設定しやすく、培養を長時間継続した場合でも培養液を継ぎ足す回数を抑制できる。 Also, in the present invention, the apparatus includes a first container defining the first containing compartment and a second container defining the second containing compartment, wherein the first container includes the first container. It is preferable that the first culture solution is accommodated so that the liquid surface of the culture solution is arranged above the upper end of the second container. According to this, the liquid level of the first culture solution can be set relatively high. Therefore, it is easy to set a large initial value of the water level difference, and the number of times of replenishing the culture solution can be suppressed even when the culture is continued for a long time.
　本発明の別の観点に係るスクリーニング方法は、複数の前記装置を用いて細胞を培養すると共に、前記複数の装置のうち少なくともいずれか１つの細胞に対しては化合物を投与し、少なくとも他のいずれか１つの細胞に対しては前記化合物とは別の化合物を投与するか前記化合物のいずれも投与しない。これによると、基材に加わる圧力を調整しやすく、例えば、圧力を小さくできる。これにより、培養液等に用いる試薬の量を減らすことが可能である。このため、本発明は、複数の装置（例えば、数十個を超える多数の装置）を用いるスクリーニング方法に特に適した培養方法である。 In a screening method according to another aspect of the present invention, cells are cultured using a plurality of the devices, a compound is administered to cells of at least one of the plurality of devices, and at least any other or one cell is administered another compound than said compound or none of said compounds. According to this, it is easy to adjust the pressure applied to the base material, and for example, the pressure can be reduced. As a result, it is possible to reduce the amount of reagents used in the culture medium and the like. For this reason, the present invention is a culture method that is particularly suitable for screening methods that employ multiple devices (eg, a large number of devices exceeding tens of devices).
（ａ）本発明の第１実施形態に係る培養方法に使用する培養装置の概略構成図である。（ｂ）（ａ）から一定時間経過した培養装置の概略構成図である。（ｃ）（ｂ）から一定時間経過した培養装置の概略構成図である。(a) It is a schematic block diagram of the culture|cultivation apparatus used for the culture|cultivation method based on 1st Embodiment of this invention. (b) is a schematic configuration diagram of the culture apparatus after a certain period of time has passed since (a). (c) is a schematic configuration diagram of the culture apparatus after a certain period of time has passed since (b). 本実施形態を利用したスクリーニング方法の内容を示す概略図である。It is a schematic diagram showing the content of the screening method using this embodiment. 本発明の別の実施形態に係る培養装置の概略構成図である。FIG. 4 is a schematic configuration diagram of a culture device according to another embodiment of the present invention; ２次元培養による解毒酵素の発現量を基準とした、実施例１による解毒酵素の相対発現量を示すグラフである。2 is a graph showing the relative expression level of the detoxification enzyme according to Example 1, based on the expression level of the detoxification enzyme obtained by two-dimensional culture. 高圧ガスによって培養液が灌流する従来の培養装置の概略図である。1 is a schematic diagram of a conventional culture apparatus in which a culture solution is perfused by high-pressure gas; FIG. （ａ）従来の培養装置に使用する灌流前の基材の画像である。（ｂ）（ａ）の基材を使用した培養における培養液の灌流の様子を示す模式図を含む基材の画像である。（ｃ）（ｂ）の培養を３日間継続した後の基材の画像である。(a) Image of a pre-perfusion substrate used in a conventional culture device. (b) is an image of the substrate including a schematic diagram showing the perfusion of the culture solution in the culture using the substrate of (a). (c) is an image of the substrate after continuing the culture of (b) for 3 days. （ａ）水位差がない培養による基材の中心部における細胞の分布を示す画像である。（ｂ）実施例２の培養による基材の中心部における細胞の分布を示す画像である。(a) is an image showing the distribution of cells in the center of the substrate by culturing without water level difference. (b) is an image showing the distribution of cells in the central part of the base material by culturing in Example 2; 水位差がない培養と実施例２の培養における細胞の生存率を示すグラフである。4 is a graph showing cell viability in the culture without water level difference and the culture in Example 2. FIG. 実施例５において、基材のコラーゲン濃度を変更して培養を実施した場合のアルブミンの相対発現量を示すグラフである。10 is a graph showing the relative expression level of albumin when culturing was performed by changing the collagen concentration of the base material in Example 5. FIG. 実施例６において、基材のコラーゲン濃度を変更して培養を実施した場合の解毒酵素の相対発現量を示すグラフである。10 is a graph showing the relative expression levels of detoxification enzymes when culturing was carried out by changing the collagen concentration of the base material in Example 6. FIG. 水位差がない培養と実施例７の培養における細胞の生存率を示すグラフである。10 is a graph showing cell viability in culture without water level difference and culture in Example 7. FIG.
［第１実施形態（培養方法）］
　本発明の一実施形態に係る第１実施形態について図面を参照しつつ説明する。図１（ａ）に示すように、本実施形態に係る細胞の培養方法に使用される培養装置１は、内側容器１０、外側容器２０、基材５０、上部培養液６０（本発明でいう第１の培養液）、及び下部培養液７０（本発明でいう第２の培養液）を有する。培養装置１は単独で使用されてもよいし、複数同時に使用されてもよい。なお、以下において、図１に示すように、培養装置１の横方向を左右方向、紙面に沿った方向であって左右方向と直交する方向を上下方向という。また、上下方向及び左右方向の両方に直交する方向（紙面に対して直交する方向）を前後方向という。 [First embodiment (culture method)]
A first embodiment according to one embodiment of the present invention will be described with reference to the drawings. As shown in FIG. 1(a), the culture apparatus 1 used in the method for culturing cells according to the present embodiment includes an inner container 10, an outer container 20, a substrate 50, and an upper culture solution 60 (the first 1 culture solution) and a lower culture solution 70 (the second culture solution referred to in the present invention). The culturing device 1 may be used alone, or may be used in combination. In the following, as shown in FIG. 1, the horizontal direction of the culture apparatus 1 is called the left-right direction, and the direction along the plane of the paper and orthogonal to the left-right direction is called the up-down direction. A direction perpendicular to both the up-down direction and the left-right direction (direction perpendicular to the plane of the paper) is referred to as the front-rear direction.
　内側容器１０は上方に開口したカップ状の容器である。内側容器１０の内部には、基材５０及び上部培養液６０を収容する区画（本発明でいう第１収容区画）が形成されている。内側容器１０は、容器の内部空間を規定する側壁１１及び底壁１５を有する。側壁１１は下方に向かってやや窄まった筒状の構造である。側壁１１の上端の左端部には左方向に突出した引掛け部１２ａが形成されている。側壁１１の上端の右端部には右方向に突出した引掛け部１２ｂが形成されている。引掛け部１２ａ及び１２ｂは外側容器２０の上端に上方から掛けられる大きさ及び形状を有している。 The inner container 10 is a cup-shaped container that opens upward. Inside the inner container 10, a compartment (first storage compartment referred to in the present invention) for containing the base material 50 and the upper culture solution 60 is formed. The inner container 10 has side walls 11 and a bottom wall 15 that define the interior space of the container. The side wall 11 has a cylindrical structure that narrows slightly downward. A hook portion 12a projecting leftward is formed at the left end portion of the upper end of the side wall 11 . A right end portion of the upper end of the side wall 11 is formed with a hook portion 12b projecting rightward. The hooks 12a and 12b are sized and shaped to be hooked on the upper end of the outer container 20 from above.
　底壁１５は複数の貫通孔１６を有する膜によって構成されている。底壁１５は側壁１１の下端部に配置されている。なお、底壁１５は側壁１１の上端部から下端部までの途中のいずれかに配置されていてもよい。貫通孔１６は上下方向に底壁１５を貫通している。これにより、貫通孔１６が内側容器１０内の空間と外側容器２０内の空間とを連通させている。貫通孔１６の孔径は、細胞が通過できないように調整されている。例えば、貫通孔１６の孔径は、０．１μｍ以上であることが好ましく、０．４～８．０μｍがより好ましい。また、貫通孔１６は、１×１０５～１×１０８個／ｃｍ２程度の密度で底壁１５に形成されている。なお、貫通孔１６のサイズ及び数は、必要な培地の流通量に応じて適宜調整されてよい。例えば、貫通孔１６は、そのサイズに応じて１つ以上形成されていればよい。 The bottom wall 15 is composed of a membrane having a plurality of through holes 16 . A bottom wall 15 is arranged at the lower end of the side wall 11 . In addition, the bottom wall 15 may be arranged somewhere in the middle from the upper end to the lower end of the side wall 11 . The through hole 16 penetrates the bottom wall 15 in the vertical direction. Thereby, the through hole 16 allows the space inside the inner container 10 and the space inside the outer container 20 to communicate with each other. The diameter of the through-hole 16 is adjusted so that cells cannot pass through it. For example, the hole diameter of the through holes 16 is preferably 0.1 μm or more, more preferably 0.4 to 8.0 μm. The through-holes 16 are formed in the bottom wall 15 at a density of about 1×10 5 to 1×10 8 holes/cm 2 . The size and number of the through-holes 16 may be appropriately adjusted according to the required flow rate of culture medium. For example, one or more through-holes 16 may be formed according to their size.
　内側容器１０の大きさは、外側容器２０の大きさ、培養する細胞数、スクリーニング条件等によって適宜選択される。内側容器１０には市販されている細胞培養用のインサートが使用されてもよい。 The size of the inner container 10 is appropriately selected according to the size of the outer container 20, the number of cells to be cultured, screening conditions, and the like. A commercially available insert for cell culture may be used for the inner container 10 .
　外側容器２０は、上方に開口したカップ状の容器である。外側容器２０の内部には、下部培養液７０を収容する区画（本発明でいう第２収容区画）が形成されている。外側容器２０は、上下、左右、及び前後方向に関して側壁１１より大きい。外側容器２０の大きさは、培養する細胞数、スクリーニング条件等によって適宜選択される。外側容器２０には、６、１２、２４、４８、９６ウェル等の市販されている細胞培養用のプレートが用いられてもよい。この場合、１つのウェルが１つの外側容器２０に対応する。 The outer container 20 is a cup-shaped container that opens upward. Inside the outer container 20, a compartment (second storage compartment referred to in the present invention) for containing the lower culture solution 70 is formed. The outer container 20 is larger than the side walls 11 in the vertical, horizontal, and front-to-rear directions. The size of the outer container 20 is appropriately selected depending on the number of cells to be cultured, screening conditions, and the like. For the outer container 20, commercially available plates for cell culture such as 6-, 12-, 24-, 48-, and 96-well plates may be used. In this case, one well corresponds to one outer container 20 .
　外側容器２０には、内側容器１０の側壁１１が上方から挿入されている。内側容器１０は、引掛け部１２ａ及び１２ｂが外側容器２０の上端に掛けられることで外側容器２０に支持されている。なお、１つの外側容器２０に対して、１つの内側容器１０が挿入されてもよいし、複数の内側容器１０が挿入されてもよい。また、内側容器１０を外側容器２０に支持させる構造は、本実施形態に係るものの他、どのようなものが採用されてもよい。 The side wall 11 of the inner container 10 is inserted into the outer container 20 from above. The inner container 10 is supported by the outer container 20 by hooking the hooks 12 a and 12 b on the upper end of the outer container 20 . One inner container 10 may be inserted into one outer container 20, or a plurality of inner containers 10 may be inserted. Also, the structure for supporting the inner container 10 on the outer container 20 may be of any type other than the structure according to the present embodiment.
　内側容器１０及び外側容器２０の材質には、基材５０、上部培養液６０及び下部培養液７０を変質させたり、細胞に悪影響を与えたりしないものが使用されていることが好ましい。例えば、内側容器１０の側壁１１及び外側容器２０には、例えば、ポリスチレン、ポリプロピレン、ポリエチレン等のプラスチック、ガラス、ステンレス等の金属が使用される。また、底壁１５には、例えば、ポリエチレンテレフタレート等のポリエステルやポリカーボネート等の合成樹脂、ステンレス等の金属が使用される。内側容器１０及び外側容器２０の材質は互いに共通していてもよいし、異なっていてもよい。 The materials used for the inner container 10 and the outer container 20 preferably do not degrade the base material 50, the upper culture solution 60 and the lower culture solution 70, or adversely affect the cells. For example, the sidewall 11 of the inner container 10 and the outer container 20 are made of plastic such as polystyrene, polypropylene, polyethylene, etc., or metal such as glass or stainless steel. The bottom wall 15 is made of, for example, polyester such as polyethylene terephthalate, synthetic resin such as polycarbonate, or metal such as stainless steel. The materials of the inner container 10 and the outer container 20 may be the same or different.
　基材５０は培養対象の細胞が埋め込まれる足場材である。基材５０は、内側容器１０の底部に配置され、底壁１５に支持されている。基材５０は、上下方向に厚みを有し、例えば、０．２ｍｍを超える厚みを有する。基材５０の厚みは、なるべく厚みを確保する観点では、好ましくは１ｍｍを超え、より好ましくは２ｍｍ以上、４ｍｍ以上、７ｍｍ以上又は１０ｍｍ以上である。また、基材５０の厚みは、なるべく細胞の生存率を確保する等の観点では、２０ｍｍ以下、好ましくは１５ｍｍ以下、より好ましくは１０ｍｍ以下、８ｍｍ以下又は５ｍｍ以下である。つまり、基材５０の厚みは、０．２～５ｍｍ、０．２～８ｍｍ、０．２～１０ｍｍ、０．２～１５ｍｍ、０．２～２０ｍｍ、１～５ｍｍ、１～８ｍｍ、１～１０ｍｍ、１～１５ｍｍ、１～２０ｍｍ、２～５ｍｍ、２～８ｍｍ、２～１０ｍｍ、２～１５ｍｍ、２～２０ｍｍ、４～５ｍｍ、４～８ｍｍ、４～１０ｍｍ、４～１５ｍｍ、４～２０ｍｍ、７～８ｍｍ、７～１０ｍｍ、７～１５ｍｍ、７～２０ｍｍ、１０～１５ｍｍ及び１０～２０ｍｍのいずれかの範囲であってよい。基材５０の厚みは、培養の条件、例えば、培養する細胞の厚みや所望する生存率の条件に応じて、上記下限値及び上限値を適宜組み合わせた範囲に設定される。基材５０は、組織の再現性を確保するため、細胞外マトリックスを構成するコラーゲン、フィブロネクチン、ラミニン等を含んだゲルからなることが好ましい。基材５０に使用される物質として、１種類の物質が選択されてもよいし、数種類が混合されてもよい。基材５０を構成する物質の成分比は用途に応じて適宜調整されてよい。例えば、コラーゲンを使用する場合は、骨、軟骨、腱、皮膚、魚鱗等のいずれに由来するものでもよい。 The base material 50 is a scaffold material in which cells to be cultured are embedded. The base material 50 is arranged at the bottom of the inner container 10 and supported by the bottom wall 15 . The base material 50 has a thickness in the vertical direction, and has a thickness exceeding 0.2 mm, for example. The thickness of the base material 50 preferably exceeds 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more, from the viewpoint of ensuring the thickness as much as possible. Moreover, the thickness of the substrate 50 is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less from the viewpoint of ensuring cell viability as much as possible. That is, the thickness of the base material 50 is 0.2 to 5 mm, 0.2 to 8 mm, 0.2 to 10 mm, 0.2 to 15 mm, 0.2 to 20 mm, 1 to 5 mm, 1 to 8 mm, 1 to 10 mm. , 1-15mm, 1-20mm, 2-5mm, 2-8mm, 2-10mm, 2-15mm, 2-20mm, 4-5mm, 4-8mm, 4-10mm, 4-15mm, 4-20mm, 7 ˜8 mm, 7-10 mm, 7-15 mm, 7-20 mm, 10-15 mm and 10-20 mm. The thickness of the base material 50 is set to a range obtained by appropriately combining the above lower limit and upper limit according to the culture conditions, for example, the thickness of the cells to be cultured and the desired viability. The substrate 50 is preferably made of a gel containing collagen, fibronectin, laminin, etc., which constitute an extracellular matrix, in order to ensure tissue reproducibility. As the substance used for the base material 50, one type of substance may be selected, or several types may be mixed. The component ratio of the substances forming the base material 50 may be appropriately adjusted according to the application. For example, when collagen is used, it may be derived from bone, cartilage, tendon, skin, fish scale, or the like.
　基材５０中のコロイドの濃度は、培養期間中、例えば、細胞に関する試験を実施する期間に亘って、細胞の生存率が十分高い範囲（例えば、５０％以上、好ましくは６０％以上、より好ましくは７０％以上、さらに好ましくは８０％以上、最も好ましくは９０％以上）内に留まるように調整可能である。また、基材５０中のコロイドの濃度は、培養する細胞の機能が発現する範囲内で調整されることが好ましい。細胞の機能とは、例えば、スクリーニング等の各種試験において分析の対象となる機能であり、特定の物質を産生する機能や特定の化合物に対して特定の反応を示す機能等をいう。基材５０中のコロイドの濃度を変更することでゲルの硬さ、物質の浸透や拡散しやすさ等の特性を調整可能である。これによって、肝臓等の正常臓器、がん等の病的な組織といった生体内の様々な状況を再現しつつ細胞の試験が可能である。一例として、基材５０中のコラーゲンの濃度が０．５～３．０ｍｇ／ｍｌ、好ましくは１．０～２．５ｍｇ／ｍｌの範囲で調整される。なお、濃度の上限は、基材５０の調製上、可能な値に設定されればよい。 The concentration of the colloid in the base material 50 is in a range in which the viability of the cells is sufficiently high (for example, 50% or more, preferably 60% or more, more preferably 60% or more, more preferably is 70% or more, more preferably 80% or more, and most preferably 90% or more). Moreover, it is preferable that the concentration of the colloid in the base material 50 is adjusted within a range in which the functions of the cells to be cultured are expressed. The function of a cell is, for example, a function to be analyzed in various tests such as screening, and refers to a function of producing a specific substance, a function of exhibiting a specific reaction to a specific compound, and the like. By changing the concentration of the colloid in the base material 50, it is possible to adjust properties such as hardness of the gel and ease of permeation and diffusion of substances. This makes it possible to test cells while reproducing various in vivo conditions such as normal organs such as the liver and pathological tissues such as cancer. As an example, the concentration of collagen in the base material 50 is adjusted in the range of 0.5-3.0 mg/ml, preferably 1.0-2.5 mg/ml. Note that the upper limit of the concentration may be set to a possible value in terms of preparation of the base material 50 .
　基材５０には培地が添加されている。培地には培養する細胞に通常用いられるものが適宜選択される。基材５０を作製するに当たり、必要に応じて緩衝液を加えてもよい。また、ストレプトマイシン、ペニシリン等の抗生物質、ウシ胎児血清等の血清といった細胞培養に必要な物質が適宜添加されてもよい。 A culture medium is added to the base material 50 . A medium commonly used for cells to be cultured is appropriately selected. In producing the base material 50, you may add a buffer solution as needed. In addition, substances required for cell culture, such as antibiotics such as streptomycin and penicillin, and serum such as fetal bovine serum, may be added as appropriate.
　培養する細胞は基材５０内に埋め込まれる。基材５０は、上記の通りの厚みを有し、且つ、細胞外マトリックスを含んでいる。このため、基材５０に埋め込まれた細胞と基材５０の全体が、実際の生体内の厚みがある組織を再現する。このような基材５０を用いて細胞を培養することで、３次元的な構造を再現した状態での培養が可能である。 The cells to be cultured are embedded in the substrate 50. The base material 50 has the thickness as described above and contains an extracellular matrix. Therefore, the cells embedded in the base material 50 and the entire base material 50 reproduce an actual in-vivo thick tissue. By culturing cells using such a substrate 50, it is possible to culture cells in a state in which the three-dimensional structure is reproduced.
　使用される細胞は、正常及び病的な状態の肝臓、すい臓、神経、腎臓、血管等に由来するものが考えられるが、特に限定されない。細胞の生物種は特に限定されず、ヒト、サル、マウス、ラット、イヌ、ネコ、ブタ、ウシ等の動物に由来する細胞を使用することができる。細胞の播種数は、培養する細胞の種類や培養の目的によって適宜決められ、培養装置１つ当たり１×１０２～１×１０７個とすることが好ましい。また、本発明における「細胞」とは隔離した細胞のほか、組織片を構成するものも含む。組織片の厚さは、基材５０内に収まる範囲であればよい。 Cells to be used may be derived from normal or pathological liver, pancreas, nerve, kidney, blood vessel, etc., but are not particularly limited. The biological species of the cells is not particularly limited, and cells derived from animals such as humans, monkeys, mice, rats, dogs, cats, pigs, and cows can be used. The seeding number of cells is appropriately determined according to the type of cells to be cultured and the purpose of culture, and is preferably 1×10 2 to 1×10 7 cells per culture apparatus. In addition, the term "cell" in the present invention includes isolated cells as well as cells that constitute tissue pieces. The thickness of the piece of tissue may be within a range within the substrate 50 .
　上部培養液６０は、内側容器１０内に収容され、基材５０上に貯留している。上部培養液６０には、培養する細胞に通常用いられる培地が適宜選択される。培地は基材５０に使用される培地と同様であっても異なっていてもよい。抗生物質、血清、ｐＨ調整剤等の細胞の培養に必要な物質が上部培養液６０に適宜添加されてもよい。また、スクリーニングの候補物質等、その他の化合物が上部培養液６０に添加されてもよい。 The upper culture solution 60 is housed in the inner container 10 and retained on the substrate 50 . For the upper culture medium 60, a medium commonly used for cells to be cultured is appropriately selected. The medium may be the same as or different than the medium used for substrate 50 . Substances necessary for cell culture, such as antibiotics, serum, and pH adjusters, may be added to the upper culture solution 60 as appropriate. Other compounds such as screening candidate substances may also be added to the upper culture solution 60 .
　下部培養液７０には、培養する細胞に通常用いられる培地が適宜選択される。培地は基材５０又は上部培養液６０に使用される培地と同様であっても異なっていてもよい。抗生物質、血清、ｐＨ調整剤等の細胞培養に必要な物質が下部培養液７０に適宜添加されてもよい。また、スクリーニングの候補物質等、その他の化合物が下部培養液７０に添加されてもよい。 A medium normally used for cells to be cultured is appropriately selected for the lower culture solution 70 . The medium may be the same as or different than the medium used for substrate 50 or top culture 60 . Substances necessary for cell culture, such as antibiotics, serum, and pH adjusters, may be added to the lower culture solution 70 as appropriate. Also, other compounds such as screening candidate substances may be added to the lower culture solution 70 .
　以下、培養装置１を用いた細胞の培養方法について説明する。まず、培養装置１の設置方法として以下を実施する。細胞外マトリックス、５倍又は１０倍等の濃縮培地、緩衝液等を混合した混合溶液中にさらに細胞を播種して混合する。これによって得られた細胞懸濁液を内側容器１０に分注し、内側容器１０を静置して、細胞懸濁液をゲル化させることで基材５０を作製する。 A method for culturing cells using the culture apparatus 1 will be described below. First, the installation method of the culture apparatus 1 is as follows. Cells are further seeded and mixed in a mixed solution containing an extracellular matrix, a 5-fold or 10-fold concentrated medium, a buffer solution, and the like. The cell suspension thus obtained is dispensed into the inner container 10, and the inner container 10 is allowed to stand still to gel the cell suspension, thereby producing the substrate 50.
　次に、外側容器２０に下部培養液７０を収容させる。下部培養液７０は、下方から基材５０に接するように、底壁１５が配置される高さ以上に液面が位置するまで外側容器２０に収容させる。次に、外側容器２０内に内側容器１０を挿入する。次に、上部培養液６０を内側容器１０に収容させる。これにより、内側容器１０内の基材５０に上部培養液６０が上方から接触する。このとき、上部培養液６０の液面が下部培養液７０の液面より高くなるようにする。これにより、上部培養液６０と下部培養液７０の間に水位差が生じる。この水位差により上部培養液６０と下部培養液７０の間に圧力差が生じ、もって、上部培養液６０が基材５０に浸透する。圧力の大きさは、０．０１～１．０ｋＰａが好ましく、０．０１～０．６ｋＰａがより好ましく、０．０１～０．２ｋＰａの範囲がさらに好ましい。水位差は、基材５０に掛かる圧力が、培養期間中、例えば、細胞に関する試験を実施する期間に亘って、基材５０を変形させない範囲とする。基材５０を変形させない期間の長さは、培養する細胞や試験の内容によって異なるが、例えば数時間から数日間が想定される。ここでいう変形は、圧力による形状の変化が生じていることが基材５０の目視観察によって明らかであることを示す。以上の設置方法における各工程の順序は、細胞の培養に問題が生じなければ入れ替わってもよい。 Next, the lower culture solution 70 is accommodated in the outer container 20 . The lower culture medium 70 is accommodated in the outer container 20 until the liquid surface is positioned at or above the height at which the bottom wall 15 is arranged so as to come into contact with the base material 50 from below. Next, insert the inner container 10 into the outer container 20 . Next, the upper culture medium 60 is accommodated in the inner container 10 . As a result, the upper culture solution 60 comes into contact with the substrate 50 in the inner container 10 from above. At this time, the liquid level of the upper culture solution 60 is made higher than the liquid level of the lower culture solution 70 . This causes a water level difference between the upper culture solution 60 and the lower culture solution 70 . Due to this water level difference, a pressure difference is generated between the upper culture solution 60 and the lower culture solution 70 , so that the upper culture solution 60 permeates the substrate 50 . The magnitude of the pressure is preferably 0.01 to 1.0 kPa, more preferably 0.01 to 0.6 kPa, even more preferably 0.01 to 0.2 kPa. The water level difference is such that the pressure applied to the substrate 50 does not deform the substrate 50 during the culture period, for example, the period during which the test on the cells is performed. The length of the period during which the base material 50 is not deformed varies depending on the cells to be cultured and the content of the test, but is assumed to be several hours to several days, for example. The term "deformation" as used herein indicates that it is evident from visual observation of the substrate 50 that the shape has changed due to pressure. The order of the steps in the installation method described above may be changed as long as there is no problem in culturing the cells.
　次に、図１（ｂ）及び（ｃ）に示すように、時間の経過に伴って上部培養液６０の基材５０への浸透が進む。基材５０に含まれている混合溶液や基材５０に浸透した上部培養液６０は、基材５０を徐々に通過し、底壁１５の貫通孔１６を通じて下部培養液７０に流入する。内側容器１０には、上部培養液６０が減るのに応じて新しい培地を適宜のタイミングで継ぎ足す。これにより、基材５０には上部培養液６０から新鮮な培地が流入し、古い培地が下部培養液７０へと流出する。よって、細胞の培養を適切に継続することができる。上部培養液６０の液面が下部培養液７０の液面より高く水位差が生じている間は、上部培養液６０が基材５０に浸透し、基材５０には新鮮な培地が流入することとなる。 Next, as shown in FIGS. 1(b) and (c), the permeation of the upper culture solution 60 into the substrate 50 progresses over time. The mixed solution contained in the base material 50 and the upper culture medium 60 permeating the base material 50 gradually pass through the base material 50 and flow into the lower culture medium 70 through the through holes 16 of the bottom wall 15 . The inner container 10 is replenished with new culture medium at an appropriate timing as the upper culture medium 60 decreases. As a result, fresh medium flows from the upper culture medium 60 into the substrate 50 and old medium flows out into the lower culture medium 70 . Therefore, cell culture can be appropriately continued. While the liquid level of the upper culture solution 60 is higher than the liquid level of the lower culture solution 70 and a water level difference is generated, the upper culture solution 60 permeates the base material 50 and fresh medium flows into the base material 50. becomes.
　以上説明した本実施形態によると、基材５０にゲルを用いることで基材５０に３次元構造を持たせることができる。また、基材５０の厚さは、０．２ｍｍを超えており、好ましくは１ｍｍを超え、より好ましくは２ｍｍ以上である。なお、基材５０の厚みは、好ましくは１ｍｍを超え、より好ましくは２ｍｍ以上、４ｍｍ以上、７ｍｍ以上又は１０ｍｍ以上である。また、基材５０の厚みは、２０ｍｍ以下、好ましくは１５ｍｍ以下、より好ましくは１０ｍｍ以下、８ｍｍ以下又は５ｍｍ以下である。基材５０の厚みは、培養の条件、例えば、培養する細胞の厚みや所望する生存率の条件に応じて、上記下限値及び上限値を適宜組み合わせた範囲に設定されてよい。 According to the present embodiment described above, it is possible to give the base material 50 a three-dimensional structure by using a gel for the base material 50 . Also, the thickness of the base material 50 exceeds 0.2 mm, preferably exceeds 1 mm, and more preferably is 2 mm or more. The thickness of the base material 50 preferably exceeds 1 mm, more preferably 2 mm or more, 4 mm or more, 7 mm or more, or 10 mm or more. Also, the thickness of the base material 50 is 20 mm or less, preferably 15 mm or less, more preferably 10 mm or less, 8 mm or less, or 5 mm or less. The thickness of the substrate 50 may be set to a range obtained by appropriately combining the above lower limit and upper limit depending on the culture conditions, for example, the thickness of the cells to be cultured and the desired viability.
　これに対し、従来技術によると基材に厚みを持たせるのが困難である。例えば、培養液の供給に加圧を用いない方法によると、基材内に培養液を浸透させにくいことから、基材の厚みはせいぜい０．１～０．２ｍｍ程度までである。また、培養液の供給に加圧を用いる特許文献１によると、組織の厚みをせいぜい１ｍｍ程度にできる旨の記載がある。これに対し、本実施形態に係る基材５０によると、上記の通り、これら従来技術を超える厚みを実現できる。このため、組織内の３次元的な構造を再現しやすくなる。また、基材５０の厚みが増すことにより、１つの培養装置１あたりに培養できる細胞量が増やせるため、細胞機能の発現を高感度に検出しやすくなる。 On the other hand, according to the conventional technology, it is difficult to make the base material thick. For example, according to a method that does not use pressurization to supply the culture medium, the thickness of the base material is at most about 0.1 to 0.2 mm because it is difficult for the culture medium to permeate into the base material. Moreover, according to Patent Document 1, which uses pressurization to supply the culture medium, there is a description that the thickness of the tissue can be reduced to about 1 mm at most. On the other hand, according to the base material 50 according to the present embodiment, as described above, it is possible to realize a thickness exceeding these conventional techniques. Therefore, it becomes easier to reproduce the three-dimensional structure in the tissue. Moreover, since the amount of cells that can be cultured per culture apparatus 1 can be increased by increasing the thickness of the substrate 50, it becomes easier to detect the expression of cell functions with high sensitivity.
　さらに、本実施形態によると、水位差によって上部培養液６０と下部培養液７０の間に圧力差を生じさせ、もって、基材５０中に上部培養液６０を浸透させる。これに関し、特許文献１は、高圧ガスを用いて容器内を加圧する構成上、基材を変形させない範囲に圧力を保つことが困難である。基材が変形すると、３次元的な構造を再現させた状態が維持できない。このため、仮に基材に１ｍｍ程度の厚みを持たせたとしても、これを用いた３次元的な培養を維持できないおそれがある。これに対し、本実施形態は、水位差の調整によって圧力差を調整できるので、基材５０に加わる圧力を調整しやすい。よって、基材５０に係る圧力を基材５０の変形を抑制できる大きさに保ちやすい。このため、基材５０に厚みを持たせつつ３次元的な培養を継続することが可能となる。 Furthermore, according to the present embodiment, a pressure difference is generated between the upper culture solution 60 and the lower culture solution 70 due to the water level difference, and thus the upper culture solution 60 permeates into the substrate 50 . In relation to this, in Patent Literature 1, it is difficult to keep the pressure within a range that does not deform the base material due to the structure in which the inside of the container is pressurized using a high-pressure gas. If the base material is deformed, the state in which the three-dimensional structure is reproduced cannot be maintained. Therefore, even if the substrate has a thickness of about 1 mm, it may not be possible to maintain three-dimensional culture using this substrate. In contrast, in the present embodiment, the pressure difference can be adjusted by adjusting the water level difference, so the pressure applied to the base material 50 can be easily adjusted. Therefore, it is easy to keep the pressure applied to the base material 50 at a level that can suppress the deformation of the base material 50 . Therefore, three-dimensional culture can be continued while the base material 50 has a certain thickness.
　なお、本実施形態に係る培養装置１は、内側容器１０、外側容器２０、基材５０、上部培養液６０及び下部培養液７０を一組とするキットの態様で提供されてもよい。当該キットが組み立てられることで培養装置１が作製される。 The culture device 1 according to this embodiment may be provided in the form of a kit including the inner container 10, the outer container 20, the substrate 50, the upper culture solution 60 and the lower culture solution 70 as a set. The culture device 1 is produced by assembling the kit.
［第２実施形態（スクリーニング方法）］
　以下、上述の実施形態に係る培養方法をスクリーニング方法に応用した第２実施形態について説明する。本実施形態では、図２に示すように、外側容器２０として、市販の複数のウェル（１つのウェルが１つの外側容器２０に相当）を有する培養プレート１０００が使用される。培養プレート１０００には、６ウェル、１２ウェル、２４ウェル、４８ウェル、７２ウェル、９６ウェル等、目的の用途に応じたウェル数の培養プレートが選択される。例えば、薬剤、食品、化粧品、等に用いられる、種類や濃度が異なる化合物Ａ、Ｂ、Ｃ、・・・・、Ｘをそれぞれ添加した上部培養液６０と、化合物Ａ～Ｘを添加していないコントロールの上部培養液６０とを準備し、各ウェルに対応する各培養装置１に分注する。そして、各培養装置１で細胞を培養後、細胞の機能や生存率を評価する。 [Second embodiment (screening method)]
A second embodiment in which the culture method according to the above embodiment is applied to a screening method will be described below. In this embodiment, as shown in FIG. 2, a commercially available culture plate 1000 having a plurality of wells (one well corresponds to one outer container 20) is used as the outer container 20. FIG. As the culture plate 1000, a culture plate having a number of wells such as 6 wells, 12 wells, 24 wells, 48 wells, 72 wells, 96 wells, etc. is selected according to the intended use. For example, an upper culture solution 60 to which compounds A, B, C, . A control upper culture solution 60 is prepared and dispensed into each culture device 1 corresponding to each well. After the cells are cultured in each culture apparatus 1, the functions and viability of the cells are evaluated.
　培養装置１によると、基材に加わる圧力を調整しやすく、例えば、圧力を小さくできるため、上部培養液６０に用いる試薬等の化合物の量を減らすことが可能である。よって、培養装置１は、複数の装置が必要となるスクリーニング方法に特に適した培養方法である。また、仮に、高圧ガスやポンプ式の加圧用の機械を用いるとすると、ウェルごとに機械を設けたり各ウェルに対して個別に圧力を調整するための機構を追加したりする必要があり、装置の構成が複雑になったり、各ウェルや装置全体の規模が過大になったりするおそれがある。これに対し、培養装置１は各ウェルの大きさを抑えることができると共に、全体の構成を複雑にしたり、規模を大きくしたりすることなく装置ごとの加圧が可能である。よって、この観点でもスクリーニング方法に適している。 According to the culture device 1, the pressure applied to the substrate can be easily adjusted, for example, the pressure can be reduced, so the amount of compounds such as reagents used in the upper culture solution 60 can be reduced. Therefore, the culturing device 1 is a culturing method particularly suitable for screening methods that require a plurality of devices. In addition, if a high-pressure gas or pump-type pressurization machine is used, it is necessary to provide a machine for each well or add a mechanism for adjusting the pressure individually for each well. , and the scale of each well or the entire device may become too large. On the other hand, the culture apparatus 1 can suppress the size of each well and pressurize each apparatus without complicating the overall configuration or increasing the scale. Therefore, from this point of view as well, it is suitable for screening methods.
［第３実施形態（別構造の容器）］
　本発明のさらに別の実施形態である第３実施形態について、図３を参照しつつ説明する。上述の実施形態と同様の装置構成については同じ符号を用いて説明するか、説明を適宜省略する。 [Third Embodiment (Container with Different Structure)]
A third embodiment, which is still another embodiment of the present invention, will be described with reference to FIG. Apparatus configurations similar to those of the above-described embodiments will be described using the same reference numerals, or description thereof will be omitted as appropriate.
　本実施形態に係る培養装置２は、内側容器３０、外側容器４０、基材５０、上部培養液６０、及び下部培養液７０を有する。培養装置２は単独で使用されてもよいし、複数同時に使用されてもよい。なお、以下において、図３に示すように、培養装置２の横方向を左右方向、紙面に沿った方向であって左右方向と直交する方向を上下方向という。 The culture device 2 according to this embodiment has an inner container 30, an outer container 40, a substrate 50, an upper culture solution 60, and a lower culture solution 70. The culture device 2 may be used singly or in combination. In the following, as shown in FIG. 3, the lateral direction of the culture apparatus 2 is referred to as the left-right direction, and the direction along the plane of the paper and orthogonal to the left-right direction is referred to as the up-down direction.
　内側容器３０は、上部容器８０、下部容器９０及び支持体９３を有する。内側容器３０の内部には、基材５０及び上部培養液６０を収容する区画（本発明でいう第１収容区画）が形成されている。内側容器３０の材質は内側容器１０の材質と同様である。 The inner container 30 has an upper container 80 , a lower container 90 and a support 93 . Inside the inner container 30, a compartment (first storage compartment referred to in the present invention) for containing the substrate 50 and the upper culture medium 60 is formed. The material of the inner container 30 is the same as the material of the inner container 10 .
　上部容器８０はシリンジ形状をしている。上部容器８０は、筒状部８１、シリンジ先端の凸部８３、及び凸部８３につながるテーパ部８２を有している。筒状部８１の上下方向の大きさは使用する上部培養液６０の量によって決められる。テーパ部８２の上端部８２ａにおいては、左右方向の下部容器９０の開口の径とほぼ同じである。上部容器８０は凸部８３が下側に配置されつつ下部容器９０に上方から挿入される。上部容器８０は上部培養液６０を収容している。この上部培養液６０は、凸部８３の下端開口を通じて下部容器９０へと流下する。 The upper container 80 has a syringe shape. The upper container 80 has a cylindrical portion 81 , a convex portion 83 at the tip of the syringe, and a tapered portion 82 connected to the convex portion 83 . The size of the tubular portion 81 in the vertical direction is determined by the amount of the upper culture solution 60 to be used. The diameter of the upper end portion 82a of the tapered portion 82 is substantially the same as the diameter of the opening of the lower container 90 in the left-right direction. The upper container 80 is inserted into the lower container 90 from above with the convex portion 83 arranged on the lower side. The upper container 80 contains the upper culture solution 60 . This upper culture medium 60 flows down into the lower container 90 through the lower end opening of the projection 83 .
　下部容器９０は上方に開口したカップ状の容器である。下部容器９０は上部培養液６０及び基材５０を収容する。下部容器９０は側壁９１及び底壁９５を有する。側壁９１は下方に向かってやや窄まった筒状の構造である。側壁９１の上端の左端部には左方向に突出した引掛け部９２ａが形成されている。側壁９１の上端の右端部には右方向に突出した引掛け部９２ｂが形成されている。 The lower container 90 is a cup-shaped container that opens upward. The lower container 90 contains the upper culture medium 60 and the substrate 50 . Lower container 90 has side walls 91 and bottom wall 95 . The side wall 91 has a cylindrical structure that narrows slightly downward. A hook portion 92a projecting leftward is formed at the left end portion of the upper end of the side wall 91 . A right end portion of the upper end of the side wall 91 is formed with a hook portion 92b projecting rightward.
　底壁９５は貫通孔９６を有する膜によって構成されている。底壁９５は側壁９１の下端部に配置されている。なお、底壁９５は側壁９１の上端部から下端部までの途中のいずれかに配置されていてもよい。貫通孔９６は上下方向に底壁９５を貫通している。これにより、貫通孔９６が下部容器９０内の空間と外側容器４０内の空間とを連通させている。貫通孔９６の孔径は、細胞が通過できないように調整されている。例えば、貫通孔９６の孔径は、０．１μｍ以上であることが好ましく、０．４～８．０μｍがより好ましい。また、貫通孔９６は、１×１０５～１×１０８個／ｃｍ２程度の密度で底壁９５に形成されている。なお、貫通孔９６のサイズ及び数は、必要な培地の流通量に応じて適宜調整されてよい。例えば、貫通孔９６は、そのサイズに応じて１つ以上形成されていればよい。 The bottom wall 95 is composed of a membrane having through holes 96 . A bottom wall 95 is arranged at the lower end of the side wall 91 . In addition, the bottom wall 95 may be arranged somewhere in the middle from the upper end to the lower end of the side wall 91 . The through hole 96 vertically penetrates the bottom wall 95 . Thereby, the through hole 96 allows the space inside the lower container 90 and the space inside the outer container 40 to communicate with each other. The diameter of the through-hole 96 is adjusted so that cells cannot pass through it. For example, the hole diameter of the through holes 96 is preferably 0.1 μm or more, more preferably 0.4 to 8.0 μm. Also, the through holes 96 are formed in the bottom wall 95 at a density of about 1×10 5 to 1×10 8 holes/cm 2 . Note that the size and number of the through-holes 96 may be appropriately adjusted according to the required medium flow rate. For example, one or more through-holes 96 may be formed according to their size.
　下部容器９０には上部容器８０のテーパ部８２及び凸部８３が上方から挿入されている。テーパ部８２の上端部８２ａが下部容器９０の側壁９１上端部の内面に接触することで下部容器９０が上部容器８０を支持している。 The tapered portion 82 and the convex portion 83 of the upper container 80 are inserted into the lower container 90 from above. The lower container 90 supports the upper container 80 by contacting the inner surface of the upper end of the side wall 91 of the lower container 90 with the upper end 82 a of the tapered portion 82 .
　支持体９３は、筒部９３ａ及びスペーサー９３ｂを有する。支持体９３は上部容器８０と下部容器９０を外側容器４０の底面上で支持し、且つ下部容器９０の上下方向の位置を調節する役割を果たす。筒部９３ａは上下方向に延びた筒形状を有する。支持体９３の上下方向の長さは、下部容器９０における側壁９１の上下方向の長さより大きい。筒部９３ａは、外側容器４０の底面に置かれた板状のスペーサー９３ｂ上に設置されている。筒部９３ａの内部は下部容器９０の側壁９１が挿入できる大きさである。筒部９３ａの径は、筒部９３ａの上端に引掛け部９２ａ及び９２ｂが引っ掛けられる程度の大きさである。筒部９３ａ内部に下部容器９０の側壁９１が挿入されつつ筒部９３ａの上端に引掛け部９２ａ及び９２ｂが掛けられることで下部容器９０が支持体９３に支持されている。 The support 93 has a tubular portion 93a and a spacer 93b. The support 93 supports the upper container 80 and the lower container 90 on the bottom surface of the outer container 40, and serves to adjust the position of the lower container 90 in the vertical direction. The tubular portion 93a has a tubular shape extending in the vertical direction. The vertical length of the support 93 is greater than the vertical length of the side wall 91 of the lower container 90 . The tubular portion 93 a is placed on a plate-like spacer 93 b placed on the bottom surface of the outer container 40 . The inside of the cylindrical portion 93a is large enough to allow the side wall 91 of the lower container 90 to be inserted therein. The diameter of the cylindrical portion 93a is large enough to hook the hooks 92a and 92b on the upper end of the cylindrical portion 93a. The lower container 90 is supported by the support 93 by inserting the side wall 91 of the lower container 90 into the cylindrical portion 93a and hooking the hooks 92a and 92b on the upper end of the cylindrical portion 93a.
　外側容器４０は上方に開口したカップ状の容器である。外側容器４０の内部には、下部培養液７０を収容する区画（本発明でいう第２収容区画）が形成されている。外側容器４０の底面上には、上記の通り支持体９３によって内側容器３０が支持されている。内側容器３０は、下部容器９０の側壁９１が外側容器４０に挿入されつつ支持体９３によって支持されている。内側容器３０の全体は、外側容器４０の上端より上方へと大きく突出している。外側容器４０の材質は外側容器２０の材質と同様である。 The outer container 40 is a cup-shaped container that opens upward. Inside the outer container 40, a compartment (second storage compartment referred to in the present invention) for containing the lower culture solution 70 is formed. The inner container 30 is supported on the bottom surface of the outer container 40 by the supports 93 as described above. The inner container 30 is supported by supports 93 while the side walls 91 of the lower container 90 are inserted into the outer container 40 . The entire inner container 30 protrudes greatly upward from the upper end of the outer container 40 . The material of the outer container 40 is the same as the material of the outer container 20 .
　培養装置２の設置方法は以下の通りである。培養装置１同様に細胞を播種した細胞懸濁液を下部容器９０に分注し、下部容器９０を静置して、細胞懸濁液をゲル化させることで基材５０を作製する。次に、外側容器４０の底面上に支持体９３を設置する。次に、側壁９１を筒部９３ａ内に挿入しつつ引掛け部９２ａ及び９２ｂを筒部９３ａの上端に掛けることで、支持体９３に下部容器９０を支持させる。次に、外側容器４０に下部培養液７０を収容させる。下部培養液７０は、下方から基材５０に接するように、底壁９５が配置される高さ以上に液面が位置するまで外側容器４０に収容させる。次に、下部容器９０内の基材５０の上方から接触するように上部培養液６０を入れて下部容器９０を培養液で満たし、上部容器８０を下部容器９０に上方から挿入し、下部容器９０に上部容器８０を支持させる。その際に、上部容器８０および下部容器９０にあらかじめ真空用グリースなどからなるシール材を塗布しておき、上部容器８０の上端部８２ａと下部容器９０の引掛け部９２ａ及び９２ｂとの間を密閉する。次に、上部培養液６０の追加分を上部容器８０に収容させる。このとき、上部培養液６０の液面が外側容器４０の上端より上方に配置されるようにする。以上の設置方法における各工程の順序は、細胞の培養に問題が生じなければ入れ替わってもよい。なお、培養装置２は、培養装置１同様に化合物のスクリーニング方法に利用することができる。 The installation method of the culture device 2 is as follows. A cell suspension in which cells are seeded in the same manner as in the culture apparatus 1 is dispensed into the lower container 90, and the lower container 90 is allowed to stand still to gel the cell suspension, thereby producing the substrate 50. FIG. Next, a support 93 is placed on the bottom surface of the outer container 40 . Next, the lower container 90 is supported by the support 93 by hooking the hooks 92a and 92b on the upper end of the cylindrical portion 93a while inserting the side wall 91 into the cylindrical portion 93a. Next, the lower culture solution 70 is accommodated in the outer container 40 . The lower culture medium 70 is accommodated in the outer container 40 until the liquid surface is positioned at a height equal to or higher than the bottom wall 95 so as to come into contact with the base material 50 from below. Next, the upper container 90 is filled with the culture solution by filling the lower container 90 with the upper culture solution 60 so as to come into contact with the base material 50 in the lower container 90 from above, and the upper container 80 is inserted into the lower container 90 from above. to support the upper container 80 . At this time, a sealing material made of vacuum grease or the like is applied in advance to the upper container 80 and the lower container 90, and the upper end portion 82a of the upper container 80 and the hook portions 92a and 92b of the lower container 90 are sealed. do. Next, an additional portion of the upper culture solution 60 is accommodated in the upper container 80 . At this time, the liquid surface of the upper culture solution 60 is arranged above the upper end of the outer container 40 . The order of the steps in the installation method described above may be changed as long as there is no problem in culturing the cells. It should be noted that the culture apparatus 2 can be used for a compound screening method, like the culture apparatus 1 .
　培養装置２によると、上部培養液６０の液面を比較的高く設定できる。このため、水位差の初期値を大きく設定しやすく、培養を長時間継続した場合でも上部培養液６０を継ぎ足す回数を抑制できる。なお、圧力の大きさは、０．０１～２．５ｋＰａの範囲であればよく、０．０１～１．０ｋＰａの範囲が好ましく、０．１～０．６ｋＰａがより好ましい。 According to the culture device 2, the liquid level of the upper culture solution 60 can be set relatively high. Therefore, it is easy to set a large initial value of the water level difference, and the number of times of replenishing the upper culture solution 60 can be suppressed even when the culture is continued for a long time. The pressure may be in the range of 0.01 to 2.5 kPa, preferably in the range of 0.01 to 1.0 kPa, more preferably in the range of 0.1 to 0.6 kPa.
　以下、実施例を挙げて本発明を詳細に説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.
　（実施例１）
　図４に示すように、２次元培養による肝細胞の解毒酵素の発現量（比較例）と培養装置１による肝細胞の解毒酵素の発現量（実施例１）について評価した。 (Example 1)
As shown in FIG. 4, the expression level of the hepatocyte detoxification enzyme by the two-dimensional culture (comparative example) and the expression level of the hepatocyte detoxification enzyme by the culture apparatus 1 (Example 1) were evaluated.
　培養装置１の作製に当たり、内側容器１０は２４ウェルインサート（ＣＯＲＮＩＮＧ社製）を使用した。底壁１５の貫通孔１６の孔径は０．４μｍであった。外側容器２０は２４ウェルの細胞培養プレート（日本ジェネティクスまたはＣＯＲＮＩＮＧ社製）を使用した。細胞は肝臓モデル細胞としてＨｅｐＧ２細胞（ヒト肝臓癌由来細胞株）を使用した。基材５０は３．０ｍｇ／ｍｌコラーゲン溶液（Ｃｅｌｌｍａｔｒｉｘ　Ｔｙｐｅ　Ｉ－Ｐ、新田ゼラチン社製）、１０倍濃縮ＤＭＥＭ培地（ダルベッコ改変イーグル培地、Ｔｈｅｒｍｏ社製）、及び緩衝液（再構成緩衝液、新田ゼラチン社製）を使用した。上部培養液６０及び下部培養液７０にはＤＭＥＭ培地（Ｓｉｇｍａ社製）を使用した。 In producing the culture device 1, the inner container 10 used a 24-well insert (manufactured by CORNING). The hole diameter of the through hole 16 of the bottom wall 15 was 0.4 μm. A 24-well cell culture plate (manufactured by Nippon Genetics or CORNING) was used as the outer container 20 . HepG2 cells (human liver cancer-derived cell line) were used as liver model cells. The base material 50 is a 3.0 mg/ml collagen solution (Cellmatrix Type IP, manufactured by Nitta Gelatin), a 10-fold concentrated DMEM medium (Dulbecco's Modified Eagle Medium, manufactured by Thermo), and a buffer solution (reconstitution buffer, Nitta Gelatin Co., Ltd.) was used. A DMEM medium (manufactured by Sigma) was used for the upper culture medium 60 and the lower culture medium 70 .
　次の通り、コラーゲン溶液、１０倍濃縮ＤＭＥＭ培地及び緩衝液の混合溶液から、２．４ｍｇ／ｍｌのコラーゲンを含有する基材５０を作製した。コラーゲン溶液：１０倍濃縮ＤＭＥＭ培地：緩衝液＝８：１：１の体積比率で氷上にて混合した。次に、混合溶液約２００μｌに細胞２．０×１０６個を混合し、細胞懸濁液を内側容器１０に収容させた。次に、３７℃のインキュベーターに内側容器１０を約３０分間静置して、細胞懸濁液をゲル化させて基材５０を作製した。基材５０の上下方向の厚さは約３～５ｍｍであった。次に、１００μｌ程度の下部培養液７０を外側容器２０に充填し、上方から外側容器２０に内側容器１０を挿入した。次に、１０００μｌの上部培養液６０を内側容器１０に収容させた。上部培養液６０と下部培養液７０の間に水位差を生じさせ、これによって基材５０に掛ける圧力を０．１～０．６ｋＰａとした。 A base material 50 containing 2.4 mg/ml collagen was prepared from a mixed solution of collagen solution, 10-fold concentrated DMEM medium, and buffer as follows. Collagen solution: 10-fold concentrated DMEM medium: buffer = 8:1:1 mixed on ice. Next, about 200 μl of the mixed solution was mixed with 2.0×10 6 cells, and the cell suspension was placed in the inner container 10 . Next, the inner container 10 was allowed to stand in an incubator at 37° C. for about 30 minutes to gel the cell suspension to produce the substrate 50 . The thickness of the substrate 50 in the vertical direction was about 3-5 mm. Next, the outer container 20 was filled with about 100 μl of the lower culture solution 70, and the inner container 10 was inserted into the outer container 20 from above. Next, 1000 μl of the upper culture medium 60 was placed in the inner container 10 . A water level difference was generated between the upper culture solution 60 and the lower culture solution 70, and the pressure applied to the substrate 50 was set to 0.1 to 0.6 kPa.
　比較例として２次元培養の培養装置を次の通り作製した。上記同様の混合溶液約５００μｌを内径３５ｍｍのプラスチックディッシュ（Ｆａｌｃｏｎ社製）に収容させ、ゲル化させて基材を作製した。基材の上面に２．０×１０６個の細胞を播種し、細胞の上方から約２０００μｌの上部培養液６０と同様の培養液を容器に収容させた。 As a comparative example, a culture apparatus for two-dimensional culture was produced as follows. About 500 μl of the mixed solution similar to the above was placed in a plastic dish (manufactured by Falcon) having an inner diameter of 35 mm, and gelled to prepare a substrate. 2.0×10 6 cells were seeded on the upper surface of the substrate, and about 2000 μl of the same culture solution as the upper culture solution 60 was placed in a container from above the cells.
　次に、３７℃のインキュベーター内に培養装置１及び２次元培養の培養装置を静置して、３日間細胞培養を行った。この間、培養装置１の上部培養液６０については１２時間おきに継ぎ足すとともに、外側容器４０内の培養液については増加分を除去した。２次元培養では1日おきに培養液を新鮮なものに交換した。次にＫＡＰＡ　ＳＹＢＲ　Ｆａｓｔ　ｑＰＣＲ　Ｋｉｔ（日本ジェネティクス社製）を使用して、ＲＴ－ｑＰＣＲ（ｒｅｖｅｒｓｅ　ｔｒａｎｓｃｒｉｐｔｉｏｎ　ｑｕａｎｔｉｔａｔｉｖｅ　ＰＣＲ、定量的逆転写ＰＣＲ法）にて、解毒酵素ＣＹＰ１Ａ１の発現量を定量した。各培養装置の細胞からＲＮＡを抽出し、逆転写酵素によりＣＹＰ１Ａ１のｍＲＮＡをｃＤＮＡに逆転写し、ｃＤＮＡを増幅してＣＹＰ１Ａ１の発現量を定量した。ＣＹＰ１Ａ１の発現量は内在性コントロール（Ｂｅｔａ－ａｃｔｉｎ）で補正した。２次元培養のＣＹＰ１Ａ１の発現量を基準として、培養装置１のＣＹＰ１Ａ１の相対発現量を評価した。培養装置１の相対発現量は２次元培養における発現量の４倍程度であった。培養装置１によって、生体内の厚みのある組織の内部環境を再現しつつ細胞の培養が可能となったと考えられる。 Next, the culture device 1 and the culture device for two-dimensional culture were placed in an incubator at 37°C, and cell culture was performed for 3 days. During this period, the upper culture medium 60 of the culture apparatus 1 was replenished every 12 hours, and the increased culture medium in the outer container 40 was removed. In the two-dimensional culture, the culture medium was replaced with fresh one every other day. Next, the expression level of the detoxification enzyme CYP1A1 was quantified by RT-qPCR (reverse transcription quantitative PCR, quantitative reverse transcription PCR method) using KAPA SYBR Fast qPCR Kit (manufactured by Nippon Genetics). RNA was extracted from the cells in each culture apparatus, CYP1A1 mRNA was reverse transcribed into cDNA using reverse transcriptase, and the cDNA was amplified to quantify the expression level of CYP1A1. The expression level of CYP1A1 was corrected with an endogenous control (Beta-actin). Based on the expression level of CYP1A1 in the two-dimensional culture, the relative expression level of CYP1A1 in the culture apparatus 1 was evaluated. The relative expression level in culture apparatus 1 was about four times the expression level in two-dimensional culture. It is considered that the culture apparatus 1 enables cell culture while reproducing the internal environment of a thick tissue in vivo.
　（従来例）
　図５及び６に示すように、高圧ガスによって培養液が灌流する従来の培養装置３００を使用して、灌流実験を行った。 (conventional example)
As shown in FIGS. 5 and 6, perfusion experiments were performed using a conventional culture apparatus 300 in which the culture solution is perfused by high pressure gas.
　培養装置３００は、図５に示すように、圧力装置３１０、培養液ボトル３２０、加圧デバイス３３０、及び廃液ボトル３４０を有している。加圧デバイス３３０内には基材３５０が収容されている。基材３５０は、実施例１の基材５０と同様の成分であるコラーゲン濃度２．４mｇ／ｍｌの混合溶液とＨｅｐＧ２細胞２．０×１０６個を含有する。基材３５０は実施例１の基材５０と同様に作製されたゲル状の基材である。 The culture device 300 has a pressure device 310, a culture solution bottle 320, a pressure device 330, and a waste solution bottle 340, as shown in FIG. A substrate 350 is housed within the pressure device 330 . The base material 350 contains a mixed solution with a collagen concentration of 2.4 mg/ml and 2.0×10 6 HepG2 cells, which are the same components as the base material 50 of Example 1. A base material 350 is a gel-like base material produced in the same manner as the base material 50 of the first embodiment.
　培養装置３００は次の通り使用した。圧力装置３１０から３ｋＰａの高圧ガスを培養液ボトル３２０に供給させた。次に、培養液ボトル３２０に収容された培養液を、高圧ガスにより培養液ボトル３２０から加圧デバイス３３０に供給させた。これにより、図６（ｂ）に示すように、基材３５０の左方向から右方向へ培養液が灌流した。次に、灌流により基材３５０を通過した培養液を加圧デバイス３３０から廃液ボトル３４０に排出させた。このようにして、基材３５０に新鮮な培養液を灌流しながら、３７℃のインキュベーター内で３日間細胞培養を行った。 The culture device 300 was used as follows. A high-pressure gas of 3 kPa was supplied from the pressure device 310 to the culture solution bottle 320 . Next, the culture solution contained in the culture solution bottle 320 was supplied from the culture solution bottle 320 to the pressurizing device 330 by high-pressure gas. As a result, as shown in FIG. 6(b), the culture solution was perfused from the left to the right of the substrate 350. As shown in FIG. Next, the culture solution passed through the substrate 350 by perfusion was discharged from the pressure device 330 to the waste solution bottle 340 . In this manner, cell culture was performed for 3 days in an incubator at 37° C. while perfusing the substrate 350 with a fresh culture medium.
　灌流前、図６（ａ）に実線で示すように基材３５０は矩形の平面形状を呈していたが、培養開始３日後には図６（ｃ）に実線で示したように基材３５０に窪みが生じた。これに対し、本発明に係る実施例１～７では、目視観察でこのような変形が生じなかった。 Before perfusion, the substrate 350 had a rectangular planar shape as indicated by the solid line in FIG. 6(a). A dent was formed. In contrast, in Examples 1 to 7 according to the present invention, such deformation did not occur by visual observation.
　（実施例２）
　図７及び８に示すように、水位差がない培養（比較例）と培養装置１による水位差がある培養（実施例２）の細胞の生存率について評価した。 (Example 2)
As shown in FIGS. 7 and 8, cell viability was evaluated for culture with no water level difference (comparative example) and culture with water level difference by the culture apparatus 1 (example 2).
　水位差がある場合として、実施例１の培養方法を実施した。比較例として、水位差がない場合については以下の条件で培養方法を実施した。１０００μｌの下部培養液７０を外側容器２０に収容させ、上部培養液６０と下部培養液７０の間に水位差が生じないように培養装置を作製した。その他の構成及び条件については、水位差がある場合と同様とした。 The culture method of Example 1 was implemented assuming that there was a water level difference. As a comparative example, the culture method was performed under the following conditions when there was no water level difference. 1000 μl of the lower culture solution 70 was placed in the outer container 20, and the culture apparatus was prepared so that the water level difference between the upper culture solution 60 and the lower culture solution 70 was not generated. Other configurations and conditions were the same as when there was a water level difference.
　次に、３日間培養後の各培養装置の基材５０において、４％パラホルムアルデヒド溶液（溶媒はリン酸緩衝液）に基材５０を浸し、４℃で一晩静置し、リン酸緩衝液で３回洗浄したのちに、上下方向及び左右方向の中心部のゲルを採取した。次に、０．５％ＴｒｉｔｏｎＸ１００溶液（溶媒はリン酸緩衝液）にゲルを浸して室温で１５分振とうした。その後リン酸緩衝液で３回洗浄したのちに、２～１０μｇ/ｍｌに希釈したＨｏｅｃｈｓｔ３３３４２（Ｓｉｇｍａ社製）及び５００倍希釈したＡｌｅｘａＦｌｕｏｒ－４８８Ｐｈａｌｌｏｉｄｉｎ（Ｔｈｅｒｍｏ社製）溶液（溶媒はリン酸緩衝液）に浸し、４℃で一晩反応させた。その後リン酸緩衝液で３回洗浄したのちに、共焦点レーザー顕微鏡（Ａ１、Ｎｉｋｏｎ社製）で画像を撮影した。生細胞としてＡｌｅｘａＦｌｕｏｒ－４８８Ｐｈａｌｌｏｉｄｉｎを、全細胞として、Ｈｏｅｃｈｓｔ３３３４２及び反射干渉像を取得した。これらの画像から生細胞と全細胞の数を測定し、その比から生存率を評価した。図７（ａ）は水位差がない場合を示し、図７（ｂ）は水位差がある場合の画像を示す（ここでの全細胞は、反射干渉像である）。これらの画像が示す通り、水位差がある場合（培養装置１を用いた場合）については、水位差がない場合と比較して生細胞が多く見られた。図８に示すように、３日間培養後、水位差がない場合は生存率２０％程度であり、培養装置１の場合は生存率９０％以上であった。培養装置１の場合は、上部培養液６０と下部培養液７０の水位差により、常に新鮮な培養液が基材５０に供給されたためと考えられる。 Next, after culturing for 3 days, the substrate 50 of each culture apparatus is immersed in a 4% paraformaldehyde solution (the solvent is a phosphate buffer), allowed to stand overnight at 4° C., and then treated with a phosphate buffer. After washing three times with , the gel in the center in the vertical and horizontal directions was collected. Next, the gel was immersed in a 0.5% Triton X100 solution (solvent: phosphate buffer) and shaken at room temperature for 15 minutes. After washing three times with a phosphate buffer, Hoechst 33342 (manufactured by Sigma) diluted to 2 to 10 μg/ml and AlexaFluor-488 Phalloidin (manufactured by Thermo) diluted 500 times (solvent is phosphate buffer). Immerse and react overnight at 4°C. After washing three times with a phosphate buffer, images were taken with a confocal laser microscope (A1, manufactured by Nikon). AlexaFluor-488 Phalloidin was acquired as live cells, Hoechst 33342 as whole cells, and reflectance interference images were acquired. From these images, the numbers of viable cells and total cells were measured, and the viability was evaluated from the ratio. FIG. 7(a) shows the case without the water level difference, and FIG. 7(b) shows the image with the water level difference (here all cells are reflection interference images). As these images show, when there is a water level difference (when the culture apparatus 1 is used), more viable cells were observed than when there is no water level difference. As shown in FIG. 8, after culturing for 3 days, the survival rate was about 20% when there was no water level difference, and in the case of the culture apparatus 1, the survival rate was 90% or more. In the case of the culture apparatus 1, it is considered that fresh culture medium was always supplied to the substrate 50 due to the water level difference between the upper culture medium 60 and the lower culture medium .
　（実施例３）
　基材５０の上下方向の厚さを４．９ｍｍ、７．８ｍｍ、１０．０ｍｍとした場合の細胞の生存率を評価した。 (Example 3)
Cell viability was evaluated when the thickness of the substrate 50 in the vertical direction was 4.9 mm, 7.8 mm, and 10.0 mm.
　基材５０の上下方向の厚さを４．９ｍｍとした場合は、実施例２と同様の構成及び条件で実施例２の培養方法を実施した。基材５０の上下方向の厚さが７．８ｍｍとした場合については、混合溶液を４００μｌ使用し、その他の構成及び条件を同様にして実施例２の培養方法を実施した。基材５０の上下方向の厚さが１０．０ｍｍとした場合については、混合溶液を６００μｌ使用し、その他の構成及び条件を同様にして実施例２の培養方法を実施した。また、実施例２と同様に各場合についての生存率を評価した。厚さ４．９ｍｍの基材５０の場合は生存率９９％、厚さ７．８ｍｍの場合は生存率８４％、厚さ１０．０ｍｍの場合は生存率５０％であった。基材５０の上下方向の厚さが大きくなるにつれて細胞の生存率は低下したが、１０．０ｍｍの厚さでも５０％の細胞が生存していた。 When the thickness of the substrate 50 in the vertical direction was 4.9 mm, the culture method of Example 2 was carried out under the same configuration and conditions as in Example 2. When the thickness of the substrate 50 in the vertical direction was 7.8 mm, 400 μl of the mixed solution was used, and the culturing method of Example 2 was performed with the other configurations and conditions being the same. When the thickness of the substrate 50 in the vertical direction was 10.0 mm, the culture method of Example 2 was performed using 600 μl of the mixed solution, and the other configurations and conditions were the same. In addition, the survival rate was evaluated in each case in the same manner as in Example 2. The survival rate was 99% for the substrate 50 with a thickness of 4.9 mm, the survival rate was 84% for the substrate 50 with a thickness of 7.8 mm, and the survival rate was 50% for the thickness of 10.0 mm. Although the survival rate of cells decreased as the thickness of the substrate 50 in the vertical direction increased, 50% of the cells survived even at a thickness of 10.0 mm.
　（実施例４）
　基材５０のコラーゲン濃度を２．４ｍｇ／ｍｌ、１．６ｍｇ／ｍｌ、１．２ｍｇ／ｍｌとした場合の細胞の生存率を評価した。 (Example 4)
Cell viability was evaluated when the base material 50 had a collagen concentration of 2.4 mg/ml, 1.6 mg/ml, and 1.2 mg/ml.
　コラーゲン濃度を２．４ｍｇ／ｍｌとした場合は、実施例２と同様に基材５０を作製した。コラーゲン濃度を１．６ｍｇ／ｍｌとした場合は、コラーゲン溶液：ＤＭＥＭ培地：緩衝液＝７：５：１の混合比の混合溶液から実施例２と同様に基材５０を作製した。コラーゲン濃度を１．２ｍｇ／ｍｌとした場合は、コラーゲン溶液：ＤＭＥＭ培地：緩衝液＝７：１０：１の混合比の混合溶液から実施例２と同様に基材５０を作製した。いずれのコラーゲン濃度の場合についても混合溶液がゲル化し、そのゲルが崩れずに維持された。 A base material 50 was produced in the same manner as in Example 2 when the collagen concentration was 2.4 mg/ml. When the collagen concentration was 1.6 mg/ml, the base material 50 was produced in the same manner as in Example 2 from a mixed solution having a mixing ratio of collagen solution:DMEM medium:buffer=7:5:1. When the collagen concentration was 1.2 mg/ml, the base material 50 was produced in the same manner as in Example 2 from a mixed solution having a mixing ratio of collagen solution:DMEM medium:buffer=7:10:1. At any collagen concentration, the mixed solution gelled, and the gel was maintained without collapsing.
　上記各コラーゲン濃度の基材５０を用いてその他の構成及び条件を同様にした培養装置１において、実施例２の培養方法を実施した。また、実施例２と同様に各場合についての生存率を評価した。コラーゲン濃度２．４ｍｇ／ｍｌの基材５０の場合は生存率９９％、１．６ｍｇ／ｍｌの場合は生存率８３％、１．２ｍｇ／ｍｌの場合は生存率９４％であった。いずれのコラーゲン濃度の培養装置１においても８０％以上の細胞が生存していた。 The culture method of Example 2 was carried out in the culture apparatus 1 having the same configuration and other conditions as the base material 50 with each of the collagen concentrations described above. In addition, the survival rate was evaluated in each case in the same manner as in Example 2. The survival rate was 99% for the substrate 50 with a collagen concentration of 2.4 mg/ml, 83% for 1.6 mg/ml, and 94% for 1.2 mg/ml. 80% or more of the cells survived in the culture device 1 with any collagen concentration.
　（実施例５）
　図９に示すように、実施例４の各コラーゲン濃度の基材５０におけるアルブミンの相対発現量を評価した。 (Example 5)
As shown in FIG. 9, the relative expression level of albumin in the substrate 50 with each collagen concentration in Example 4 was evaluated.
　各コラーゲン濃度の基材５０を用いた培養装置１において、細胞数のみ１．５×１０５個とし、その他の構成及び条件を同様にして、実施例１の培養方法を実施した。アルブミンの評価方法も同様にＲＴ－ｑＰＣＲである（内在性コントロールとしてＳ１８を使用）。コラーゲン濃度２．４ｍｇ／ｍｌの基材５０におけるアルブミンの発現量を基準として、各コラーゲン濃度の基材５０におけるアルブミンの相対発現量を評価した。図９に示すように、アルブミンの発現量には各濃度間で違いが表れなかった。 In the culture apparatus 1 using the base material 50 of each collagen concentration, the culture method of Example 1 was performed with only the number of cells set to 1.5×10 5 and the other configurations and conditions being the same. The albumin evaluation method is also RT-qPCR (using S18 as an endogenous control). Based on the expression level of albumin in the base material 50 with a collagen concentration of 2.4 mg/ml, the relative expression level of albumin in the base material 50 with each collagen concentration was evaluated. As shown in FIG. 9, there was no difference in the expression level of albumin between the concentrations.
　（実施例６）
　図１０に示すように、実施例４の各コラーゲン濃度の基材５０における解毒酵素の相対発現量を評価した。 (Example 6)
As shown in FIG. 10, the relative expression levels of detoxification enzymes in the substrate 50 of each collagen concentration in Example 4 were evaluated.
　各コラーゲン濃度の基材５０を用いた培養装置１において、細胞数のみ１．５×１０５個とし、実施例１同様に細胞を培養した。実施例１同様に、解毒酵素ＣＹＰ１Ａ１の発現量を定量した（内在性コントロールとしてＳ１８を使用）。コラーゲン濃度２．４ｍｇ／ｍｌの基材５０を用いた場合のＣＹＰ１Ａ１の発現量を基準として、各コラーゲン濃度の基材５０におけるＣＹＰ１Ａ１の相対発現量を評価した。図１０に示すように、解毒酵素の発現量はコラーゲン濃度が小さいと低下する傾向が見られた。 Cells were cultured in the same manner as in Example 1 except that the number of cells was set to 1.5×10 5 in the culture apparatus 1 using the substrate 50 with each collagen concentration. The expression level of the detoxification enzyme CYP1A1 was quantified in the same manner as in Example 1 (S18 was used as an endogenous control). Based on the expression level of CYP1A1 when using the base material 50 with a collagen concentration of 2.4 mg/ml, the relative expression level of CYP1A1 in the base material 50 with each collagen concentration was evaluated. As shown in FIG. 10, the expression level of the detoxification enzyme tended to decrease when the collagen concentration was low.
　（実施例７）
　図１１に示すように、水位差がない培養（比較例）と培養装置２による水位差がある培養（実施例７）の細胞の生存率を評価した。 (Example 7)
As shown in FIG. 11, cell viability was evaluated for culture with no water level difference (comparative example) and culture with water level difference by culture apparatus 2 (example 7).
　培養装置１の代わりにシリンジ形状を有する上部容器８０を備えた培養装置２を用いて上部培養液６０の初期量を変えたこと、上部培養液６０の継ぎ足しと外側容器４０内で増加した培養液の除去を約２４時間毎にしたこと以外、実施例２と同様に、水位差がある場合（実施例７）と水位差がない場合（比較例）とのそれぞれについて細胞の生存率を取得した。なお、シリンジ形状を有する上部容器８０は、円柱の形状のものを使用し、上部培養液６０の初期量は約５ｍｌとした。図１１に示すように、水位差がない場合は生存率１０％未満であり、培養装置２の場合は生存率９０％以上であった。 Changing the initial amount of the upper culture medium 60 by using the culture apparatus 2 provided with the upper container 80 having a syringe shape instead of the culture apparatus 1, replenishing the upper culture medium 60, and increasing the culture medium in the outer container 40 In the same manner as in Example 2, except that the was removed about every 24 hours, the survival rate of cells was obtained for each of the case where there was a water level difference (Example 7) and the case where there was no water level difference (Comparative Example). . The syringe-shaped upper container 80 was cylindrical, and the initial volume of the upper culture solution 60 was about 5 ml. As shown in FIG. 11, the survival rate was less than 10% when there was no water level difference, and the survival rate was 90% or more in the case of the culture apparatus 2.
　（変形例）
　以上は、本発明の好適な実施形態についての説明であるが、本発明は上述の実施形態に限られるものではなく、課題を解決するための手段に記載された範囲の限りにおいて様々な変更が可能なものである。以下、上述の実施形態に係る変形例について説明する。 (Modification)
Although the preferred embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope described in the Means for Solving the Problems. It is possible. Modifications of the above-described embodiment will be described below.
　上述の第１実施形態及び第２実施形態においては、内側容器１０及び外側容器２０としてそれぞれ独立の容器が使用されているが、これらの容器が一体に形成されているものが使用されてもよい。 In the above-described first and second embodiments, independent containers are used as the inner container 10 and the outer container 20, respectively, but these containers formed integrally may also be used. .
　上述の第３実施形態においては、内側容器３０が上部容器８０及び下部容器９０によって構成されているが、内側容器３０が一体に形成されていてもよい。 In the above-described third embodiment, the inner container 30 is composed of the upper container 80 and the lower container 90, but the inner container 30 may be integrally formed.
　上述の第１実施形態～第３実施形態に加え、内側容器へと上部培養液６０を単位時間当たり適宜の量で供給する供給装置が設けられてもよい。また、外側容器から外部へと下部培養液７０を排出させる排出装置が設けられてもよい。これらの供給装置や排出装置として点滴装置が用いられてもよい。このような構成により、上部培養液６０と下部培養液７０の水位差の変化を抑制できる。 In addition to the first to third embodiments described above, a supply device may be provided that supplies the upper culture medium 60 to the inner container in an appropriate amount per unit time. A discharge device may also be provided for discharging the lower culture solution 70 from the outer container to the outside. A drip device may be used as these supply device and discharge device. With such a configuration, a change in the water level difference between the upper culture solution 60 and the lower culture solution 70 can be suppressed.
　上述の第１実施形態～第３実施形態に係る培養装置が複数段に連結されてもよい。例えば、前段の培養装置１の外側容器２０の下部培養液７０を後段の培養装置１の内側容器１０へと流出させることで、前段の培養装置１の下部培養液７０を後段の培養装置１の上部培養液６０として使用するように複数段の培養装置１が設置されてもよい。 The culture apparatuses according to the first to third embodiments described above may be connected in multiple stages. For example, by causing the lower culture solution 70 in the outer container 20 of the culture device 1 in the preceding stage to flow into the inner container 10 of the culture device 1 in the latter stage, A plurality of stages of the culture apparatus 1 may be installed so as to be used as the upper culture solution 60 .
　また、上述の第３実施形態において、支持体９３が筒状以外の構造を有していてもよい。 Also, in the above-described third embodiment, the support 93 may have a structure other than a tubular shape.
　また、上述の第１実施形態～第３実施形態に係る内側容器１０及び３０並びに外側容器２０及び４０のそれぞれは、適宜の形状に構成されてよい。例えば、円柱状の他、角柱状、円錐台状、半球状等の形状に構成されてもよい。 Also, each of the inner containers 10 and 30 and the outer containers 20 and 40 according to the first to third embodiments described above may be configured in an appropriate shape. For example, in addition to a columnar shape, it may be configured in a prismatic shape, a truncated cone shape, a hemispherical shape, or the like.
　また、上述の第１実施形態～第３実施形態では、上部培養液６０の液面が下部培養液７０の液面より高くなるように水位差を生じさせている。これに対し、下部培養液７０の液面が上部培養液６０の液面より高くなるように水位差を生じさせてもよい。 Further, in the first to third embodiments described above, the water level difference is generated so that the liquid level of the upper culture solution 60 is higher than the liquid level of the lower culture solution 70 . On the other hand, the water level difference may be generated so that the liquid level of the lower culture solution 70 is higher than the liquid level of the upper culture solution 60 .
　１、２　培養装置
　１０、３０　内側容器
　２０、４０　外側容器
　１１、９１　側壁
　１５、９５　底壁
　１６、９６　貫通孔
　５０　基材
　６０　上部培養液
　７０　下部培養液 Reference Signs List 1, 2 culture apparatus 10, 30 inner container 20, 40 outer container 11, 91 side wall 15, 95 bottom wall 16, 96 through hole 50 substrate 60 upper culture solution 70 lower culture solution

Claims (14)

  1. 　基材中の細胞を培養する方法であって、
    　前記基材に上方から接触するように第１の培養液を収容する第１収容区画と、前記基材に下方から接触するように第２の培養液を収容する第２収容区画とを有する装置において、前記第１の培養液と前記第２の培養液の間に水位差を生じさせることで前記第１及び第２の培養液の少なくともいずれかを前記基材中に浸透させることを特徴とする培養方法。     A method of culturing cells in a substrate, comprising:
    An apparatus having a first storage compartment that accommodates a first culture solution so as to contact the substrate from above and a second storage compartment that accommodates a second culture solution so as to contact the substrate from below. wherein at least one of the first and second culture solutions is permeated into the substrate by creating a water level difference between the first culture solution and the second culture solution. culture method.
  2. 　前記装置が、前記第１収容区画を規定する側壁及び底壁を有する容器を備えており、
    　前記底壁に前記第１収容区画と前記第２収容区画を連通させる貫通孔が形成されていることを特徴とする請求項１に記載の培養方法。     said apparatus comprising a container having side walls and a bottom wall defining said first containment compartment;
    2. The culture method according to claim 1, wherein a through hole is formed in the bottom wall for communicating the first accommodation compartment and the second accommodation compartment.
  3. 　前記水位差を前記基材が変形しない範囲とすることを特徴とする請求項１又は２に記載の培養方法。 The culture method according to claim 1 or 2, wherein the water level difference is within a range in which the substrate is not deformed.
  4. 　前記基材の上下方向に関する厚さが０．２ｍｍを超えることを特徴とする請求項１～３のいずれか１項に記載の培養方法。 The culture method according to any one of claims 1 to 3, wherein the thickness of the substrate in the vertical direction exceeds 0.2 mm.
  5. 　基材がゲルであることを特徴とする、請求項１～４のいずれか１項に記載の培養方法。 The culture method according to any one of claims 1 to 4, wherein the substrate is a gel.
  6. 　前記基材におけるコロイドの濃度を、細胞の機能が発現する範囲内で調整することを特徴とする請求項５に記載の培養方法。 The culture method according to claim 5, wherein the colloid concentration in the substrate is adjusted within a range in which cell functions are expressed.
  7. 　前記基材におけるコロイドの濃度を、前記基材中の細胞の生存率が８０％以上となる範囲内で調整することを特徴とする請求項５又は６に記載の培養方法。 The culture method according to claim 5 or 6, characterized in that the colloid concentration in the base material is adjusted within a range in which the viability of cells in the base material is 80% or higher.
  8. 　前記基材におけるコロイドの濃度を１．０～２．５ｍｇ／ｍｌの範囲内で調整することを特徴とする請求項５～７のいずれか１項に記載の培養方法。 The culture method according to any one of claims 5 to 7, wherein the colloid concentration in the base material is adjusted within the range of 1.0 to 2.5 mg/ml.
  9. 　前記基材がゲルであり、
    　前記基材におけるコロイドの濃度を１．０ｍｇ／ｍｌ以上となる範囲内で調整することを特徴とする請求項３に記載の培養方法。     the base material is a gel,
    4. The culture method according to claim 3, wherein the concentration of the colloid in the substrate is adjusted within a range of 1.0 mg/ml or more.
  10. 　前記基材がコラーゲンを含有したゲルであることを特徴とする請求項５～９のいずれか１項に記載の培養方法。 The culture method according to any one of claims 5 to 9, wherein the base material is a gel containing collagen.
  11. 　前記装置が、前記第１収容区画を規定する第１容器と、前記第２収容区画を規定する第２容器とを備えており、
    　前記第１容器に、前記第１の培養液の液面が前記第２容器の上端より上方に配置されるように前記第１の培養液を収容することを特徴とする請求項１～１０のいずれか１項に記載の培養方法。     said apparatus comprising a first container defining said first containment compartment and a second container defining said second containment compartment;
    The method according to any one of claims 1 to 10, wherein the first container contains the first culture solution so that the surface of the first culture solution is positioned above the upper end of the second container. The culture method according to any one of the items.
  12. 　請求項１～１１のいずれか１項に記載の培養方法において、複数の前記装置を用いて細胞を培養すると共に、前記複数の装置のうち少なくともいずれか１つの細胞に対しては化合物を投与し、少なくとも他のいずれか１つの細胞に対しては前記化合物とは別の化合物を投与するか前記化合物のいずれも投与しないことを特徴とするスクリーニング方法。 In the culture method according to any one of claims 1 to 11, cells are cultured using a plurality of the devices, and a compound is administered to cells in at least one of the plurality of devices. , a screening method comprising administering a compound other than said compound or none of said compound to at least any one other cell.
  13. 　細胞が埋め込まれた基材と、
    　前記基材に上方から接触するように第１の培養液を収容する第１収容区画と、
    　前記基材に下方から接触するように第２の培養液を収容する第２収容区画とを備えており、
    　前記第１の培養液と前記第２の培養液の間に水位差を生じさせることで前記第１及び第２の培養液の少なくともいずれかを前記基材中に浸透させることを特徴とする細胞の培養装置。     a substrate in which cells are embedded;
    a first accommodation compartment that accommodates a first culture solution so as to contact the substrate from above;
    a second storage compartment that stores a second culture medium so as to contact the base material from below;
    Cells characterized in that at least one of the first culture medium and the second culture medium is permeated into the substrate by creating a water level difference between the first culture medium and the second culture medium. culture equipment.
  14. 　請求項１３の培養装置と、前記第１及び第２の培養液とを備えていることを特徴とする細胞の培養キット。 A cell culture kit comprising the culture apparatus according to claim 13 and the first and second culture solutions.
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