Cell culture body production method, cell culture member production method, cell culture body, and cell culture member

Abstract

A cell culture body production method for producing a cell culture body used for culturing cells, the method comprising a decellularization step of obtaining an extracellular matrix secreted from human-derived cells by decellularizing a culture substrate obtained by culturing the human-derived cells.

Description

Method for producing cell culture medium, method for producing cell culture member, cell culture medium, and cell culture member

[0001] The present disclosure relates to a method for producing a cell culture construct, a method for producing a cell culture component, a cell culture construct, and a cell culture component.

[0002] Conventionally, techniques for culturing cells in a highly biocompatible environment close to that of a living body have been known. For example, Patent Literature 1 discloses a method for decellularizing various animal-derived organs (e.g., pig jugular veins) by contacting them with a treatment solution containing a surfactant, thereby obtaining the extracted interstitial structure as a cell culture medium (cell culture substrate). When cells such as vascular endothelial cells, smooth muscle cells, and stem cells, e.g., vascular smooth muscle cells, are seeded and cultured in this cell culture medium, cells with a shape similar to the long spindle-shaped smooth muscle cells found in living organisms can be grown, making them suitable for transplantation and regenerative medicine.

[0003] Patent Literature 2 discloses a method for producing a cell-support complex, which includes the steps of coating at least a portion of a substrate made of an artificial material with a cell culture medium (a basement membrane matrix mixture), seeding cultured cells onto the cell culture medium adhered to the substrate, and culturing the cultured cells to form a monolayer structure of cultured cells. It is disclosed that the cell culture medium is made of Matrigel (registered trademark), a mixture of extracellular matrix proteins extracted from mouse sarcoma.

[0004] JP 2011-135836 A JP 2017-86479 A

[0005] For example, in drug discovery research, tissue engineering, cell transplantation, etc., when conducting research and development using cells in human fields or using existing technologies, it is desirable to efficiently perform cell culture, testing, etc. by improving the accuracy of test results and reproducibility. If this could be achieved, it would also lead to a reduction in the use of animals in the use and production of cell cultures. Therefore, there is a need to produce cell cultures that more closely mimic the human biological environment and are more suitable for cell culture than the cell cultures obtained from non-human animals disclosed in Patent Documents 1 and 2.

[0006] Therefore, an object of the present disclosure is to provide a method for producing a cell culture medium, a cell culture medium, and a cell culture medium that more closely mimic the human biological environment, as well as a method for producing a cell culture medium, a cell culture medium, and a cell culture medium that are suitable for culturing cells.

[0007] The method for producing a cell culture construct according to the present disclosure is a method for producing a cell culture construct used for culturing cells, and includes a decellularization step of decellularizing a culture substrate obtained by culturing human-derived cells to obtain an extracellular matrix secreted from the human-derived cells.

[0008] Furthermore, the method for producing a cell culture component according to the present disclosure comprises culturing the cell culture medium to a density of 10 to 1000 μg / cm ２ and applying the resultant to the surface of the base to produce a cell culture element.

[0009] Furthermore, the cell culture medium according to the present disclosure is a cell culture medium used for culturing cells, and contains an extracellular matrix secreted from the human-derived cells by decellularizing a culture substrate obtained by culturing human-derived cells.

[0010] Furthermore, a cell culture component according to the present disclosure includes the cell culture medium and a base to which the cell culture medium is attached.

[0011] The present disclosure makes it possible to provide a method for producing a cell culture medium, a method for producing a cell culture component, a cell culture medium, and a cell culture component that more closely mimic the human biological environment. It also makes it possible to provide a method for producing a cell culture medium, a method for producing a cell culture component, a cell culture medium, and a cell culture component that are suitable for culturing cells.

[0012] Graph showing DNA residual rate for Examples 1-1 to 1-8 and Comparative Example 1-1. Graph showing DNA residual rate for Examples 2-1 to 2-8 and Comparative Example 2-1. Graph showing cell migration rate (horizontal direction) for Examples 4-1 to 4-6 and Comparative Examples 4-1 and 4-2. Graph showing cell growth rate for Examples 4-1 to 4-6 and Comparative Examples 4-1 and 4-2. Graph showing cell adhesion rate for Examples 4-1 to 4-6 and Comparative Examples 4-1 and 4-2. Graph showing cell perimeter for Examples 4-1 to 4-6 and Comparative Examples 4-1 and 4-2. Graph showing cell growth rate for Examples 5-1 to 5-6 and Comparative Examples 4-1 and 4-2. Graph showing cell migration rate (vertical direction) for Examples 5-1 to 5-6 and Comparative Examples 4-1 and 4-2.

[0013] <Embodiments> Embodiments of the present disclosure will be described in detail. Note that the following description is intended to embody the technical idea of ​​the present disclosure, and unless otherwise specified, the present disclosure is not limited to the following.

[0014] (Cell Culture) The cell culture of this embodiment is a cell culture used for culturing cells, and contains an extracellular matrix secreted from the human-derived cells by decellularizing a culture substrate obtained by culturing one or more types of human-derived cells. When two or more types of human-derived cells are used, the cell culture may contain a first extracellular matrix obtained by decellularizing the first human-derived cells (first human-derived cells) and a second extracellular matrix obtained by decellularizing the second human-derived cells (second human-derived cells). The cell culture may be liquid or solid, or may be a dispersion system such as a sol or gel. The cell culture may be a culture solution or medium for culturing cells. The cell culture may also be called dECM (Decellularized ECM).

[0015] The cell culture contains components that constitute the interstitium, basement membrane, etc., and are primarily present outside the cells (extracellular matrix). These components include proteins such as laminin, collagen IV, entactin (night gene), and heparan sulfate proteoglycan (perlecan), as well as growth factors. Growth factors include VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, and HGF. Other components contained in the cell culture include fibronectin, fibrillin, elastin, other collagens (e.g., collagen I, collagen III, etc.), glycosaminoglycans, matrix, reticular fibers, and thrombospondin.

[0016] (Human-derived cells) Human-derived cells are not particularly limited as long as they are derived from humans, but may be, for example, one or more of stem cells, progenitor cells, mature cells, or cancer cells derived from these. Examples of pluripotent cells include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, skin stem cells, epidermal keratinocyte stem cells, retinal stem cells, retinal epithelial stem cells, chondrocyte stem cells, amniotic cells, umbilical cord blood cells, bone marrow-derived cells, adipose-derived stem cells, ectodermal stem cells, mesodermal stem cells, and endodermal stem cells. Examples of progenitor cells and mature cells include one or more of ectodermal cells, mesodermal cells, and endodermal cells. Examples of ectodermal cells include sensory organ cells (cells of the retina and inner ear), nervous system cells (neurons (e.g., forebrain neurons, midbrain neurons, cerebellar neurons, hindbrain neurons, spinal cord neurons, etc.), neural tube cells, neural crest cells), epidermal cells (epidermal cells, lens epithelial cells), etc. Examples of mesodermal cells include blood and lymphocyte cells (hematopoietic stem cells, erythrocytes, platelets, macrophages, granulocytes, helper T cells, killer T cells, B lymphocytes, etc.), vascular cells (vascular endothelial cells, etc.), cardiomyocytes (e.g., atrial myocytes, ventricular myocytes, etc.), osteoblasts, osteocytes, chondrocytes, tendon cells, adipocytes, skeletal muscle cells, smooth muscle cells, etc. Endodermal cells include digestive system cells (hepatocytes, hepatic sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, pit cells, bile duct cells, mesothelial cells, pancreatic endocrine cells, acinar cells, duct cells, absorptive cells, goblet cells, Paneth cells, enteroendocrine cells, etc.), and cells of tissues such as the lung and thyroid gland. Furthermore, human-derived cells can be one or more of epithelial cells, which are cells derived from epithelial tissue, and mesenchymal stem cells. Examples of epithelial cells include digestive system cells from the oral cavity, lower pharynx, esophagus, stomach, duodenum, colon, and rectum; respiratory system cells from the nasal cavity, nasal pharynx, trachea, bronchi, and alveoli; urinary system cells from the ureter, bladder, and urethra; reproductive system cells from the uterus and fallopian tubes; circulatory system cells from blood vessels, lymphatic vessels, and endocardium; and skin cells from the epidermis.Mesenchymal stem cells can be derived from a variety of sources, including stem cells derived from tissues such as skin, fat, bone marrow, umbilical cord, and umbilical cord blood. As human-derived cells, from the viewpoint of mimicking the human biological environment and creating a cell culture suitable for cell cultivation, it is preferable to use one or more types of ectodermal cells and stem cells. It is more preferable to use one or more types of epithelial cells, endothelial cells, mesothelial cells, neurons, adipocytes, and somatic stem cells. It is even more preferable to use one or more types of epithelial cells and mesenchymal stem cells. Human colon carcinoma-derived cells (Caco-2) are preferably used as epithelial cells. Human adipose tissue-derived stem cells (hADSCs) are preferably used as mesenchymal stem cells. When two types of human-derived cells, epithelial cells and mesenchymal stem cells, are used, the epithelial cells (human colon carcinoma-derived cells) are referred to as the first human-derived cells, and the mesenchymal stem cells (human adipose tissue-derived stem cells) are referred to as the second human-derived cells.

[0017] (Cell Culture Component) The cell culture component comprises a base (sometimes called a substrate) and a cell culture medium applied (attached) to the surface of the base. The material for the base is not particularly limited, and examples thereof include glass, resins (polystyrene, polyethylene, polypropylene, poly(acrylic acid ester), poly(methacrylic acid ester), polyacrylamide, polyacrylonitrile, polyethylene terephthalate, polycarbonate, polysulfone, polyethersulfone, poly(L-lactic acid), poly(glycolic acid), poly(ε-caprolactone), poly(ethylene glycol), and copolymers thereof), polypeptides (for example, collagen, gelatin, casein, fibroin, keratin, laminin, integrin, fibronectin, vitronectin), polysaccharides (cellulose, hyaluronic acid, chondroitin sulfate, starch, chitin, chitosan, and derivatives thereof), silica, silicon, and metals (gold, silver, copper, iron, zinc, aluminum, nickel, and alloys or oxides thereof), and a combination of two or more selected from the group consisting of these may be used. The shape of the base is not particularly limited as long as it is a shape that allows cells to be cultured, and examples include a cell culture plate, a cell culture dish (petri dish), a cell culture flask, a cell culture bag, etc. In addition, a porous body may be used as the base, and for example, a filter (porous membrane) such as a membrane filter or a nonwoven fabric may be used. Note that bases that are not porous, such as cell culture dishes (petri dishes), are sometimes called non-porous bodies.

[0018] When the base is a filter (porous membrane) such as a membrane filter, its thickness, average pore diameter, etc. can be changed as appropriate. The average pore diameter of the base is not particularly limited, but may be, for example, 0.1 μm or more and 30 μm or less, or 3 μm to 10 μm. The water contact angle of the base is not particularly limited, but may be, for example, 50° or more and 95° or less. Note that, as the base, a member called a cell culture insert, in which the bottom of a concave container is a membrane filter (porous membrane), may be used.

[0019] The cell culture medium and cell culture device can be used to culture cells of human origin, for example, in drug discovery research, organ culture, cell transplantation, cancer research, gene therapy, cellular agriculture, and the like.

[0020] (Method for producing a cell culture medium and method for producing a cell culture component) The method for producing a cell culture medium is a method for producing a cell culture medium used for culturing cells, and includes a decellularization step of obtaining extracellular matrix secreted from human-derived cells by decellularizing a culture substrate obtained by culturing human-derived cells.

[0021] The decellularization method is not particularly limited. The decellularization process may include at least one of a surfactant treatment process (chemical treatment) using a surfactant, an enzyme treatment process (biological treatment) using an enzyme, or a freeze-thaw treatment process (physical treatment) performing freezing and thawing. These processes may include adding or removing a solvent or buffer solution.

[0022] In the surfactant treatment step, decellularization may be performed using a surfactant with a concentration of 0.06 to 10% by mass (0.06% by mass or more and 10% by mass or less). The type of surfactant is not particularly limited, but may include at least one surfactant selected from the group consisting of Triton X ("Triton" is a registered trademark), Lubrol PX ("Lubrol" is a registered trademark), deoxycholic acid, cholic acid, Tween (registered trademark), and Emulgen. The surfactant concentration may be 0.08% by mass or more and 5% by mass or less, 0.1% by mass or more and 1% by mass or less, or 0.6% by mass or less.

[0023] In the enzyme treatment step, decellularization may be performed using 10 to 500 U (10 U or more and 500 U or less) of enzyme. The type of enzyme is not particularly limited, but for example, nucleases such as DNase and RNase can be used. The amount of enzyme (enzyme unit: unit) may be 50 U or more and 400 U or less, or 100 U or more and 300 U or less.

[0024] The method for producing a cell culture construct may also include a mixing step of mixing a first extracellular matrix obtained by decellularizing first human-derived cells selected from the above-mentioned exemplified examples of human-derived cells with a second extracellular matrix obtained by decellularizing second human-derived cells selected from the above-mentioned exemplified examples of human-derived cells but different from the first human-derived cells. This mixing step may further include mixing three or more types of human-derived cells, such as a third extracellular matrix obtained by decellularizing third human-derived cells selected from the above-mentioned exemplified examples of human-derived cells but different from the first and second human-derived cells. In the mixing step, epithelial cells (human colon cancer-derived cells) may be used as the first human-derived cells, and mesenchymal stem cells (human adipose tissue-derived stem cells) may be used as the second human-derived cells. In this mixing step, the first extracellular matrix (extracellular matrix obtained by decellularizing epithelial cells (human colon cancer-derived cells)) and the second extracellular matrix (extracellular matrix obtained by decellularizing mesenchymal stem cells (human adipose tissue-derived stem cells)) may be mixed so that their mass ratio (protein mass ratio) falls within a predetermined range.

[0025] This mass ratio can be varied depending on the material and shape of the base. For example, when a non-porous substrate (e.g., a polystyrene Petri dish) is used as the base on which the cell culture is applied, the first extracellular matrix and the second extracellular matrix may be mixed at a mass ratio ranging from 2:8 to 10:0. In this case, from the viewpoint of mimicking the human biological environment and producing a cell culture suitable for cell culture, the mass ratio is preferably in the range of 2:8 to 9:1, more preferably 2:8 to 8:2, even more preferably 2:8 to 7:3, even more preferably 3:7 to 7:3, particularly preferably 4:6 to 7:3, even more preferably 5:5 to 7:3, even more preferably 5.5:4.5 to 6.5:3.5, and even more preferably 6:4.

[0026] On the other hand, when a porous body (e.g., a membrane filter) is used as the base on which the cell culture medium is applied, the mass ratio at which the first extracellular matrix and the second extracellular matrix are mixed is preferably in the range of 0:10 to 5.5:4.5, more preferably in the range of 0.5:9.5 to 5.5:4.5, even more preferably in the range of 1:9 to 5:5, still more preferably in the range of 1.5:8.5 to 4.5:5.5, and particularly preferably in the range of 2:8 to 4:6, from the viewpoint of simulating the human biological environment and producing a cell culture medium suitable for cell culture.

[0027] The method for producing the cell culture device comprises culturing the above-mentioned cell culture mass to a density of 10 to 1000 μg / cm ２ (10 μg / cm ２ More than 1000μg / cm ２ The cell culture element is manufactured by applying the coating to the surface of the base so that the density is 20 μg / cm ２ More than 500μg / cm ２ It may be less than 50 μg / cm ２ More than 200μg / cm ２ It may be less than 80 μg / cm ２ 150 μg / cm or more ２ The following is also acceptable.

[0028] In relation to the above-described embodiment, the present disclosure further discloses the following techniques.

[0029] <1> A method for producing a cell culture construct used for culturing cells, comprising a decellularization step of decellularizing a culture substrate obtained by culturing human-derived cells to obtain an extracellular matrix secreted by the human-derived cells.

[0030] <2> The method for producing a cell culture construct according to <1>, wherein the decellularization step is performed using a surfactant having a concentration of 0.06 to 10% by mass.

[0031] <3> The method for producing a cell culture construct according to <1> or <2>, wherein the decellularization step uses 10 to 500 U of an enzyme to perform the decellularization.

[0032] <4> The method for producing a cell culture construct according to any one of <1> to <3>, comprising a mixing step of mixing a first extracellular matrix obtained by decellularizing a first human-derived cell with a second extracellular matrix obtained by decellularizing a second human-derived cell different from the first human-derived cell.

[0033] <5> The method for producing a cell culture construct according to claim <4>, wherein in the mixing step, epithelial cells are used as the first human-derived cells and mesenchymal stem cells are used as the second human-derived cells.

[0034] <6> The method for producing a cell culture construct according to <4> or <5>, wherein, when the cell culture construct is subjected to a non-porous body, in the mixing step, the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells are mixed at a mass ratio in the range of 2:8 to 10:0.

[0035] <7> The method for producing a cell culture construct according to <4> or <5>, wherein, when the cell culture construct is subjected to a porous body, in the mixing step, the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells are mixed at a mass ratio in the range of 0:10 to 5.5:4.5.

[0036] <8> The method for producing a cell culture construct according to any one of <4> to <7>, wherein in the mixing step, human colon cancer-derived cells are used as the epithelial cells and human adipose tissue-derived stem cells are used as the mesenchymal stem cells.

[0037] <9> The cell culture according to any one of <1> to <8>, wherein the cell culture has a density of 10 to 1000 μg / cm ２ The method for producing a cell culture member includes applying the above-mentioned solution to the surface of a base so as to form a cell culture member.

[0038] <10> A cell culture used for culturing cells, the cell culture comprising an extracellular matrix secreted from human-derived cells by decellularizing a culture substrate obtained by culturing human-derived cells.

[0039] <11> A cell culture device comprising the cell culture construct according to <10> and a base to which the cell culture construct is attached.

[0040] <12> The cell culture device according to <11>, wherein the base is a non-porous body.

[0041] <13> The cell culture component according to <12>, wherein the cell culture comprises the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells in a mass ratio ranging from 2:8 to 10:0.

[0042] <14> The cell culture device according to <11>, wherein the base is a porous body.

[0043] <15> The cell culture component according to <14>, wherein the cell culture comprises the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells in a mass ratio ranging from 0:10 to 5.5:4.5.

[0044] Specific examples of the present disclosure will be shown below, but the present disclosure is not limited to these.

[0045] The materials used are summarized below. (Materials) Human-derived cells (first human-derived cells): Caco-2 cells (human colon carcinoma-derived cells), RIKEN BRC (RCB0988) Human-derived cells (second human-derived cells): hADSC (human adipose tissue-derived stem cells), Lonza Co., Ltd. (PT-5006) Medium A: DMEM, manufactured by Gibco, containing the following components: Fetal bovine serum (10% by volume), non-essential amino acids (1% by volume), penicillin-streptomycin (antibiotics: 1% by volume) Medium B: MEMα, manufactured by Gibco, containing the following components: Fetal bovine serum (10% by volume), penicillin-streptomycin (antibiotics: 1% by volume)

[0046] Example 1-1 (Preparation of culture substrate) Caco-2 cells (first human-derived cells) and DMEM (medium A) were seeded in a 100 mm cell culture dish at a seeding density of 0.5 × 10 ６The cells were seeded at 100 cells / dish. Culture was carried out for 7 days under conditions of a temperature of 37°C and a carbon dioxide concentration of 5% by volume to obtain a culture substrate. During the culture, the medium was changed every 2 to 3 days.

[0047] (Decellularization) The medium was removed from the culture substrate, and the cells were decellularized with PBS(-) (Dulbecco's Phosphate Buffered Saline, manufactured by Nacalai Tesque, Inc.: Mg ２＋ , Ca ２＋ The cells were washed with a phosphate buffer solution (containing no phosphate buffer), to which a 0.05% by mass Triton X-100 solution (manufactured by Alfa Aesar) was added as a nonionic surfactant, and the cells were immersed at 37°C for 5 minutes. The Triton X-100 solution was then removed, and the cells were washed once with PBS(-), to give the cell culture construct of Example 1-1.

[0048] <Examples 1-2 and 1-3> Cell culture constructs of Examples 1-2 and 1-3 were obtained in the same manner as in Example 1-1, except that the immersion time of the culture substrate in the Triton X-100 solution in the decellularization step was changed to 10 minutes or 30 minutes.

[0049] Examples 1-4 to 1-6 Cell cultures of Examples 1-4 to 1-6 were obtained in the same manner as in Example 1-1, except that the concentration of the Triton X-100 solution in the decellularization step was changed to 0.1% by mass and the immersion time under that concentration condition was changed to 5 minutes, 10 minutes, or 30 minutes.

[0050] Examples 1-7 to 1-9 Cell cultures of Examples 1-7 to 1-9 were obtained in the same manner as in Example 1-1, except that the concentration of the Triton X-100 solution in the decellularization step was changed to 0.5% by mass and the immersion time under that concentration condition was changed to 5 minutes, 10 minutes, or 30 minutes.

[0051] (Evaluation of DNA Residual Rate) If DNA from human-derived cells remains in the cell culture, there is concern that it may affect the cultured cells. Therefore, for the above-mentioned Examples and Comparative Examples, the DNA residual rate (%) was evaluated by measuring the DNA amount before and after decellularization. The DNA amounts before and after decellularization were each calculated as the average of multiple samples (three samples). The DNA amount before decellularization was obtained by measuring the cell substrate with a plate reader (SpectraMax iD3, Molecular Devices Japan, Inc., ID3-STD). The DNA amount after decellularization was obtained by measuring the cell culture suspension with a plate reader. The DNA residual rate (%) was calculated using the following formula: DNA residual rate (%) = (DNA amount after decellularization / DNA amount before decellularization) × 100. The results are shown in Table 1 and Figure 1.

[0052]

[0053] In Example 1-1 and Examples 1-3 to 1-9, the DNA residual rate can be reduced to 60% or less. According to Examples 1-4 to 1-9, if the concentration of the Triton X-100 solution is 0.1% by mass or more, the DNA residual rate can be reduced to 25% or less for any immersion time. According to Examples 1-7 and 1-8, if the concentration of the Triton X-100 solution is 0.5% by mass and the immersion time is 10 minutes or less, the DNA residual rate can be reduced to 10% or less.

[0054] Example 2-1: The culture medium was removed from a culture substrate prepared using the same procedure as in Example 1-1, and the substrate was washed with PBS(-). A 0.5% by mass Triton X-100 solution was added as a nonionic surfactant, and the substrate was immersed at 37°C for 10 minutes. The Triton X-100 solution was then removed, the substrate was washed once with PBS(-), and the substrate was again immersed in PBS(-). This was left at -80°C for 30 minutes, and then a freeze-thaw process of leaving the substrate at 37°C for 30 minutes was repeated three times (3 cycles). The PBS(-) was removed from the culture substrate after the freeze-thaw process, and the substrate was washed once with fresh PBS(-). 50 U of DNase I solution (manufactured by Nippon Gene Co., Ltd.) was added as an enzyme, and the substrate was immersed at 37°C for 90 minutes. The DNase I solution was removed from this culture substrate, and the substrate was washed three times with PBS(-) to obtain the cell culture mass of Example 2-1.

[0055] <Examples 2-2 and 2-3> Cell culture constructs of Examples 2-2 and 2-3 were obtained in the same manner as in Example 2-1, except that the immersion time of the culture substrate in the DNase I solution in the decellularization step was changed to 120 minutes or 180 minutes, respectively.

[0056] Examples 2-4 to 2-6 Cell cultures of Examples 2-4 to 2-6 were obtained in the same manner as in Example 2-1, except that the amount of enzyme in the DNase I solution in the decellularization step was changed to 100 U and the immersion time under that enzyme amount was changed to 90 minutes, 120 minutes, or 180 minutes.

[0057] <Examples 2-7 to 2-9> Cell cultures of Examples 2-7 to 2-9 were obtained in the same manner as in Example 2-1, except that the amount of enzyme in the DNase I solution in the decellularization step was changed to 300 U and the immersion time under that enzyme amount was changed to 90 minutes, 120 minutes, or 180 minutes.

[0058] (Evaluation of DNA Residual Rate) For the above Examples and Comparative Examples, the DNA residual rate (%) was evaluated by measuring the DNA amount before decellularization (before surfactant treatment) and after decellularization (after enzyme treatment). The DNA amount before and after decellularization was each the average value of multiple samples (two samples). The results are shown in Table 2 and Figure 2.

[0059]

[0060] In Example 2-1 and Examples 2-3 to 2-9, the DNA residual rate could be reduced to 10% or less. As shown in Examples 2-7 to 2-9, when the amount of DNase I enzyme was 300 U, the DNA residual rate could be reduced to 6% or less. Looking at each example, it is possible to sufficiently reduce the DNA residual rate even with an immersion time of 90 minutes.

[0061] Example 3-1: The culture medium was removed from a culture substrate prepared using the same procedure as in Example 1-1, and the substrate was washed with PBS(-). A 0.5% by mass Triton X-100 solution was added as a nonionic surfactant, and the substrate was immersed at 37°C for 10 minutes. The Triton X-100 solution was then removed, the substrate was washed once with PBS(-), and the substrate was again immersed in PBS(-). This was left standing at -80°C for 30 minutes, and then a freeze-thaw process of leaving the substrate standing at 37°C for 30 minutes was repeated three times (3 cycles). The PBS(-) was removed from the culture substrate after the freeze-thaw process, and the substrate was washed once with fresh PBS(-). 200 U of DNase I solution was added as an enzyme, and the substrate was immersed at 37°C for 90 minutes. The DNase I solution was removed from this culture substrate, and the substrate was washed three times with PBS(-) to obtain the cell culture mass of Example 3-1.

[0062] Example 3-2: The second human-derived cells, hADSCs, and MEMα of medium B were seeded in a 100 mm cell culture dish at a seeding density of 0.5 × 10 ６The cells were seeded at 100 cells / dish. The cells were cultured for 7 days under conditions of a temperature of 37°C and a carbon dioxide concentration of 5% by volume, yielding a culture substrate. During culture, the medium was changed every 2-3 days. When changing the medium before decellularization (the final medium change in the culture substrate preparation process), a medium containing 100 μM L-ascorbic acid was used. Subsequent decellularization was carried out in the same manner as in Example 3-1, yielding the cell culture construct of Example 3-2.

[0063] (Qualitative Evaluation of Cell Cultures) The cell cultures prepared in Examples 3-1 and 3-2 were each scraped together with a scraper and collected in a tube, and then dispersed using a sonicator under ice cooling to obtain a dispersion (suspension). This suspension was subjected to immunostaining to confirm the presence or absence of specific proteins. As a result, it was confirmed that collagen IV was present in the cell culture of Example 3-1. It was confirmed that laminin α5 was present in the cell culture of Example 3-2.

[0064] Examples 4-1 to 4-6: A cell culture suspension prepared in Example 3-1 (a first cell culture comprising a first extracellular matrix obtained by decellularizing Caco-2 cells, a first human-derived cell) and a cell culture suspension prepared in Example 3-2 (a second cell culture comprising a second extracellular matrix obtained by decellularizing hADSCs, a second human-derived cell) were prepared. The protein content (μg / ml) of each cell culture suspension was measured using a plate reader (SpectraMax iD3, ID3-STD, manufactured by Molecular Devices Japan, Inc.). The first extracellular matrix suspension and the second extracellular matrix suspension were mixed to obtain protein ratios of 0:10 (Example 4-1), 2:8 (Example 4-2), 4:6 (Example 4-3), 6:4 (Example 4-4), 8:2 (Example 4-5), and 10:0 (Example 4-6), respectively, to prepare multiple mixed suspensions (mixing step).

[0065] The mixed suspensions were each mixed in a solution of 100 μg / cm ２The coated body was prepared by applying the solution to the surface of a polystyrene petri dish serving as a base so that the solution was uniform. The coated body was left to stand in an incubator for 1 hour, and excess liquid was removed to prepare the cell culture devices of Examples 4-1 to 4-6. These cell culture devices include the petri dish and the coated body (cell culture body) applied to the petri dish.

[0066] <Comparative Examples 4-1 and 4-2> Matrigel (manufactured by Corning, 8 mg / mL) was used to prepare a matrix with a protein density of 100 μg / cm ２ The coated body was left to stand in an incubator for 1 hour, and excess liquid was removed to obtain a cell culture component of Comparative Example 4-1. The polystyrene Petri dish itself (a Petri dish with no coating on its surface) was used as Comparative Example 4-2.

[0067] (Evaluation of Cell Migration (Lateral)) In Examples 4-1 to 4-6 and Comparative Example 4-1, a glass tube was placed on the upper surface of the cell culture component (a cell culture cultured on the upper surface of a Petri dish), and in Comparative Example 4-2, a glass tube was placed directly on the upper surface of the Petri dish. A549 cells (human alveolar basal epithelial adenocarcinoma cells) were seeded on the inside and outside of the portion of each upper surface where the glass tube abutted. The cells seeded on the inside and outside were referred to as the inner cells and the outer cells, respectively. After confirming that each cell had adhered to each upper surface, the glass tube was removed. Photographs were taken immediately after removal of the glass tube (after 0 hours), after 36 hours, and after 72 hours, and the area of ​​the region between the inner cells and the outer cells (intercellular area) was measured. The intercellular area was calculated as the average value of multiple samples (three samples). The cell migration rate was calculated by subtracting the intercellular area at each elapsed time from the intercellular area immediately after removal of the glass tube (after 0 hours), and dividing the result by the intercellular area immediately after removal of the glass tube, as shown in the following formula: Cell migration rate (horizontal direction) = (intercellular area immediately after removal of the glass tube - intercellular area at each elapsed time) / intercellular area immediately after removal of the glass tube.

[0068] (Evaluation of Cell Growth Rate, Cell Adhesion, and Cell Perimeter) In Examples 4-1 to 4-6 and Comparative Example 4-1, A549 cells were seeded on the upper surface of the cell culture component (cell cultures cultured on the upper surface of a Petri dish), and in Comparative Example 4-2, A549 cells were seeded directly on the upper surface of the Petri dish. These were cultured for 3 hours and 24 hours under conditions of a temperature of 37°C and a carbon dioxide concentration of 5% by volume, allowing the A549 cells to adhere to the upper surface. For the 3-hour and 24-hour cultures, the medium was removed and the non-adherent cells were washed three times with PBS(-). The number of cells adhered to each upper surface (adherent cell count) was then measured. Images were taken with an inverted microscope and analyzed using analysis software (cellSens). The number of adherent cells was calculated as the average of multiple samples (five samples). Based on this adherent cell count, the cell growth rate and cell adhesion were calculated using the following formula: Cell increase rate = number of adherent cells after 24 hours of culture / number of adherent cells after 3 hours of culture. Cell adhesion degree = number of adherent cells after 3 hours of culture in each Example etc. / number of adherent cells after 3 hours of culture in Comparative Example 4-2. Furthermore, for the samples cultured for 3 hours (after removal of the medium and washing with PBS(-)), the cells adhered to the upper surface of each sample were photographed with an inverted microscope, and the cell perimeter was measured using analysis software (cellSens).

[0069] The results are shown in Table 3 and Figures 3 to 6. In Table 3, MX means matrix, and the column "1st MX: 2nd MX" indicates the protein amount ratio between the first extracellular matrix and the second extracellular matrix. In the graphs of each figure, the parentheses in the legend indicate the protein amount ratio between the first extracellular matrix and the second extracellular matrix, and the error bars indicate the standard deviation.

[0070]

[0071] The cell migration rate is higher in all Examples than in the Comparative Examples. In particular, the cell migration rate after 36 hours is higher in each Example than in the Comparative Examples. The cell growth rate is higher in each Example than in Comparative Example 4-2, and in Examples 4-2 to 4-6, the values ​​are equal to or higher than that of Comparative Example 4-1. The cell adhesion rate is higher in each Example than in Comparative Example 4-2. The cell perimeter is higher in each Example than in Comparative Example 4-2, and the values ​​are equal to or higher than that of Comparative Example 4-1.

[0072] Examples 5-1 to 5-6 A plurality of mixed suspensions prepared in the same proportions as in Examples 4-1 to 4-6 were each mixed to a concentration of 100 μg / cm ２ The coating was applied to the inner bottom surface (upper surface of the membrane filter) of a cell culture insert serving as a base so that the coating was formed. This cell culture insert is a concave container as a whole, and the bottom of the container is a membrane filter (porous membrane). The membrane filter constituting the bottom has a pore diameter (average pore diameter) of 8 μm and a pore density of 6±2×10 ４ pieces / cm ２ The material of this membrane filter was polyethylene terephthalate. Except for this, the cell culture devices of Examples 5-1 to 5-6 were prepared in the same manner as in Examples 4-1 to 4-6, respectively. These cell culture devices included an insert and a coating (cell culture) coated on the membrane filter of the insert.

[0073] Comparative Examples 5-1 to 5-2: Matrigel (manufactured by Corning, 8 mg / mL) was used to prepare a matrix with a protein density of 100 μg / cm ２ The solution was applied to the inner bottom surface (upper surface of the membrane filter) of the cell culture insert so that the solution was applied to the inner bottom surface of the cell culture insert (upper surface of the membrane filter) to obtain a coated body. The insert was left to stand in an incubator for 1 hour, and excess solution was removed to obtain the cell culture member of Comparative Example 5-1. The cell culture insert itself (an insert with nothing applied to the upper surface of the membrane filter) was used as Comparative Example 5-2.

[0074] (Evaluation of Cell Proliferation Rate) In Examples 5-1 to 5-6 and Comparative Example 5-1, A549 cells were seeded on the upper surface of the cell culture component (cell culture mass coated on the upper surface of the membrane filter of the insert), and in Comparative Example 5-2, A549 cells were seeded on the upper surface of the membrane filter of the insert. These were cultured for 3 hours and 24 hours under conditions of a temperature of 37°C and a carbon dioxide concentration of 5% by volume, and A549 cells were allowed to adhere to each upper surface. For the 3-hour and 24-hour cultures, the medium was removed and the cells were washed three times with PBS(-) to remove non-adherent cells that did not adhere to the upper surface. The number of cells adhered to each upper surface (adherent cell count) was then measured. The number of adherent cells was measured by staining cell nuclei and using analysis software (ImageJ). The number of adherent cells was calculated as the average value of multiple samples (5 samples). Based on this number of adherent cells, the cell growth rate and cell adhesion degree were calculated using the following formula: Cell growth rate = Number of adherent cells after 24 hours of culture / Number of adherent cells after 3 hours of culture.

[0075] (Evaluation of Cell Migration (Vertical)) In Examples 5-1 to 5-6 and Comparative Example 5-1, A549 cells were seeded on the upper surface of the cell culture component (cell cultures cultured on the upper surface of the membrane filter of the insert), and in Comparative Example 5-2, A549 cells were seeded on the upper surface of the membrane filter of the insert. These inserts were immersed in a container containing serum medium (Gibco, product name MEMα, 10% by volume of fetal bovine serum, penicillin-streptomycin (antibiotics: 1% by volume)), and serum-free medium (Gibco, product name MEMα) was poured into the insert. That is, the lower side of the membrane filter in the insert was immersed in serum medium, and the upper side of the membrane filter was immersed in serum-free medium, creating a nutrient concentration gradient. This allowed the degree of migration of A549 cells through the membrane filter downward (to a container containing serum medium) to be evaluated. 24 hours after seeding the A549 cells, the number of A549 cells that had migrated downward through the membrane filter (number of migrating cells) was counted by staining the cell nuclei and using analysis software (ImageJ). The number of migrating cells in each Example etc. was divided by the number of migrating cells in Comparative Example 5-2, as shown in the following formula, to obtain the degree of cell migration (vertical direction). Cell migration degree (vertical direction) = number of migrating cells in each Example etc. / number of migrating cells in Comparative Example 5-2

[0076] The results are shown in Table 4 and Figures 7 and 8. In Table 4, MX stands for matrix, and the column "1st MX: 2nd MX" indicates the protein amount ratio between the first extracellular matrix and the second extracellular matrix. In the graphs of each figure, the parentheses in the legend indicate the protein amount ratio between the first extracellular matrix and the second extracellular matrix, and the error bars indicate the standard deviation.

[0077]

[0078] As a result, it has become possible to produce cell cultures that more closely mimic the human biological environment and cell cultures that are suitable for cell cultivation.

Claims

1. A method for producing a cell culture construct used for culturing cells, comprising a decellularization step of decellularizing a culture substrate obtained by culturing human-derived cells, thereby obtaining an extracellular matrix secreted by the human-derived cells.

2. The method for producing a cell culture construct according to claim 1, wherein the decellularization step is performed using a surfactant at a concentration of 0.06 to 10% by mass.

3. The method for producing a cell culture construct according to claim 1 or 2, wherein the decellularization step uses 10 to 500 U of an enzyme to perform the decellularization.

4. A method for producing a cell culture construct according to claim 1 or 2, comprising a mixing step of mixing a first extracellular matrix obtained by decellularizing a first human-derived cell with a second extracellular matrix obtained by decellularizing a second human-derived cell different from the first human-derived cell.

5. The method for producing a cell culture construct according to claim 4, wherein in the mixing step, epithelial cells are used as the first human-derived cells and mesenchymal stem cells are used as the second human-derived cells.

6. The method for producing a cell culture construct according to claim 5, wherein, when the cell culture construct is applied to a non-porous body, in the mixing step, the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells are mixed at a mass ratio in the range of 2:8 to 10:

0.

7. The method for producing a cell culture construct according to claim 5, wherein, when the cell culture construct is applied to a porous body, in the mixing step, the first extracellular matrix obtained from the epithelial cells and the second extracellular matrix obtained from the mesenchymal stem cells are mixed at a mass ratio ranging from 0:10 to 5.5:4.

5.

8. The method for producing a cell culture according to claim 5, wherein in the mixing step, human colon cancer-derived cells are used as the epithelial cells and human adipose tissue-derived stem cells are used as the mesenchymal stem cells.

9. The cell culture according to claim 1 or 2 is grown at a density of 10 to 1000 μg / cm ２ a method for producing a cell culture member by applying the above-mentioned solution to a surface of a base so as to form a cell culture member.

10. A cell culture used for culturing cells, comprising an extracellular matrix secreted by human-derived cells, the cell culture being obtained by decellularizing a culture substrate obtained by culturing human-derived cells.

11. A cell culture device comprising: the cell culture medium according to claim 10; and a base on which the cell culture medium is attached.

12. The cell culture device according to claim 11, wherein the base is a non-porous body.

13. The cell culture device according to claim 12, wherein the cell culture comprises a first extracellular matrix obtained from epithelial cells and a second extracellular matrix obtained from mesenchymal stem cells in a mass ratio ranging from 2:8 to 10:

0.

14. The cell culture device according to claim 11, wherein the base is a porous body.

15. The cell culture device according to claim 14, wherein the cell culture comprises a first extracellular matrix obtained from epithelial cells and a second extracellular matrix obtained from mesenchymal stem cells in a mass ratio ranging from 0:10 to 5.5:4.5.

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