WO2018168955A1 - 細胞のin vivoでの特性を反映した細胞の反応の評価 - Google Patents
細胞のin vivoでの特性を反映した細胞の反応の評価 Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Definitions
- Non-patent Document 1 It is known that cells existing in a living body recognize and respond to the surrounding physical environment (Non-patent Document 1).
- Non-Patent Document 2 describes that mesenchymal stem cells are induced into muscle cells and osteoblasts by culturing them in a gel matrix having the same rigidity as the microenvironment of living tissue.
- Patent Documents 1 and 2 describe that mesenchymal stem cells are induced to a dormant state by culturing them on a gel matrix having the same rigidity as the microenvironment of living tissue.
- a cell changes a state and the direction of differentiation according to the microenvironment of the surrounding tissue which the cell contacts.
- JP 2010-532167 A Special table 2010-532166
- An object of the present invention is to provide an evaluation system capable of expressing in vitro a cell reaction closer to a cell reaction occurring in a living body based on the above-mentioned problem newly found by the inventor. .
- the present inventor has cultivated cells on a gel or gel matrix having the same rigidity as the microenvironment of the living tissue from which the cells are derived, so It was found that a cell reaction closer to the reaction can be expressed in vitro.
- the support In order to express the in vivo characteristics of cells in vitro, the support has a rigidity reflecting the rigidity of the surrounding environment recognized by the cells in vivo, and the rigidity is 100 kPa or less in terms of shear modulus Contacting the cells; maintaining the properties of cells that have been introduced and / or expressed in vitro with the properties of cells in vivo; Inputting a signal into a cell in which said property is introduced and / or maintained; Measuring a cell's response to an input signal; (However, the cells do not include mesenchymal stem cells).
- Item 2. Item 2.
- the method according to Item 1 wherein the signal is input to the cell by loading the cell with at least one factor selected from the group consisting of a chemical factor, a physical factor, and a biological factor.
- the chemical factor is at least one selected from the group consisting of compounds, ions, gases, nucleic acids, carbohydrates, lipids, glycoproteins, glycolipids, lipoproteins, amino acids, peptides, proteins, polyphenols, cytokines, and chemokines.
- Item 3. The method according to Item 1 or 2.
- the method according to Item 1 or 2 wherein the physical factor is at least one selected from the group consisting of rigidity, pressure, tension, light, radiation, oxygen concentration, pH, and temperature of the environment surrounding the cell.
- Item 3 The method according to Item 1 or 2, wherein the biological factor is at least one selected from bacteria, fungi, viruses, allergens, human cells, non-human animal cells, and components contained therein.
- Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the cell is a hepatocyte, and the rigidity of the support is 0.2 to 5 kpa.
- Item 7. Item 7. The method according to Item 6, wherein the cell response indicates induction of drug-metabolizing enzyme, non-alcoholic steatohepatitis mechanism, or idiopathic liver injury.
- Item 8. Item 6. The method according to any one of Items 1 to 5, wherein the cell response is the efficacy or toxicity of the factor.
- Item 9. Item 6.
- the support has a rigidity reflecting the rigidity of the surrounding environment recognized by the cells in vivo, and the rigidity is 100 kPa or less in terms of shear modulus Contacting the cells; maintaining the properties of cells that have been introduced and / or expressed in vitro with the properties of cells in vivo; Inputting a signal into a cell in which said property is introduced and / or maintained; Contacting a cell with a candidate substance for preventing, treating or ameliorating a disease or disorder prior to the input of a signal, simultaneously with the input of the signal or after the input of the signal; Measuring a cell's response to an input signal; A method for screening a candidate substance for preventing, treating or ameliorating a disease or disorder (however, the cells do not include mesenchymal stem cells).
- Item 13 The method according to Item 12, wherein the signal is input to the cell by loading the cell with at least one factor selected from the group consisting of a chemical factor, a physical factor, and a biological factor.
- the chemical factor is at least one selected from the group consisting of compounds, ions, gases, nucleic acids, carbohydrates, lipids, glycoproteins, glycolipids, lipoproteins, amino acids, peptides, proteins, polyphenols, cytokines, and chemokines.
- the physical factor is at least one selected from the group consisting of rigidity, pressure, tension, light, radiation, oxygen concentration, pH, and temperature of the environment surrounding the cell.
- the biological factor is at least one selected from bacteria, fungi, viruses, allergens, human cells, non-human animal cells, and components contained therein.
- Item 17. The method according to any one of Items 12 to 16, wherein the cell is a hepatocyte and the rigidity of the support is 0.2 to 5 kPa.
- Item 17 wherein the cell response indicates induction of drug-metabolizing enzyme, non-alcoholic steatohepatitis mechanism, or idiopathic liver injury.
- Item 19. Item 17. The method according to any one of Items 12 to 16, wherein the cellular response is the efficacy or toxicity of the factor.
- Item 20. Item 17. The method according to any one of Items 12 to 16, wherein the cell is a cardiomyocyte, and the rigidity of the support is 5 to 100 kPa.
- Item 21 Item 21. The method according to Item 20, wherein the chemical factor is an oxidative stress inducer and the cellular response is an oxidative stress response.
- Item 22. Item 21. The method according to Item 20, wherein the oxidative stress inducer is glucose.
- Item 23 In order to express the in vivo characteristics of the cells in vitro, a support having a rigidity reflecting the rigidity of the surrounding environment recognized by the cells in vivo and having a shear elastic modulus of 100 kPa or less is provided.
- Item 24. Item 24. The kit according to Item 23, further comprising a cell suitable for performing an evaluation or screening of a target cell response.
- A is a figure which shows the rigidity of the microenvironment with which the typical cell is contacting in the living body.
- B is a diagram showing the rigidity of normal tissue or abnormal tissue (quoted from data from Soft Matter, 2007, 3, 299-306. And Am J Physiol Gastroint Liver Physiol 293: G1147-G1154, 2007.) It is a figure describing an example of a method for producing a polyacrylamide support (the figure is quoted from Cell, 2006, Vol. 126, pp. 677-689). It is a graph which shows the change of the rigidity of the polyacrylamide gel according to the mixing ratio of acrylamide and bisacrylamide (cited from Cell Motil Cytoskeleton. 2005 Jan; 60 (1): 24-34.).
- FIG. It is a figure showing the mitochondrial membrane potential of the cultured cardiomyocytes at normal glucose concentration and 15 mM glucose concentration. It is a figure showing the mitochondrial membrane potential of the cultured cardiomyocytes at normal glucose concentration and 15 mM glucose concentration.
- A indicates ROS accumulation.
- B shows mitochondrial membrane potential. It is a figure which shows the comparison of the rifampicin reactivity of the primary culture hepatocyte on a support body of 500 Pa, and a three-dimensional culture Spheroid. It is a figure which shows the effect of the support body of 500 Pa in idiopathic drug-induced liver injury. It is the figure which expressed the accumulation
- FIG. 2 is a graph showing the inflammation-inducing action of hepatocytes on macrophages evaluated by the amount of TNF- ⁇ .
- the evaluation method evaluates a cell response to a signal input to the cell in vitro.
- the evaluation method includes a step of bringing a cell into contact with a support having rigidity that reflects the rigidity of the surrounding environment that the cell recognizes in vivo.
- the evaluation method includes a step of using cells that have introduced and / or expressed in vivo cell characteristics in vitro and maintaining the characteristics in vitro.
- the evaluation method includes a step of inputting a signal to a cell in which the characteristic is introduced and / or maintained.
- the evaluation method includes a step of measuring a cell response to an input signal.
- the cell in the evaluation method, by bringing a cell into contact with the support under the above conditions, the cell can express the original characteristic of the cell in vivo or a characteristic close to the characteristic in vivo.
- the cell is not limited as long as it originates from a living organism.
- mammalian cells avian cells, insect cells, amphibian cells, fish cells and the like can be mentioned.
- Mammalian cells are preferably cells derived from humans, monkeys, sheep, goats, cattle, horses, pigs, cats, dogs, rabbits, mice, rats, guinea pigs, and the like.
- the avian cell is a cell derived from a chicken.
- the cell is a cell that can be cultured in vitro.
- the cell may be a cultured cell line.
- the cell may be a cell collected from a living body when culturing a primary cultured cell or the like.
- the cell may be a cell obtained by infecting a primary cultured cell or the like with a virus and acquiring a certain proliferation ability (having a higher proliferation ability or frequency of division than the primary culture cell, preferably immortalization).
- a primary cultured cell refers to any cell that has been subcultured after collection of the cell or tissue, until the culture starts and the proliferation ability ceases.
- primary cultured cells include hepatocytes, muscle cells (including cardiomyocytes, smooth muscle cells, skeletal muscle cells) and liver non-parenchymal cells, neurons, glial cells, vascular endothelial cells, lymphatic endothelial cells, octopus podocytes Mesangial cells, tubule cells, skin epithelial cells, mucosal epithelial cells, corneal epithelial cells, photoreceptors, bipolar cells, horizontal cells, Muller cells, alveolar epithelial cells, bronchial epithelial cells, esophageal epithelial cells, gastrointestinal epithelial cells, Examples thereof include mammary gland cells, duct epithelial cells, pancreatic islet cells, pancreatic duct cells, adipocytes, macrophages, monocyte cells, fibroblasts, and precursor cells thereof, and cells after mutation.
- the cells exclude mesenchymal stem cells. More preferably, stem cells such as somatic stem cells and
- the cells include cells differentiated into specific cells from pluripotent stem cells (including embryonic stem cells, induced pluripotent stem cells, etc.). In addition, cells differentiated into specific cells from tissue stem cells such as mesenchymal stem cells are also included. For example, a method for differentiating embryonic stem cells into mesenchymal stem cells is disclosed in Stem Cell Rev and Rep (2017) 13: 68-78. A method for differentiating mesenchymal stem cells into hepatocytes is disclosed in World J Stem Cells 2011 December 26; 3 (12): 113-121.
- the cells may be normal cells or abnormal cells such as diseased cells.
- collected from the genetically modified animal and the disease model animal may be sufficient.
- the ambient environment that the cell recognizes in vivo is the environment around the target cell in the tissue from which the cell is collected if the cell is a primary cultured cell or the like. If the cell is a cultured cell line, it is the microenvironment of the tissue that would have been placed if the cell was originally in vivo.
- the tissue may be normal or have some abnormality, such as a disease or disorder. For example, when a cell is collected from a lesion, the tissue means a tissue in the lesion.
- the microenvironment is generally used as a term indicating a state of a cell surrounding that controls the behavior of the cell through biophysical or biochemical factors.
- the rigidity of the surrounding environment recognized by the cells in the living body may be the rigidity of the tissue itself, or may be the environment of the micro environment itself in contact with the cells in the tissue.
- ⁇ Rigidity is not limited as long as it represents hardness. Preferably, it is represented by shear modulus (displacement modulus). There are several measuring methods based on various different principles (Sportsmedicine, 2014, No. 166, 1-30). The rigidity can be measured, for example, by the following method.
- the structure or support is regarded as an elastic regular quadrangular column and shear stress S acts on the bottom surface and a plane parallel thereto, the side surface of the regular quadrangular column is deformed into a rhombus with an apex angle of 90 ° ⁇ ⁇ .
- S shear stress
- the dynamic stiffness of the tissue is measured with a strain-controlled rheometrics fluids spectrometer III (Rheometrics, Piscataway, NJ).
- the stiffness (G ′) which is a value representing elastic resistance, is calculated from the shear stress of the same phase at a vibration (1 rad / s) shear strain of 2%.
- G ′ a value representing elastic resistance
- a stainless steel punch for example, a sample having a thickness of 5 to 10 mm and a diameter of 5 to 10 mm is cut out and placed between the plates.
- the short-term G ′ ( ⁇ ) is measured by oscillating at a shear strain of 2%.
- FIG. 1A shows the rigidity of the microenvironment with which typical cells are in contact.
- FIG. 1B shows the rigidity of a typical tissue in a normal state and an abnormal state.
- the liver, blood vessels, and the like become stiff due to fibrosis and calcification.
- the rigidity of the microenvironment can be measured using an atomic force microscope (AFM) described in, for example, Biophysical Journal, Volume 93, December 2007, p4453-4461.
- AFM atomic force microscope
- the support is not limited as long as it can reflect the rigidity of the surrounding environment that cells recognize in vivo.
- a gel and a gel matrix can be mentioned.
- Supports include those that can provide a two-dimensional culture environment and those that can provide a three-dimensional culture environment.
- the gel examples include a gel having a skeleton composed of a gelling agent such as agarose, acrylamide, collagen, fibrin, silicone, glycosaminoglycan, carrageenan, and locust bean gum.
- a gelling agent such as agarose, acrylamide, collagen, fibrin, silicone, glycosaminoglycan, carrageenan, and locust bean gum.
- Preferred examples of the gelling agent include acrylamide, collagen, and fibrin.
- Acrylamide is particularly preferable because it can produce both a low-rigidity gel and a high-rigidity gel and can easily adjust the rigidity of the gel.
- Collagen is preferable because it can express an environment close to that in the living body.
- the gel can be prepared by a known method.
- Gelling agents such as agarose, acrylamide, collagen, fibrin, silicone, glycosaminoglycan, carrageenan, and locust bean gum are suitably used for preparing a gel for two-dimensional culture.
- a gelling agent for three-dimensional culture collagen, fibrin, silicone, glycosaminoglycan, VitroGel TM 3D, VitroGel TM 3D-RGB (TheWell Bioscience), BD Matrigel TM matrix (BD Bioscience), etc. are used. be able to.
- the gel matrix contains components other than the gel.
- components other than the gel include an adhesion protein and an adhesion protein crosslinking agent for crosslinking the adhesion protein to the gel.
- the gel matrix is preferably coated on its surface with adhesion proteins. Gels such as polyacrylamide gels and silicone gels that are not biologically derived are preferably used as the gel matrix.
- adhesion protein examples include at least one selected from the group consisting of collagen, fibronectin, integrin, cadherin, laminin, proteoglycan and the like.
- at least one selected from the group consisting of collagen, fibronectin, and a mixture of collagen and fibronectin can be used.
- the mixing ratio of collagen and fibronectin is a mass (g) ratio, and is 15 to 3, preferably 10 to 5, with respect to fibronectin 1.
- limit especially as collagen Preferably it is type I collagen or type IV collagen.
- the collagen those derived from human, rat, mouse, kangaroo, cow and fish (for example, shark) can be used.
- Collagen may be produced by gene recombination.
- fibronectin those derived from fish, human, cow, kangaroo, mouse, rat and the like can be preferably used.
- Fibronectin may be produced by gene recombination.
- Heterobifunctional crosslinking agent can be used as the adhesive protein crosslinking agent.
- the heterobifunctional cross-linking agent is preferably sulfo-SANPAH (sulfosuccinimidyl 6 (4′-azido-2′-nitrophenyl-amino) hexanoate, Pierce No. 22589), or acrylic acid N-hydroxysuccinimide ester (NHS).
- the preparation method of the gel matrix is not particularly limited.
- it can be prepared by mixing components other than the gel and gelling.
- Collagen final concentration of about 0.3 to 1.0 mg / ml
- collagen final concentration of about 0.3 to 1.0 mg / ml
- fibronectin final concentration of 0.05 to 0.5 mg / ml
- a heterobifunctional cross-linking agent final concentration of 0.5 to 3 mg / ml
- an acrylamide solution including bisacrylamide
- ammonium persulfate, N, N, N ′, N '-Tetramethylethylenediamine (TEMED) is added for gelation.
- TEMED ammonium persulfate
- a polyacrylamide gel is prepared. For example, 0.2 to 1 mg / ml heterobifunctional cross-linking agent is added to dimethyl sulfoxide (DMSO) and a HEPES buffer. And the solution is dropped onto the gel surface with a pipette. Subsequently, the polyacrylamide gel is placed, for example, 6 inches below the ultraviolet lamp and irradiated for 8-15 minutes. Next, the gel after ultraviolet irradiation is washed.
- DMSO dimethyl sulfoxide
- HEPES buffer a HEPES buffer
- the polyacrylamide gel is placed, for example, 6 inches below the ultraviolet lamp and irradiated for 8-15 minutes.
- FIG. 2 shows a schematic diagram for preparing a polyacrylamide gel coated with an adhesion protein.
- the stiffness of the support reflects the stiffness of the surrounding environment that the cells recognize in vivo. “Reflecting the rigidity of the surrounding environment” intends that the rigidity of the support is included in the range of the rigidity of the surrounding environment, or that the rigidity of the support is comparable to the rigidity of the surrounding environment. Accordingly, the rigidity of the support is determined according to the rigidity of the tissue from which the cells are derived, and is 100 kPa or less, 50 kPa or less, 10 kPa or less, 5 kPa or less, 1 kPa or less, 500 Pa or less. The stiffness of the support preferably depends on the stiffness of the gel.
- the rigidity of the gel can be adjusted by the concentration of the gelling agent in the gel preparation solution containing the gelling agent of each gel.
- the rigidity of a gel can also be adjusted with the mixing ratio of a gelling agent and a gel crosslinking agent.
- the gelling agent is acrylamide
- a gel having a rigidity of 10 Pa to 100,000 Pa can be prepared.
- the gelling agent is collagen
- a gel having a rigidity of 1 Pa to 1 kPa can be prepared.
- the gelling agent is fibrin
- a rigid gel having a viscosity of 50 Pa to 4 kPa can be prepared.
- the gelling agent is silicone, a gel having a rigidity of 400 Pa to 300 kPa can be prepared.
- a gel having a desired rigidity can be obtained by changing the concentration of total acrylamide (total amount of acrylamide and bisacrylamide) contained in the gel preparation solution.
- the total acrylamide concentration in the gel preparation may be 0.5-50% by weight depending on the desired stiffness.
- FIG. 3 shows the rigidity according to the concentration of acrylamide and bisacrylamide.
- the rigidity of a polyacrylamide gel prepared from a 3% by mass acrylamide solution is about 50 to 300 Pa.
- the acrylamide: bisacrylamide ratio of the acrylamide gel is preferably in the range of 100: 1 to 5: 1 by mass ratio.
- the mixing ratio of acrylamide with respect to 1 part by mass of bisacrylamide can be selected from 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5 parts by mass.
- Examples of the solvent for the acrylamide solution include water, PBS, 150 mM NaCl-added Tris-HCl buffer, a cell culture medium to which no serum is added or serum is added.
- Acrylamide can be gelled using ammonium persulfate and TEMED. Addition amounts of ammonium persulfate and TEMED are known. For example, ammonium persulfate and TEMED may be added in an amount of about 15 ⁇ l of 10% ammonium persulfate and about 4.5 ⁇ l of TEMED per 1,500 ⁇ l of acrylamide solution regardless of the total acrylamide concentration.
- Fibrin gel can be prepared by reacting fibrinogen with thrombin and calcium ions in vitro.
- the fibrinogen content contained in the fibrin gel preparation may be 1 to 50 mg / mL.
- salmon fibrinogen Searun Holdings, Freeport, ME
- 50 mM Tris-HCl buffer pH 7.4
- 150 mM NaCl at a concentration of 3 mg / mL or 18 mg / mL.
- Add 2 units / mL fish thrombin (Searun Holdings) to 400 ⁇ l fibrinogen solution.
- the fibrin gel prepared from the 3 mg / mL and 18 mg / mL fibrinogen solutions has a rigidity of about 250 Pa and 2150 Pa, respectively.
- Fibrinogen can be a fibrinogen derived from a variable temperature animal or a constant temperature animal.
- a fish is preferable, and a salmon is more preferable.
- the homeothermic animal is preferably a mammal, more preferably a human or a cow. Fibrinogen may be produced by gene recombination.
- Examples of the solvent for preparing the fibrinogen solution include water, PBS, 150 mM NaCl-added Tris-HCl buffer, cell culture medium to which serum is not added or serum is added, and the like.
- the thrombin added to polymerize fibrinogen into fibrin is not limited as long as fibrinogen can be polymerized. Fibrinogen and thrombin are preferably the same species.
- calcium ions can be added to promote fibrinogen polymerization by thrombin. Calcium ions are supplied from, for example, calcium chloride.
- the collagen gel can be prepared by adding a cross-linking agent to the collagen solution and allowing to stand, for example, at 3 to 8 ° C. for 12 to 24 hours.
- the collagen content contained in the collagen solution can be 0.05 mass% to 0.8 mass%.
- a gel having a rigidity of about 60 to 100 kPa in terms of compressive modulus (20 to 33 kPa in terms of shear modulus) can be prepared.
- Collagen is not limited as long as a gel can be prepared, but is preferably type I collagen or type IV collagen.
- Type I collagen is preferred as the collagen.
- the collagen those derived from human, rat, mouse, kangaroo, cow and fish (for example, shark) can be used.
- Collagen may be produced by gene recombination.
- the solvent for preparing the collagen solution is preferably an acidic (pH about 1 to 4, preferably about pH 2.5 to 3.5) aqueous solution.
- an acidic (pH about 1 to 4, preferably about pH 2.5 to 3.5) aqueous solution for example, 10 ⁇ 3 mol of diluted hydrochloric acid can be used as the solvent. Collagen dissolved in dilute hydrochloric acid may be neutralized.
- Cross-linking agents include N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC), glutaraldehyde, 1,4-dibutandiol etheryl (BDDDGE), N-hydroxysuccinimide (NHS) and water-soluble carbodiimide (NHS). Selected from etc. Preferably, it is WSC.
- EDC N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide
- BDDDGE 1,4-dibutandiol etheryl
- NHS N-hydroxysuccinimide
- NHS water-soluble carbodiimide
- Silicone gel can be obtained from Shin-Etsu Silicone (Shin-Etsu Chemical Co., Ltd.) or the like.
- KE-104Gel Cat-104 complex elastic modulus 2 ⁇ 10 4 N / m 2
- KE-1051J complex elastic modulus 2 ⁇ 10 4 N / m 2
- KE-1052 A / B
- complex elastic modulus 2 ⁇ 10 4 N / m 2 KE-110Gel Cat-110
- complex elastic modulus 2 ⁇ 10 4 N / m 2 KE-1056 (complex elastic modulus 4 ⁇ 10 3 N / m) 2 ), FE-57 (complex elastic modulus 3 ⁇ 10 4 N / m 2 ) and the like.
- 1 N / m 2 is approximately 0.3 Pa.
- the cells are brought into contact with the support.
- Expression means that a state closer to the characteristics of a cell in vivo (for example, the response of the cell to the input of a signal) is derived in vitro.
- Preferably expression is reproducible.
- the in vivo characteristics have been brought out is, for example, between cells cultured in a petri dish or the like and cells cultured using a support having rigidity reflecting the rigidity of the surrounding environment recognized by the cells in vivo.
- the response to the input of the signal was compared, and it was determined that in vivo characteristics were extracted (expressed) when the responsiveness of the cells cultured on the support was closer to the response of the cells in vivo. can do. Evaluation of signals and responsiveness to signals will be described later.
- Contacting the cell and the support includes temporarily contacting the cell and the support, or continuing contact for a certain time.
- contacting the cell with the support includes culturing the cell on the support. It is preferable that the primary cultured cells and the like are brought into contact with a rigid support reflecting the rigidity of the surrounding environment recognized by the cells in the living body without being brought into contact with a rigid rigidity such as a plastic petri dish after being collected. This is because when cells are brought into contact with a hard support such as plastic, the in vivo characteristics of the cells may be lost at that time.
- the contact is preferably performed in an environment where cells can be cultured.
- the contact is performed in the presence of a culture medium corresponding to the cells.
- the culture medium may be selected according to the cells. For example, Minimum Essential Medium (MEM), Eagle MEM, ⁇ -MEM, high glucose (4.5 g / L) -containing DMEM, low glucose (1.0 g / L) Containing DMEM, Iskov modified Dulbecco medium (IMDM), Glasgow MEM (GMEM), RPMI-1640, McCoy 5A medium, MCDB medium, Ham's F-12, Ham's F-10, Williams Medium E and mixed media thereof Etc.
- the culture medium may contain about 5 to 30% serum as necessary.
- the culture medium may contain an antibiotic.
- at least one selected from fatty acids, albumin, cell growth factors, amino acids, nucleic acids, and vitamins may be additionally added to the culture medium.
- the contact between the cell and the support is carried out at a temperature of, for example, 25 to 38 ° C., preferably in the presence of 4 to 10% carbon dioxide gas, depending on the type of cell.
- the support is preferably washed or equilibrated with PBS, Hank's buffer salt, culture medium for target cells (serum or the like may be included), and then contacted with the cells.
- the evaluation method also includes maintaining the characteristics of the expressed in vivo cells in vitro. Maintaining the properties of the expressed cells in vitro may include using the support to grow the cells in the presence of a culture medium. In addition, maintaining the characteristics of the expressed cells in vitro may include passaging the cells.
- the cells in contact with the support are maintained using the above-mentioned culture medium, depending on the cell type, for example, at a temperature of 25 to 38 ° C., preferably in the presence of 4 to 10% carbon dioxide gas.
- a state where there is no input of signals other than signals necessary for introduction and / or maintenance of cell characteristics in vivo is also referred to as a “basal state”.
- the basal state is preferably obtained by bringing the cell into contact with a support having a rigidity reflecting the rigidity of the surrounding environment recognized by the cell in vivo, and preferably in a state in which the cell is in contact with the support in the presence of a culture medium.
- the evaluation method includes a step of inputting a signal to a cell in which the characteristics are introduced and / or maintained.
- the signal is not limited as long as it elicits a reaction that is to be evaluated using cells.
- the signal is preferably an external stimulator.
- External stimuli include chemical factors, physical factors, biological factors and combinations thereof.
- Chemical factors include compounds, ions, gases, nucleic acids, carbohydrates, lipids, glycoproteins, glycolipids, lipoproteins, amino acids, peptides, proteins (including antibodies), polyphenols, cytokines, and chemokines. It is preferable that it is at least one selected. More preferably, the chemical factor is an oxidative stress inducer.
- Oxidative stress is preferably high glucose load (7 mM to 30 mM, preferably 10 mM to 20 mM, more preferably 10 mM to 15 mM in final concentration), high fatty acid load (preferably saturated or unsaturated fatty acid having 14 to 18 carbon atoms, More preferably, oleic acid, palmitic acid or a mixture thereof is added at a final concentration in the range of 0.01 mM to 10 mM, preferably in the range of 0.1 mM to 7 mM, hydrogen peroxide (final concentration of 1 ⁇ M to 100 ⁇ M, preferably 5 ⁇ M). To 80 ⁇ M, more preferably 10 ⁇ M to 50 ⁇ M).
- the physical factor is preferably at least one selected from the group consisting of rigidity, pressure, tension, light, radiation, oxygen concentration, pH, and temperature of the surrounding environment of the cell.
- Biological factors include bacteria, fungi, viruses, allergens (pollen, animal skin, ticks, etc.), human cells, non-human animal cells (preferably non-human mammalian cells), and components contained therein (lipopoly Saccharides, allergen extracts, bacterial extracts, fungal extracts, virus capsid proteins, virus core proteins, virus envelope proteins, vaccine components, etc.), extracellular secretory vesicles (conditioning medium, cell culture supernatant, etc.) It is preferable that it is at least one kind selected.
- the above signals can be input to the cells for a necessary period at a concentration or intensity depending on the type of each signal.
- a physical factor the above-mentioned culture medium is used. Except for the case where the physical factor is a temperature, depending on the type of cell, for example, preferably at a temperature of 25 to 38 ° C. Is carried out in the presence of 4-10% carbon dioxide.
- each factor is added to a culture medium containing cells and a support. Depending on the type of cells, for example, at a temperature of 25-38 ° C., preferably 4-10. The cells are brought into contact with each factor in the presence of% carbon dioxide.
- the signal input may be transient, may continue for a certain period, or may be intermittent.
- the culture medium used when contacting the cell with the chemical agent may be a culture medium used when contacting the cell and the support, and is used when contacting the cell and the support according to the signal. A culture medium different from the culture medium to be used may be used.
- the evaluation method includes a step of measuring a cell response to the input signal.
- the measurement method can be measured by a method according to the input signal. For example, genome modification (including DNA modification and histone modification), DNA mutation, RNA expression / degradation, protein expression / degradation, protein phosphorylation, enzyme activity, lipid metabolism, sugar metabolism, generation of active oxygen Detoxification reaction (eg, activation of P450), activation of stress response protein (such as heat shock protein), expression or release, activation of deinger signal protein (such as HMGB1), expression or release, phagocytic reaction, apoptotic reaction, Cellular responses can be evaluated by measuring ubiquitination, mitochondrial function, endoplasmic reticulum function, immune response, and the like.
- the cell reaction can also be evaluated by staining the cells with a staining substance and observing them with a microscope or the like. These measuring methods are known. Evaluation of the response of the cell includes evaluating that the input signal works favorably on the cell and / or evaluating that it adversely affects the cell. In addition, evaluating the cellular response includes elucidating the mechanism of the cellular response to the input signal.
- a candidate substance for preventing, treating or ameliorating a disease or disorder may be used as a physical factor, chemical factor or biological factor. Further, the cell response may be evaluated by combining the input of a signal (preferably an input of a signal that causes damage to the cell) and the addition of a candidate substance for preventing, treating, or improving the disease or disorder. . That is, the evaluation method can also be used as a screening method for candidate substances for preventing, treating or ameliorating a disease or disorder.
- a method for screening a candidate substance for preventing, treating or ameliorating a disease or disorder has a rigidity reflecting the rigidity of the surrounding environment recognized by a cell in vivo, and the rigidity is 100 kPa or less in terms of shear modulus.
- a step of contacting a support with a cell a step of in vivo introducing and / or maintaining the characteristics of a cell that has been introduced and / or expressed in vitro, and the property is introduced and / or maintained.
- a step of measuring a response of a cell to an input signal When the cell response to the input signal is measured, the response of the cell not in contact with the candidate substance and the cell in contact with the candidate substance are compared.
- the candidate substance is suggested or determined to be able to prevent, treat or ameliorate the disease or disorder.
- the myocyte injury model evaluation method relates to an evaluation method using muscle cells.
- the present invention also relates to a myocyte injury model and a screening method for candidate substances for preventing, treating or improving myocyte injury.
- the description in the section of the evaluation method is incorporated herein.
- Muscle cells refer to cardiomyocytes, smooth muscle cells or skeletal muscle cells. Preferred examples include primary cultured muscle cells, smooth muscle cells, and skeletal muscle cells. The animal from which the myocytes are collected is not particularly limited, and examples thereof include the animals described in the evaluation method section.
- a myocyte disorder preferably derived from metabolism and inflammation.
- the “muscle cell disorder derived from metabolism and inflammation” means, for example, cardiomyocytes, smooth muscle cells or skeletals among cell disorders derived from hyperglycemia, mitochondrial metabolism abnormality, oxidative stress, endoplasmic reticulum stress, lipid metabolism abnormality, etc. This refers to cell damage that occurs in muscle cells.
- cardiomyocyte disorders in hyperglycemia include increased oxidative stress, mitochondrial dysfunction (metabolic abnormalities), lipid metabolism abnormalities, angiotensin aldosterone activation, calcium homeostasis and ionic metabolism abnormalities, etc.
- Mitochondrial dysfunction (metabolic abnormality) first increases the concentration of reactive oxygen species (ROS) derived from mitochondria, which causes damage to the mitochondria, which causes a decrease in membrane potential in the mitochondria.
- ROS reactive oxygen species
- Abnormal lipid metabolism means that ATP production in mitochondria is impaired, energy production using long-chain fatty acid as a substrate is inhibited, and cardiac contraction effect is reduced (Monthly Diabetes, 2013/2, Vol. 5, No. .2, 6-7).
- the “muscle cell damage model” is an in vitro damage model using primary cultured cardiomyocytes, smooth muscle cells or skeletal muscle cells.
- the muscle cells are brought into contact with the same physical environment as the in vivo myocytes (the rigidity of the support reflects the stiffness of the surrounding environment of the in vivo myocytes), and the physiological function is It includes muscle cells that have been introduced or maintained so as to have properties similar to those of vivo muscle cells.
- the myocyte injury model includes a myocyte in which a signal is input to a myocyte that has been introduced or maintained so as to have characteristics similar to those of an in vivo myocyte.
- the signal is high glucose or hydrogen peroxide
- the myocyte damage model includes a myocyte damage model in which cell damage is caused by a high glucose load.
- the accumulation amount of oxidative stress is small in the basal state, but oxidative stress accumulates upon receiving a signal input (preferably high glucose load).
- This reaction can be reversible when subjected to high glucose load on a rigid support that reflects the stiffness of in vivo myocardial tissue or skeletal muscle tissue.
- a signal input preferably high glucose load
- This reaction can be reversible when subjected to high glucose load on a rigid support that reflects the stiffness of in vivo myocardial tissue or skeletal muscle tissue.
- it is irreversible on a very rigid support such as glass, and the cells undergo apoptosis due to the accumulation of oxidative stress.
- the support described in the section of the evaluation method can be used.
- the culture medium for contacting or maintaining the myocytes with the support is, for example, 5 to 20% (preferably 8 to 12%, more preferably 5 to 10%) fetal bovine serum-added DMEM ( Low glucose) and the like.
- the conditions for maintaining the muscle cells in contact with the support or on the support follow the conditions described in the section of the evaluation method.
- the rigidity of the support body in contact with the muscle cells is preferably about 5 kPa to 100 kPa, which is the same as that of the heart or skeletal muscle tissue. More preferred is 10 kPa to 30 kPa, and still more preferred is 10 kPa to 15 kPa. More preferably, 15 kPa can be mentioned.
- High glucose means that glucose in the culture medium is added at a high concentration.
- the glucose concentration in the culture medium it is preferable to use a glucose concentration of 10 to 25 mM or 10 to 15 mM corresponding to the blood glucose level of a general human diabetic patient.
- a glucose concentration 10 to 25 mM or 10 to 15 mM corresponding to the blood glucose level of a general human diabetic patient.
- ROS mitochondrial membrane potential
- oxidative stress is accumulated even if the glucose concentration is about 10 mM.
- primary cultured cardiomyocytes cultured on a 15 kPa support are stable and difficult to dedifferentiate.
- the accumulation of oxidative stress in the present invention refers to the accumulation (concentration increase) of intracellular ROS, and the change (responsiveness) of the accumulation amount can be detected by a ROS labeling reagent.
- Various reagents are commercially available as ROS labeling reagents, and can be appropriately used according to the purpose.
- Preferable examples include fluorescein derivatives such as H2DCFDA, carboxy-H2DCFDA, and chloromethyl-H2DCFDA.
- the increase in the concentration of reactive oxygen species can be measured using a known kit for measuring reactive oxygen species.
- Mitochondrial metabolic disorders refers to a diverse group of diseases caused by dysfunction of the mitochondrial respiratory chain. The disease is caused by mutations in nuclear DNA or mitochondrial DNA (mtDNA), affecting multiple internal organs and causing significant nerve and muscle damage. Major symptoms of mitochondrial metabolism include eyelid drooping, extraocular muscle palsy, proximal myopathy and exercise intolerance, cardiomyopathy, sensorineural hearing loss, optic nerve atrophy, retinitis pigmentosa, and diabetes. Symptoms to the central nervous system include variable encephalopathy, epilepsy, dementia, migraine, stroke-like seizures, ataxia, and convulsions.
- Oxidative stress refers to a state in which the oxidative damage power of reactive oxygen species (ROS) generated in vivo exceeds the antioxidant capacity of the antioxidant system in vivo.
- ROS reactive oxygen species
- Lipids, proteins / enzymes responsible for structure and function, and genetic DNA responsible for genetic information are oxidized and damaged. As a result, the structure and function of the living body are disturbed, causing disease, premature aging, and becoming susceptible to cancer and lifestyle-related diseases.
- “Abnormal lipid metabolism” refers to a state in which lipid contained in blood is excessive or insufficient. Generally refers to hyperlipidemia.
- the dyslipidemia (hyperlipidemia) is classified into types such as hypercholesterolemia, high LDL cholesterolemia, low HDL cholesterolemia, and hypertriglyceridemia.
- “Muscle cell disorders derived from metabolism or inflammation” include metabolic diseases related to lifestyle-related diseases such as obesity, diabetes, dyslipidemia, and hypertension, and autoimmunity such as asthma and rheumatoid arthritis, allergies, immunodeficiencies, etc. It refers to a myocyte disorder derived from. Particularly preferred are myocyte disorders derived from metabolic diseases related to lifestyle-related diseases such as hypertension.
- a myocyte injury model is input by bringing a myocyte into contact with a candidate substance for preventing, treating or ameliorating myocyte injury before or after inputting a signal, or after inputting a signal.
- candidate substance for preventing, treating or ameliorating myocyte injury before or after inputting a signal, or after inputting a signal.
- Candidate substances for preventing, treating or ameliorating a disease or disorder can be screened by measuring the response of cells to the signal. Details of the screening method are as follows. The description in the section of screening method is incorporated herein.
- a candidate substance for preventing, treating or ameliorating metabolic and inflammatory myocyte damage using muscular cell damage model with increased oxidative stress Drugs can be evaluated and created.
- Drugs can be evaluated and created.
- the screening method can also be used for screening candidate substances for the prevention, treatment or improvement of sarcopenia or locomotive syndrome in patients with increased oxidative stress, particularly diabetic patients.
- One aspect of the hepatocyte injury model evaluation method relates to an evaluation method using hepatocytes.
- the evaluation method includes evaluation of hepatocellular injury (particularly idiopathic drug-induced liver injury), analysis method of non-alcoholic steatohepatitis, and screening method of candidate substances for preventing, treating or improving hepatocellular injury. including.
- hepatocellular injury particularly idiopathic drug-induced liver injury
- analysis method of non-alcoholic steatohepatitis analysis method of non-alcoholic steatohepatitis
- screening method of candidate substances for preventing, treating or improving hepatocellular injury. including.
- Hepatocyte refers to a cell derived from the liver parenchyma. Preferred examples include primary cultured hepatocytes. The animal from which hepatocytes are collected is not particularly limited, and can include the animals described in the section of the evaluation method.
- “Evaluation of hepatocyte damage” means evaluation of hepatocyte damage in vitro using primary cultured hepatocytes.
- Hepatocytes used for the evaluation of hepatocellular injury are the same as in vivo hepatocytes in vitro (the surrounding environment recognized by hepatocytes in normal or abnormal liver tissues where the stiffness of the support is in vivo) Hepatocytes are brought into contact with the environment (reflecting stiffness) and include cells that have been introduced or maintained so that their physiological function is similar to that of in vivo hepatocytes.
- hepatocytes used for evaluation of hepatocellular injury include hepatocytes that have been input or maintained so as to have characteristics similar to those of in vivo hepatocytes, in which a signal is input.
- the stiffness of the surrounding environment recognized by hepatocytes in normal or abnormal liver tissue in vivo is about 0.2 to 5 kpa. Therefore, the rigidity of the support is preferably within this range.
- the rigidity of the liver tissue is increased by fibrosis or the like as shown in FIG. 1B.
- the stiffness of the microenvironment of normal liver tissue is about 0.2 to 0.7 kPa, preferably about 0.3 to 0.6 kPa. Therefore, when using normal hepatocytes and evaluating normal cell responses, or when evaluating normal cell responses at an early stage of abnormalities, use this rigid support. It is preferable to do.
- a support having a rigidity of 0.7 kPa or more and about 2 to 3 kPa can be used.
- the culture medium for contacting or maintaining the hepatocytes with the support is, for example, 5 to 20% (preferably 8 to 12%) fetal bovine serum and Hepatocyte Maintenance Supplement Pack (Thermo Fisher Co., Ltd.). Addition Williams Medium E etc. can be used.
- the conditions for contacting or maintaining the hepatocytes on the support are in accordance with the conditions described in the evaluation method section.
- the signal is preferably a signal that causes induction of drug-metabolizing enzymes, idiopathic drug-induced liver injury or non-alcoholic steatohepatitis.
- Signals that cause idiopathic drug-induced liver injury are, for example, chemical factors or biological factors. In particular, it may be a candidate substance administered to a human or animal to prevent, treat or ameliorate a disease or disorder.
- a method for measuring a cell response to an input signal can be selected according to the signal. For example, measurement of the detoxification function of hepatocytes (measurement of the activity of P450 family such as CYP3A4), method of measuring the accumulation of oxidative stress, method of measuring the expression of proteins released into the critical state of cells such as HMGB1 Can be mentioned.
- a signal is idiopathic if the measurement of the response of the cell to the input signal determines that the cell is or can be damaged compared to a cell that did not receive the signal. It can be determined to cause drug-induced liver injury.
- the signal that induces drug-metabolizing enzymes or idiopathic drug-induced liver injury is, for example, a chemical factor or a biological factor.
- it may be a drug or a candidate substance administered to a human or animal to prevent, treat or improve a disease or disorder.
- a method for measuring a cell response to an input signal can be selected according to the signal. For example, measurement of detoxification function of hepatocytes (measurement of activity of P450 family such as CYP3A4), method of measuring accumulation of oxidative stress, method of measuring expression of proteins expressed in critical state of cells such as HMGB1 Can be mentioned.
- a signal is idiopathic if the measurement of the response of the cell to the input signal determines that the cell is or can be damaged compared to a cell that did not receive the signal. It can be determined to cause drug-induced liver injury.
- the signal can be input to the cell for a required period at a concentration or intensity depending on the type of each signal.
- the signal preferably indicates the mechanism of non-alcoholic steatohepatitis.
- the signal indicating the mechanism causing nonalcoholic steatohepatitis is, for example, a chemical factor or a biological factor.
- carbohydrates and / or lipids that can cause overnutrition causing adult diseases can be used.
- the carbohydrate is preferably fructose
- the lipid is preferably a fatty acid (preferably a saturated or unsaturated fatty acid having 14 to 18 carbon atoms), particularly a saturated fatty acid.
- Carbohydrates or lipids may be added to the culture medium containing hepatocytes at a final concentration in the range of 0.01 mM to 10 mM, preferably in the range of 0.1 mM to 7 mM, if necessary.
- the culture medium is preferably a low glucose (5 mM) -containing culture medium containing about 5% fetal calf serum.
- the contact period between the cells and the signal is about 4 hours to 30 days, preferably about 1 to 7 days, and about 1 to 3 days.
- a method for measuring a cell response to an input signal can be selected according to the signal. Examples thereof include microscopic observation of lipid droplet accumulation in hepatocytes, a method for measuring accumulation of oxidative stress, and a method for measuring expression and release of proteins released into a critical state of cells such as HMGB1.
- the measurement of the response of the cell to the input signal is determined to cause nonalcoholic steatohepatitis or can cause nonalcoholic steatohepatitis compared to cells that did not receive the signal Can be determined that the signal causes non-alcoholic steatohepatitis.
- the signal can be input to the cell for a required period at a concentration or intensity depending on the type of each signal.
- the candidate signal for preventing, treating or ameliorating hepatocellular injury is contacted with the hepatocyte, and the input signal
- Candidate substances for preventing, treating or ameliorating a disease or disorder can be screened by measuring the response of cells to. Details of the screening method are as follows. The description in the section of screening method is incorporated herein.
- Kit In the present invention, the above-described 1. ⁇ 4. And the kit for realizing the cell reaction evaluation method, the screening method, the myocyte damage model, and the hepatocyte damage model described above.
- the kit is used in vivo so that the cells necessary for carrying out the cell response evaluation method, screening method, myocyte injury model, and hepatocyte injury model express in vivo characteristics in vivo. It includes a support (the support described in the above 1 to 4) having a rigidity reflecting the rigidity of the surrounding environment to be recognized and having a shear elastic modulus of 100 kPa or less.
- the kit may also contain cells suitable for carrying out the target cell reaction evaluation method, screening method, myocyte injury model, and hepatocyte injury model.
- the kit may contain a medium suitable for the cells, additives such as cytokines and inhibitors, antibiotics, buffers and the like.
- additives such as cytokines and inhibitors, antibiotics, buffers and the like.
- the dynamic rigidity of the support was measured with a strain-controlled rheometrics-fluids-spectrometer-III (Rheometrics, Piscataway, NJ).
- the stiffness (G ′) which is a value representing elastic resistance, was calculated from the shear stress of the same phase at a vibration (1 rad / s) shear strain of 2%.
- the short-term rigidity G ′ ( ⁇ ) was measured by vibrating at a strain of 2%.
- a steady strain of 10% was applied, the sample was relaxed for 30 seconds, and the long-term rigidity G ′ (t) was measured. The results are shown in FIG.
- a copolymer of acrylamide and bisacrylamide was prepared, and a polyamide gel support of 15 kPa (for muscle cell culture) or 500 Pa (for hepatocytes) was prepared.
- a mixed solution of acrylamide and bisacrylamide was polymerized using N, N, N, N-tetramethylethylenediamine and 10% ammonium persulfate. The solution was placed on a glass plate having a diameter of 22 mm and pretreated with 3-aminopropyltrimethoxysilane and glutaraldehyde.
- polyacrylamide gel does not have cell adhesion
- 50 mM HEPES buffer solution of N-sulfosuccinimidyl-6- (4′-azido-2′-nitrophenylamino) hexanoate (0.5 mg / ml) as a cross-linking agent (PH 8) was dropped and coated. The top was covered with a cover glass, and then the cover glass was removed.
- the gel for muscle cell culture was coated with a mixed solution of 0.05 mg / ml fibronectin and 0.1 mg / ml type I collagen.
- the gel for hepatocyte culture was coated with a solution of 0.1 mg / ml type I collagen (rat hepatocytes) or type IV collagen (human hepatocytes).
- Covering the gel with the adhesive protein was performed by covering the gel containing the heterobifunctional cross-linking agent with a solution containing the adhesive protein, and allowing the adhesive protein to bind to the heterobifunctional cross-linking agent.
- Example 1 Functional evaluation of primary cultured cardiomyocytes cultured in the same rigid support environment (15 kPa) as in vivo
- Culture of primary cultured cardiomyocytes on a 15 kPa polyamide gel support As shown in FIG.
- newborn ventricles from Wistar rats 1 to 3 days old were collected and digested with type IV collagenase and dispase.
- Enzymatically isolated cells were seeded in plastic culture dishes and cultured at 37 ° C. in a cell culture incubator containing 5% carbon dioxide for 40 minutes.
- the coexisting fibroblasts were fixed on the bottom of the culture dish and removed, then the supernatant was collected, centrifuged, and the collected cardiomyocytes were seeded on the polyacrylamide support, and 5 mM glucose (corresponding to normoglycemia) And 5% FCS-containing DMEM (low glucose) (containing L-glutamine and phenol red).
- Example 2 Functional evaluation of primary cultured cardiomyocytes loaded with high glucose in the same rigid environment (15 kPa) as in vivo (1) Method As in Example 1, cardiomyocytes were cultured on a 15 kPa support. Finally, the medium was changed to a medium having a glucose concentration of 5 mM to 25 mM and cultured for 1-2 days. The cytoskeleton was evaluated by fluorescent immunostaining using ⁇ -actinin antibody and Phalloidin binding to F-actin that were fluorescently labeled.
- Example 3 Functional evaluation of primary cultured cardiomyocytes loaded with hydrogen peroxide in the same rigid environment (15 kPa) as in vivo (1) Method After isolating primary cultured cardiomyocytes as in Example 1, 1-2 The cells were cultured in DMEM supplemented with 10% FCS containing normal concentration of glucose (5 mM) for a day, and finally hydrogen peroxide was loaded for 1 hour (10 ⁇ M, 50 ⁇ M). The cytoskeleton was evaluated by fluorescent immunostaining using an ⁇ -actinin antibody.
- Example 4 Effect of 15 mM glucose concentration on ROS of primary cultured cardiomyocytes To confirm events that lead to chronic complications in cardiomyocytes, 10-15 mM glucose concentration, a common hyperglycemia in type 2 diabetic patients, was The effect on mitochondria was compared between cultured cardiomyocytes on a 15 kPa gel and cultured cardiomyocytes on glass.
- N-acetylcysteine (NAC) was used as a scavenger for ROS.
- NAC N-acetylcysteine
- CM-H2DCFDA is a fluorescent indicator of ROS having cell membrane permeability, and is known to increase green fluorescence when oxidized by intracellular ROS, particularly hydrogen peroxide and hydroxy radicals.
- CM-H2DCFDA dye was incorporated into cardiomyocytes (37 ° C., 50 minutes). Thereafter, green light emission (wavelength: 535 nm) when excited with blue light (wavelength: 485 nm) is acquired as a fluorescent image on a confocal laser microscope (Nikon).
- Example 5 Effect of 15 mM glucose concentration on mitochondrial membrane potential of primary cultured cardiomyocytes
- JC-I staining is evaluated by JC-I staining.
- the JC-1 dye which is a probe for detecting the mitochondrial membrane potential, is positively charged and accumulates inside the electronegative mitochondria.
- Mitochondrial membrane potential-dependent accumulation inside the mitochondria by JC-I dye is indicated by a fluorescence wavelength shift from green (about 529 nm) to red (about 590 nm). That is, when the mitochondria are damaged, the accumulation of JC-I dye inside the mitochondria decreases, so the ratio of red color decreases and green becomes dominant. From this, the membrane potential of mitochondria can be shown by a decrease in the red / green fluorescence intensity ratio (Circulation.2005; 111: p2752-2759).
- JC-1 dye was incorporated (37 ° C., 30 minutes), and fluorescence images were acquired and evaluated with a confocal laser microscope.
- cultured cardiomyocytes on a 15 kPa gel support have a higher mitochondrial activity in the basal state than a glass support, and the mitochondrial activity decreases in response to ROS accumulation accompanying an increase in glucose concentration. It shows that myocardial cells are close to the behavior when exposed to oxidative stress. This difference will be discussed together with the results of the following apoptosis assay.
- Example 6 Effect of different culture supports on apoptosis (cell death) of primary cultured cardiomyocytes (1) Method Comparison of differences in apoptosis of primary cultured cardiomyocytes on 15 kPa gel support and glass support Therefore, TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling) assay was performed using In situ Cell Death Detection kit-FITC (Roche). The TUNEL assay is a general method for detecting DNA fragments derived in the apoptotic signal transduction pathway (Cell Death and Disease (2014) 5, e1479; doi: 10.1038 / cddis. 2014.430).
- TUNEL positive nuclei / total number of nuclei dead cells per field of view
- cardiomyocytes were loaded with high glucose, TUNEL dye was incorporated and reacted (37 ° C., 60 minutes). After that, it was sealed with a fluorescence degradation inhibitor containing DAPI, and an image was acquired and analyzed with an upright fluorescence microscope.
- TUNEL positive cells on 15 kPa gel support As shown in FIGS. 14 and 15, TUNEL positive nuclei in cultured cardiomyocytes on 15 kPa gel support are cultured on glass support. There were significantly fewer than cardiomyocytes.
- FIG. 15 which quantifies FIGS. 13 to 14, it is shown that apoptosis on a 15 ⁇ kPa gel support is less likely to occur than on a glass support. It was suggested that obstacles could be reduced and better condition could be maintained. Furthermore, when ROS accumulates with hyperglycemia in cardiomyocytes on glass supports, irreversible damage occurs, mitochondrial membrane potential decreases, and apoptosis begins. Therefore, even if it is removed with a ROS remover, apoptosis is not suppressed. On the other hand, in myocardial cells on a 15 kPa gel support, the damage caused by the accumulation of ROS is reversible and cell death is unlikely to occur. It was. Thus, the cardiomyocyte model of the present invention can reduce irreversible cell damage related to oxidative stress during long-term culture, and is a model suitable for evaluating the influence of chronic oxidative stress load on cardiomyocytes. it is conceivable that.
- Example 7 Evaluation of mitochondrial membrane potential of cultured cardiomyocytes when ROS remover was administered after high glucose load (1) Method The same procedure as in Example 5 was performed. Primary cultured cardiomyocytes on 15 kPa gel support and glass support were cultured and subjected to high glucose load (15 mM, 24 hours). After that, the culture solution is replaced with an ROS remover or normal glucose concentration (5 mM) and cultured for 24 hours, and then the JC-1 dye is incorporated (37 ° C, 30 minutes) with a confocal laser microscope. was obtained and evaluated.
- Example 8 Effect of hyperglycemia in skeletal muscle cells
- the effect of 10-15 mM glucose concentration which is common hyperglycemia in type 2 diabetic patients, on mitochondria
- (1) Method Rat skeletal myoblasts were obtained from Cosmo Bio. Using the differentiation medium provided with the cells (contents not shown), the cells were differentiated into skeletal muscle cells according to the protocol designated by Cosmo Bio. The skeletal muscle cells were seeded on a cover glass prepared in the same manner as described in Example 1 or on a 15 kPa gel.
- Hyperglycemia treatment and ROS removal agent (NAC) treatment were performed by the method described in Example 5, ROS accumulation was stained with the fluorescent reagent CM-H2DCFDA (FIG. 17A), and mitochondrial membrane potential was stained with JC-1 (FIG. 17B). ).
- FIG. 17 in cultured skeletal muscle cells, as in cultured cardiomyocytes, ROS accumulation at normal glucose concentration is greater when cultured on a 15 kPa gel support than when cultured on a glass support. It was found that the mitochondrial membrane potential was significantly lower and that the culture on the 15 kPa gel support was significantly higher than the culture on the glass support.
- Example 9 Comparison of rifampicin reactivity between primary cultured hepatocytes on 500 Pa support and three-dimensional cultured Spheroid (1) Method In human primary cultured hepatocytes, the most in vivo functions of conventional primary cultured hepatocytes We evaluated the presence or absence of the superiority of the support of 500 Pa that matches the stiffness of normal liver tissue over spheroid culture, which is thought to be able to faithfully reflect the above. Human primary hepatocytes were seeded on a 500 Pa support surface coated with type IV collagen or glass that is usually used in cell culture but has high non-physiological rigidity, and cultured in the same manner as in Example 9.
- spheroids were formed from primary human cultured hepatocytes using Cell-able (Sumitomo Bakelite). After culturing for 12 days, rifampicin known to induce CYP3A4 was used and stimulated with 0 or 40 ⁇ M rifampicin for 46 hours. Increase of CYP3A4 activity by rifampicin by measuring CYP3A4 activity with P450-Glo CYP3A4 Assay (Luciferin-IPA) (Promega), dividing by cell number, and dividing CYP3A4 activity upon rifampicin stimulation by ground-state CYP3A4 activity I asked for a degree.
- Example 10 Prediction of idiopathic drug-induced liver injury Diclofenac, troglitazone, ranitidine and the like are known to cause idiopathic drug-induced liver injury. On the other hand, acetaminophen, ethanol, etc. show hepatotoxicity when taken in excess, but do not cause idiopathic drug liver injury. The following experiment was conducted to search for an evaluation system for screening the risk of idiopathic drug liver injury.
- HMGB1 concentration released into the culture medium after 24 hours of incubation with acetaminophen (known as a compound that does not cause idiopathic drug-induced liver injury) or acetaminophen (known as a compound that does not cause idiopathic drug-induced liver injury) was quantified by ELISA using HMGB1 ELISA Kit2 (Sinotest).
- HMGB1 can be a marker for predicting the occurrence of idiopathic drug-induced liver injury by the compound, and primary cultured hepatocytes cultured on a support equivalent to the stiffness of normal liver tissue are useful in the prediction. Was suggested (FIG. 19).
- Example 11 Mechanism 1 of non-alcoholic steatohepatitis
- Nonalcoholic steatohepatitis is thought to be caused by damage to hepatocytes, even if hepatocytes do not die due to lifestyle.
- the following experiment was conducted.
- the cell viability was confirmed by trypan blue staining. Those with a survival rate of 75% or less were excluded.
- the number of cells is counted and cells are spread on a 500 Pa support or glass support at 3.5x10 ⁇ 5 cells / well (6-well plate), 5% FCS and Hepatocyte Maintenance Supplement Pack (Thermo Fisher CM4000) The cells were cultured in D-MEM (low glucose) (containing L-glutamine and phenol red) for 3 days.
- D-MEM low glucose
- fructose 5.5 mM
- palmitic acid 0.5 mM
- lipid droplet staining and ROS 24 hours later.
- the culture medium was replaced with 10% FCS-added DMEM containing 5 mM glucose. None was added to the control. In the control group, 10% bovine serum albumin used for dissolving lipid was added.
- LipiDye is a fluorescent dye that stains intracellular lipid droplets with high sensitivity (reference: Yamaguchi E, et al., Angew. Chem. Int. Ed., 54: 4539-4543 (2015)).
- LipiDye was diluted with DMEM (without FCS and antibiotics added) to a final concentration of 1 ⁇ M, and added to each 6-well dish at 1-2 ml. After incubating at 37 ° C. for 2 hours, washed once with HBSS (+), the sample was observed with a Nikon A1 confocal laser microscope.
- the control of primary cultured hepatocytes cultured on a glass support was compared with the control of primary cultured hepatocytes cultured on a 500 Pa support. Many accumulations of ROS were observed. Even in primary cultured hepatocytes cultured on a glass support, accumulation of ROS was observed in cells to which a mixture of palmitic acid and palmitic acid-oleic acid was added. However, the primary cultured hepatocytes cultured on a 500 Pa support rather than these cells, and the cells to which fructose, palmitic acid, oleic acid-palmitic acid mixed solution were added, were cultured on the glass support. There was more ROS accumulation than primary cultured hepatocytes.
- Example 12 Mechanism 2 of non-alcoholic steatohepatitis In order to elucidate the mechanism of nonalcoholic steatohepatitis, the following experiment was conducted.
- rat primary cultured hepatocytes were analyzed using a polyacrylamide gel of 500 Pa that matches the hardness of normal liver tissue. Or cultured on glass that is normally used in cell culture but has non-physiologically high hardness. In order to promote cell adhesion, the surface of each culture support was coated with type IV collagen. On the next day, 10 6 cells / well of mouse peritoneal macrophages were added to wells containing rat primary cultured hepatocytes, and co-culture was started.
- Mouse peritoneal macrophages were stimulated with thioglyconate and peritoneal macrophages were collected by the method described in “Investigation of gene transfer into mouse primary peritoneal macrophages” of invitrogen. Specifically, 2 ml of 5% thioglycollate medium (Sigma) was injected into the abdominal cavity of C57 / BL6J mice, and decapitated 3.5 days later, using a syringe and a syringe needle with a total of 15 ml of PBS (7 ml + 8 ml). The peritoneal macrophages were collected by washing the peritoneal cavity twice.
- the collected peritoneal washing solution was centrifuged at 1000 rpm, 4 ° C. for 5 minutes, washed twice with PBS, and the number of cells was calculated. Fructose stimulation was applied 24 hours after the start of the same hepatocyte-macrophage co-culture, and the culture medium was collected 24 hours later, and the amount of TNF ⁇ secretion was measured by ELISA.
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Abstract
Description
このように、細胞は、その細胞が接する周辺組織の微小環境に応じて、状態や分化の方向性を変える。
発明者らの検討により、このような人工的な剛性条件では、本来生体内で細胞に起きている反応が観察できない場合があることが明らかとなった。
項1.細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体と細胞とを接触させる工程と、
in vivoの細胞の特性をin vitroで導入及び/又は発現した細胞の特性をin vitroで維持する工程と、
前記特性が導入、及び/又は維持されている細胞に、シグナルを入力する工程と、
入力されたシグナルに対する細胞の反応を測定する工程と、
を含む、細胞の反応の評価方法(ただし、前記細胞に間葉系幹細胞は含まない)。
項2.前記シグナルは、化学的因子及、物理的因子及び生物学的因子よりなる群から選択される少なくとも一つの因子を細胞に負荷することによって細胞に入力される、項1に記載の方法。
項3.化学的因子が、化合物、イオン、気体、核酸、糖質、脂質、糖タンパク質、糖脂質、リポタンパク質、アミノ酸、ペプチド、タンパク質、ポリフェノール類、サイトカイン類及びケモカインよりなる群から選択される少なくとも一種である、項1又は2に記載の方法。
項4.物理的因子が、細胞の周囲環境の剛性、圧力、張力、光、放射線、酸素濃度、pH及び温度よりなる群から選択される少なくとも一種である、項1又は2に記載の方法。
項5.生物学的因子が、細菌、真菌、ウイルス、アレルゲン、ヒト細胞、ヒト以外の動物細胞及びこれらに含まれる成分より選択される少なくとも一種である、項1又は2に記載の方法。
項6.細胞が肝細胞であり、支持体の剛性が0.2~5kpaである、項1~5のいずれか一項に記載の方法。
項7.細胞の反応が薬物代謝酵素の誘導、非アルコール性脂肪肝炎のメカニズム、又は特発性肝障害を示す、項6に記載の方法。
項8.細胞の反応が前記因子の効能又は毒性である、項1~5のいずれか一項に記載の方法。
項9.細胞が心筋細胞であり、支持体の剛性が5~100kpaである、項1~5のいずれか一項に記載の方法。
項10.化学的因子が酸化ストレス誘導物質であり、細胞の反応が酸化ストレス応答である、項9に記載の方法。
項11.酸化ストレス誘導物質がグルコースである、項10に記載の方法。
項12.細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体と細胞とを接触させる工程と、
in vivoの細胞の特性をin vitroで導入及び/又は発現した細胞の特性をin vitroで維持する工程と、
前記特性が導入、及び/又は維持されている細胞に、シグナルを入力する工程と、
シグナルの入力に先立って、シグナルの入力と同時に、又はシグナルの入力の後に、疾患、又は障害を予防、治療又は改善するための候補物質と細胞とを接触させる工程と、
入力されたシグナルに対する細胞の反応を測定する工程と、
を含む、疾患、又は障害を予防、治療又は改善するための候補物質のスクリーニング方法(ただし、前記細胞に間葉系幹細胞は含まない)。
項13.前記シグナルは、化学的因子及、物理的因子及び生物学的因子よりなる群から選択される少なくとも一つの因子を細胞に負荷することによって細胞に入力される、項12に記載の方法。
項14.化学的因子が、化合物、イオン、気体、核酸、糖質、脂質、糖タンパク質、糖脂質、リポタンパク質、アミノ酸、ペプチド、タンパク質、ポリフェノール類、サイトカイン類及びケモカインよりなる群から選択される少なくとも一種である、項12又は13に記載の方法。
項15.物理的因子が、細胞の周囲環境の剛性、圧力、張力、光、放射線、酸素濃度、pH及び温度よりなる群から選択される少なくとも一種である、項12又は13に記載の方法。
項16.生物学的因子が、細菌、真菌、ウイルス、アレルゲン、ヒト細胞、ヒト以外の動物細胞及びこれらに含まれる成分より選択される少なくとも一種である、項12又は13に記載の方法。
項17.細胞が肝細胞であり、支持体の剛性が0.2~5kPaである、項12~16のいずれか一項に記載の方法。
項18.細胞の反応が薬物代謝酵素の誘導、非アルコール性脂肪肝炎のメカニズム、又は特発性肝障害を示す、項17に記載の方法。
項19.細胞の反応が前記因子の効能又は毒性である、項12~16のいずれか一項に記載の方法。
項20.細胞が心筋細胞であり、支持体の剛性が5~100kPaである、項12~16のいずれか一項に記載の方法。
項21.化学的因子が酸化ストレス誘導物質であり、細胞の反応が酸化ストレス応答である、項20に記載の方法。
項22.酸化ストレス誘導物質がグルコースである、項20に記載の方法。
項23.細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体を含む、請求項12~22に記載の細胞の反応の評価方法、又は請求項1~11に記載のスクリーニング方法を実施するためのキット。
項24.さらに目的とする細胞の反応の評価又はスクリーニングを実施するために適した細胞を含む、項23に記載のキット。
細胞の反応の評価方法(以下、単に「評価方法」とする)について説明する。
評価方法において、物理的因子、化学的因子又は生物学的因子として、例えば疾患、又は障害を予防、治療又は改善するための候補物質を用いてもよい。また、シグナルの入力(好ましくは細胞に障害を来すシグナルの入力)と、疾患、又は障害を予防、治療又は改善するための候補物質の添加を組み合わせて細胞の反応の評価を行ってもよい。すなわち評価方法は、疾患、又は障害を予防、治療又は改善するための候補物質のスクリーニング方法としても使用できる。
評価方法の一態様は、筋細胞を使用した評価方法に関する。また、筋細胞障害モデル、及び筋細胞障害を予防、治療又は改善するための候補物質のスクリーニング方法に関する。評価方法の項に記載された用語で、本項でも使用される用語については、評価方法の項の説明をここに援用する。
評価方法の一態様は、肝細胞を使用した評価方法に関する。また、評価方法は、肝細胞障害(特に特発性薬剤性肝障害)の評価、非アルコール性脂肪肝炎のメカニズムの解析方法、及び肝細胞障害を予防、治療又は改善するための候補物質のスクリーニング方法を含む。評価方法の項に記載された用語で、本項でも使用される用語については、評価方法の項の説明をここに援用する。
「剛性」の定義は、評価方法の項の説明にしたがう。
本発明には、上記1.~4.で述べた細胞の反応の評価方法、スクリーニング方法、筋細胞障害モデル、及び肝細胞障害モデルを実現するためのキットを含む。キットは、細胞の反応の評価方法、スクリーニング方法、筋細胞障害モデル、及び肝細胞障害モデルを実施するために必要な細胞が、in vivoにける特性をin vitroで発現するように、生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体(上記1.~4.で述べた支持体)を含む。また、キットは、目的とする細胞の反応の評価方法、スクリーニング方法、筋細胞障害モデル、及び肝細胞障害モデルを実施するために適した細胞を含んでいてもよい。この他、キットは、前記細胞に適した培地、サイトカインやインヒビター等の添加物、抗生物質、バッファー等を含んでいてもよい。上記1.~4.に記載の細胞の反応の評価方法、スクリーニング方法、筋細胞障害モデル、及び肝細胞障害モデルの説明は、ここに援用される。
(1)ポリアクリルアミドゲルの剛性
ゲルの剛性を変化させるために、ポリマー質量を一定の7.5%とし、ビスアクリルアミド濃度を0.01%、0.03%または0.3%に変化させて、アクリルアミドおよびビスアクリルアミド(Fisher Biotech, Loughborough, Leicestershire, UK)溶液を調製した。アクリルアミド及びビスアクリルアミドを含む溶液を後述するひずみ制御型rheometrics fluids spectrometer IIIのプレート間に流し込み、過硫酸アンモニウム及びN,N,N’,N’-テトラメチルエチレンジアミン(TEMED)を添加してとのまま重合させ、動的剛性率を測定した。
0.1NのNaOHを200μlのピペットで滴下し、直径22 mmのガラス製カバーガラス(Fisherbrandカタログ番号12-545-101;Fisher Scientific, Pittsburgh, PA)の表面を5分間覆った。NaOH溶液を吸引し、3-APTMS(3-アミノプロピルトリメトキシシラン、Sigma社のNo.28-1778、Sigma, St. Louis, MO)200μlを3分間適用した。このガラス製カバーガラスを脱イオン水で十分にすすいで残っている3-APTMS溶液を洗い流し、0.5%vグルタルアルデヒド(Sigma社のNo.G7651)水溶液200μlを20分間カバーガラスに加えた。
カバーグラスを培養皿中に置き、0.05mg/mlのフィブロネクチン及び0.1mg/mlのI型コラーゲンの混合液またはIV型コラーゲン溶液を加えて、30分間UV照射の後、室温で2-3時間静置し、表面を被覆した。
(1)15 kPaのポリアミドゲル支持体での初代培養心筋細胞の培養
図4に示すように、Wistarラットの生後1~3日の新生児の心室を採取し、IV型のコラゲナーゼとディスパーゼで消化した。酵素的に単離した細胞を、プラスチックの培養皿に播種し、37℃で5%炭酸ガスを含む細胞培養インキュベーター内で40分間培養した。そして共存する線維芽細胞を培養皿の底に定着させて除去した後に上清を回収、遠心分離して回収した心筋細胞を、上記ポリアクリルアミド支持体に播種し、5 mMグルコース(正常血糖に相当)を含む5%FCS加DMEM(低グルコース)(L-グルタミン、フェノールレッド含有)で培養した。
図5に示すように、心筋収縮に寄与する細胞骨格系を評価した。ガラスの上では、心筋細胞は不整列なアクチン・ネットワークと筋線維分節を示した。生体の心臓の剛性に近い、15 kPaの支持体上で培養された場合にのみ、心筋細胞は整列した筋線維分節を作り上げて行くことが分かった。生理学的な剛性の15 kPaであれば、心筋細胞は細長い形状を形成することが示された。
以上の結果は、心筋細胞が整列した筋線維分節の構造を形成するには、生理学的な剛性を持った支持体が必要であることを示している。
(1)方法
実施例1と同様に心筋細胞の培養を15 kPaの支持体上で行い、最後に5 mM~25 mMのグルコース濃度の培地に交換して1~2日の培養を行った。細胞骨格の評価は、α-アクチニン抗体と、F-アクチンに結合するPhalloidinを蛍光ラベルされたものを用いて蛍光免疫染色を行い評価した。
図6に示すように、25 mMの高グルコース濃度で培養すると、細胞骨格構造(α-アクチニン、及びF-アクチン)は15 kPaゲル上の培養心筋細胞で崩壊したが、ガラス上の培養細胞ではその崩壊は認められなかった。また、高グルコース刺激と同様の浸透圧刺激を与える高マンニトール刺激では15 kPaゲル上の培養心筋細胞における細胞骨格構造の崩壊が見られず、高グルコースでの細胞骨格の崩壊は浸透圧による影響ではないことが明らかとなった。
(1)方法
実施例1と同様に初代培養心筋細胞を単離した後1~2日間正常な濃度(5 mM)のグルコースを含む10%FCS加DMEMで培養し、最後に1時間過酸化水素を負荷した(10μM、50μM)。細胞骨格の評価は、α-アクチニン抗体を用いて蛍光免疫染色を行い評価した。
図7に示すように、10μM、50μMの過酸化水素を曝露すると、細胞骨格構造(α-アクチニン)は15 kPaゲル上の培養心筋細胞で崩壊したが、ガラス上ではその崩壊は認められなかった。高グルコース負荷で細胞骨格の崩壊が起きることの原因の一つに酸化ストレスの蓄積が考えられるため、過酸化水素を直接負荷したが、やはり高グルコース負荷と同様の変化がみられた。
心筋細胞における慢性合併症に繋がるイベントを確認するため、2型糖尿病患者のありふれた高血糖である10~15 mMグルコース濃度がミトコンドリアに及ぼす影響を、15 kPaのゲル上の培養心筋細胞と、ガラス上の培養心筋細胞とで比較することを行った。
図8に示すような方法で行った。N-アセチルシステイン(NAC)をROSの除去剤(scavenger)として使用した。まず、新たに調製された初代培養心筋細胞のミトコンドリアにおけるROS蓄積を、蛍光試薬CM-H2DCFDAを指示薬として評価した。CM-H2DCFDAは、細胞膜透過性を有するROSの蛍光指示薬であり細胞内のROS、特に過酸化水素やヒドロキシラジカルによって酸化されることにより緑色の蛍光が増加することが知られている。
図9に示されるように、正常なグルコース濃度におけるROSの蓄積量は、ガラス支持体よりも15 kPaゲル支持体での培養心筋細胞で顕著に低い値を示した。一方、高グルコース濃度では、ガラス支持体と15 kPaゲル支持体上の培養心筋細胞は共にROS蓄積量が増加した。しかし、ROS除去剤であるNACの作用で15 kPa支持体上の培養心筋細胞では、ガラス上の培養心筋細胞に比較し、高グルコース負荷によるROS蓄積が極めて効率よく除去された。
6.実施例5:15 mMグルコース濃度が初代培養心筋細胞のミトコンドリア膜電位に及ぼす影響
ミトコンドリアの膜電位の評価は、JC-I染色により評価する。ミトコンドリア膜電位を検出するプローブであるJC-1色素は、正に荷電しているため、電気陰性的なミトコンドリア内部に蓄積する。JC-I色素によるミトコンドリア膜電位依存的なミトコンドリア内部への蓄積は、緑色(約529 nm)から赤色(約590 nm)への蛍光波長シフトによって示される。すなわち、ミトコンドリアが障害を受けた時には、JC-I色素のミトコンドリア内部への蓄積が減少するため、赤色の比率が低下し、緑色が支配的になってくる。このことから、ミトコンドリアの膜電位は、赤色/緑色の蛍光強度比の減少によって示すことができる(Circulation.2005;111:p2752-2759)。そこで、心筋細胞に24時間の高グルコース負荷を行った後、JC-1色素を取り込ませて(37℃、30分)共焦点レーザー顕微鏡で蛍光画像を取得し評価した。
図10及び図11に示されるように、正常グルコース濃度でのミトコンドリア膜電位は、ガラス支持体での培養心筋細胞よりも、15 kPaゲル支持体上の培養心筋細胞の方がより顕著に高くなっていることが見出された。更に、15 kPaゲル支持体上の培養心筋細胞においては、中程度の高グルコース濃度(15 mM)でミトコンドリア膜電位が、有意に減少するが、ROS除去剤NAC(1 mM)によってROSを除去すると顕著に回復した。この結果は、ミトコンドリア内でのROS蓄積の結果と相関していた。一方でガラス上の培養心筋細胞では、高グルコース負荷で低下した膜電位がROS除去剤によって回復せず、ROS蓄積レベルとミトコンドリア膜電位の減少との間に相関性は見られなかった。即ち、ガラス支持体よりも、15 kPaゲル支持体上の培養心筋細胞の方が、基底状態におけるミトコンドリア活性が高く、かつグルコース濃度上昇に伴うROS蓄積に応じてミトコンドリア活性が低下し、生体内で心筋細胞が酸化ストレスに暴露された際の挙動に近いことを示している。この相違点については次のアポトーシスアッセイの結果と合わせて考察する。
(1)方法
15 kPaゲル支持体上とガラス支持体上での初代培養心筋細胞のアポトーシスの相違を比較するため、In situ Cell Death Detection kit-FITC (Roche)を用いてTUNEL(terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling)アッセイを行った。なお、TUNELアッセイは、アポトーシスのシグナル伝達経路の中で派生してくるDNA断片を検知する一般的な方法である(Cell Death and Disease(2014)5, e1479; doi:10.1038/cddis. 2014.430)。DNAの断片化を起こした細胞をラベル化し緑色の蛍光として捕捉することができる。細胞核を染めるDAPI色素と二重染色することで、一視野あたりの死細胞(TUNEL陽性核/全核数)を算出することができる。心筋細胞を高グルコース負荷した後、TUNEL色素を取り込ませ反応させた(37℃、60分)。その後、DAPIを含んだ蛍光劣化防止剤で封入し正立蛍光顕微鏡で画像を取得して解析した。
a)培養支持体の相違による初代培養心筋細胞のアポトーシス確認
図12に示すように、DNaseI処理によるTUNEL陽性核は、ガラス支持体と15 kPaゲル支持体のどちらでも同様に観察された。この結果から、15 kPaゲル支持体上でのアポトーシス細胞を評価できると考えられた。
図13及び図15に示すように、高グルコース濃度がアポトーシスにもたらす影響を、TUNELアッセイで評価した。その結果、ガラス支持体上での培養心筋細胞では正常グルコース濃度においてもアポトーシスを認め、高グルコース濃度に曝露するとアポトーシスが著明に誘導された。更にROS除外剤NAC(1 mM)を添加してもアポトーシスは抑制されなかった。
図14及び図15に示すように、15 kPaゲル支持体上の培養心筋細胞中のTUNEL陽性核は、ガラス支持体上の培養心筋細胞と比較すると顕著に少なかった。
(1) 方法
実施例5と同様に行った。15 kPaゲル支持体上とガラス支持体上での初代培養心筋細胞を培養し、高グルコース負荷を行った(15 mM、24時間)。その後、培養液をROS除去剤、あるいは正常グルコース濃度(5 mM)のものに交換して24時間培養した後JC-1色素を取り込ませて(37℃、30分)共焦点レーザー顕微鏡で蛍光画像を取得し評価した。
図16に示すように、15 kPaゲル上の培養心筋細胞のミトコンドリア膜電位は、高グルコース濃度(15 mM)による培養の後にROS除去剤の入った培養液に交換することで回復した。また、正常グルコース濃度の培養液に交換すると、部分的にミトコンドリア膜電位が回復することがわかった。一方、ガラス支持体上の心筋細胞のミトコンドリア膜電位は、ROS除去剤入りの培養液に交換、あるいは正常グルコース濃度の培養液に交換しても回復しないことがわかった。
骨格筋細胞における高血糖の影響を確認するため、2型糖尿病患者のありふれた高血糖である10~15 mMグルコース濃度がミトコンドリアに及ぼす影響を、15 kPaのゲル上の培養骨格筋細胞と、ガラス上の培養骨格筋細胞とで比較することを行った。
(1)方法
ラット骨格筋芽細胞はコスモバイオから入手した。細胞と共に提供された分化用培地(内容非公開)を用い、コスモバイオ指定のプロトコールに則って骨格筋細胞に分化させた。この骨格筋細胞を実施例1に記載と同様の方法で準備したカバーグラス上、または15 kPaのゲル上に蒔いた。実施例5に記載の方法で高血糖処理およびROS除去剤(NAC)処理を行い、ROS蓄積を蛍光試薬CM-H2DCFDAによる染色(図17A)で、ミトコンドリア膜電位をJC-1による染色(図17B)で評価した。
(2)結果
図17に示すように、培養心筋細胞と同様、培養骨格筋細胞においても正常グルコース濃度でのROS蓄積はガラス支持体での培養よりも15 kPaゲル支持体上の培養の方がより顕著に低く、またミトコンドリア膜電位は、ガラス支持体での培養よりも15 kPaゲル支持体上の培養の方がより顕著に高くなっていることが見出された。更に、15 kPaゲル支持体上の培養骨格筋細胞においては、中程度の高グルコース濃度(15 mM)でROSが優位に蓄積してミトコンドリア膜電位が有意に減少するが、ROS除去剤NAC(1 mM)によってROSが除去されると同時にミトコンドリア膜電位も顕著に回復した。一方ガラス支持体上の骨格筋細胞においてはROS除去剤によるROSの低下やミトコンドリア膜電位の回復は見られなかった。この結果は、培養心筋細胞の場合と同様に、ガラス支持体よりも15 kPaゲル支持体上の培養骨格筋細胞の方が、生体内で骨格筋細胞が酸化ストレスに暴露された際の挙動に近いことを示している。
(1)方法
ヒト初代培養肝細胞において、従来の初代培養肝細胞培養で最も生体内の機能を忠実に反映しうると考えられているスフェロイド培養に対し、正常肝組織の剛性に一致する500 Paの支持体の優位性の有無を評価した。IV型コラーゲンで表面をコーティングした500 Paの支持体または細胞培養で通常使用されるが非生理的に高い剛性であるガラスにヒト初代培養肝細胞を蒔き、実施例9と同様に培養した。またCell-able(住友ベークライト)を用い、ヒト初代培養肝細胞でスフェロイドを形成させた。12日間培養後、CYP3A4を誘導することで知られているリファンピシンを用い、0または40 μMのリファンピシンで46時間刺激した。CYP3A4活性をP450-Glo CYP3A4 Assay (Luciferin-IPA) (Promega)で測定して細胞数で除し、リファンピシン刺激時のCYP3A4活性を基底状態のCYP3A4活性で除することにより、リファンピシンによるCYP3A4活性の上昇度を求めた。
ヒト初代培養細胞においてリファンピシン刺激によるCYP3A4活性上昇度は、500 Paの支持体が最高であった。したがって正常肝組織の剛性に近い支持体上での培養が、スフェロイドによる三次元培養に比較し、CYP3A4発現を誘導する薬剤刺激時でCYP3A4活性上昇において優位であることが示唆された(図18)。
ジクロフェナク、トログリタゾン、ラニチジン等は、特発性薬剤肝障害を起こすことが知られている。一方、アセトアミノフェン、エタノール等は、過剰摂取すれば肝毒性を示すものの、特発性薬剤肝障害を惹起することはない。特発性薬剤肝障害のリスクをスクリーニングするための評価系を探索するため、以下の実験を行った。
正常肝組織の硬度に一致する500 Paの支持体上で培養されている初代培養肝細胞は、細胞培養で通常使用されるが非生理的に高い硬度であるガラス上で培養されている初代培養肝細胞に比較し、生体内における肝細胞機能をより正確に発現すると想定される。そこで化合物による特発性薬剤性肝障害の発生予測において、500 Paの支持体上で培養されている初代培養肝細胞の優位性の証明を目指した。High-mobility group box 1 protein (HMGB1)は細胞死の過程で細胞外に放出され、免疫細胞を活性化する作用を持つ(Immunol Rev. 2017 Nov;280(1):74-82)。そこで特発性薬剤性肝障害の過程の早期検出マーカーに、肝細胞が放出するHMGB1が有用であるとの仮説を設定し、その証明を行った。
ジクロフェナクは250 μM以上の高濃度で、アセトアミノフェンは2.5 mM以上の高濃度で細胞毒性を示すことが報告されている(J Toxicol Sci. 2016;41(5):605-15)。特発性薬剤性肝障害を起こしうるジクロフェナク刺激の場合、500 Paの支持体において既報の毒性領域と一致して濃度依存性に肝細胞によるHMGB1放出が見られた。ガラスにおいては毒性領域より低濃度のジクロフェナクでHMGB1放出が見られた。また特発性薬剤性肝障害を起こさないアセトアミノフェン刺激の場合、500 Paの支持体、ガラス支持体のいずれでも毒性領域である高濃度でもHMGB1放出は見られなかった。したがってHMGB1は化合物による特発性薬剤性肝障害の発生を予測するマーカーになりうること、またその予測において正常肝組織の剛性と同等な支持体上で培養された初代培養肝細胞が有用であることが示唆された(図19)。
非アルコール性脂肪肝炎は、生活習慣が原因で肝細胞が死滅しないまでも、肝細胞が障害され引き起こされると考えられる。
非アルコール性脂肪肝炎のメカニズムを解明するため、以下の実験を行った。
ラットの初代培養肝細胞は以下の方法で採取した。
5~6週齢のWistar rat(オス)に麻酔をした後、腹部を切開し門脈がよくみえるように結合組織類を剥離した。20 Gのサーフローで門脈をクランプし、クリップで固定した。37℃に加温したEGTA灌流液で灌流し肝臓の血液を洗い流した。肝臓が黄土色に変わった後、37℃に加温したコラゲナーゼ液で灌流した。十分にコラゲナーゼを作用させた後、肝臓を摘出しシャーレに移し、ハサミやメスで細断した。その後、遠心分離、セルストレイナーでろ過し、再度遠心分離した。沈殿に培養液を加えピペッティング後、トリパンブルー染色で細胞の生存率を確認した。生存率が75%以下のものは除外した。同時に細胞数のカウントも行い3.5x10^5個/well(6ウェルプレート)ずつ細胞を500 Paの支持体、またはガラス支持体に蒔き、5%FCS及び Hepatocyte Maintenance Supplement Pack(サーモフィッシャー株式会社CM4000)加D-MEM(低グルコース)(L-グルタミン、フェノールレッド含有)で3日間培養した。 その後、被験物質として、フルクトース(5.5mM)、パルミチン酸(0.5mM)、又はオレイン酸-パルミチン酸(0.5mM、OA:PA=2:1)を添加し、24時間後に脂肪滴染色、及びROSの定量を行った。被験物質を添加する際、培養培地は5 mMグルコースを含む10%FCS加DMEMに交換した。コントロールには何も加えなかった。コントロール群は脂質を溶解するときに使用した10%ウシ血清アルブミンを添加した。
図20に示すように脂肪滴の蓄積は、ガラス支持体上で培養された初代培養肝細胞においては、コントロール(control)と、フルクトース(fructose)、パルミチン酸(PA)、又はオレイン酸-パルミチン酸混合液(OA:PA(2:1))との間に差は認められなかった。一方500Paの支持体上で培養された初代培養肝細胞では、フルクトース、パルミチン酸、オレイン酸-パルミチン酸混合液を添加された細胞は、コントロールと比較して顕著な脂肪滴の蓄積が認められた。このことから、ガラス支持体では、細胞への脂肪の蓄積は観察できないが、正常肝組織の剛性と同等な支持体上で培養された初代培養肝細胞では、脂肪の蓄積をin vitroで発現できることが示された。
非アルコール性脂肪肝炎のメカニズムを解明するため、以下の実験を行った。
培養支持体の硬度の相違による、肝細胞のマクロファージに対する炎症惹起作用への影響を評価するため、ラット初代培養肝細胞を、正常肝組織の硬度に一致する500 Paのポリアクリルアミドゲル、または細胞培養で通常使用されるが非生理的に高い硬度であるガラス上で培養した。細胞の接着を促すため、それぞれの培養支持体の表面をIV型コラーゲンでコーティングした。翌日、ラット初代培養肝細胞を含むウェルに106個/ウェルのマウス腹腔マクロファージを加え、共培養を開始した。マウス腹腔マクロファージは、invitrogenの「マウス初代腹腔マクロファージへの遺伝子導入の検討」に記載の方法でチオグリコネート刺激、および腹腔マクロファージの採取を行った。具体的には、C57/BL6J マウス腹腔内に5%thioglycollate medium (Sigma) 2 mlを注入し、3.5日後に断頭の上、シリンジ及び注射針を用いて計15mlのPBS(7 ml+8 ml) にて2 回腹腔内を洗浄し腹腔マクロファージを回収した。回収した腹腔洗浄液は1000rpm、4℃、5 分間遠心後、PBSにて2 回洗浄し、細胞数を算定した。同肝細胞-マクロファージ共培養開始24時間後にフルクトース刺激を加え、その24時間後に培養液を採取してTNFα分泌量をELISAにて測定した。
図22に示すように、500 Paのポリアクリルアミドゲルを用いた場合、マクロファージ共培養でTNFα分泌量が増加しており、マクロファージによるTNFα分泌を観測できた。フルクトース刺激による過栄養状態でTNFα分泌がさらに有意に増加しており、脂肪肝の状態を模倣した肝細胞がマクロファージを刺激し、マクロファージによるTNFα分泌を増加させたものと考えられた。一方ガラスを用いた場合でも、マクロファージ共培養でTNFα分泌量が増加しており、マクロファージによるTNFα分泌を観測できているが、フルクトース刺激によるTNFα分泌増加は見られなかった。
Claims (24)
- 細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体と細胞とを接触させる工程と、
in vivoの細胞の特性をin vitroで導入及び/又は発現した細胞の特性をin vitroで維持する工程と、
前記特性が導入、及び/又は維持されている細胞に、シグナルを入力する工程と、
入力されたシグナルに対する細胞の反応を測定する工程と、
を含む、細胞の反応の評価方法(ただし、前記細胞に間葉系幹細胞は含まない)。 - 前記シグナルは、化学的因子及、物理的因子及び生物学的因子よりなる群から選択される少なくとも一つの因子を細胞に負荷することによって細胞に入力される、請求項1に記載の方法。
- 化学的因子が、化合物、イオン、気体、核酸、糖質、脂質、糖タンパク質、糖脂質、リポタンパク質、アミノ酸、ペプチド、タンパク質、ポリフェノール類、サイトカイン類及びケモカインよりなる群から選択される少なくとも一種である、請求項1又は2に記載の方法。
- 物理的因子が、細胞の周囲環境の剛性、圧力、張力、光、放射線、酸素濃度、pH及び温度よりなる群から選択される少なくとも一種である、請求項1又は2に記載の方法。
- 生物学的因子が、細菌、真菌、ウイルス、アレルゲン、ヒト細胞、ヒト以外の動物細胞及びこれらに含まれる成分より選択される少なくとも一種である、請求項1又は2に記載の方法。
- 細胞が肝細胞であり、支持体の剛性が0.2~5kpaである、請求項1~5のいずれか一項に記載の方法。
- 細胞の反応が薬物代謝酵素の誘導、非アルコール性脂肪肝炎のメカニズム、又は特発性肝障害を示す、請求項6に記載の方法。
- 細胞の反応が前記因子の効能又は毒性である、請求項1~5のいずれか一項に記載の方法。
- 細胞が心筋細胞であり、支持体の剛性が5~100kpaである、請求項1~5のいずれか一項に記載の方法。
- 化学的因子が酸化ストレス誘導物質であり、細胞の反応が酸化ストレス応答である、請求項9に記載の方法。
- 酸化ストレス誘導物質が、グルコースである、請求項10に記載の方法。
- 細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体と細胞とを接触させる工程と、
in vivoの細胞の特性をin vitroで導入及び/又は発現した細胞の特性をin vitroで維持する工程と、
前記特性が導入、及び/又は維持されている細胞に、シグナルを入力する工程と、
シグナルの入力に先立って、シグナルの入力と同時に、又はシグナルの入力の後に、疾患、又は障害を予防、治療又は改善するための候補物質と細胞とを接触させる工程と、
入力されたシグナルに対する細胞の反応を測定する工程と、
を含む、疾患、又は障害を予防、治療又は改善するための候補物質のスクリーニング方法(ただし、前記細胞に間葉系幹細胞は含まない)。 - 前記シグナルは、化学的因子及、物理的因子及び生物学的因子よりなる群から選択される少なくとも一つの因子を細胞に負荷することによって細胞に入力される、請求項12に記載の方法。
- 化学的因子が、化合物、イオン、気体、核酸、糖質、脂質、糖タンパク質、糖脂質、リポタンパク質、アミノ酸、ペプチド、タンパク質、ポリフェノール類、サイトカイン類及びケモカインよりなる群から選択される少なくとも一種である、請求項12又は13に記載の方法。
- 物理的因子が、細胞の周囲環境の剛性、圧力、張力、光、放射線、酸素濃度、pH及び温度よりなる群から選択される少なくとも一種である、請求項12又は13に記載の方法。
- 生物学的因子が、細菌、真菌、ウイルス、アレルゲン、ヒト細胞、ヒト以外の動物細胞及びこれらに含まれる成分より選択される少なくとも一種である、請求項12又は13に記載の方法。
- 細胞が肝細胞であり、支持体の剛性が0.2~5kpaである、請求項12~16のいずれか一項に記載の方法。
- 細胞の反応が薬物代謝酵素の誘導、非アルコール性脂肪肝炎のメカニズム、又は特発性肝障害を示す、請求項17に記載の方法。
- 細胞の反応が前記因子の効能又は毒性である、請求項12~16のいずれか一項に記載の方法。
- 細胞が心筋細胞であり、支持体の剛性が5~100kPaである、請求項12~16のいずれか一項に記載の方法。
- 化学的因子が酸化ストレス誘導物質であり、細胞の反応が酸化ストレス応答である、請求項20に記載の方法。
- 酸化ストレス誘導物質が、グルコースである、請求項20に記載の方法。
- 細胞のin vivoでの特性をin vitroで発現するために、細胞が生体内で認識する周囲環境の剛性を反映した剛性を有し、かつ前記剛性が剪断弾性率で100kPa以下である支持体を含む、請求項1~11に記載の細胞の反応の評価方法、又は請求項12~22に記載のスクリーニング方法を実施するためのキット。
- さらに目的とする細胞の反応の評価又はスクリーニングを実施するために適した細胞を含む、請求項23に記載のキット。
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