WO2024162464A1 - Culture device, culture tissue, production method for culture tissue, and evaluation method - Google Patents

Culture device, culture tissue, production method for culture tissue, and evaluation method Download PDF

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Publication number
WO2024162464A1
WO2024162464A1 PCT/JP2024/003437 JP2024003437W WO2024162464A1 WO 2024162464 A1 WO2024162464 A1 WO 2024162464A1 JP 2024003437 W JP2024003437 W JP 2024003437W WO 2024162464 A1 WO2024162464 A1 WO 2024162464A1
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tissue
mesh
cultured
culture
medium
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PCT/JP2024/003437
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French (fr)
Japanese (ja)
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誠 井上
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株式会社インファーマシア
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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 culture device, a cultured tissue, and a method for producing and evaluating the cultured tissue.
  • Patent Documents 1 and 2 disclose an apparatus and method for culturing free-floating tissue.
  • the target tissue is cultured while sandwiched between a first feeder cell layer and a second feeder cell layer. Specifically, the target tissue is cultured while immersed in a culture medium while sandwiched between a lower base to which the first feeder cell layer is adhered and an upper base to which the second feeder cell layer is adhered.
  • the culture device has a mesh for pressing the tissue into the culture medium.
  • the method for manufacturing a culture device includes culturing tissue while pressing the tissue into a medium using a mesh.
  • the cultured tissue is obtained by culturing the tissue while pressing it into a medium using a mesh.
  • the evaluation method includes providing a reagent to a cultured tissue obtained by culturing the tissue in a medium while the tissue is pressed down by a mesh, and obtaining a functional evaluation of the reagent and/or the cultured tissue depending on the response of the cultured tissue to the reagent.
  • FIG. 1 is a perspective view of a culture device according to one embodiment.
  • FIG. 2 is a perspective view of a holder that constitutes a part of the culture device according to one embodiment.
  • FIG. 3 is a perspective view of a first cylindrical part of a holder that constitutes a part of the culture device according to one embodiment.
  • FIG. 4 is a perspective view of a second cylindrical part of a holder that constitutes a part of the culture device according to one embodiment.
  • FIG. 5 is a cross-sectional view of the culture device according to one embodiment taken along line 5A-5A in FIG.
  • FIG. 6 is a cross-sectional view of the culture device according to one embodiment taken along line 6A-6A in FIG.
  • FIG. 7 is a graph showing the amount of glycerol in the culture supernatant in Examples 1 to 5 and Reference Example 1.
  • FIG. 8 is a graph showing the amount of adiponectin secreted in the culture supernatant in Examples 1 to 5 and Reference Example 1.
  • FIG. 9 is a graph showing the reduced amount of glucose in the culture supernatant in Example 6.
  • Fig. 1 is a perspective view of the culture device according to one embodiment.
  • Fig. 2 is a perspective view of a holding section constituting a part of the culture device according to one embodiment.
  • Fig. 3 is a perspective view of a first cylindrical section of the holding section constituting a part of the culture device according to one embodiment.
  • Fig. 4 is a perspective view of a second cylindrical section of the holding section constituting a part of the culture device according to one embodiment.
  • Fig. 5 is a cross-sectional view of the culture device according to one embodiment taken along line 5A-5A in Fig. 1.
  • Fig. 6 is a cross-sectional view of the culture device according to one embodiment taken along line 6A-6A in Fig. 1.
  • the culture device 100 is a device for tissue culture.
  • the tissue to be cultured is preferably buoyant.
  • buoyant may mean the property of floating in a culture medium.
  • tissue to be cultured may be tissue harvested from a living organism.
  • tissue here includes a "tissue piece” harvested from a living organism.
  • the living organism from which the tissue is harvested is not particularly limited, but is preferably, for example, an animal or human body.
  • the tissue to be cultured may be, for example, adipose tissue or brain tissue.
  • the tissue to be cultured is preferably animal adipose tissue or brain tissue, more preferably human adipose tissue or brain tissue.
  • the adipose tissue may be collected from any organ.
  • the adipose tissue may be, for example, subcutaneous fat, visceral fat, or fat pieces collected from bone marrow, thymus, blood vessels, heart, mesentery, pancreas, liver, retroorbital or mammary tissue, etc.
  • the tissue to be cultured may be tissue collected from a healthy organ, or may be tissue collected from an abnormal organ. Examples of abnormal organs include lipoma and cancer tissue.
  • the size of the tissue to be cultured is not particularly limited, but may be, for example, 1 mg to 1000 mg, 5 mg to 500 mg, 10 mg to 200 mg, or 20 mg to 100 mg.
  • the culture device 100 has a mesh 50 for pressing the tissue into the culture medium.
  • the mesh 50 has a lattice shape.
  • the mesh 50 is located below the transition R1 of the culture medium during tissue culture.
  • the culture device 100 may have a storage section 10 capable of storing the culture medium and a holding section 20 that holds the mesh 50.
  • a tissue culture plate may be used for the storage section 10.
  • the culture device 100 is configured to be able to form a culture space S1 for accommodating tissue below the mesh 50.
  • the culture space S1 is a space for accommodating tissue during culture.
  • the mesh 50 is provided at least above the culture space S1 (see Figures 5 and 6).
  • the mesh 50 presses the tissue into the medium during tissue culture. As a result, even if the tissue is buoyant, the tissue is cultured in a state where it is submerged from the interface of the medium during culture. This allows the tissue to be cultured with the entire surface of the tissue in contact with the medium.
  • the inventors of the present application have found that by culturing the tissue by pressing it into the medium using the mesh 50, the morphology and/or function of the tissue can be maintained as long as possible. It is generally known that the use of foreign structures in internal tissues can cause inflammation, fibrosis, etc., but the inventors of the present application have unexpectedly found that culture using a mesh results in less significant changes in gene expression related to inflammation and fibrosis than when a mesh is not used.
  • the mesh 50 preferably has an opening of, for example, 10 ⁇ m or more, 20 ⁇ m or more, 30 ⁇ m or more, 40 ⁇ m or more, or 55 ⁇ m or more.
  • an opening of the mesh 50 By making the opening of the mesh 50 larger than the above desired value, oxygen and nutrients can be more easily distributed through the mesh 50 into the culture space S1 in the medium, and the material produced from the tissue can be prevented from clogging the mesh 50. It is believed that this makes it possible to maintain the morphology and/or function of the tissue for as long as possible.
  • the mesh 50 preferably has an opening size of, for example, 200 ⁇ m or less, or 188 ⁇ m or less. If the opening size of the mesh 50 is smaller than the desired value, the tissue pressed down by the mesh, or the cells separated from the tissue, will be less likely to pass through the holes in the mesh 50 and escape upward.
  • the size of fat cells is a maximum of about 200 ⁇ m. Therefore, when the cells that make up the tissue are fat cells, it is preferable that the mesh 50 has mesh openings of, for example, 200 ⁇ m or less or 188 ⁇ m or less.
  • the mesh size of the mesh 50 may be, for example, 188 ⁇ m or less, preferably 108 ⁇ m or less, more preferably 77 ⁇ m or less, and even more preferably 55 ⁇ m or less. If the mesh size of the mesh 50 is sufficiently smaller than the size of the cultured tissue, it is presumed that the mechanical load from the mesh 50 to the cultured tissue will be small, providing a good culture environment.
  • the opening rate of the mesh 50 is not particularly limited, but may be, for example, in the range of 5 to 70%, preferably in the range of 10 to 60%, and more preferably in the range of 25 to 50%. From the viewpoint of facilitating the distribution of oxygen and nutrients through the mesh 50 into the culture space S1 in the medium, it is preferable that the opening rate of the mesh 50 is not too low.
  • the wire diameter of the mesh 50 is not particularly limited, but may be, for example, 10 ⁇ m or more, preferably 20 ⁇ m or more, and more preferably 30 ⁇ m or more.
  • the thickness of the mesh 50 is not particularly limited, but may be, for example, 20 ⁇ m or more, preferably 30 ⁇ m or more, and more preferably 40 ⁇ m or more.
  • the wire diameter of the mesh 50 is not particularly limited, but may be, for example, 200 ⁇ m or less, preferably 170 ⁇ m or less, and more preferably 130 ⁇ m or less.
  • the thickness of the mesh 50 is not particularly limited, but may be, for example, 380 ⁇ m or less, preferably 320 ⁇ m or less, and more preferably 240 ⁇ m or less. It is believed that the smaller the wire diameter and/or thickness of the mesh 50, the softer the mesh 50 will be, and therefore it is presumed that the mechanical load on the cultured tissue may be reduced.
  • the mesh 50 is not particularly limited, but may have a lattice structure of, for example, three layers or less, two layers or less, or a single layer. From the standpoint of ensuring the softness of the mesh 50 and/or the permeability of oxygen and nutrients, it is preferable that the number of layers constituting the mesh 50 is small.
  • the material constituting the mesh 50 is not particularly limited, but is preferably a material that has low adhesiveness to the tissue to be cultured.
  • the material constituting the mesh may be a polymer that can be sterilized by high-pressure steam sterilization, radiation sterilization, or EOG sterilization, such as silk, hyaluronic acid, cellulose, polyethylene, polypropylene, polyester, polylactic acid, polymethacrylic acid derivatives, polyvinyl chloride, polyphenylene sulfide, polyethylene terephthalate, or nylon.
  • the mesh 50 may have a biocompatible coating layer.
  • the material constituting the biocompatible coating layer may be, for example, a hydrogel.
  • the hydrogel may contain at least one selected from various compounds such as collagen, laminin, fibronectin, vitronectin, and gelatin.
  • the holding portion 20 may have a first cylindrical portion 30 and a second cylindrical portion 40.
  • the second cylindrical portion 40 is configured to be able to fit inside (the hollow portion of) the first cylindrical portion 30.
  • the first cylindrical portion 30 and the second cylindrical portion 40 may be formed as a single, inseparable structure.
  • the shape of the first tubular portion 30 is not particularly limited, but may be, for example, a cylindrical shape or a rectangular tube shape.
  • the first tubular portion 30 may have a wall portion 32 that is cylindrical or rectangular tube shape.
  • the space inside the wall portion 32 may form a hollow portion 38.
  • the inner surface of the wall portion 32 constituting the first cylindrical portion 30 has a step 33. Due to this step 33, the inner diameter L1 of the upper side of the first cylindrical portion 30 is larger than the inner diameter L2 of the lower side of the first cylindrical portion 30.
  • the outer diameter L3 of the second cylindrical portion 40 may be smaller than the inner diameter L1 of the upper side of the first cylindrical portion 30 and larger than the inner diameter L2 of the lower side of the first cylindrical portion 30.
  • the mesh 50 is provided at the lower end of the second cylindrical portion 40.
  • the end of the mesh 50 abuts against the step 33 of the first cylindrical portion 30.
  • the end of the mesh 50 is sandwiched between the step 33 of the first cylindrical portion 30 and the wall portion 42 that constitutes the second cylindrical portion 40 (see Figures 5 and 6).
  • the portion of the wall 32 constituting the first cylindrical section 30 below the step 33 constitutes a protrusion 34 that protrudes downward from the mesh 50.
  • the protrusion 34 may define the boundary of the culture space S1 described above.
  • the culture space S1 is defined by the space surrounded by the mesh 50 and the protrusion 34.
  • the protrusion 34 may be configured to completely or partially surround the culture space S1. When the protrusion 34 protrudes downward from the mesh 50, the tissue pressed down by the mesh 50 can be prevented from moving laterally and flowing into an area where the mesh 50 is not present.
  • the protrusion 34 has an opening 36 through which the culture medium can flow. This allows the culture medium to move from the inside to the outside of the culture space S1, or from the outside to the inside, through the opening 36. This makes it easier for the culture medium in the culture space S1 to be maintained in a fresh state, and makes it easier for oxygen and nutrients to flow into the culture space S1.
  • the shape of the first tubular portion 40 is not particularly limited, but may be, for example, a cylindrical shape or a square tube shape.
  • the second tubular portion 40 may have a wall portion 42 that is cylindrical or square tube shape.
  • the second tubular portion 40 preferably has a hollow portion 48 that is accessible to the mesh 50. In this case, the space inside the wall portion 42 constitutes the hollow portion 48. This allows the worker culturing the tissue to easily remove oil and other substances floating on the surface of the culture medium through the mesh 50.
  • the culture device 100 may have a lid for sealing the culture space S1.
  • the lid (not shown) may be attached to the storage unit, for example, and configured to surround the entire holding unit 20.
  • the method for producing a cultured tissue according to this embodiment can be carried out using the culture device 100 described above, for example.
  • the method for producing cultured tissue first involves harvesting the tissue to be cultured from a living organism.
  • the harvested tissue may be cut to a desired size.
  • the types of tissue cultured in this embodiment are as described above, and therefore will not be described here.
  • the tissue cut to the desired size is cultured in a medium with the tissue pressed down by a mesh.
  • a medium There are no particular limitations on the type of medium, and it may be selected appropriately depending on the tissue to be cultured.
  • the tissue is not subjected to cell dispersion treatment such as collagenase treatment, and is cultured as a tissue mass.
  • the mesh preferably has an opening of, for example, 10 ⁇ m or more, 20 ⁇ m or more, 30 ⁇ m or more, 40 ⁇ m or more, or 55 ⁇ m or more.
  • the mesh preferably has an opening of, for example, 200 ⁇ m or less, or 188 ⁇ m or less. Other preferred parameters regarding the mesh are as described above.
  • tissue culture can be carried out using the culture device 100 described above.
  • the configuration of the culture device 100 is as described above, so a description thereof will be omitted.
  • tissue culture is carried out without using foreign cells such as supporting cells.
  • the medium used for culture may be DMEM, MEM, RPMI, F12, M199, or a mixture of these media, which may contain additives such as serum albumin (such as BSA or HSA), polyvinyl alcohol, lipoproteins (such as LDL or HDL), free fatty acids, insulin, dexamethasone, cortisol, glucose, pyruvic acid, glutamine, fatty acids, biotin, pantothenic acid, transferrin, selenic acid, vanadate, as well as antibiotics such as gentamicin, penicillin, and streptomycin.
  • serum albumin such as BSA or HSA
  • polyvinyl alcohol polyvinyl alcohol
  • lipoproteins such as LDL or HDL
  • free fatty acids insulin
  • dexamethasone cortisol
  • glucose pyruvic acid
  • glutamine fatty acids
  • biotin biotin
  • pantothenic acid transferrin
  • selenic acid vanadate
  • vanadate
  • the number of days for which the tissue is cultured is not particularly limited. However, the tissue can be cultured for, for example, 14 days or more, 24 days or more, 32 days or more, 42 days or more, 49 days or more, or 56 days or more.
  • the above-described culture device 100 allows the tissue to be cultured while maintaining its morphology for a long period of time.
  • the number of days for which the tissue is cultured may be, for example, 120 days or less, 90 days or less, 80 days or less, 70 days or less, or 60 days or less.
  • the medium may be changed every desired number of days from the start of tissue culture. It should be noted that there are no particular limitations on the timing or frequency of medium changes. In each medium change, the medium may be removed and added once, for example, or may be repeated multiple times.
  • a cultured tissue is produced by culturing the tissue as described above.
  • the cultured tissue according to this embodiment may be a tissue produced by the above-described production method.
  • the gene expression function and/or physiological activity function of the cultured tissue can be maintained as long as possible.
  • the number of genes that change significantly in tissue on the 32nd day of culture is smaller than that in tissue on the 4th day of culture, or there are fewer significant gene changes related to tissue alterations such as those related to tissue fibrosis and inflammation.
  • the above culture method can suppress the decline in physiological activity of adipose tissue, such as the amount of adiponectin secreted.
  • the above-mentioned culture device, cultured tissue, and/or cultured tissue manufacturing method can be used as a tool for a method of evaluating the function of various tissues.
  • the evaluation method includes providing a reagent to a cultured tissue obtained by culturing the tissue in a medium while pressing the tissue with a mesh, and obtaining an evaluation of the reagent and/or the cultured tissue according to the response of the cultured tissue to the reagent.
  • the mesh may be as described above. Specifically, it may be the mesh provided in the culture device described above.
  • the reagent given to the tissue cultured by the culture method described above may be appropriately selected depending on the item to be evaluated.
  • the evaluation method can be used as a pharmacological test and/or a drug efficacy test to measure the response of tissues to a reagent.
  • the evaluation method can also be used for screening purposes in drug discovery.
  • the reagent may be a candidate drug substance.
  • Example 1 First, relatively large blood vessels and connective tissue were removed from human abdominal adipose tissue (hereinafter, simply referred to as "adipose tissue") surgically removed from a 71-year-old female patient without metabolic disease, and the adipose tissue was then cut into small pieces of 20 mg to 100 mg with surgical scissors. Red blood cells were removed from the cut pieces of adipose tissue by centrifugation to obtain adipose tissue (tissue) to be cultured.
  • adipose tissue human abdominal adipose tissue
  • the adipose tissue cut into small pieces was cultured in the above-mentioned culture device 100 (see also Figures 1 to 6).
  • the adipose tissue was pressed into the medium (culture fluid) by a mesh.
  • the mesh was coated with collagen. As shown in Table 1 below, in Example 1, the mesh opening was 30 ⁇ m, the mesh opening rate was 25%, the mesh wire diameter was 30 ⁇ m, and the mesh thickness was 47 ⁇ m.
  • the culture solution was DMEM containing 0.1% BSA, 100 mg/mL glucose, 3 nM dexamethasone, 1 ng/mL insulin, and 10 ⁇ g/mL gentamicin.
  • the 24-well tissue culture plate in which the culture device 100 was immersed was placed in a CO2 incubator and cultured at 37° C. in a 5% carbon dioxide atmosphere.
  • Example 2 Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 2, the mesh opening was 55 ⁇ m, the mesh opening ratio was 34%, the mesh wire diameter was 39 ⁇ m, and the mesh thickness was 62 ⁇ m.
  • Example 3 Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 3, the mesh opening was 77 ⁇ m, the mesh opening ratio was 37%, the mesh wire diameter was 50 ⁇ m, and the mesh thickness was 83 ⁇ m.
  • Example 4 Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 4, the mesh opening was 108 ⁇ m, the mesh opening ratio was 41%, the mesh wire diameter was 61 ⁇ m, and the mesh thickness was 106 ⁇ m.
  • Example 5 Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 5, the mesh opening was 188 ⁇ m, the mesh opening ratio was 35%, the mesh wire diameter was 130 ⁇ m, and the mesh thickness was 235 ⁇ m.
  • Figure 7 is a graph showing the amount of glycerol in the culture supernatant on Day 4, Day 18, and Day 32 in Examples 1 to 5 and Reference Example 1. In all cases, an increase in the amount of glycerol was observed from Day 4 to Day 18. This is presumably due to improved lipid breakdown in response to the low glucose concentration. Furthermore, the amount of glycerol was roughly the same on Day 18 and Day 32. This result suggests that lipid breakdown function is maintained in the cultured tissue.
  • FIG. 8 is a graph showing the amount of adiponectin secreted in the culture supernatant on Day 4, Day 18, and Day 32 in Examples 1 to 5 and Reference Example 1.
  • Reference Example 1 a significant decrease in the amount of adiponectin secreted was observed with the number of days of culture.
  • the amount of adiponectin secreted tended to be maintained, and the decrease in the amount of adiponectin secreted was suppressed.
  • a culture device 100 equipped with a mesh having openings in the range of 30 ⁇ m and 77 ⁇ m, more specifically 30 ⁇ m and 55 ⁇ m was used, the amount of secretion was maintained very well. This result indicates that the use of a culture device 100 equipped with a mesh allows the adipocyte function in the cultured tissue to be well maintained.
  • RNA extraction treatment On Day 4 and Day 32, tissues (100-200 mg wet weight) were homogenized in QIAzol Lysis Reagent (QIAGEN) and RNA extracts were obtained according to the manufacturer's protocol. From this RNA extract, purified RNA solution was obtained using "ISOSPIN Cell & Tissue RNA (Nippon Gene; 314-08211)" according to the manufacturer's protocol. The obtained RNA showed sufficient quality with an RIN value of 7 or more when analyzed by a bioanalyzer. An example of the actual measured values is shown in Table 2 below.
  • RNA solutions purified on Day 4 and Day 32 were used to obtain raw data (fastq files) using an Illumina NovaSeq 6000 (standard read length: PE 150 (150 bp ⁇ 2 paired-end)) in a test commissioned by Rhelixa (RNA-seq). After that, the fastq files were mapped to the genome sequence using fastp/STAR/featureCount, and gene expression variation analysis was performed using EdgeR.
  • log FC log fold-change
  • FDR False Discovery Ratio
  • significance rank for genes that showed a significant increase in expression in Reference Example 1 are shown in Table 3 below.
  • log FC is defined as the logarithm of the value obtained by dividing the expression level (signal value) of a specific gene on Day 32 by the expression level (signal value) of the gene on Day 4, with the base being 2.
  • FDR is the false detection rate in multiple comparison testing.
  • the significance rank corresponds to the rank when genes are arranged in ascending order of p-value.
  • a "significant increase in expression" of a gene is defined as an "FDR value of 0.01 or less” and a "log FC value of 1.0 or more.”
  • the gene group shown in Table 3 (EGR1, FOSB, PLTP, FADS1, COL1A1, LTBP2, FADS2) should be avoided from the viewpoint of maintaining healthy tissue function.
  • Examples 1 to 5 few of these genes showed significant expression increases. Specifically, in Examples 1 and 2, the only gene that showed significant expression increases was EGR1 from the gene group listed in Table 3. In Example 4, the only gene that showed significant expression increases was PTLP from the gene group listed in Table 3. In Examples 3 and 5, there were no genes that showed significant expression increases in the gene group listed in Table 3. Thus, in Examples 1 to 5, there was only 0 or 1 gene that showed significant expression increases in the gene group shown in Table 3. This means that the morphology and/or function of the adipose tissue is maintained for a long period in Examples 1 to 5 compared to Reference Example 1.
  • Table 4 shows the number of genes that showed significant changes in expression between Day 4 and Day 32 among all the gene groups analyzed.
  • a "significant change in expression" of a gene is defined as an "FDR value of 0.01 or less” and an “absolute value of log FC of 1.0 or more.”
  • Example 4 From Table 4, the number of genes that showed significant changes in expression in Examples 1 to 3 was smaller than in Examples 4 and 5, and Example 2 had the smallest number of genes that showed significant changes in expression. Therefore, it can be seen that tissue culture using a mesh that is 30 ⁇ m or more and 77 ⁇ m or less is more preferable from the perspective of maintaining the functional expression of genes. However, it should be noted that since the number of genes that showed significant changes in expression was also sufficiently small in Examples 4 and 5, the mesh opening of the mesh according to the present invention may be outside the above numerical range.
  • Example 6 20-100 mg of human abdominal adipose tissue (hereinafter referred to as "adipose tissue") from a 72-year-old woman without metabolic disease cut into small pieces was cultured in the above-mentioned culture device 100 (see also Figures 1 to 6).
  • the adipose tissue was pressed into the medium (culture solution) by a mesh during culture.
  • a collagen-coated mesh with a mesh opening of 77 ⁇ m was used for the culture device 100.
  • the mesh was the same as that used in Example 3.
  • the medium composition was DMEM containing 0.2% BSA, 3 nM dexamethasone, 1 ng/mL insulin, 300 mg/dL glucose, 0.18 mM palmitic acid, and 10 ⁇ g/mL gentamicin.
  • the 24-well tissue culture plate immersed in the culture device 100 was placed in a CO2 incubator and cultured at 37°C under a 5% carbon dioxide atmosphere for 21 days. The day when the culture was started was designated as Day 0, and the medium was exchanged on Days 1, 2, 5, 7, 9, 12, 14, 16, and 19. The medium exchange on Day 19 involved removing the medium and then adding the medium three times to completely exchange the medium.
  • 100 ⁇ L of the medium was collected and frozen (frozen sample 1). After 24 hours of culture from the medium exchange on Day 19 (Day 20), 100 ⁇ L of the medium was collected and frozen (frozen sample 2).
  • the medium was replaced with a medium containing an insulin concentration of 20 ng/mL. On day 20, the medium was replaced three times, similar to day 19. Immediately after the medium replacement on day 20, 100 ⁇ L of medium was collected and frozen (frozen storage sample 3). After 24 hours of culture from the medium replacement (day 21), 100 ⁇ L of medium was collected and frozen (frozen storage sample 4).
  • the glucose concentration in the medium was quantified using a glucose measurement kit (Dojindo Laboratories; G264). By comparing the glucose concentrations in the medium of frozen samples 1 and 2, the amount of change in the glucose concentration in the medium after 24 hours at an insulin concentration of 1 ng/mL was determined. Similarly, by comparing the glucose concentrations in the medium of frozen samples 3 and 4, the amount of change in the glucose concentration in the medium after 24 hours at an insulin concentration of 20 ng/mL was determined. These changes in glucose concentration are considered to correspond to the amount of glucose uptake into adipose tissue.
  • Figure 9 is a graph showing the amount of glucose reduction in the culture supernatant in Example 6 evaluated by the above procedure.
  • the graph showing an insulin concentration of 1 ng/mL corresponds to the difference between the glucose concentration of frozen-preserved sample 2 and the glucose concentration of frozen-preserved sample 1.
  • the graph showing an insulin concentration of 20 ng/mL corresponds to the difference between the glucose concentration of frozen-preserved sample 4 and the glucose concentration of frozen-preserved sample 1.
  • Figure 9 confirms that there is a significant increase in glucose uptake at an insulin concentration of 20 ng/mL compared to 1 ng/mL. This confirmed that insulin-responsive adipose tissue function was maintained even after tissue culture.
  • [Appendix 1] A culture device having a mesh for pressing the tissue into the medium.
  • [Appendix 2] The culture device of claim 1, wherein the mesh has an opening size of 10 ⁇ m or more and 200 ⁇ m or less.
  • [Appendix 3] 3. The culture device of claim 1 or 2, wherein the mesh has a biocompatible coating layer.
  • [Appendix 4] The culture device according to any one of claims 1 to 3, wherein the tissue is buoyant.
  • [Appendix 5] The culture device according to any one of claims 1 to 4, wherein the tissue is adipose tissue or brain tissue.
  • [Appendix 7] The protrusion is formed to surround the tissue culture space, The culture device according to any one of claims 1 to 6, wherein the protrusion has an opening through which culture medium can flow.
  • [Appendix 8] A second cylindrical portion extends upward while holding the mesh, The culture device according to any one of claims 1 to 7, wherein the second cylindrical portion has a hollow portion through which the mesh is accessible.
  • [Appendix 9] A method for producing a cultured tissue, comprising culturing the tissue while pressing the tissue into a medium with a mesh.
  • [Appendix 10] The method for producing a cultured tissue described in Appendix 9, wherein the mesh has an opening of 10 ⁇ m or more and 200 ⁇ m or less.
  • [Appendix 11] The method for producing cultured tissue described in Appendix 9 or 10, wherein the mesh has a biocompatible coating layer.
  • [Appendix 12] The method for producing a cultured tissue according to any one of claims 9 to 11, wherein the cultured tissue has floating properties.
  • [Appendix 13] The method for producing a cultured tissue according to any one of Appendices 9 to 12, wherein the cultured tissue is an adipose tissue.
  • [Appendix 14] The method for producing a cultured tissue according to any one of claims 9 to 13, wherein the tissue is cultured for 14 days or more.
  • [Appendix 15] A cultured tissue obtained by culturing a tissue while the tissue is pressed into a medium using a mesh.
  • [Appendix 16] The cultured tissue of claim 15, wherein the tissue is free-floating.
  • An evaluation method comprising: applying a reagent to a cultured tissue obtained by culturing the tissue while it is pressed into a medium by a mesh; and obtaining a functional evaluation of the reagent and/or the cultured tissue depending on the response of the cultured tissue to the reagent.

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Abstract

Provided are a culture device and/or culture method for tissue with which the form and/or function of the tissue can be maintained as much as possible over the long term. This culture device (100) has a mesh (50) for pressing tissue down into a culture medium.

Description

培養装置、培養組織、培養組織の製造方法及び評価方法Culture device, cultured tissue, manufacturing method and evaluation method of cultured tissue
　本発明は、培養装置、培養組織、培養組織の製造方法及び評価方法に関する。 The present invention relates to a culture device, a cultured tissue, and a method for producing and evaluating the cultured tissue.
　近年、生体内での微小環境を保持した状態で細胞を培養することを目的として、採取した組織（組織片を含む）を細胞分散させることなく培養する組織培養が行われることがある。下記の特許文献１及び特許文献２は、浮遊性の組織を培養する装置及び方法を開示する。 In recent years, tissue culture has been performed in which collected tissue (including tissue fragments) is cultured without dispersing the cells, with the aim of culturing cells while maintaining the in vivo microenvironment. The following Patent Documents 1 and 2 disclose an apparatus and method for culturing free-floating tissue.
　特許文献１，２では、標的の組織は、第１の支持細胞層と第２の支持細胞層との間に挟まれた状態で培養される。具体的には、標的の組織は、第１の支持細胞層が接着された下部ベースと、第２の支持細胞層が接着された上部ベースと、の間に挟まれた状態で、培地に浸されて培養される。 In Patent Documents 1 and 2, the target tissue is cultured while sandwiched between a first feeder cell layer and a second feeder cell layer. Specifically, the target tissue is cultured while immersed in a culture medium while sandwiched between a lower base to which the first feeder cell layer is adhered and an upper base to which the second feeder cell layer is adhered.
国際公開第２０１７／０６６４７１号International Publication No. 2017/066471 米国特許出願公開第２０２１－０２８４９６９号明細書US Patent Publication No. 2021-0284969
　動物ないし人体のような生体から採取された組織の形態及び／又は機能は、時間の経過とともに変化する。したがって、組織の形態及び／又は機能を長期にわたってなるべく健全に維持することが可能な組織の培養装置及び／又は培養方法が望まれる。培養組織の維持を目的として、特許文献１，２の様に、脂肪由来幹細胞のような支持細胞を用いる手法がある。一方で、培養組織内の細胞間相互作用という微小環境を保持したまま培養するためには、支持細胞のような外来細胞を用いずに培養することが求められる。外来細胞を用いない培養は、特に培養組織の機能解析としてパスウェイ解析などを行う場合には重要である。 The morphology and/or function of tissues harvested from living organisms such as animals or humans changes over time. Therefore, there is a need for a tissue culture device and/or culture method that can maintain the morphology and/or function of tissues as healthy as possible for a long period of time. For the purpose of maintaining cultured tissues, there are methods that use support cells such as adipose-derived stem cells, as described in Patent Documents 1 and 2. On the other hand, in order to culture the tissue while maintaining the microenvironment of cell-cell interactions within the cultured tissue, it is necessary to culture the tissue without using foreign cells such as support cells. Culturing without using foreign cells is important, particularly when performing pathway analysis as a functional analysis of the cultured tissue.
　一態様に係る培養装置は、組織を培地中に押下するためのメッシュを有する。 In one embodiment, the culture device has a mesh for pressing the tissue into the culture medium.
　一態様に係る培養装置の製造方法は、メッシュにより培地中に組織を押下させた状態で組織を培養することを含む。 In one embodiment, the method for manufacturing a culture device includes culturing tissue while pressing the tissue into a medium using a mesh.
　一態様に係る培養組織は、メッシュにより培地中に組織を押下させた状態で前記組織を培養することによって得られたものである。 In one embodiment, the cultured tissue is obtained by culturing the tissue while pressing it into a medium using a mesh.
　一態様に係る評価方法は、メッシュにより培地中に組織を押下させた状態で培養することによって得られた培養組織に試薬を与え、前記試薬に対する前記培養組織の応答に応じて、前記試薬、及び／又は前記培養組織に関する機能評価を得ること、を含む。 The evaluation method according to one embodiment includes providing a reagent to a cultured tissue obtained by culturing the tissue in a medium while the tissue is pressed down by a mesh, and obtaining a functional evaluation of the reagent and/or the cultured tissue depending on the response of the cultured tissue to the reagent.
図１は、一実施形態に係る培養装置の斜視図である。FIG. 1 is a perspective view of a culture device according to one embodiment. 図２は、一実施形態に係る培養装置の一部を構成する保持部の斜視図である。FIG. 2 is a perspective view of a holder that constitutes a part of the culture device according to one embodiment. 図３は、一実施形態に係る培養装置の一部を構成する保持部の第１筒状部の斜視図である。FIG. 3 is a perspective view of a first cylindrical part of a holder that constitutes a part of the culture device according to one embodiment. 図４は、一実施形態に係る培養装置の一部を構成する保持部の第２筒状部の斜視図である。FIG. 4 is a perspective view of a second cylindrical part of a holder that constitutes a part of the culture device according to one embodiment. 図５は、一実施形態に係る培養装置における図１の５Ａ－５Ａ線に沿った断面図である。FIG. 5 is a cross-sectional view of the culture device according to one embodiment taken along line 5A-5A in FIG. 図６は、一実施形態に係る培養装置における図１の６Ａ－６Ａ線に沿った断面図である。FIG. 6 is a cross-sectional view of the culture device according to one embodiment taken along line 6A-6A in FIG. 図７は、実施例１～５及び参考例１における培養上清中のグリセロール量を示すグラフである。FIG. 7 is a graph showing the amount of glycerol in the culture supernatant in Examples 1 to 5 and Reference Example 1. 図８は、実施例１～５及び参考例１における培養上清中の分泌アディポネクチン量を示すグラフである。FIG. 8 is a graph showing the amount of adiponectin secreted in the culture supernatant in Examples 1 to 5 and Reference Example 1. 図９は、実施例６における培養上清中のグルコース低下量を示すグラフである。FIG. 9 is a graph showing the reduced amount of glucose in the culture supernatant in Example 6.
　以下、図面を参照して、実施形態について説明する。以下の図面において、同一又は類似の部分には、同一又は類似の符号を付している。 Below, the embodiments will be described with reference to the drawings. In the following drawings, the same or similar parts are denoted by the same or similar reference numerals.
　［培養装置］
　まず、組織を培養するための培養装置について図１～６を参照して説明する。図１は、一実施形態に係る培養装置の斜視図である。図２は、一実施形態に係る培養装置の一部を構成する保持部の斜視図である。図３は、一実施形態に係る培養装置の一部を構成する保持部の第１筒状部の斜視図である。図４は、一実施形態に係る培養装置の一部を構成する保持部の第２筒状部の斜視図である。図５は、一実施形態に係る培養装置における図１の５Ａ－５Ａ線に沿った断面図である。図６は、一実施形態に係る培養装置における図１の６Ａ－６Ａ線に沿った断面図である。 [Culture device]
First, a culture device for culturing tissue will be described with reference to Figs. 1 to 6. Fig. 1 is a perspective view of the culture device according to one embodiment. Fig. 2 is a perspective view of a holding section constituting a part of the culture device according to one embodiment. Fig. 3 is a perspective view of a first cylindrical section of the holding section constituting a part of the culture device according to one embodiment. Fig. 4 is a perspective view of a second cylindrical section of the holding section constituting a part of the culture device according to one embodiment. Fig. 5 is a cross-sectional view of the culture device according to one embodiment taken along line 5A-5A in Fig. 1. Fig. 6 is a cross-sectional view of the culture device according to one embodiment taken along line 6A-6A in Fig. 1.
　一実施形態に係る培養装置１００は、組織培養をするための装置である。培養すべき組織は、浮遊性を有することが好ましい。ここで、浮遊性は、培地に浮遊する性質を有するものであってよい。 The culture device 100 according to one embodiment is a device for tissue culture. The tissue to be cultured is preferably buoyant. Here, buoyant may mean the property of floating in a culture medium.
　培養すべき組織は、生体から採取された組織であってよい。ここで、「組織」という用語は、生体から採取した「組織片」を含む概念であることに留意されたい。組織を採取するための生体は、特に制限されないが、例えば動物ないし人体であることが好ましい。 The tissue to be cultured may be tissue harvested from a living organism. It should be noted that the term "tissue" here includes a "tissue piece" harvested from a living organism. The living organism from which the tissue is harvested is not particularly limited, but is preferably, for example, an animal or human body.
　培養すべき組織は、例えば脂肪組織又は脳組織であってよい。培養すべき組織は、好ましくは動物の脂肪組織又は脳組織であり、より好ましくはヒトの脂肪組織又は脳組織である。脂肪組織は、任意の臓器から採取したものであってよい。脂肪組織は、例えば、皮下脂肪、内臓脂肪だけでなく、骨髄、胸腺、血管、心臓、腸間膜、膵臓、肝臓、後眼窩又は乳腺組織等から採取した脂肪片であってよい。培養する組織は、健常の臓器から採取した組織であってもよく、異常を有する臓器から採取した組織であってもよい。異常を有する臓器は、例えば脂肪腫やがん組織などが挙げられる。 The tissue to be cultured may be, for example, adipose tissue or brain tissue. The tissue to be cultured is preferably animal adipose tissue or brain tissue, more preferably human adipose tissue or brain tissue. The adipose tissue may be collected from any organ. The adipose tissue may be, for example, subcutaneous fat, visceral fat, or fat pieces collected from bone marrow, thymus, blood vessels, heart, mesentery, pancreas, liver, retroorbital or mammary tissue, etc. The tissue to be cultured may be tissue collected from a healthy organ, or may be tissue collected from an abnormal organ. Examples of abnormal organs include lipoma and cancer tissue.
　培養する組織のサイズは、特に制限されないが、例えば１ｍｇ～１０００ｍｇ、５ｍｇ～５００ｍｇ、１０ｍｇ～２００ｍｇ、又は２０ｍｇ～１００ｍｇであってよい。 The size of the tissue to be cultured is not particularly limited, but may be, for example, 1 mg to 1000 mg, 5 mg to 500 mg, 10 mg to 200 mg, or 20 mg to 100 mg.
　培養装置１００は、組織を培地中に押下するためのメッシュ５０を有する。メッシュ５０は、格子状の形状を有している。メッシュ５０は、組織の培養中に、培地の推移Ｒ１よりも下方に位置する。培養装置１００は、培地を収容可能な収容部１０と、メッシュ５０を保持する保持部２０と、を有していてよい。収容部１０には組織培養プレートを用いてもよい。 The culture device 100 has a mesh 50 for pressing the tissue into the culture medium. The mesh 50 has a lattice shape. The mesh 50 is located below the transition R1 of the culture medium during tissue culture. The culture device 100 may have a storage section 10 capable of storing the culture medium and a holding section 20 that holds the mesh 50. A tissue culture plate may be used for the storage section 10.
　培養装置１００は、メッシュ５０の下部に、組織を収容する培養空間Ｓ１を形成可能に構成されている。培養空間Ｓ１は、培養中の組織を収容する空間である。言い換えると、メッシュ５０は、培養空間Ｓ１の少なくとも上側に設けられている（図５及び図６参照）。 The culture device 100 is configured to be able to form a culture space S1 for accommodating tissue below the mesh 50. The culture space S1 is a space for accommodating tissue during culture. In other words, the mesh 50 is provided at least above the culture space S1 (see Figures 5 and 6).
　メッシュ５０は、組織の培養中において、組織を培地中に押下させる。これにより、組織が浮遊性を有する場合であったとしても、組織は、培養中に培地の界面から沈められた状態で培養される。これにより、組織は、組織の表面全体が培地と接触した状態で培養され得る。本願の発明者は、メッシュ５０を用いて組織を培地中に押下して培養することによって、組織の形態及び／又は機能を長期にわたってなるべく維持できることを見出した。外来構造物を体内組織に使用した場合に炎症や線維化などを惹起することが一般的に知られているが、予想外にも本願の発明者はメッシュを用いた培養では、メッシュを用いない場合よりも炎症や線維化に関わる有意な遺伝子発現変動が少ないことを見出した。 The mesh 50 presses the tissue into the medium during tissue culture. As a result, even if the tissue is buoyant, the tissue is cultured in a state where it is submerged from the interface of the medium during culture. This allows the tissue to be cultured with the entire surface of the tissue in contact with the medium. The inventors of the present application have found that by culturing the tissue by pressing it into the medium using the mesh 50, the morphology and/or function of the tissue can be maintained as long as possible. It is generally known that the use of foreign structures in internal tissues can cause inflammation, fibrosis, etc., but the inventors of the present application have unexpectedly found that culture using a mesh results in less significant changes in gene expression related to inflammation and fibrosis than when a mesh is not used.
　メッシュ５０は、例えば、１０μｍ以上、２０μｍ以上、３０μｍ以上、４０μｍ以上、又は５５μｍ以上の目開きを有することが好ましい。メッシュ５０の目開きが上記の所望の値よりも大きいことにより、メッシュ５０を通して培地中の培養空間Ｓ１内に酸素や栄養素が行き渡り易くなるとともに、組織から生成された物質がメッシュ５０に詰まることを抑制することができる。これにより、組織の形態及び／又は機能をより長期にわたってなるべく維持することができると考えられる。 The mesh 50 preferably has an opening of, for example, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, or 55 μm or more. By making the opening of the mesh 50 larger than the above desired value, oxygen and nutrients can be more easily distributed through the mesh 50 into the culture space S1 in the medium, and the material produced from the tissue can be prevented from clogging the mesh 50. It is believed that this makes it possible to maintain the morphology and/or function of the tissue for as long as possible.
　メッシュ５０は、例えば２００μｍ以下、又は１８８μｍ以下の目開きを有することが好ましい。メッシュ５０の目開きが所望の値よりも小さいことによって、メッシュによって押下した組織、もしくは当該組織から分離した細胞が、メッシュ５０の穴を通って上方に抜けにくくなる。 The mesh 50 preferably has an opening size of, for example, 200 μm or less, or 188 μm or less. If the opening size of the mesh 50 is smaller than the desired value, the tissue pressed down by the mesh, or the cells separated from the tissue, will be less likely to pass through the holes in the mesh 50 and escape upward.
　脂肪細胞のサイズは、最大２００μｍ程度である。したがって、組織を構成する細胞が脂肪細胞である場合、メッシュ５０は、例えば２００μｍ以下又は１８８μｍ以下の目開きを有することが好ましい。 The size of fat cells is a maximum of about 200 μm. Therefore, when the cells that make up the tissue are fat cells, it is preferable that the mesh 50 has mesh openings of, for example, 200 μm or less or 188 μm or less.
　また、培養組織中の細胞機能の良好に維持の観点から、メッシュ５０の目開きは、例えば１８８μｍ以下、好ましくは１０８μｍ以下、より好ましくは７７μｍ以下、よりいっそう好ましくは５５μｍ以下であってよい。メッシュ５０の目開きが培養組織のサイズよりも十分に小さいと、メッシュ５０から培養組織への機械的な負荷が小さくなり、良好な培養環境が与えられると推測される。 In addition, from the viewpoint of maintaining good cellular function in the cultured tissue, the mesh size of the mesh 50 may be, for example, 188 μm or less, preferably 108 μm or less, more preferably 77 μm or less, and even more preferably 55 μm or less. If the mesh size of the mesh 50 is sufficiently smaller than the size of the cultured tissue, it is presumed that the mechanical load from the mesh 50 to the cultured tissue will be small, providing a good culture environment.
　メッシュ５０の目開きに関する上記の上限値と下限値は、任意に組み合わせることができることに留意されたい。 Please note that the above upper and lower limit values for the mesh opening of the mesh 50 can be combined in any way.
　メッシュ５０の開口率は、特に制限されないが、例えば５～７０％の範囲、好ましくは１０～６０％の範囲、より好ましくは２５～５０％の範囲であってよい。なお、メッシュ５０を通して培地中の培養空間Ｓ１内に酸素や栄養素が行き渡り易くするという観点において、メッシュ５０の開口率は、低すぎないことが好ましい。 The opening rate of the mesh 50 is not particularly limited, but may be, for example, in the range of 5 to 70%, preferably in the range of 10 to 60%, and more preferably in the range of 25 to 50%. From the viewpoint of facilitating the distribution of oxygen and nutrients through the mesh 50 into the culture space S1 in the medium, it is preferable that the opening rate of the mesh 50 is not too low.
　メッシュ５０の線径は、特に制限されないが、例えば１０μｍ以上、好ましくは２０μｍ以上、より好ましくは３０μｍ以上であってよい。また、メッシュ５０の厚みは、特に制限されないが、例えば２０μｍ以上、好ましくは３０μｍ以上、より好ましくは４０μｍ以上であってよい。 The wire diameter of the mesh 50 is not particularly limited, but may be, for example, 10 μm or more, preferably 20 μm or more, and more preferably 30 μm or more. The thickness of the mesh 50 is not particularly limited, but may be, for example, 20 μm or more, preferably 30 μm or more, and more preferably 40 μm or more.
　メッシュ５０の線径は、特に制限されないが、例えば２００μｍ以下、好ましくは１７０μｍ以下、より好ましくは１３０μｍ以下であってよい。また、メッシュ５０の厚みは、特に制限されないが、例えば３８０μｍ以下、好ましくは３２０μｍ以下、より好ましくは２４０μｍ以下であってよい。メッシュ５０の線径及び／又は厚みが小さいほどメッシュ５０が柔らかくなると考えられるため、培養組織への機械的な負荷が小さくなり得ると推測される。 The wire diameter of the mesh 50 is not particularly limited, but may be, for example, 200 μm or less, preferably 170 μm or less, and more preferably 130 μm or less. The thickness of the mesh 50 is not particularly limited, but may be, for example, 380 μm or less, preferably 320 μm or less, and more preferably 240 μm or less. It is believed that the smaller the wire diameter and/or thickness of the mesh 50, the softer the mesh 50 will be, and therefore it is presumed that the mechanical load on the cultured tissue may be reduced.
　メッシュ５０は、特に制限されないが、例えば３層以下、２層以下又は単層の格子状の構造を有していてよい。メッシュ５０の柔らかさの確保、及び／又は酸素や栄養素の通過性の観点から、メッシュ５０を構成する層の数は、小さいことが好ましい。 The mesh 50 is not particularly limited, but may have a lattice structure of, for example, three layers or less, two layers or less, or a single layer. From the standpoint of ensuring the softness of the mesh 50 and/or the permeability of oxygen and nutrients, it is preferable that the number of layers constituting the mesh 50 is small.
　メッシュ５０を構成する材料は、特に制限されないが、例えば培養する組織に対して接着性の低い材料であることが好ましい。メッシュを構成する材料は、高圧蒸気滅菌、放射線滅菌あるいはＥＯＧ滅菌が可能な高分子、例えばシルク、ヒアルロン酸、セルロース、ポリエチレン、ポリプロピレン、ポリエステル、ポリ乳酸、ポリメタクリル酸誘導体、ポリ塩化ビニル、ポリフェニレンサルファイド、ポリエチレンテレフタレート又はナイロンなどであってよい。 The material constituting the mesh 50 is not particularly limited, but is preferably a material that has low adhesiveness to the tissue to be cultured. The material constituting the mesh may be a polymer that can be sterilized by high-pressure steam sterilization, radiation sterilization, or EOG sterilization, such as silk, hyaluronic acid, cellulose, polyethylene, polypropylene, polyester, polylactic acid, polymethacrylic acid derivatives, polyvinyl chloride, polyphenylene sulfide, polyethylene terephthalate, or nylon.
　メッシュ５０は、生体適合性を有するコーティング層を有していてもよい。生体適合性を有するコーティング層を構成する材料は、例えばハイドロゲルであってよい。ハイドロゲルは、例えば、コラーゲン、ラミニン、フィブロネクチン、ビトロネクチン、ゼラチンなどの種々の化合物から選択された少なくとも１つを含んでいてよい。 The mesh 50 may have a biocompatible coating layer. The material constituting the biocompatible coating layer may be, for example, a hydrogel. The hydrogel may contain at least one selected from various compounds such as collagen, laminin, fibronectin, vitronectin, and gelatin.
　本実施形態では、保持部２０は、第１筒状部３０と、第２筒状部４０と、を有していてよい。第２筒状部４０は、第１筒状部３０の内側（中空部）に嵌合可能に構成されている。あるいは第１筒状部３０と第２筒状部４０は、一体不可分の一つの構造体として形成されていてもよい。 In this embodiment, the holding portion 20 may have a first cylindrical portion 30 and a second cylindrical portion 40. The second cylindrical portion 40 is configured to be able to fit inside (the hollow portion of) the first cylindrical portion 30. Alternatively, the first cylindrical portion 30 and the second cylindrical portion 40 may be formed as a single, inseparable structure.
　第１筒状部３０の形状は、特に制限されないが、例えば円筒形状や角筒形状等であってよい。具体的には、第１筒状部３０は、円筒形状や角筒形状の壁部３２を有していてよい。壁部３２の内側の空間は、中空部３８を構成していてよい。 The shape of the first tubular portion 30 is not particularly limited, but may be, for example, a cylindrical shape or a rectangular tube shape. Specifically, the first tubular portion 30 may have a wall portion 32 that is cylindrical or rectangular tube shape. The space inside the wall portion 32 may form a hollow portion 38.
　第１筒状部３０を構成する壁部３２の内面は段差３３を有する。この段差３３により、第１筒状部３０の上部側の内径Ｌ１は、第１筒状部３０の下部側の内径Ｌ２よりも大きくなっている。第２筒状部４０の外径Ｌ３は、第１筒状部３０の上部側の内径Ｌ１よりも小さく、かつ第１筒状部３０の下部側の内径Ｌ２よりも大きくなっていてよい。これにより、第１筒状部３０の中空部３８に上方から挿入された第２筒状部４０は、第１筒状部３０の段差３３付近まで挿入し得る（図５参照）。 The inner surface of the wall portion 32 constituting the first cylindrical portion 30 has a step 33. Due to this step 33, the inner diameter L1 of the upper side of the first cylindrical portion 30 is larger than the inner diameter L2 of the lower side of the first cylindrical portion 30. The outer diameter L3 of the second cylindrical portion 40 may be smaller than the inner diameter L1 of the upper side of the first cylindrical portion 30 and larger than the inner diameter L2 of the lower side of the first cylindrical portion 30. As a result, the second cylindrical portion 40 inserted from above into the hollow portion 38 of the first cylindrical portion 30 can be inserted up to the vicinity of the step 33 of the first cylindrical portion 30 (see FIG. 5).
　メッシュ５０は、第２筒状部４０の下端に設けられている。メッシュ５０の端部は、第１筒状部３０の段差３３に当接する。具体的には、メッシュ５０の端部は、第１筒状部３０の段差３３と、第２筒状部４０を構成する壁部４２との間に挟まれた状態になる（図５及び図６参照）。 The mesh 50 is provided at the lower end of the second cylindrical portion 40. The end of the mesh 50 abuts against the step 33 of the first cylindrical portion 30. Specifically, the end of the mesh 50 is sandwiched between the step 33 of the first cylindrical portion 30 and the wall portion 42 that constitutes the second cylindrical portion 40 (see Figures 5 and 6).
　第１筒状部３０を構成する壁部３２のうち段差３３よりも下方の部分は、メッシュ５０から下方に突出した突出部３４を構成する。突出部３４は、前述した培養空間Ｓ１の境界を規定していてよい。この場合、培養空間Ｓ１は、メッシュ５０と突出部３４により囲まれた空間によって規定される。 The portion of the wall 32 constituting the first cylindrical section 30 below the step 33 constitutes a protrusion 34 that protrudes downward from the mesh 50. The protrusion 34 may define the boundary of the culture space S1 described above. In this case, the culture space S1 is defined by the space surrounded by the mesh 50 and the protrusion 34.
　突出部３４は、培養空間Ｓ１を完全に又は部分的に取り囲むよう構成されていてよい。突出部３４がメッシュ５０から下方に突出している場合、メッシュ５０で押下された組織が、側方へ移動してメッシュ５０が存在していない領域へ流れてしまうことを抑制することができる。 The protrusion 34 may be configured to completely or partially surround the culture space S1. When the protrusion 34 protrudes downward from the mesh 50, the tissue pressed down by the mesh 50 can be prevented from moving laterally and flowing into an area where the mesh 50 is not present.
　好ましくは、突出部３４は、培地の流入が可能な開口３６を有する。これにより、培地は、開口３６を通して培養空間Ｓ１の内側から外側へ、又は外側から内側へ移動可能である。これにより、培養空間Ｓ１内の培地が新鮮な状態に維持されやすく、酸素や栄養素が培養空間Ｓ１内により流入し易くなる。 Preferably, the protrusion 34 has an opening 36 through which the culture medium can flow. This allows the culture medium to move from the inside to the outside of the culture space S1, or from the outside to the inside, through the opening 36. This makes it easier for the culture medium in the culture space S1 to be maintained in a fresh state, and makes it easier for oxygen and nutrients to flow into the culture space S1.
　第１筒状部４０の形状は、特に制限されないが、例えば円筒形状や角筒形状等であってよい。具体的には、第２筒状部４０は、円筒形状や角筒形状の壁部４２を有していてよい。第２筒状部４０は、メッシュ５０にアクセス可能な中空部４８を有することが好ましい。この場合、壁部４２の内側の空間が中空部４８を構成する。これにより、組織の培養を行う作業者は、メッシュ５０を介して培地の表面に浮かんだ油分などを容易に除去することができる。 The shape of the first tubular portion 40 is not particularly limited, but may be, for example, a cylindrical shape or a square tube shape. Specifically, the second tubular portion 40 may have a wall portion 42 that is cylindrical or square tube shape. The second tubular portion 40 preferably has a hollow portion 48 that is accessible to the mesh 50. In this case, the space inside the wall portion 42 constitutes the hollow portion 48. This allows the worker culturing the tissue to easily remove oil and other substances floating on the surface of the culture medium through the mesh 50.
　図示していないが、培養装置１００は、培養空間Ｓ１を密閉するための蓋を有していても良い。不図示の蓋は、例えば、収容部に取り付けられ、保持部２０全体を取り囲むよう構成されていてもよい。 Although not shown, the culture device 100 may have a lid for sealing the culture space S1. The lid (not shown) may be attached to the storage unit, for example, and configured to surround the entire holding unit 20.
　［培養組織及び培養組織の製造方法］
　以下、培養組織の製造方法について説明する。本実施形態に係る培養組織の製造方法は、例えば前述した培養装置１００を用いて実施することができる。 [Cultured tissue and method for producing cultured tissue]
The method for producing a cultured tissue according to this embodiment can be carried out using the culture device 100 described above, for example.
　培養組織の製造方法は、まず、生体から培養すべき組織を採取することを含む。採取された組織は、所望のサイズにカットされてもよい。本実施形態で培養する組織の種類は、前述したとおりであるため、その説明を省略する。 The method for producing cultured tissue first involves harvesting the tissue to be cultured from a living organism. The harvested tissue may be cut to a desired size. The types of tissue cultured in this embodiment are as described above, and therefore will not be described here.
　所望のサイズにカットされた組織は、メッシュにより培地中に組織を押下させた状態で培養される。培地の種類は、特に制限されず、培養する組織に応じて適切に選択されればよい。ここで、組織に対して、コラゲナーゼ処理のような細胞分散処理は行わず、組織塊のまま培養する。 The tissue cut to the desired size is cultured in a medium with the tissue pressed down by a mesh. There are no particular limitations on the type of medium, and it may be selected appropriately depending on the tissue to be cultured. Here, the tissue is not subjected to cell dispersion treatment such as collagenase treatment, and is cultured as a tissue mass.
　メッシュは、例えば、１０μｍ以上、２０μｍ以上、３０μｍ以上、４０μｍ以上又は５５μｍ以上の目開きを有することが好ましい。また、メッシュは、例えば２００μｍ以下、又は１８８μｍ以下の目開きを有することが好ましい。メッシュに関するその他の好ましいパラメータについては前述したとおりである。 The mesh preferably has an opening of, for example, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, or 55 μm or more. The mesh preferably has an opening of, for example, 200 μm or less, or 188 μm or less. Other preferred parameters regarding the mesh are as described above.
　このような組織の培養は、前述した培養装置１００を用いて実施できる。培養装置１００の構成は、前述したとおりであるため、その説明を省略する。本実施形態において、組織の培養は、支持細胞のような外来細胞を用いずに行われる。 Such tissue culture can be carried out using the culture device 100 described above. The configuration of the culture device 100 is as described above, so a description thereof will be omitted. In this embodiment, tissue culture is carried out without using foreign cells such as supporting cells.
　培養に用いる培地は、ＤＭＥＭ、ＭＥＭ、ＲＰＭＩ、Ｆ１２、Ｍ１９９あるいはこれらの混合培地に血清アルブミン（ＢＳＡやＨＳＡなど）、ポリビニルアルコール、リポタンパク質（ＬＤＬやＨＤＬなど）、遊離脂肪酸、インスリン、デキサメタゾン、コルチゾール、グルコース、ピルビン酸、グルタミン、脂肪酸、ビオチン、パントテン酸、トランスフェリン、亜セレン酸、バナジン酸などの添加剤に加えゲンタマイシン、ペニシリン、ストレプトマイシンなどの抗生物質を含んでいてよい。 The medium used for culture may be DMEM, MEM, RPMI, F12, M199, or a mixture of these media, which may contain additives such as serum albumin (such as BSA or HSA), polyvinyl alcohol, lipoproteins (such as LDL or HDL), free fatty acids, insulin, dexamethasone, cortisol, glucose, pyruvic acid, glutamine, fatty acids, biotin, pantothenic acid, transferrin, selenic acid, vanadate, as well as antibiotics such as gentamicin, penicillin, and streptomycin.
　組織の培養日数は、特に制限されない。ただし、組織は、例えば１４日以上、２４日以上、３２日以上、４２日以上、４９日以上、又は５６日以上、培養することができる。前述した培養装置１００により、長期わたって組織の形態を維持した状態で組織を培養することができる。 The number of days for which the tissue is cultured is not particularly limited. However, the tissue can be cultured for, for example, 14 days or more, 24 days or more, 32 days or more, 42 days or more, 49 days or more, or 56 days or more. The above-described culture device 100 allows the tissue to be cultured while maintaining its morphology for a long period of time.
　組織の培養日数の上限は、特に制限されない。組織の培養日数は、例えば１２０日以下、９０日以下、８０日以下、７０日以下又は６０日以下であってよい。 There is no particular upper limit to the number of days for which the tissue is cultured. The number of days for which the tissue is cultured may be, for example, 120 days or less, 90 days or less, 80 days or less, 70 days or less, or 60 days or less.
　組織の培養開始から所望の日数ごとに、培地の交換が行われることが好ましい。培地交換のタイミングや頻度は、特に制限されないことに留意されたい。各々の培地交換において、培地の除去と培地の添加は、例えば１回ずつ行われてもよく、複数回繰り返し行われてもよい。 It is preferable to change the medium every desired number of days from the start of tissue culture. It should be noted that there are no particular limitations on the timing or frequency of medium changes. In each medium change, the medium may be removed and added once, for example, or may be repeated multiple times.
　上記のように組織を培養することによって培養組織が製造される。言い換えると、本実施形態に係る培養組織は、上記の製造方法によって製造された組織であってよい。 A cultured tissue is produced by culturing the tissue as described above. In other words, the cultured tissue according to this embodiment may be a tissue produced by the above-described production method.
　上記のように、メッシュにより培地中に組織を押下させた状態で組織を培養することによって、培養組織の遺伝子発現機能及び／又は生理活性機能を長期にわたってなるべく維持できる。 As described above, by culturing the tissue while it is pressed into the medium by the mesh, the gene expression function and/or physiological activity function of the cultured tissue can be maintained as long as possible.
　例えば、培養３２日目における組織について、培養４日目における組織の遺伝子発現と比較して有意に変動する遺伝子の数が少ない、あるいは組織の線維化や炎症に関わるような組織の変質に関わる有意な遺伝子変動が少ない。 For example, the number of genes that change significantly in tissue on the 32nd day of culture is smaller than that in tissue on the 4th day of culture, or there are fewer significant gene changes related to tissue alterations such as those related to tissue fibrosis and inflammation.
　また、上記の培養方法により、例えばアディポネクチン分泌量のような脂肪組織の生理活性機能の低下を抑制することができる。 In addition, the above culture method can suppress the decline in physiological activity of adipose tissue, such as the amount of adiponectin secreted.
　上記の培養装置、培養組織、及び／又は培養組織の製造方法は、種々の組織の機能評価方法のツールとして利用することができる。当該評価方法は、メッシュにより培地中に組織を押下させた状態で培養することによって得られた培養組織に試薬を与え、試薬に対する培養組織の応答に応じて、試薬、及び／又は培養組織に関する評価を得ること、を含む。 The above-mentioned culture device, cultured tissue, and/or cultured tissue manufacturing method can be used as a tool for a method of evaluating the function of various tissues. The evaluation method includes providing a reagent to a cultured tissue obtained by culturing the tissue in a medium while pressing the tissue with a mesh, and obtaining an evaluation of the reagent and/or the cultured tissue according to the response of the cultured tissue to the reagent.
　ここで、メッシュには、前述したものであってよい。具体的には、前述した培養装置に備えられたメッシュであってよい。前述した培養方法によって培養された組織に与える試薬は、評価すべき項目に応じて適切に設定すればよい。 The mesh may be as described above. Specifically, it may be the mesh provided in the culture device described above. The reagent given to the tissue cultured by the culture method described above may be appropriately selected depending on the item to be evaluated.
　当該評価方法は、試薬に対する組織の応答を測定するための薬理試験及び／又は薬効試験として利用できる。当該評価方法は、創薬のスクリーニング目的としても利用できる。この場合、試薬は、薬剤の候補物質であってよい。 The evaluation method can be used as a pharmacological test and/or a drug efficacy test to measure the response of tissues to a reagent. The evaluation method can also be used for screening purposes in drug discovery. In this case, the reagent may be a candidate drug substance.
　［実施例］
　（実施例１）
　まず、代謝性疾患のない７１才女性患者から手術により摘出したヒト腹部脂肪組織（以下、単に「脂肪組織」と称することがある。）から比較的大きい血管及び結合組織を除去した後、手術用ハサミによって脂肪組織を２０ｍｇ～１００ｍｇの小片になるようカットした。カットした脂肪組織片から遠心分離にて赤血球を除去することによって、培養する脂肪組織（組織）を得た。 [Example]
Example 1
First, relatively large blood vessels and connective tissue were removed from human abdominal adipose tissue (hereinafter, simply referred to as "adipose tissue") surgically removed from a 71-year-old female patient without metabolic disease, and the adipose tissue was then cut into small pieces of 20 mg to 100 mg with surgical scissors. Red blood cells were removed from the cut pieces of adipose tissue by centrifugation to obtain adipose tissue (tissue) to be cultured.
　次に、小片にカットされた脂肪組織を前述した培養装置１００にて培養した（図１～図６も参照）。脂肪組織は、培養中、メッシュにより培地（培養液）中に押下された。メッシュはコラーゲンにてコーティングされた。以下表１に示すように、実施例１では、メッシュの目開きは３０μｍであり、メッシュの開口率は２５％であり、メッシュの線径は３０μｍであり、メッシュの厚みは４７μｍであった。 Then, the adipose tissue cut into small pieces was cultured in the above-mentioned culture device 100 (see also Figures 1 to 6). During culture, the adipose tissue was pressed into the medium (culture fluid) by a mesh. The mesh was coated with collagen. As shown in Table 1 below, in Example 1, the mesh opening was 30 μm, the mesh opening rate was 25%, the mesh wire diameter was 30 μm, and the mesh thickness was 47 μm.
　ここで、培養液は、０．１％のＢＳＡ、１００ｍｇ／ｍＬのグルコース、３ｎＭのデキサメタゾン、１ｎｇ／ｍＬのインスリン、１０μｇ／ｍＬのゲンタマイシンを含有したＤＭＥＭであった。培養装置１００を浸漬させた２４ウェル組織培養プレートをＣＯ２インキュベーターに静置し、３７℃で５％の二酸化炭素雰囲気下で培養した。 Here, the culture solution was DMEM containing 0.1% BSA, 100 mg/mL glucose, 3 nM dexamethasone, 1 ng/mL insulin, and 10 μg/mL gentamicin. The 24-well tissue culture plate in which the culture device 100 was immersed was placed in a CO2 incubator and cultured at 37° C. in a 5% carbon dioxide atmosphere.
　培養装置１００で組織の培養を開始した日をＤａｙ０としたとき、Ｄａｙ１，２，４，７，９，１１，１４，１６，１８，２１，２３，２５，２８，３０において培地交換が行われた。Ｄａｙ２，１６及びＤａｙ３０での培地交換においては、培地除去と培地添加を３回繰り返して行い培地を完全に交換した。なお、Ｄａｙ４，１８及びＤａｙ３２において培地交換前の培地の一部は採取し、脂肪組織機能評価のため凍結保存した。 If the day when tissue culture began in the culture device 100 is Day 0, culture medium changes were performed on Days 1, 2, 4, 7, 9, 11, 14, 16, 18, 21, 23, 25, 28, and 30. For culture medium changes on Days 2, 16, and 30, medium removal and medium addition were repeated three times to completely change the medium. Note that on Days 4, 18, and 32, a portion of the medium before medium change was collected and frozen for evaluation of adipose tissue function.
　（実施例２）
　メッシュのパラメータの違いを除き、実施例１と同様に脂肪組織（組織）を培養した。以下表１に示すように、実施例２では、メッシュの目開きは５５μｍであり、メッシュの開口率は３４％であり、メッシュの線径は３９μｍであり、メッシュの厚みは６２μｍであった。 Example 2
Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 2, the mesh opening was 55 μm, the mesh opening ratio was 34%, the mesh wire diameter was 39 μm, and the mesh thickness was 62 μm.
　（実施例３）
　メッシュのパラメータの違いを除き、実施例１と同様に脂肪組織（組織）を培養した。以下表１に示すように、実施例３では、メッシュの目開きは７７μｍであり、メッシュの開口率は３７％であり、メッシュの線径は５０μｍであり、メッシュの厚みは８３μｍであった。 Example 3
Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 3, the mesh opening was 77 μm, the mesh opening ratio was 37%, the mesh wire diameter was 50 μm, and the mesh thickness was 83 μm.
　（実施例４）
　メッシュのパラメータの違いを除き、実施例１と同様に脂肪組織（組織）を培養した。以下表１に示すように、実施例４では、メッシュの目開きは１０８μｍであり、メッシュの開口率は４１％であり、メッシュの線径は６１μｍであり、メッシュの厚みは１０６μｍであった。 Example 4
Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 4, the mesh opening was 108 μm, the mesh opening ratio was 41%, the mesh wire diameter was 61 μm, and the mesh thickness was 106 μm.
　（実施例５）
　メッシュのパラメータの違いを除き、実施例１と同様に脂肪組織（組織）を培養した。以下表１に示すように、実施例５では、メッシュの目開きは１８８μｍであり、メッシュの開口率は３５％であり、メッシュの線径は１３０μｍであり、メッシュの厚みは２３５μｍであった。 Example 5
Except for differences in mesh parameters, adipose tissue (tissue) was cultured in the same manner as in Example 1. As shown in Table 1 below, in Example 5, the mesh opening was 188 μm, the mesh opening ratio was 35%, the mesh wire diameter was 130 μm, and the mesh thickness was 235 μm.
　（表１）
Figure JPOXMLDOC01-appb-I000001
 (Table 1)
Figure JPOXMLDOC01-appb-I000001
　（参考例１）
　実施例１と同様の手法で培養するヒト腹部脂肪組織（組織）を得た。組織は、前述したメッシュを使用することなく培地に浮遊させたまま培養した。培地及びその他の培養条件は、実施例１と同様である。 (Reference Example 1)
Human abdominal adipose tissue (tissue) to be cultured was obtained in the same manner as in Example 1. The tissue was cultured floating in the medium without using the mesh described above. The medium and other culture conditions were the same as in Example 1.
　［培養上清中のグリセロール量の定量］
　実施例１～５及び参考例１において、Ｄａｙ４，１８およびＤａｙ３２で採取し凍結保存した培養液を４℃で融解し、遠心処理の後、上澄みの一部を用いて、培地上清中のグリセロール量をグリセロールアッセイキット（Sigma-Aldrich社; MAK117）にて測定した。 [Quantitative determination of glycerol content in culture supernatant]
In Examples 1 to 5 and Reference Example 1, the culture fluids collected on Day 4, 18, and 32 and stored frozen were thawed at 4° C. and centrifuged, and then the amount of glycerol in the culture supernatant was measured using a glycerol assay kit (Sigma-Aldrich; MAK117) using a portion of the supernatant.
　図７は、実施例１～５及び参考例１におけるＤａｙ４、Ｄａｙ１８及びＤａｙ３２の培養上清中のグリセロール量を示すグラフである。何れの場合においてもＤａｙ４からＤａｙ１８にかけてグリセロール量の上昇が見られた。これは低いグルコース濃度に呼応した脂質分解の向上によるものであると推察される。また、Ｄａｙ１８とＤａｙ３２ではほぼ同程度のグリセロール量を示した。この結果は培養組織における脂質分解機能が維持されていることを示唆する。 Figure 7 is a graph showing the amount of glycerol in the culture supernatant on Day 4, Day 18, and Day 32 in Examples 1 to 5 and Reference Example 1. In all cases, an increase in the amount of glycerol was observed from Day 4 to Day 18. This is presumably due to improved lipid breakdown in response to the low glucose concentration. Furthermore, the amount of glycerol was roughly the same on Day 18 and Day 32. This result suggests that lipid breakdown function is maintained in the cultured tissue.
　［培養上清中のアディポネクチン分泌量の定量］
　実施例１～５及び参考例１において、Ｄａｙ４，１８およびＤａｙ３２で採取し凍結保存した培養液を４℃で融解した後、遠心処理ののち上澄みの一部を用いて、培地中の分泌アディポネクチン量をHuman Adiponectin ELISA Kit（ABclonal社; RK00060）にて測定した。 [Quantitative determination of adiponectin secretion amount in culture supernatant]
In Examples 1 to 5 and Reference Example 1, the culture fluids collected on Day 4, 18, and 32 and stored frozen were thawed at 4°C, centrifuged, and then a portion of the supernatant was used to measure the amount of adiponectin secreted in the medium using a Human Adiponectin ELISA Kit (ABclonal; RK00060).
　図８は、実施例１～５及び参考例１におけるＤａｙ４、Ｄａｙ１８及びＤａｙ３２の培養上清中の分泌アディポネクチン量を示すグラフである。参考例１では、培養日数とともにアディポネクチン分泌量の顕著な低下が認められた。これに対して実施例１～５では、アディポネクチン分泌量は保持される傾向であり、アディポネクチン分泌量の低下は抑制された。特に３０μｍ及び７７μｍの範囲、より詳細には３０μｍ及び５５μｍの目開きを有するメッシュを備えた培養装置１００が用いられた場合、非常に良好に分泌量が保持された。この結果は、メッシュを備えた培養装置１００を用いることによって、培養組織中の脂肪細胞機能が良好に維持されることを示す。 FIG. 8 is a graph showing the amount of adiponectin secreted in the culture supernatant on Day 4, Day 18, and Day 32 in Examples 1 to 5 and Reference Example 1. In Reference Example 1, a significant decrease in the amount of adiponectin secreted was observed with the number of days of culture. In contrast, in Examples 1 to 5, the amount of adiponectin secreted tended to be maintained, and the decrease in the amount of adiponectin secreted was suppressed. In particular, when a culture device 100 equipped with a mesh having openings in the range of 30 μm and 77 μm, more specifically 30 μm and 55 μm, was used, the amount of secretion was maintained very well. This result indicates that the use of a culture device 100 equipped with a mesh allows the adipocyte function in the cultured tissue to be well maintained.
　［ＲＮＡ抽出処理］
　Ｄａｙ４及びＤａｙ３２において、組織（湿重量計１００～２００ｍｇ）をQIAzol Lysis Reagent（ＱＩＡＧＥＮ）中でホモジナイズし、メーカー規定のプロトコルに沿って、ＲＮＡ抽出液を得た。このＲＮＡ抽出液から、「ISOSPIN Cell & Tissue RNA (ニッポンジーン; 314-08211)」にて、メーカーのプロトコルに沿って、精製ＲＮＡ溶液を得た。得られたＲＮＡは、バイオアナライザーによる分析にてＲＩＮ値７以上の十分な品質を示した。以下実測値の一例を表２に示した。 [RNA extraction treatment]
On Day 4 and Day 32, tissues (100-200 mg wet weight) were homogenized in QIAzol Lysis Reagent (QIAGEN) and RNA extracts were obtained according to the manufacturer's protocol. From this RNA extract, purified RNA solution was obtained using "ISOSPIN Cell & Tissue RNA (Nippon Gene; 314-08211)" according to the manufacturer's protocol. The obtained RNA showed sufficient quality with an RIN value of 7 or more when analyzed by a bioanalyzer. An example of the actual measured values is shown in Table 2 below.
　（表２）
Figure JPOXMLDOC01-appb-I000002
 (Table 2)
Figure JPOXMLDOC01-appb-I000002
　［遺伝子発現データ］
　実施例１～５及び参考例１においてＤａｙ４及びＤａｙ３２に精製したＲＮＡ溶液を用いて、Rhelixa社委託試験（RNA-seq）により、Illumina NovaSeq 6000（標準取得リード長：PE 150(150bp×2 paired-end)）を用いて、生データ（fastqファイル）を取得した。その後、fastqファイルからfastp/STAR/featureCountを用いてゲノム配列にマッピングした後、EdgeRにて遺伝子発現変動解析を実施した。 [Gene expression data]
In Examples 1 to 5 and Reference Example 1, the RNA solutions purified on Day 4 and Day 32 were used to obtain raw data (fastq files) using an Illumina NovaSeq 6000 (standard read length: PE 150 (150 bp × 2 paired-end)) in a test commissioned by Rhelixa (RNA-seq). After that, the fastq files were mapped to the genome sequence using fastp/STAR/featureCount, and gene expression variation analysis was performed using EdgeR.
　発現変動遺伝子のうち、線維化や炎症を惹起する遺伝子の有意な発現上昇が参考例１において特異的にみられるものがあった。参考例１において有意な発現上昇をした遺伝子について、log FC（log fold-change）、ＦＤＲ（False Discovery Ratio）、有意差順位を以下の表３に示した。ここで、「log FC」は、特定の遺伝子について、底を２とし、Ｄａｙ３２における遺伝子の発現量（シグナル値）をＤａｙ４における遺伝子の発現量（シグナル値）で割った値の対数によって定義される。「ＦＤＲ」は、多重比較検定法における誤検出率である。有意差順位は、ｐ値の小さい順に遺伝子を並べたときの順位に相当する。また、本明細書において、遺伝子の「有意な発現上昇」は、「ＦＤＲの値が０．０１以下」かつ「log FCの値が１．０以上」によって規定される。 Among the genes with altered expression, some genes that induce fibrosis or inflammation showed a significant increase in expression specifically in Reference Example 1. The log FC (log fold-change), FDR (False Discovery Ratio), and significance rank for genes that showed a significant increase in expression in Reference Example 1 are shown in Table 3 below. Here, "log FC" is defined as the logarithm of the value obtained by dividing the expression level (signal value) of a specific gene on Day 32 by the expression level (signal value) of the gene on Day 4, with the base being 2. "FDR" is the false detection rate in multiple comparison testing. The significance rank corresponds to the rank when genes are arranged in ascending order of p-value. In addition, in this specification, a "significant increase in expression" of a gene is defined as an "FDR value of 0.01 or less" and a "log FC value of 1.0 or more."
　（表３）
Figure JPOXMLDOC01-appb-I000003
 (Table 3)
Figure JPOXMLDOC01-appb-I000003
　表３で示された遺伝子群（EGR1,FOSB，PLTP，FADS1，COL1A1，LTBP2，FADS2）は組織の健常な機能維持という点において、有意な発現上昇を避けるべきものである。実施例１～５ではこれらの遺伝子のうち有意な発現上昇を示したものがほとんど見られなかった。具体的には、実施例１，２では、有意な発現上昇をした遺伝子は、表３に掲載した遺伝子群のうちのEGR1のみであった。また、実施例４では、有意な発現上昇をした遺伝子は、表３に掲載した遺伝子群のうちのPTLPのみであった。実施例３，５では、有意な発現上昇をした遺伝子は、表３に掲載した遺伝子群のなかに存在しなかった。このように、実施例１～５では、表３で示された遺伝子群のなかで、有意な発現上昇をした遺伝子は、０ないし１つだけであった。このことは、実施例１～５では脂肪組織の形態及び／又は機能が、参考例１と比較すると、長期にわたって維持されていることを意味する。 The gene group shown in Table 3 (EGR1, FOSB, PLTP, FADS1, COL1A1, LTBP2, FADS2) should be avoided from the viewpoint of maintaining healthy tissue function. In Examples 1 to 5, few of these genes showed significant expression increases. Specifically, in Examples 1 and 2, the only gene that showed significant expression increases was EGR1 from the gene group listed in Table 3. In Example 4, the only gene that showed significant expression increases was PTLP from the gene group listed in Table 3. In Examples 3 and 5, there were no genes that showed significant expression increases in the gene group listed in Table 3. Thus, in Examples 1 to 5, there was only 0 or 1 gene that showed significant expression increases in the gene group shown in Table 3. This means that the morphology and/or function of the adipose tissue is maintained for a long period in Examples 1 to 5 compared to Reference Example 1.
　以下の表４は、解析したすべての遺伝子群の中で、Ｄａｙ４とＤａｙ３２との間における有意な発現変動をした遺伝子の数を示している。ここで、本明細書において、遺伝子の「有意な発現変動」は、「ＦＤＲの値が０．０１以下」かつ「log FCの絶対値が１．０以上」によって規定される。 Table 4 below shows the number of genes that showed significant changes in expression between Day 4 and Day 32 among all the gene groups analyzed. Here, in this specification, a "significant change in expression" of a gene is defined as an "FDR value of 0.01 or less" and an "absolute value of log FC of 1.0 or more."
　（表４）
Figure JPOXMLDOC01-appb-I000004
 (Table 4)
Figure JPOXMLDOC01-appb-I000004
　表４から、実施例１～３では有意な発現変動をした遺伝子の数は他の実施例４，５よりも少なく、実施例２では有意な発現変動をした遺伝子の数が最も少ない。したがって、３０μｍ以上、かつ７７μｍ以下のメッシュを用いた組織の培養は、遺伝子の機能発現の維持という観点からより好ましいことがわかる。もっとも、実施例４，５においても、有意な発現変動をした遺伝子の数は十分に小さいため、本発明に係るメッシュの目開きは、上記数値範囲の外であってもよいことに留意されたい。 From Table 4, the number of genes that showed significant changes in expression in Examples 1 to 3 was smaller than in Examples 4 and 5, and Example 2 had the smallest number of genes that showed significant changes in expression. Therefore, it can be seen that tissue culture using a mesh that is 30 μm or more and 77 μm or less is more preferable from the perspective of maintaining the functional expression of genes. However, it should be noted that since the number of genes that showed significant changes in expression was also sufficiently small in Examples 4 and 5, the mesh opening of the mesh according to the present invention may be outside the above numerical range.
　（実施例６）
　小片にカットされた代謝性疾患のない７２才女性由来ヒト腹部脂肪組織（以下、「脂肪組織」と称する。）２０－１００ｍｇを前述した培養装置１００にて培養した（図１～図６も参照）。脂肪組織は、培養中、メッシュにより培地（培養液）中に押下した。培養装置１００にはコラーゲンコーティング処置した目開き７７μｍのメッシュを用いた。当該メッシュは、実施例３で使用したものと同じである。培地組成は０．２％のＢＳＡ、３ｎＭのデキサメタゾン、１ｎｇ／ｍＬのインスリン、３００ｍｇ／ｄＬのグルコース、０．１８ｍＭのパルミチン酸、１０μｇ／ｍＬのゲンタマイシンを含有したＤＭＥＭであった。培養装置１００を浸漬させた２４ウェル組織培養プレートをＣＯ２インキュベーターに静置し、３７℃で５％の二酸化炭素雰囲気下で２１日間培養した。培養を開始した日をＤａｙ０としたとき、Ｄａｙ１，２，５，７，９，１２，１４，１６，１９において培地交換を行った。Ｄａｙ１９での培地交換は、培地除去後、培地添加を３回行い、培地を完全に交換した。培地交換直後、１００μＬの培地を採取し凍結保存した（凍結保存サンプル１）。Ｄａｙ１９での培地交換から２４時間培養後（Ｄａｙ２０）、１００μＬの培地を採取し凍結保存した（凍結保存サンプル２）。 Example 6
20-100 mg of human abdominal adipose tissue (hereinafter referred to as "adipose tissue") from a 72-year-old woman without metabolic disease cut into small pieces was cultured in the above-mentioned culture device 100 (see also Figures 1 to 6). The adipose tissue was pressed into the medium (culture solution) by a mesh during culture. A collagen-coated mesh with a mesh opening of 77 μm was used for the culture device 100. The mesh was the same as that used in Example 3. The medium composition was DMEM containing 0.2% BSA, 3 nM dexamethasone, 1 ng/mL insulin, 300 mg/dL glucose, 0.18 mM palmitic acid, and 10 μg/mL gentamicin. The 24-well tissue culture plate immersed in the culture device 100 was placed in a CO2 incubator and cultured at 37°C under a 5% carbon dioxide atmosphere for 21 days. The day when the culture was started was designated as Day 0, and the medium was exchanged on Days 1, 2, 5, 7, 9, 12, 14, 16, and 19. The medium exchange on Day 19 involved removing the medium and then adding the medium three times to completely exchange the medium. Immediately after the medium exchange, 100 μL of the medium was collected and frozen (frozen sample 1). After 24 hours of culture from the medium exchange on Day 19 (Day 20), 100 μL of the medium was collected and frozen (frozen sample 2).
　凍結保存サンプル２の取得後、Ｄａｙ２０においては、インスリン濃度を２０ｎｇ／ｍＬとした培地にて培地交換を行った。Ｄａｙ２０において、Ｄａｙ１９と同様培地交換を３回行った。Ｄａｙ２０において培地交換直後１００μＬの培地を採取し凍結保存した（凍結保存サンプル３）。培地交換から２４時間培養後（Ｄａｙ２１）、１００μＬの培地を採取し凍結保存した（凍結保存サンプル４）。 After obtaining frozen storage sample 2, on day 20, the medium was replaced with a medium containing an insulin concentration of 20 ng/mL. On day 20, the medium was replaced three times, similar to day 19. Immediately after the medium replacement on day 20, 100 μL of medium was collected and frozen (frozen storage sample 3). After 24 hours of culture from the medium replacement (day 21), 100 μL of medium was collected and frozen (frozen storage sample 4).
　採取し凍結保存した培地（凍結保存サンプル１～４）を解凍した後、培地中のグルコース濃度をグルコース測定キット（同仁化学研究所; G264）にて定量した。凍結保存サンプル１と凍結保存サンプル２における培地中のグルコース濃度を比較することによって、１ｎｇ／ｍＬのインスリン濃度における２４時間後の培地中グルコース濃度の変化量を求めた。同様に、凍結保存サンプル３と凍結保存サンプル４における培地中のグルコース濃度を比較することによって、２０ｎｇ／ｍＬのインスリン濃度における２４時間後の培地中グルコース濃度の変化量を求めた。これらのグルコース濃度の変化量は、脂肪組織へのグルコース取込量に相当すると考えられる。 After thawing the collected and frozen-stored medium (frozen samples 1 to 4), the glucose concentration in the medium was quantified using a glucose measurement kit (Dojindo Laboratories; G264). By comparing the glucose concentrations in the medium of frozen samples 1 and 2, the amount of change in the glucose concentration in the medium after 24 hours at an insulin concentration of 1 ng/mL was determined. Similarly, by comparing the glucose concentrations in the medium of frozen samples 3 and 4, the amount of change in the glucose concentration in the medium after 24 hours at an insulin concentration of 20 ng/mL was determined. These changes in glucose concentration are considered to correspond to the amount of glucose uptake into adipose tissue.
　図９は、上記手順により評価した実施例６における培養上清中のグルコース低下量を示すグラフである。図９において、１ｎｇ／ｍＬのインスリン濃度が示すグラフは、凍結保存サンプル２のグルコース濃度と凍結保存サンプル１のグルコース濃度の差分に相当する。同様に、２０ｎｇ／ｍＬのインスリン濃度が示すグラフは、凍結保存サンプル４のグルコース濃度と凍結保存サンプル１のグルコース濃度の差分に相当する。図９から、インスリン濃度２０ｎｇ／ｍＬでは１ｎｇ／ｍＬと比較して顕著なグルコース取込上昇が確認された。このことから、組織培養後もインスリン応答性の脂肪組織機能が保持されていることが確認された。 Figure 9 is a graph showing the amount of glucose reduction in the culture supernatant in Example 6 evaluated by the above procedure. In Figure 9, the graph showing an insulin concentration of 1 ng/mL corresponds to the difference between the glucose concentration of frozen-preserved sample 2 and the glucose concentration of frozen-preserved sample 1. Similarly, the graph showing an insulin concentration of 20 ng/mL corresponds to the difference between the glucose concentration of frozen-preserved sample 4 and the glucose concentration of frozen-preserved sample 1. Figure 9 confirms that there is a significant increase in glucose uptake at an insulin concentration of 20 ng/mL compared to 1 ng/mL. This confirmed that insulin-responsive adipose tissue function was maintained even after tissue culture.
　ここで、参考例１では、遺伝子EGR1及びFOSBは、有意に発現上昇している。EGR1及びFOSBはインスリン抵抗性と関連するという示唆があり、参考例１ではインスリン応答性が低下すると予想される。したがって、実施例６に係る組織の培養方法及び培養組織は、参考例１と比較してインスリン応答性の脂肪組織機能が維持されることが期待できる。 Here, in Reference Example 1, the expression of the genes EGR1 and FOSB is significantly increased. There is a suggestion that EGR1 and FOSB are associated with insulin resistance, and it is predicted that insulin responsiveness will be reduced in Reference Example 1. Therefore, it is expected that the tissue culture method and cultured tissue of Example 6 will maintain insulin-responsive adipose tissue function compared to Reference Example 1.
　［付記］
　前述した実施形態及び／又は実施例の記載により、少なくとも以下の付記として記載された発明が本明細書内に明示されていることに留意されたい。 [Additional Notes]
Please note that the above description of the embodiments and/or examples clearly indicates the invention described in at least the following appendix within this specification.
　［付記１］
　組織を培地中に押下するためのメッシュを有する培養装置。
　［付記２］
　前記メッシュは、１０μｍ以上、かつ２００μｍ以下の目開きを有する、付記１に記載の培養装置。
　［付記３］
　前記メッシュは、生体適合性を有するコーティング層を有する、付記１又は２に記載の培養装置。
　［付記４］
　前記組織は浮遊性を有する、付記１から３のいずれか１項に記載の培養装置。
　［付記５］
　前記組織は、脂肪組織又は脳組織である、付記１から４のいずれか１項に記載の培養装置。
　［付記６］
　前記メッシュから下方に突出した突出部を有する、付記１から５のいずれか１項に記載の培養装置。
　［付記７］
　前記突出部は、前記組織の培養空間を囲むよう形成されており、
　前記突出部は、培地の流入が可能な開口を有する、付記１から６のいずれか１項に記載の培養装置。
　［付記８］
　前記メッシュを保持しつつ、上方に延びた第２筒状部を有し、
　前記第２筒状部は、前記メッシュにアクセス可能な中空部を有する、付記１から７のいずれか１項に記載の培養装置。
　［付記９］
　メッシュにより培地中に組織を押下させた状態で組織を培養することを含む、培養組織の製造方法。
　［付記１０］
　前記メッシュは、１０μｍ以上、かつ２００μｍ以下の目開きを有する、付記９に記載の培養組織の製造方法。
　［付記１１］
　前記メッシュは、生体適合性を有するコーティング層を有する、付記９又は１０に記載の培養組織の製造方法。
　［付記１２］
　前記培養組織は浮遊性を有する、付記９から１１のいずれか１項に記載の培養組織の製造方法。
　［付記１３］
　前記培養組織は、脂肪組織である、付記９から１２のいずれか１項に記載の培養組織の製造方法。
　［付記１４］
　前記組織を、１４日以上培養する、付記９から１３のいずれか１項に記載の培養組織の製造方法。
　［付記１５］
　メッシュにより培地中に組織を押下させた状態で前記組織を培養することによって得られた、培養組織。
　［付記１６］
　前記組織は浮遊性を有する、付記１５に記載の培養組織。
　［付記１７］
　前記組織は、脂肪組織である、付記１５又は１６に記載の培養組織。
　［付記１８］
　前記組織を、１４日以上培養することによって得られた、付記１５から１７のいずれか１項に記載の培養組織。
　［付記１９］
　メッシュにより培地中に組織を押下させた状態で培養することによって得られた培養組織に試薬を与え、前記試薬に対する前記培養組織の応答に応じて、前記試薬、及び／又は前記培養組織に関する機能評価を得ること、を含む、評価方法。 [Appendix 1]
A culture device having a mesh for pressing the tissue into the medium.
[Appendix 2]
The culture device of claim 1, wherein the mesh has an opening size of 10 μm or more and 200 μm or less.
[Appendix 3]
3. The culture device of claim 1 or 2, wherein the mesh has a biocompatible coating layer.
[Appendix 4]
The culture device according to any one of claims 1 to 3, wherein the tissue is buoyant.
[Appendix 5]
The culture device according to any one of claims 1 to 4, wherein the tissue is adipose tissue or brain tissue.
[Appendix 6]
6. A culture device according to any one of claims 1 to 5, having a protrusion protruding downward from the mesh.
[Appendix 7]
The protrusion is formed to surround the tissue culture space,
The culture device according to any one of claims 1 to 6, wherein the protrusion has an opening through which culture medium can flow.
[Appendix 8]
A second cylindrical portion extends upward while holding the mesh,
The culture device according to any one of claims 1 to 7, wherein the second cylindrical portion has a hollow portion through which the mesh is accessible.
[Appendix 9]
A method for producing a cultured tissue, comprising culturing the tissue while pressing the tissue into a medium with a mesh.
[Appendix 10]
The method for producing a cultured tissue described in Appendix 9, wherein the mesh has an opening of 10 μm or more and 200 μm or less.
[Appendix 11]
The method for producing cultured tissue described in Appendix 9 or 10, wherein the mesh has a biocompatible coating layer.
[Appendix 12]
The method for producing a cultured tissue according to any one of claims 9 to 11, wherein the cultured tissue has floating properties.
[Appendix 13]
The method for producing a cultured tissue according to any one of Appendices 9 to 12, wherein the cultured tissue is an adipose tissue.
[Appendix 14]
The method for producing a cultured tissue according to any one of claims 9 to 13, wherein the tissue is cultured for 14 days or more.
[Appendix 15]
A cultured tissue obtained by culturing a tissue while the tissue is pressed into a medium using a mesh.
[Appendix 16]
The cultured tissue of claim 15, wherein the tissue is free-floating.
[Appendix 17]
17. The cultured tissue of claim 15 or 16, wherein the tissue is an adipose tissue.
[Appendix 18]
18. The cultured tissue according to any one of claims 15 to 17, obtained by culturing the tissue for 14 days or more.
[Appendix 19]
An evaluation method comprising: applying a reagent to a cultured tissue obtained by culturing the tissue while it is pressed into a medium by a mesh; and obtaining a functional evaluation of the reagent and/or the cultured tissue depending on the response of the cultured tissue to the reagent.
　上述したように、実施形態及び実施例を通じて本発明の内容を開示したが、この開示の一部をなす論述及び図面は、本発明を限定するものであると理解すべきではない。この開示から当業者には様々な代替の実施形態、実施例及び運用技術が明らかとなる。したがって、本発明の技術的範囲は、上述の説明から妥当な特許請求の範囲に係る発明特定事項によってのみ定められるものである。 As described above, the contents of the present invention have been disclosed through embodiments and examples, but the descriptions and drawings that form part of this disclosure should not be understood as limiting the present invention. From this disclosure, various alternative embodiments, examples and operating techniques will become apparent to those skilled in the art. Therefore, the technical scope of the present invention is determined only by the invention-specific matters related to the scope of the claims that are appropriate from the above explanation.
　本出願は２０２３年２月３日に出願された日本国特許出願２０２３－０１５０９３号に基づく優先権を主張するものであり、当該特許出願の全内容がここに参照により援用される。

  This application claims priority based on Japanese Patent Application No. 2023-015093, filed on February 3, 2023, the entire contents of which are incorporated herein by reference.

Claims (20)

  1. 　組織を培地中に押下するためのメッシュを有する培養装置。 A culture device with a mesh for pressing tissue into the medium.
  2. 　前記メッシュは、１０μｍ以上、かつ２００μｍ以下の目開きを有する、請求項１に記載の培養装置。 The culture device according to claim 1, wherein the mesh has an opening size of 10 μm or more and 200 μm or less.
  3. 　前記メッシュは、生体適合性を有するコーティング層を有する、請求項１に記載の培養装置。 The culture device of claim 1, wherein the mesh has a biocompatible coating layer.
  4. 　前記組織は浮遊性を有する、請求項１に記載の培養装置。 The culture device of claim 1, wherein the tissue is buoyant.
  5. 　前記組織は、脂肪組織又は脳組織である、請求項１に記載の培養装置。 The culture device according to claim 1, wherein the tissue is adipose tissue or brain tissue.
  6. 　前記メッシュから下方に突出した突出部を有する、請求項１に記載の培養装置。 The culture device of claim 1, having a protrusion protruding downward from the mesh.
  7. 　前記突出部は、前記組織の培養空間を囲むよう形成されており、
    　前記突出部は、培地の流入が可能な開口を有する、請求項１に記載の培養装置。     The protrusion is formed to surround the tissue culture space,
    The culture device according to claim 1 , wherein the protrusion has an opening through which the culture medium can flow.
  8. 　前記メッシュを保持しつつ、上方に延びた第２筒状部を有し、
    　前記第２筒状部は、前記メッシュにアクセス可能な中空部を有する、請求項１に記載の培養装置。     A second cylindrical portion extends upward while holding the mesh,
    The culture device according to claim 1 , wherein the second cylindrical portion has a hollow portion through which the mesh can be accessed.
  9. 　メッシュにより培地中に組織を押下させた状態で組織を培養することを含む、培養組織の製造方法。 A method for producing cultured tissue, comprising culturing the tissue while pressing the tissue into a medium using a mesh.
  10. 　前記メッシュは、１０μｍ以上、かつ２００μｍ以下の目開きを有する、請求項９に記載の培養組織の製造方法。 The method for producing cultured tissue according to claim 9, wherein the mesh has an opening size of 10 μm or more and 200 μm or less.
  11. 　前記メッシュは、生体適合性を有するコーティング層を有する、請求項９に記載の培養組織の製造方法。 The method for producing cultured tissue according to claim 9, wherein the mesh has a coating layer that is biocompatible.
  12. 　前記培養組織は浮遊性を有する、請求項９に記載の培養組織の製造方法。 The method for producing a cultured tissue according to claim 9, wherein the cultured tissue has floating properties.
  13. 　前記培養組織は、脂肪組織である、請求項９に記載の培養組織の製造方法。 The method for producing cultured tissue according to claim 9, wherein the cultured tissue is adipose tissue.
  14. 　前記組織を、１４日以上培養する、請求項９に記載の培養組織の製造方法。 The method for producing a cultured tissue according to claim 9, wherein the tissue is cultured for 14 days or more.
  15. 　メッシュにより培地中に組織を押下させた状態で前記組織を培養することによって得られた、培養組織。 A cultured tissue obtained by culturing the tissue while it is pressed into the medium using a mesh.
  16. 　前記組織は浮遊性を有する、請求項１５に記載の培養組織。 The cultured tissue of claim 15, wherein the tissue is buoyant.
  17. 　前記組織は、脂肪組織である、請求項１５に記載の培養組織。 The cultured tissue of claim 15, wherein the tissue is adipose tissue.
  18. 　前記組織を、１４日以上培養することによって得られた、請求項１５に記載の培養組織。 The cultured tissue according to claim 15, obtained by culturing the tissue for 14 days or more.
  19. 　メッシュにより培地中に組織を押下させた状態で培養することによって得られた培養組織に試薬を与え、前記試薬に対する前記培養組織の応答に応じて、前記試薬、及び／又は前記培養組織に関する機能評価を得ること、を含む、評価方法。 An evaluation method comprising: providing a reagent to a cultured tissue obtained by culturing the tissue in a medium while the tissue is pressed down by a mesh; and obtaining a functional evaluation of the reagent and/or the cultured tissue depending on the response of the cultured tissue to the reagent.
  20. 　動物ないし人体から採取した１ｍｇ～１０００ｍｇのサイズを有する脂肪組織を、１０μｍ以上かつ２００μｍ以下の目開きを有するメッシュにより培地中に押下させた状態で、脂肪組織の形態を維持しながら、１４日以上かつ１２０日以下の期間、培養することを含む、培養組織の製造方法。

          A method for producing a cultured tissue, comprising: pressing adipose tissue having a size of 1 mg to 1000 mg collected from an animal or human body into a culture medium using a mesh having an opening size of 10 μm or more and 200 μm or less, and culturing the tissue for a period of 14 days or more and 120 days or less while maintaining the morphology of the adipose tissue.
PCT/JP2024/003437 2023-02-03 2024-02-02 Culture device, culture tissue, production method for culture tissue, and evaluation method WO2024162464A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003180335A (en) * 2001-12-21 2003-07-02 Sumitomo Bakelite Co Ltd Storable culture vessel
JP2005087029A (en) * 2003-09-12 2005-04-07 Japan Tissue Engineering:Kk Culture apparatus and culture kit
WO2008059945A1 (en) * 2006-11-17 2008-05-22 Japan Tissue Engineering Co., Ltd. Tissue piece pinching device and culture kit
JP2015073520A (en) * 2013-10-11 2015-04-20 Ａｇｃテクノグラス株式会社 Cell cultivation container

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003180335A (en) * 2001-12-21 2003-07-02 Sumitomo Bakelite Co Ltd Storable culture vessel
JP2005087029A (en) * 2003-09-12 2005-04-07 Japan Tissue Engineering:Kk Culture apparatus and culture kit
WO2008059945A1 (en) * 2006-11-17 2008-05-22 Japan Tissue Engineering Co., Ltd. Tissue piece pinching device and culture kit
JP2015073520A (en) * 2013-10-11 2015-04-20 Ａｇｃテクノグラス株式会社 Cell cultivation container

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