WO2022270932A1 - Composition for culturing stomach cells - Google Patents

Composition for culturing stomach cells Download PDF

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Publication number
WO2022270932A1
WO2022270932A1 PCT/KR2022/008916 KR2022008916W WO2022270932A1 WO 2022270932 A1 WO2022270932 A1 WO 2022270932A1 KR 2022008916 W KR2022008916 W KR 2022008916W WO 2022270932 A1 WO2022270932 A1 WO 2022270932A1
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cells
concentration
composition
gastric
growth factor
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PCT/KR2022/008916
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French (fr)
Korean (ko)
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남기택
이부현
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연세대학교 산학협력단
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Publication of WO2022270932A1 publication Critical patent/WO2022270932A1/en

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/345—Gastrin; Cholecystokinins [CCK]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • the present invention relates to a composition for culturing cells isolated from a biological tissue or organ, particularly the stomach, and a cell culture method using the same.
  • the epithelial tissue of the stomach corresponds physiologically to a self-renewing tissue.
  • stem cells exist only in the upper part of the gastric gland, but recently, research on chief cells as stem cells existing in the lower part is being conducted. However, it was not possible to clearly distinguish and confirm its function or role.
  • the main cell is a cell that secretes pepsin and gestic lipase and is located at the base.
  • Homeostasis in the stomach depends on the balance of production and maintenance of various cell lineages, such as acid-producing parietal cells, mucus-secreting throat cells, and zymogen-secreting principal cells.
  • mucus-secreting throat cells transdifferentiate into principal cells as they migrate from the parietal cells to the base of the gastric gland.
  • One object of the present invention is to provide a composition for culturing cells isolated from the stomach.
  • Another object of the present invention is to provide a method for culturing gastric cells using the above composition.
  • the present invention relates to a composition for culturing stomach cells.
  • the cell may be a chief cell.
  • the "gastric chief cell” is also called a peptic cell or a gastric zymogenic cell, and is a gastric gland cell that secretes pepsinogen and gastric lipase. ), and also applies to cells that secrete chymosin in ruminants.
  • the culture composition of the present invention contains at least one of bovine serum albumin (BSA) and insulin-transferrin-selenium-sodium pyruvate (ITS-A) with respect to a basal culture medium.
  • BSA bovine serum albumin
  • ITS-A insulin-transferrin-selenium-sodium pyruvate
  • the bovine serum albumin has a working concentration of 1 to 20 w/v%, preferably 5 to 15 w/v%, more preferably 8 to 12 w/v%, based on the basal culture medium. It may be w/v%.
  • the insulin-transferrin-selenium-sodium pyruvate has a working condition of 1 to 15 ug/ml, preferably 5 to 13 ug/ml, More preferably, it may be 6 to 10 ug/ml.
  • the culture composition of the present invention may further include hepatocyte growth factor (HGF) with respect to the basal culture medium.
  • HGF hepatocyte growth factor
  • the "hepatocyte growth factor (HGF) or scatter factor (SF)” is a paracrine cell growth, motility and morphogenesis factor, secreted by mesenchymal cells and mainly epithelial It acts on cells and endothelial cells and acts on hematopoietic progenitor cells and T cells.
  • the HGF can be used without limitation as long as it is a protein encoded by the mammalian HGF gene, and HGF expressed by cells genetically engineered to express the HGF gene can be used, or chemically or biochemically. Any synthetic, commercially available (for example, HGF manufactured by RD Systems) may be used without limitation.
  • the origin of the HGF is not particularly limited as long as it is a mammal, and for example, it may be human HGF (hHGF) or mouse HGF (mHGF), but mHGF may be preferably used.
  • the hepatocyte growth factor has a working concentration of 100 to 1000 ng/ml, preferably 100 to 500 ng/ml, more preferably 100 to 300 ng/ml with respect to the basal culture medium. can be ml.
  • the culture composition of the present invention contains Wnt3a, R-spondin, Noggin, B27, N-acetyl cysteine (NAC), fibroblast growth factor (FGF) , epidermal growth factor (EGF), gastrin (gastrin), Y-27632, and hydrocortisone (hydrocortisone) may further include one or more selected from the group consisting of.
  • composition for culture of the present invention may further include Wnt3a or conditioned media containing the same, wherein the working concentration is 10 to 80 v/v%, preferably with respect to the basal culture medium. It may be 30 to 70 v/v%, more preferably 40 to 60 v/v%.
  • the culture composition of the present invention may further include R-spondin or a conditioned medium containing the same, wherein the basal culture medium has a working concentration of 0.1 to 30 It may be v/v%, preferably 1 to 20 v/v%, and more preferably 5 to 15 v/v%.
  • the culture composition of the present invention may further include Noggin.
  • the Noggin can be used without limitation as long as it is a protein encoded by the mammalian NOG gene, and Noggin expressed by cells genetically engineered to express the NOG gene can be used, or chemically or biochemically synthesized Noggin can be used. Any one, such as a commercially available one (for example, Noggin manufactured by RD Systems), can be used without limitation.
  • the origin of the Noggin is not particularly limited as long as it is a mammal, and for example, it may be derived from a human or a mouse, but preferably a mouse-derived one can be used.
  • the working concentration of Noggin in the basal culture medium may be 10 to 200 ng/ml, preferably 50 to 150 ng/ml, and more preferably 80 to 120 ng/ml.
  • the composition for culture of the present invention may further contain B27.
  • the B27 may have a working condition of 0.005 to 0.1 v/v%, preferably 0.01 to 0.05 v/v%, and more preferably 0.01 to 0.03 v/v% with respect to the basal culture medium. .
  • composition for culture of the present invention may further include N-acetyl cysteine (NAC), wherein the N-acetyl cysteine (NAC) has a working concentration of 0.1 to 5 mM, preferably with respect to the basal culture medium. Preferably it may be 0.5 to 2.5 mM, more preferably 1 to 1.5 mM.
  • NAC N-acetyl cysteine
  • the composition for culture of the present invention may further include fibroblast growth factor (FGF).
  • FGF fibroblast growth factor
  • the fibroblast growth factor (FGF) may be one or more selected from the group consisting of FGF1, FGF2 (bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF17, and FGF18.
  • FGF fibroblast growth factor
  • the fibroblast growth factor (FGF) is not particularly limited as long as it is a mammal, and for example, a human-derived one can be used.
  • the fibroblast growth factor has a working concentration of 100 to 1000 ng/ml, preferably 100 to 500 ng/ml, more preferably 100 to 1000 ng/ml with respect to the basal culture medium. 300 ng/ml.
  • the fibroblast growth factor may be FGF2 (bFGF), and the working concentration is 50 to 200 ng/ml, preferably 50 to 150 ng/ml with respect to the basal culture medium. ml, more preferably 80 to 120 ng/ml.
  • the fibroblast growth factor (FGF) may be hFGF10, and the working concentration (working condition) with respect to the basal culture medium is 50 to 200 ng / ml, preferably 50 to 150 ng / ml, more Preferably it may be 80 to 120 ng/ml.
  • the culture composition of the present invention may further include epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • the origin of the epidermal growth factor (EGF) is not particularly limited as long as it is a mammal, and for example, it may be derived from a human or a mouse, but preferably a mouse-derived one may be used.
  • the epidermal growth factor has a working concentration of 5 to 100 ng/ml, preferably 10 to 50 ng/ml, more preferably 20 to 30 ng/ml with respect to the basal culture medium. can be ml.
  • composition for culture of the present invention may further include gastrin, and the gastrin has a working condition of 0.01 to 10 uM, preferably 0.1 to 5 uM, more preferably 0.1 to 5 uM with respect to the basal culture medium. may be 0.5 to 1.5 uM.
  • composition for culture of the present invention may further include Y-27632, and the Y-27632 has a working concentration of 1 to 50 mM, preferably 5 to 30 mM, more preferably 5 to 30 mM, with respect to the basal culture medium. It may be 5 to 15 mM.
  • composition for culture of the present invention may further include hydrocortisone.
  • the hydrocortisone may have a working concentration of 0.01 to 10 ug/ml, preferably 0.1 to 5 ug/ml, and more preferably 0.5 to 1.5 ug/ml, based on the basal culture medium.
  • the composition for culture of the present invention is MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified EagleMedium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM as the basic culture medium (Glasgow's MEM), F12 (Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5 A , Media 199, MEM ⁇ Media, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM/F-12, Advanced RPMI1640, etc. may be used, but is not limited thereto.
  • the basal culture medium may have a working condition of 10 to 50 v/v%, preferably 20 to 40 v/v%, and more preferably 30 to 40 v/v%.
  • the basal culture medium may further include at least one of glutamax, hydroxyethyl piperazine ethane sulfonic acid (HEPES), and an antibiotic.
  • glutamax hydroxyethyl piperazine ethane sulfonic acid (HEPES)
  • HEPES hydroxyethyl piperazine ethane sulfonic acid
  • the working concentration of glutamax in the basal culture medium is 0.01 to 10 v/v%, preferably 0.1 to 5 v/v%, more preferably 0.5 to 1.5 v It can be /v%.
  • the hydroxyethylpiperazineethanesulfonic acid is 0.01 to 10 mM, preferably 0.1 to 5 mM, more preferably 0.5 to 1.5 mM at a working condition in the basal culture medium.
  • the antibiotic may include at least one of penicillin and streptomycin.
  • the working concentration of the antibiotic in the basal culture medium may be 50 to 200 ng/ml, preferably 50 to 150 ng/ml, and more preferably 80 to 120 ng/ml.
  • composition for culture of the present invention can be used for inducing differentiation of stomach cells into gastric mucosal cells.
  • the gastric mucosal cells may be at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells.
  • the "surface mucous cells (mucous foveolar cells)" is a cell that secretes mucus, and serves to protect the stomach from hydrochloric acid by covering the inside of the stomach.
  • the “parietal cell” is a gastric epithelial cell that secretes hydrochloric acid (HCl) and intrinsic factor, and is known to be distributed in gastric glands.
  • HCl hydrochloric acid
  • the "mucus neck cell” is a cell that secretes mucin, and is mostly located at the top of the gastric gland.
  • composition for culturing of the present invention can be used as a composition for culturing gastric organoids.
  • the "organoid” of the present invention refers to a cell having a 3D three-dimensional structure, and refers to a model similar to a tissue prepared through an artificial culture process that is not collected or obtained from an animal or the like. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro.
  • the gastric organoid may be a gastric mucosal organoid.
  • it relates to a method for culturing gastric main cells.
  • the culturing method of the present invention may include culturing the isolated chief cells in the composition for culturing according to the present invention.
  • the culturing may be performed at a temperature of 35 to 40 °C for 1 hour to 120 days, but is not limited thereto.
  • it relates to a method for preparing gastric organoids.
  • the manufacturing method of the present invention may include forming an organoid by putting the isolated chief cells into Matrigel and the composition for culture according to the present invention.
  • the matrigel may be growth factor reduced matrigel, but is not limited thereto.
  • the gastric organoid prepared in the present invention may be a gastric mucosal organoid containing at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells. there is.
  • a gastric organoid prepared by the method for preparing a gastric organoid of the present invention is provided.
  • the gastric organoid of the present invention is a gastric mucosal organoid containing at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells, Not only can it be used to effectively screen gastric injury therapeutic agents, but it can also be used as a cell therapy directly used for gastric injury.
  • cell therapy refers to cells and tissues prepared from humans through isolation, culture, and special chewing, which are medicines used for the purpose of treatment, diagnosis, and prevention. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating or selecting allogeneic or heterogeneous cells in vitro, or changing the biological characteristics of cells in other ways.
  • composition for living body transplantation comprising the gastric organoid of the present invention as an active ingredient.
  • the "bio implanting” may refer to a phenomenon that is administered to a subject and becomes a transplant site.
  • the “individual” may be an individual who has suffered gastric damage or needs to reconstruct, restore, or form the stomach by transplanting organoids to at least a portion of the stomach through gastrectomy or the like.
  • the gastric organoid is 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 4 ⁇ 10 8 , 4 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 8 ⁇ 10 8 , 8 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 2 ⁇ 10 9 , 2 ⁇ 10 9 to 4 ⁇ 10 9 , 4 ⁇ 10 9 to 1 ⁇ 10 10 , 2 ⁇ 10 8 to 6 ⁇ 10 8 , 6 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 8 to 2 ⁇ 10 8 , 2 ⁇ 10 8 to 2 ⁇ 10 9 , 1 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 1 ⁇ 10 10 or 1 ⁇ 10 7 to 1 ⁇ 10 9 may be transplanted in an amount of any one cell / kg, but is limited thereto no.
  • the pharmaceutical composition of the present invention may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.
  • compositions of the present invention are not limited thereto, but are formulated in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration.
  • buffers, preservatives, painless A topical agent, a solubilizer, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, and the like may be used.
  • Formulations of the pharmaceutical composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above.
  • the pharmaceutically acceptable carrier for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is.
  • it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like can be used.
  • fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
  • the route of administration of the pharmaceutical composition of the present invention is not limited thereto, but is not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal administration is included, and may be, for example, oral or parenteral administration.
  • the "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition may be administered by direct injection into the kidney, but is not limited thereto.
  • the pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. It can vary widely, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, drug form, administration route and period, but can be appropriately selected by those skilled in the art, 0.0001 to 50 mg/day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
  • chief cells of the stomach can be differentiated into mucous foveolar cells, parietal cells, and mucus neck cells. Furthermore, gastric mucosal organoids can be formed, so that it can be used as a cell therapy.
  • FIG. 1 is a schematic diagram of a method for separating chief cells, pit cells, parietal cells, and isthmus cells after each treatment in the gastric organ according to the present invention.
  • Figure 2 shows a photograph of tying the junction of the esophagus and the stomach of the mouse using a sterilized suture in Example 1.
  • Figure 3 shows a photograph of washing by immersing in a PBS solution after extracting the stomach of a mouse in Example 1.
  • Figure 4 shows a photograph after turning the forestomach of the mouse excised with a sterilized cotton swab in Example 1 so that the gastric mucosa is exposed to the outside.
  • Example 5 shows a photograph after tying the fundus of the inverted stomach with a sterilized suture in Example 1 so that the mucous membrane is exposed to the outside.
  • FIG. 6 shows a photograph of injecting a solution for first cell isolation using an insulin syringe into a forestomach region of a mouse in Example 1.
  • Example 7 is a photograph showing a fraction 1 solution obtained by immersing the stomach in the second cell isolation solution in Example 1 and stirring for 30 minutes at a speed of 60 RPM in a 37 ° C incubator.
  • FIG. 8 shows a photograph of obtaining a fraction 2 solution by taking the stomach out of the fraction 1 solution in Example 1, immersing it in a third cell separation solution and stirring for 30 minutes under the same conditions.
  • Figure 9 shows a photograph of the fraction 5 solution in Example 1 passing through a 100 ⁇ m sieve filter.
  • FIG. 10 shows a photograph in which the main cells were precipitated by centrifugation of the Fraction 5 solution passed through the sieve filter in Example 1.
  • H&E hematoxylin & eosin
  • the present invention relates to a composition for culturing isolated chief cells for research on the function and role of chief cells present in conventional gastric glands. Unlike conventional methods, the present invention provides a culture composition for culturing differentiated main cells by specifically isolating only main cells by exposing the gastric mucosa to the outside.
  • a culture composition for differentiating the separated main cells it can differentiate all lineages, especially parietal cells, which have been difficult to differentiate in the past, and form gastric mucosal organoids, thereby providing an important foothold for the development of therapeutic compositions related to gastrointestinal diseases. there is.
  • a basic solution was prepared by adding each component in the composition shown in Table 1 to sterilized tertiary distilled water.
  • a first cell separation solution was prepared by adding the components shown in Table 2 to the basic solution prepared in Preparation Example 1.
  • the solution prepared in this way was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 ⁇ m before use.
  • working concentration 1st Cell Separation Solution
  • EDTA 2mM/basic solution
  • BSA 1w/v% / basic solution
  • Proteinase E 2.5mg/ml basic solution
  • Collagenase Type 1 4mg/ml basic solution
  • a second cell separation solution was prepared by adding the components shown in Table 3 to the basic solution prepared in Preparation Example 1.
  • the solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 ⁇ m before use.
  • a third cell separation solution was prepared by adding the ingredients shown in Table 4 to the basic solution prepared in Preparation Example 1 above.
  • the solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 ⁇ m before use.
  • the above main cell culture solution was prepared by adding each component to the basal culture medium according to the composition shown in Table 5 below.
  • the solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 ⁇ m before use.
  • FIG. 1 is a schematic diagram of a method for separating chief cells, pit cells, parietal cells, and isthmus cells after each treatment in the gastric organ according to the present invention.
  • 8-week-old C57BL/6 mice were euthanized using a CO 2 chamber, the abdominal cavity was incised, and the junction of the esophagus and stomach was tied using a sterilized suture (FIG. 2).
  • the stomach was removed, it was immersed in a PBS solution and washed (FIG. 3), and the anterior part (Antrum/Pylorus) except for the fundus part was removed using scissors.
  • the forestomach part was pushed with a sterilized cotton swab and turned inside out so that the gastric mucosa was exposed to the outside (FIG. 4).
  • the incised fundus was tied with a sterile suture (FIG. 5).
  • the stomach was inflated by injecting the first cell isolation solution into the Forestomach region using an insulin syringe (FIG. 6). Thereafter, the stomach was immersed in the second cell separation solution and stirred at 60 RPM for 30 minutes in an incubator at 37 ° C (Fig. 7) (fraction 1 solution), and after 30 minutes, the stomach was taken out of the obtained fraction 1 solution to separate the third cells.
  • Fraction 5 solution was passed through a 100 ⁇ m sieve filter (FIG. 9) and centrifuged at 12000 RPM to precipitate main cells contained in the solution (FIG. 10). The supernatant was removed and only the main precipitated cells were obtained.
  • Hematoxylin & eosin (H&E) staining was performed on the gastric tissue excised from the mouse in Example 1 and the gastric tissue filtered from the Fraction 5 solution, and the results are shown in FIG. 11 .
  • Gif and GPR43 which are major cell-specific proteins, were expressed in all the isolated cells.
  • Example 1 the cells precipitated and recovered from the Fraction 5 solution obtained in Example 1 were the main cells derived from the stomach.
  • Example 1 the 8-week-old C57BL/6 mouse was euthanized using a CO 2 chamber, the abdominal cavity was incised, and the junction of the esophagus and stomach was tied using a sterile suture, and the stomach was removed and then placed in a PBS solution. Washed by immersion. Thereafter, the extracted stomach was finely chopped (chopping), cultured with EDTA, and single cells were isolated through trypsin treatment.
  • Figure 14 shows a schematic diagram of this experiment, after subcutaneously administering 5 mg of Tamoxifen 3 times to a reporter mouse (Mist1creEr; R26-tdTomato) capable of expressing RFP fluorescent protein specifically in the main cell present in the stomach. , The stomach was removed to obtain a fraction 5 solution in the same manner as in Example 1, and cells precipitated and recovered therefrom were observed under a microscope. The results are shown in FIG. 15 .
  • a reporter mouse Mist1creEr; R26-tdTomato
  • RFP fluorescent protein was expressed as a whole in the cells precipitated and recovered from the fraction 5 solution.
  • a fraction 1 solution, a fraction 3 solution, and a fraction 5 solution were obtained in the same manner as in Example 1 for germ free (GF) mice and specific pathogen free (SPF) mice. Thereafter, mRNA was extracted from cells present in each fraction solution, and expression levels of various gastric epithelial cell markers were confirmed by qRT-PCR, and the results are shown in FIG. 16 .
  • Ki67 a proliferative cell marker, in Fraction 5 solution cells derived from SPF mice and in Fraction 5 solution cells derived from GF mice, Ki67 was expressed more strongly in Fraction 5 solution cells derived from GF mice. As confirmed, it was found that the stem cell activity of the GF mouse-derived Fraction 5 solution cells was superior to that of the Fraction 5 solution cells derived from SPF mice.
  • the main cells derived from the Fraction 5 solution obtained from the SPF mouse and the GF mouse in Experimental Example 4 were cultured for 7 days after passing one passage each, and immunostaining of gastric epithelial cells differentiated from these main cells was performed. 17.
  • Example 4 After diluting the main cell pellet isolated in Example 1 in 1 ml of RPMI1640 culture medium, counting the cells, centrifuging again, mixing the cell aggregates with Matrigel (15 ⁇ l/well), dispensing into 24-well plates, and then at 37 ° C. Incubated for 1 hour. Then, the main cell culture solution of Preparation Example 4 was added and cultured in an incubator at 37° C., 5 vol% gas CO 2 . The culture medium was freshly replaced every 2-3 days, and differentiation into other epithelial cells was observed after 5 days of culture. A photograph of the prepared gastric organoid under a microscope is shown in FIG. 18 .
  • the cell pellet isolated in Comparative Example 1 was diluted in 1 ml of RPMI1640 culture medium, the cells were counted, centrifuged again, the cell aggregates were mixed with Matrigel, and then dispensed into a 24-well plate and incubated at 37 ° C. for 1 hour. Then, a culture medium (ENRGFW medium) supplemented with EGF, gastrin, FGF10, Noggin, Wnt3a, and R-spondin was added and cultured in a 37°C CO 2 incubator. The culture medium was freshly replaced every 2-3 days. A photo of the prepared gastric organoid under a microscope is shown in FIG. 19 .
  • the present invention relates to a composition for culturing isolated chief cells for research on the functions and roles of chief cells present in conventional gastric glands.
  • a culture composition capable of differentiating main cells by specifically isolating only main cells by exposing the gastric mucosa to the outside.
  • a culture composition for differentiating the isolated main cells it differentiates all lineages, especially parietal cells, which were difficult to differentiate in the past, and forms gastric mucosal organoids, which can be used very effectively for treatment in the development of therapeutic compositions related to gastrointestinal diseases. It is expected that

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Abstract

The present invention relates to a composition for culturing chief cells isolated from living tissues or organs, particularly the stomach, and a method for culturing cells using the composition.

Description

위 세포 배양용 조성물Composition for culturing gastric cells
본 발명은 생체 조직 또는 기관, 특히는 위(stomach)로부터 분리된 세포를 배양하기 위한 조성물 및 이를 이용한 세포 배양 방법에 관한 것이다. The present invention relates to a composition for culturing cells isolated from a biological tissue or organ, particularly the stomach, and a cell culture method using the same.
위의 상피 조직은 생리학적으로 자가 재생 조직에 해당한다. 종래에는 위선의 상부에만 줄기세포가 존재하는 것으로 파악되었으나, 최근 하부에 존재하는 줄기세포로서 주 세포(Chief cell)에 대한 연구가 진행되고 있다. 그러나, 그 기능이나 역할에 대해 뚜렷하게 구분되어 확인할 수 없었다. 위의 루멘을 구성하고 있는 세포 형태 중, 주 세포는 펩신과 게스트릭 리파제를 분비하는 세포로서, 하부(base)에 위치한다.The epithelial tissue of the stomach corresponds physiologically to a self-renewing tissue. Conventionally, it was recognized that stem cells exist only in the upper part of the gastric gland, but recently, research on chief cells as stem cells existing in the lower part is being conducted. However, it was not possible to clearly distinguish and confirm its function or role. Among the cell types constituting the lumen of the stomach, the main cell is a cell that secretes pepsin and gestic lipase and is located at the base.
위에서 항상성은 다양한 세포 계통으로 예를 들면 산 생성 벽 세포, 점액 분비 목 세포 및 지모겐 분비 주 세포의 생성 및 유지의 균형에 의존한다. 정상 위에서 점액 분비 목 세포는 벽 세포로부터 위선의 기저부로 이동하면서 주 세포로 전이 분화(transdifferentiate)한다. Homeostasis in the stomach depends on the balance of production and maintenance of various cell lineages, such as acid-producing parietal cells, mucus-secreting throat cells, and zymogen-secreting principal cells. In the normal stomach, mucus-secreting throat cells transdifferentiate into principal cells as they migrate from the parietal cells to the base of the gastric gland.
벽 세포의 부재 시 주 세포 또한 소멸하는 것으로 알려져 있으므로, 이러한 주 세포의 성숙을 조절하는 인자를 벽 세포가 분비할 것으로 예측되었다. 이러한 인자 및 신호 기작들을 밝히려는 많은 시도가 있었지만, 주 세포의 항상성 및 기능의 복잡한 기작을 구성하는 요소에 대한 연구는 아직까지 많이 진행된 바 없다. Since principal cells are also known to die in the absence of parietal cells, it was predicted that parietal cells would secrete factors that regulate the maturation of these principal cells. Although many attempts have been made to elucidate these factors and signaling mechanisms, studies on the elements constituting the complex mechanisms of homeostasis and function of principal cells have not yet been conducted.
따라서, 최근에는 위 벽 세포 및 주 세포 등의 상관 관계를 조사하고 위의 기작 등을 연구하기 위하여, 위 기관으로부터 이들 세포를 높은 효율로 분리할 수 있는 방법이나 분리 후 이를 배양하기 위한 기술이 요구되고 있는 실정이다. Therefore, recently, in order to investigate the correlation between gastric parietal cells and main cells and to study the mechanism of the stomach, a method capable of isolating these cells from the gastric organ with high efficiency or a technique for culturing them after separation is required. It is becoming.
본 발명의 일 목적은 위로부터 분리된 세포를 배양하기 위한 조성물을 제공하고자 한다. One object of the present invention is to provide a composition for culturing cells isolated from the stomach.
본 발명의 다른 목적은 상기한 조성물을 이용하여 위 세포를 배양하는 방법을 제공하고자 한다. Another object of the present invention is to provide a method for culturing gastric cells using the above composition.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
이하, 본원에 기재된 다양한 구체예가 도면을 참조로 기재된다. 하기 설명에서, 본 발명의 완전한 이해를 위해서, 다양한 특이적 상세 사항, 예컨대, 특이적 형태, 조성물 및 공정 등이 기재되어 있다. 그러나, 특정의 구체예는 이들 특이적 상세 사항 중 하나 이상 없이, 또는 다른 공지된 방법 및 형태와 함께 실행될 수 있다. 다른 예에서, 공지된 공정 및 제조 기술은 본 발명을 불필요하게 모호하게 하지 않게 하기 위해서, 특정의 상세사항으로 기재되지 않는다. "한 가지 구체예" 또는 "구체예"에 대한 본 명세서 전체를 통한 참조는 구체예와 결부되어 기재된 특별한 특징, 형태, 조성 또는 특성이 본 발명의 하나 이상의 구체예에 포함됨을 의미한다. 따라서, 본 명세서 전체에 걸친 다양한 위치에서 표현된 "한 가지 구체예에서" 또는 "구체예"의 상황은 반드시 본 발명의 동일한 구체예를 나타내지는 않는다. 추가로, 특별한 특징, 형태, 조성, 또는 특성은 하나 이상의 구체예에서 어떠한 적합한 방법으로 조합될 수 있다.Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.
본 발명 내 특별한 정의가 없으면 본 명세서에 사용된 모든 과학적 및 기술적인 용어는 본 발명이 속하는 기술분야에서 당 업자에 의하여 통상적으로 이해되는 것과 동일한 의미를 가진다.Unless there is a specific definition within the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.
본 발명의 일 구현 예에 따르면, 위(stomach) 세포의 배양용 조성물에 관한 것이다. According to one embodiment of the present invention, it relates to a composition for culturing stomach cells.
본 발명에서 상기 세포는 주 세포(chief cell)일 수 있다. 본 발명에서 상기 “위 주 세포(gastric chief cell)”는 소화성 세포(peptic cell) 또는 위 효소원 세포(gastric zymogenic cell)이라고도 불리우며 펩시노겐 및 위장 리파아제(gastric lipase)를 분비하는 위선 세포(gastric gland cell)에 해당하며, 반추 동물에서 키모신(chymosin)을 분비하는 세포에도 해당된다. In the present invention, the cell may be a chief cell. In the present invention, the "gastric chief cell" is also called a peptic cell or a gastric zymogenic cell, and is a gastric gland cell that secretes pepsinogen and gastric lipase. ), and also applies to cells that secrete chymosin in ruminants.
본 발명의 배양용 조성물은 기본 배양 배지에 대하여 소 혈청 알부민(bovine serum albumin; BSA) 및 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(insulin-transferrin-selenium-sodium pyruvate; ITS-A) 중 적어도 하나를 포함할 수 있다. The culture composition of the present invention contains at least one of bovine serum albumin (BSA) and insulin-transferrin-selenium-sodium pyruvate (ITS-A) with respect to a basal culture medium. can include
본 발명에서 상기 소 혈청 알부민(BSA)은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 1 내지 20 w/v%, 바람직하게는 5 내지 15 w/v%, 보다 바람직하게는 8 내지 12 w/v%일 수 있다. In the present invention, the bovine serum albumin (BSA) has a working concentration of 1 to 20 w/v%, preferably 5 to 15 w/v%, more preferably 8 to 12 w/v%, based on the basal culture medium. It may be w/v%.
본 발명에서 상기 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(ITS-A)는 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 1 내지 15 ug/ml, 바람직하게는 5 내지 13 ug/ml, 보다 바람직하게는 6 내지 10 ug/ml일 수 있다. In the present invention, the insulin-transferrin-selenium-sodium pyruvate (ITS-A) has a working condition of 1 to 15 ug/ml, preferably 5 to 13 ug/ml, More preferably, it may be 6 to 10 ug/ml.
본 발명의 배양용 조성물은 기본 배양 배지에 대하여 간세포 성장 인자(hepatocyte growth factor; HGF)를 더 포함할 수 있다. The culture composition of the present invention may further include hepatocyte growth factor (HGF) with respect to the basal culture medium.
본 발명에서 상기 “간세포 성장 인자(hepatocyte growth factor; HGF) 또는 산란 인자(scatter factor; SF)”는 주변 분비(paracrine) 세포 성장, 운동성 및 형태 형성 인자로, 중간엽 세포에 의해 분비되며 주로 상피 세포 및 내피 세포에 작용하고 조혈 전구 세포 및 T 세포에 작용한다.In the present invention, the "hepatocyte growth factor (HGF) or scatter factor (SF)" is a paracrine cell growth, motility and morphogenesis factor, secreted by mesenchymal cells and mainly epithelial It acts on cells and endothelial cells and acts on hematopoietic progenitor cells and T cells.
본 발명에서 상기 HGF는 포유 동물의 HGF 유전자에 의해 코딩되는 단백질이라면 제한없이 사용될 수 있고, 상기 HGF 유전자를 발현하도록 유전적으로 조작된 세포 등에 의해 발현된 HGF를 사용할 수 있고, 혹은 화학적 혹은 생화학적으로 합성한 것이나, 시판되는 것(예를 들면 RD Systems 사제의 HGF) 등 어느 것이라도 제한없이 사용될 수 있다. 이때 상기 HGF의 유래로서는 포유 동물인 한 특히 제한되지 않으며, 예를 들면, 사람 HGF(hHGF)나 마우스 HGF(mHGF)일 수 있으나, 바람직하게는 mHGF를 이용할 수 있다. In the present invention, the HGF can be used without limitation as long as it is a protein encoded by the mammalian HGF gene, and HGF expressed by cells genetically engineered to express the HGF gene can be used, or chemically or biochemically. Any synthetic, commercially available (for example, HGF manufactured by RD Systems) may be used without limitation. In this case, the origin of the HGF is not particularly limited as long as it is a mammal, and for example, it may be human HGF (hHGF) or mouse HGF (mHGF), but mHGF may be preferably used.
본 발명에서 상기 간세포 성장 인자(HGF)는 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 100 내지 1000 ng/ml, 바람직하게는 100 내지 500 ng/ml, 보다 바람직하게는 100 내지 300 ng/ml일 수 있다. In the present invention, the hepatocyte growth factor (HGF) has a working concentration of 100 to 1000 ng/ml, preferably 100 to 500 ng/ml, more preferably 100 to 300 ng/ml with respect to the basal culture medium. can be ml.
본 발명의 배양용 조성물은 HGF 외에도, Wnt3a, R-스폰딘(R-spondin), Noggin, B27, N-아세틸 시스테인(N-acetyl cysteine; NAC), 섬유아세포 성장 인자(fibroblast growth factor; FGF), 표피 성장 인자(epidermal growth factor; EGF), 가스트린(gastrin), Y-27632, 및 하이드로코르티손(hydrocortisone)으로 이루어진 군에서 선택된 1종 이상을 더 포함할 수 있다. In addition to HGF, the culture composition of the present invention contains Wnt3a, R-spondin, Noggin, B27, N-acetyl cysteine (NAC), fibroblast growth factor (FGF) , epidermal growth factor (EGF), gastrin (gastrin), Y-27632, and hydrocortisone (hydrocortisone) may further include one or more selected from the group consisting of.
본 발명의 배양용 조성물은 Wnt3a 또는 이를 포함하는 조건 배지(conditioned media)를 더 포함할 수 있는데, 이때 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 10 내지 80 v/v%, 바람직하게는 30 내지 70 v/v%, 보다 바람직하게는 40 내지 60 v/v%일 수 있다. The composition for culture of the present invention may further include Wnt3a or conditioned media containing the same, wherein the working concentration is 10 to 80 v/v%, preferably with respect to the basal culture medium. It may be 30 to 70 v/v%, more preferably 40 to 60 v/v%.
본 발명의 배양용 조성물은 R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지(conditioned media)를 더 포함할 수 있는데, 이때 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 0.1 내지 30 v/v%, 바람직하게는 1 내지 20 v/v%, 보다 바람직하게는 5 내지 15 v/v%일 수 있다. The culture composition of the present invention may further include R-spondin or a conditioned medium containing the same, wherein the basal culture medium has a working concentration of 0.1 to 30 It may be v/v%, preferably 1 to 20 v/v%, and more preferably 5 to 15 v/v%.
본 발명의 배양용 조성물은 Noggin을 더 포함할 수 있다. 이때 상기 Noggin은 포유 동물의 NOG 유전자에 의해 코딩되는 단백질이라면 제한없이 사용될 수 있고, 상기 NOG 유전자를 발현하도록 유전적으로 조작된 세포 등에 의해 발현된 Noggin을 사용할 수 있고, 혹은 화학적 혹은 생화학적으로 합성한 것이나, 시판되는 것(예를 들면 RD Systems 사제의 Noggin) 등 어느 것이라도 제한없이 사용될 수 있다. 이때 상기 Noggin의 유래로서는 포유 동물인 한 특히 제한되지 않으며, 예를 들면, 사람 유래 또는 마우스 유래일 수 있으나, 바람직하게는 마우스 유래인 것을 이용할 수 있다.The culture composition of the present invention may further include Noggin. At this time, the Noggin can be used without limitation as long as it is a protein encoded by the mammalian NOG gene, and Noggin expressed by cells genetically engineered to express the NOG gene can be used, or chemically or biochemically synthesized Noggin can be used. Any one, such as a commercially available one (for example, Noggin manufactured by RD Systems), can be used without limitation. At this time, the origin of the Noggin is not particularly limited as long as it is a mammal, and for example, it may be derived from a human or a mouse, but preferably a mouse-derived one can be used.
본 발명에서 상기 Noggin은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 10 내지 200 ng/ml, 바람직하게는 50 내지 150 ng/ml, 보다 바람직하게는 80 내지 120 ng/ml일 수 있다. In the present invention, the working concentration of Noggin in the basal culture medium may be 10 to 200 ng/ml, preferably 50 to 150 ng/ml, and more preferably 80 to 120 ng/ml.
본 발명의 배양용 조성물은 B27을 더 포함할 수 있다. 이때 상기 B27은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 0.005 내지 0.1 v/v%, 바람직하게는 0.01 내지 0.05 v/v%, 보다 바람직하게는 0.01 내지 0.03 v/v%일 수 있다.The composition for culture of the present invention may further contain B27. In this case, the B27 may have a working condition of 0.005 to 0.1 v/v%, preferably 0.01 to 0.05 v/v%, and more preferably 0.01 to 0.03 v/v% with respect to the basal culture medium. .
본 발명의 배양용 조성물은 N-아세틸 시스테인(NAC)을 더 포함할 수 있고, 이때 상기 N-아세틸 시스테인(NAC)은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 0.1 내지 5 mM, 바람직하게는 0.5 내지 2.5 mM, 보다 바람직하게는 1 내지 1.5 mM일 수 있다. The composition for culture of the present invention may further include N-acetyl cysteine (NAC), wherein the N-acetyl cysteine (NAC) has a working concentration of 0.1 to 5 mM, preferably with respect to the basal culture medium. Preferably it may be 0.5 to 2.5 mM, more preferably 1 to 1.5 mM.
본 발명의 배양용 조성물은 섬유아세포 성장 인자(FGF)를 더 포함할 수 있다. 이때 상기 섬유아세포 성장 인자(FGF)는 FGF1, FGF2(bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF17, 및 FGF18로 이루어진 군에서 선택된 1종 이상일 수 있다. 또한, 상기 섬유아세포 성장 인자(FGF) 유래로서는 포유 동물인 한 특히 제한되지 않으며, 예를 들면, 사람 유래의 것을 이용할 수 있다.The composition for culture of the present invention may further include fibroblast growth factor (FGF). In this case, the fibroblast growth factor (FGF) may be one or more selected from the group consisting of FGF1, FGF2 (bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF17, and FGF18. In addition, the fibroblast growth factor (FGF) is not particularly limited as long as it is a mammal, and for example, a human-derived one can be used.
또한, 본 발명에서 상기 섬유아세포 성장 인자(FGF)는 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 100 내지 1000 ng/ml, 바람직하게는 100 내지 500 ng/ml, 보다 바람직하게는 100 내지 300 ng/ml일 수 있다. In addition, in the present invention, the fibroblast growth factor (FGF) has a working concentration of 100 to 1000 ng/ml, preferably 100 to 500 ng/ml, more preferably 100 to 1000 ng/ml with respect to the basal culture medium. 300 ng/ml.
또한, 본 발명에서 상기 섬유아세포 성장 인자(FGF)는 FGF2(bFGF)일 수 있고, 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 50 내지 200 ng/ml, 바람직하게는 50 내지 150 ng/ml, 보다 바람직하게는 80 내지 120 ng/ml일 수 있다.In addition, in the present invention, the fibroblast growth factor (FGF) may be FGF2 (bFGF), and the working concentration is 50 to 200 ng/ml, preferably 50 to 150 ng/ml with respect to the basal culture medium. ml, more preferably 80 to 120 ng/ml.
또한, 본 발명에서 상기 섬유아세포 성장 인자(FGF)는 hFGF10일 수 있고, 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 50 내지 200 ng/ml, 바람직하게는 50 내지 150 ng/ml, 보다 바람직하게는 80 내지 120 ng/ml일 수 있다.In addition, in the present invention, the fibroblast growth factor (FGF) may be hFGF10, and the working concentration (working condition) with respect to the basal culture medium is 50 to 200 ng / ml, preferably 50 to 150 ng / ml, more Preferably it may be 80 to 120 ng/ml.
본 발명의 배양용 조성물은 표피 성장 인자(EGF)를 더 포함할 수 있다. 이때 상기 표피 성장 인자(EGF)의 유래로서는 포유 동물인 한 특히 제한되지 않으며, 예를 들면, 사람 유래 또는 마우스 유래일 수 있으나, 바람직하게는 마우스 유래인 것을 이용할 수 있다.The culture composition of the present invention may further include epidermal growth factor (EGF). At this time, the origin of the epidermal growth factor (EGF) is not particularly limited as long as it is a mammal, and for example, it may be derived from a human or a mouse, but preferably a mouse-derived one may be used.
본 발명에서 상기 표피 성장 인자(EGF)는 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 5 내지 100 ng/ml, 바람직하게는 10 내지 50 ng/ml, 보다 바람직하게는 20 내지 30 ng/ml일 수 있다.In the present invention, the epidermal growth factor (EGF) has a working concentration of 5 to 100 ng/ml, preferably 10 to 50 ng/ml, more preferably 20 to 30 ng/ml with respect to the basal culture medium. can be ml.
본 발명의 배양용 조성물은 가스트린(gastrin)을 더 포함할 수 있고, 상기 가스트린은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 0.01 내지 10 uM, 바람직하게는 0.1 내지 5 uM, 보다 바람직하게는 0.5 내지 1.5 uM일 수 있다. The composition for culture of the present invention may further include gastrin, and the gastrin has a working condition of 0.01 to 10 uM, preferably 0.1 to 5 uM, more preferably 0.1 to 5 uM with respect to the basal culture medium. may be 0.5 to 1.5 uM.
본 발명의 배양용 조성물은 Y-27632을 더 포함할 수 있고, 상기 Y-27632은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 1 내지 50 mM, 바람직하게는 5 내지 30 mM, 보다 바람직하게는 5 내지 15 mM일 수 있다. The composition for culture of the present invention may further include Y-27632, and the Y-27632 has a working concentration of 1 to 50 mM, preferably 5 to 30 mM, more preferably 5 to 30 mM, with respect to the basal culture medium. It may be 5 to 15 mM.
본 발명의 배양용 조성물은 하이드로코르티손(hydrocortisone)을 더 포함할 수 있다. 이때 상기 하이드로코르티손은 상기 기본 배양 배지에 대하여 작업 농도(working condition)가 0.01 내지 10 ug/ml, 바람직하게는 0.1 내지 5 ug/ml, 보다 바람직하게는 0.5 내지 1.5 ug/ml일 수 있다.The composition for culture of the present invention may further include hydrocortisone. In this case, the hydrocortisone may have a working concentration of 0.01 to 10 ug/ml, preferably 0.1 to 5 ug/ml, and more preferably 0.5 to 1.5 ug/ml, based on the basal culture medium.
본 발명의 배양용 조성물은 상기 기본 배양 배지로 MEM(Minimum Essential Medium), BME(Basal Medium Eagle), DMEM(Dulbecco's Modified EagleMedium), EMEM(Eagle's minimal essential medium), IMDM(Iscove's Modified Dulbecco's Medium), GMEM(Glas- gow's MEM), F12(Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 배지(Leibovitz's L-15 medium), McCoy's 5 A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM/F-12, Advanced RPMI1640, 등의 배지를 이용할 수 있으나, 이에 제한되는 것은 아니다. The composition for culture of the present invention is MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified EagleMedium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM as the basic culture medium (Glasgow's MEM), F12 (Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5 A , Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM/F-12, Advanced RPMI1640, etc. may be used, but is not limited thereto.
본 발명에서 상기 기본 배양 배지는 작업 농도(working condition)가 10 내지 50 v/v%, 바람직하게는 20 내지 40 v/v%, 보다 바람직하게는 30 내지 40 v/v%일 수 있다. In the present invention, the basal culture medium may have a working condition of 10 to 50 v/v%, preferably 20 to 40 v/v%, and more preferably 30 to 40 v/v%.
본 발명에서 상기 기본 배양 배지는 글루타맥스(glutamax), 하이드록시에틸피페라진에테인설폰산(Hydroxyethyl piperazine Ethane Sulfonicacid; HEPES) 및 항생제 중 적어도 하나를 더 포함할 수 있다. In the present invention, the basal culture medium may further include at least one of glutamax, hydroxyethyl piperazine ethane sulfonic acid (HEPES), and an antibiotic.
본 발명에서 상기 글루타맥스(glutamax)는 상기 기본 배양 배지 내 작업 농도(working condition)가 0.01 내지 10 v/v%, 바람직하게는 0.1 내지 5 v/v%, 보다 바람직하게는 0.5 내지 1.5 v/v%일 수 있다.In the present invention, the working concentration of glutamax in the basal culture medium is 0.01 to 10 v/v%, preferably 0.1 to 5 v/v%, more preferably 0.5 to 1.5 v It can be /v%.
본 발명에서 상기 하이드록시에틸피페라진에테인설폰산(HEPES)은 상기 기본 배양 배지 내 작업 농도(working condition)가 0.01 내지 10 mM, 바람직하게는 0.1 내지 5 mM, 보다 바람직하게는 0.5 내지 1.5 mM일 수 있다.In the present invention, the hydroxyethylpiperazineethanesulfonic acid (HEPES) is 0.01 to 10 mM, preferably 0.1 to 5 mM, more preferably 0.5 to 1.5 mM at a working condition in the basal culture medium. can
본 발명에서 상기 항생제는 페니실린(penicillin) 및 스트렙토마이신(streptomycin) 중 적어도 하나를 포함할 수 있다. 여기서 상기 항생제는 상기 기본 배양 배지 내 작업 농도(working condition)가 50 내지 200 ng/ml, 바람직하게는 50 내지 150 ng/ml, 보다 바람직하게는 80 내지 120 ng/ml일 수 있다.In the present invention, the antibiotic may include at least one of penicillin and streptomycin. Here, the working concentration of the antibiotic in the basal culture medium may be 50 to 200 ng/ml, preferably 50 to 150 ng/ml, and more preferably 80 to 120 ng/ml.
본 발명의 배양용 조성물은 위(stomach) 주 세포(chief cell)의 위 점막 세포로의 분화 유도의 용도로 사용될 수 있다. The composition for culture of the present invention can be used for inducing differentiation of stomach cells into gastric mucosal cells.
본 발명에서 상기 위 점막 세포는 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상일 수 있다. In the present invention, the gastric mucosal cells may be at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells.
본 발명에서 상기 “표면 점액 세포(mucous foveolar cells)”는 점액을 분비하는 세포로, 위의 내부를 덮어 위를 염산으로부터 보호하는 역할을 한다. In the present invention, the "surface mucous cells (mucous foveolar cells)" is a cell that secretes mucus, and serves to protect the stomach from hydrochloric acid by covering the inside of the stomach.
본 발명에서 상기 “벽 세포(parietal cell)”는 염산(HCl) 및 내인자를 분비하는 위의 상피 세포로 위저선(gastric glands)에 분포하는 것으로 알려져 있다. In the present invention, the “parietal cell” is a gastric epithelial cell that secretes hydrochloric acid (HCl) and intrinsic factor, and is known to be distributed in gastric glands.
본 발명에서 상기 “목 점액 세포(mucus neck cell)”는 점액(mucin)을 분비하는 세포로, 대부분 위저선의 상부에 위치한다. In the present invention, the "mucus neck cell" is a cell that secretes mucin, and is mostly located at the top of the gastric gland.
본 발명의 배양용 조성물은 위 오가노이드 배양용 조성물로 사용될 수 있다. The composition for culturing of the present invention can be used as a composition for culturing gastric organoids.
본 발명의 상기 “오가노이드”는 3D 입체구조를 가지는 세포를 의미하며, 동물 등에서 수집, 취득하지 않은 인공적인 배양 과정을 통해 제조한 조직과 유사한 모델을 의미한다. 2D 배양과는 달리, 3D 세포 배양은 체외에서 세포가 모든 방향으로 성장할 수 있다.The "organoid" of the present invention refers to a cell having a 3D three-dimensional structure, and refers to a model similar to a tissue prepared through an artificial culture process that is not collected or obtained from an animal or the like. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro.
본 발명에서 상기 위 오가노이드는 위 점막 오가노이드일 수 있다. In the present invention, the gastric organoid may be a gastric mucosal organoid.
본 발명의 다른 구현 예에 따르면, 위 주 세포의 배양 방법에 관한 것이다. According to another embodiment of the present invention, it relates to a method for culturing gastric main cells.
본 발명의 배양 방법은 분리된 위 주 세포(chief cell)를 본 발명에 따른 배양용 조성물에서 배양하는 단계를 포함할 수 있다. The culturing method of the present invention may include culturing the isolated chief cells in the composition for culturing according to the present invention.
본 발명에서 상기 배양은 35 내지 40 ℃의 온도 하에서 1 시간 내지 120 일 동안 수행될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the culturing may be performed at a temperature of 35 to 40 °C for 1 hour to 120 days, but is not limited thereto.
본 발명의 또 다른 구현 예에 따르면, 위 오가노이드의 제조 방법에 관한 것이다. According to another embodiment of the present invention, it relates to a method for preparing gastric organoids.
본 발명의 제조 방법은 분리된 위 주 세포(chief cell)를 마트리겔 및 본 발명에 따른 배양용 조성물에 넣고 오가노이드를 형성하는 단계;를 포함할 수 있다. The manufacturing method of the present invention may include forming an organoid by putting the isolated chief cells into Matrigel and the composition for culture according to the present invention.
본 발명에서 상기 마트리겔은 성장인자 감소 마트리겔인 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the matrigel may be growth factor reduced matrigel, but is not limited thereto.
본 발명에서 제조되는 위 오가노이드는 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상을 포함하는 위 점막 오가노이드일 수 있다. The gastric organoid prepared in the present invention may be a gastric mucosal organoid containing at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells. there is.
본 발명의 또 다른 구현 예에 따르면, 본 발명의 상기 위 오가노이드의 제조 방법에 의해 제조된 위 오가노이드를 제공한다.According to another embodiment of the present invention, a gastric organoid prepared by the method for preparing a gastric organoid of the present invention is provided.
본 발명의 상기 위 오가노이드는 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상을 모두 포함하는 위 점막 오가노이드로서, 위 손상 치료제를 효과적으로 스크리닝하는 데에 사용될 수 있을 뿐만 아니라, 직접 위 손상에 사용되는 세포 치료제로서 사용될 수 있다.The gastric organoid of the present invention is a gastric mucosal organoid containing at least one selected from the group consisting of mucous foveolar cells, parietal cells, and mucus neck cells, Not only can it be used to effectively screen gastric injury therapeutic agents, but it can also be used as a cell therapy directly used for gastric injury.
본 명세서에서 용어 "세포 치료제"는 사람으로부터 분리, 배양 및 특수한 저작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종세포를 체외에서 증식, 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 치료, 진단 및 예방의 목적으로 사용되는 의약품을 지칭한다.As used herein, the term "cell therapy" refers to cells and tissues prepared from humans through isolation, culture, and special chewing, which are medicines used for the purpose of treatment, diagnosis, and prevention. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating or selecting allogeneic or heterogeneous cells in vitro, or changing the biological characteristics of cells in other ways.
본 발명의 또 다른 구현 예에 따르면, 본 발명의 위 오가노이드를 유효성분으로 포함하는 생체 이식용 약학 조성물에 관한 것이다. According to another embodiment of the present invention, it relates to a pharmaceutical composition for living body transplantation comprising the gastric organoid of the present invention as an active ingredient.
본 발명에서 상기 “생체 이식(bio implanting)”은 개체에 투여되어 이식 부위에 되는 현상을 의미할 수 있다.In the present invention, the "bio implanting" may refer to a phenomenon that is administered to a subject and becomes a transplant site.
본 발명에서 상기 “개체”는 위 손상이 발생하였거나 위 절제술 등으로 위의 적어도 일부의 부위에 대하여 오가노이드를 이식하여 위를 재건, 복원 또는 형성시킬 필요가 있는 개체일 수 있다. In the present invention, the “individual” may be an individual who has suffered gastric damage or needs to reconstruct, restore, or form the stomach by transplanting organoids to at least a portion of the stomach through gastrectomy or the like.
본 발명의 약학 조성물에서 상기 위 오가노이드는 1×107 내지 1×108, 1×108 내지 2×108, 2×108 내지 4×108, 4×108 내지 6×108, 6×108 내지 8×108, 8×108 내지 1×109, 1×109 내지 2×109, 2×109 내지 4×109, 4×109 내지 1×1010, 2×108 내지 6×108, 6×108 내지 1×109, 1×108 내지 2×108, 2×108 내지 2×109, 1×107 내지 1×108, 1×108 내지 1×109, 1×109 내지 1×1010 또는 1×107 내지 1×109개 중 어느 하나의 세포/㎏의 양으로 이식될 수 있으나, 이에 제한되는 것은 아니다.In the pharmaceutical composition of the present invention, the gastric organoid is 1×10 7 to 1×10 8 , 1×10 8 to 2×10 8 , 2×10 8 to 4×10 8 , 4×10 8 to 6×10 8 , 6×10 8 to 8×10 8 , 8×10 8 to 1×10 9 , 1×10 9 to 2×10 9 , 2×10 9 to 4×10 9 , 4×10 9 to 1×10 10 , 2×10 8 to 6×10 8 , 6×10 8 to 1×10 9 , 1×10 8 to 2×10 8 , 2×10 8 to 2×10 9 , 1×10 7 to 1×10 8 , 1 × 10 8 to 1 × 10 9 , 1 × 10 9 to 1 × 10 10 or 1 × 10 7 to 1 × 10 9 may be transplanted in an amount of any one cell / kg, but is limited thereto no.
본 발명의 상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다.The pharmaceutical composition of the present invention may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.
본 발명의 상기 약학 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사 용액의 형태로 제형화되어 사용될 수 있다. 본 발명의 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등이 사용될 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등이 혼합되어 사용될 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등이 사용될 수 있다. 본 발명의 약학 조성물의 제형은 상술한 바와 같은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수 회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형화할 수 있다.The pharmaceutical compositions of the present invention are not limited thereto, but are formulated in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration. In the case of injections, buffers, preservatives, painless A topical agent, a solubilizer, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, and the like may be used. Formulations of the pharmaceutical composition of the present invention may be variously prepared by mixing with the pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항 응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like can be used. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.
본 발명의 상기 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함되며, 예를 들면, 경구 또는 비경구 투여일 수 있다.The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but is not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal administration is included, and may be, for example, oral or parenteral administration.
본 발명의 상기 "비경구"란, 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 목적상 상기 약학 조성물은 신장에 직접 주사되는 방식으로 투여될 수 있으나, 이에 제한되는 것은 아니다.The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. For the purposes of the present invention, the pharmaceutical composition may be administered by direct injection into the kidney, but is not limited thereto.
본 발명의 상기 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50 mg/kg 또는 0.001 내지 50 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. It can vary widely, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, drug form, administration route and period, but can be appropriately selected by those skilled in the art, 0.0001 to 50 mg/day kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
본 발명에서 제공하는 조성물을 이용하는 경우 위의 주 세포(chief cell)을 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell) 등으로 분화시킬 수 있고, 이에 따라 더 나아가서는 위 점막 오가노이드를 형성할 수 있어 세포 치료제로의 이용 또한 가능하다. When using the composition provided by the present invention, chief cells of the stomach can be differentiated into mucous foveolar cells, parietal cells, and mucus neck cells. Furthermore, gastric mucosal organoids can be formed, so that it can be used as a cell therapy.
도 1은 본 발명에 따라 위 기관에 각 처리 후 주 세포(chief cell), 벽공 세포(pit cell), 벽 세포(parietal cell) 및 협부 세포(isthmus cell)를 분리하는 방법의 모식도를 나타낸 것이다.1 is a schematic diagram of a method for separating chief cells, pit cells, parietal cells, and isthmus cells after each treatment in the gastric organ according to the present invention.
도 2는 실시예 1에서 멸균된 봉합사를 이용하여 마우스의 식도와 위의 접합 부위를 묶어준 사진을 나타낸 것이다. Figure 2 shows a photograph of tying the junction of the esophagus and the stomach of the mouse using a sterilized suture in Example 1.
도 3은 실시예 1에서 마우스의 위를 적출한 뒤 PBS 용액에 침지하여 세척하는 사진을 나타낸 것이다. Figure 3 shows a photograph of washing by immersing in a PBS solution after extracting the stomach of a mouse in Example 1.
도 4는 실시예 1에서 멸균된 면봉으로 적출된 마우스의 전위(Forestomach)를 밀어 위 점막이 외부로 노출되도록 뒤집어준 뒤의 사진을 나타낸 것이다. Figure 4 shows a photograph after turning the forestomach of the mouse excised with a sterilized cotton swab in Example 1 so that the gastric mucosa is exposed to the outside.
도 5는 실시예 1에서 점막이 외부로 노출되도록 뒤집어진 위의 위 저 부분(Fundus)을 멸균된 봉합사로 묶어 준 후의 사진을 나타낸 것이다. 5 shows a photograph after tying the fundus of the inverted stomach with a sterilized suture in Example 1 so that the mucous membrane is exposed to the outside.
도 6은 실시예 1에서 마우스의 전위(Forestomach) 부위에 인슐린 주사기를 이용하여 제1 세포 분리용 용액을 주입하는 사진을 나타낸 것이다. FIG. 6 shows a photograph of injecting a solution for first cell isolation using an insulin syringe into a forestomach region of a mouse in Example 1. FIG.
도 7은 실시예 1에서 위를 제2 세포 분리용 용액에 침지하여 37도씨 인큐베이터에서 60RPM 속도로 30분간 교반 시켜 분획1 용액을 얻은 사진을 나타낸 것이다. 7 is a photograph showing a fraction 1 solution obtained by immersing the stomach in the second cell isolation solution in Example 1 and stirring for 30 minutes at a speed of 60 RPM in a 37 ° C incubator.
도 8은 실시예 1에서 분획1 용액에서 위를 꺼내어 제3 세포 분리용 용액에 침지하여 동일한 조건으로 30분간 교반 시켜 분획2 용액을 얻은 사진을 나타낸 것이다. FIG. 8 shows a photograph of obtaining a fraction 2 solution by taking the stomach out of the fraction 1 solution in Example 1, immersing it in a third cell separation solution and stirring for 30 minutes under the same conditions.
도 9는 실시예 1에서 분획5 용액을 100 μm 거름망 필터를 통과시키는 사진을 나타낸 것이다. Figure 9 shows a photograph of the fraction 5 solution in Example 1 passing through a 100 μm sieve filter.
도 10은 실시예 1에서 거름망 필터를 통과시킨 분획5 용액을 원심분리하여 주 세포를 침전시킨 사진을 나타낸 것이다. FIG. 10 shows a photograph in which the main cells were precipitated by centrifugation of the Fraction 5 solution passed through the sieve filter in Example 1.
도 11은 실험예 1에서 마우스로부터 적출된 위 조직과, 분획5 용액에서 여과된 위 조직에 대하여 헤마톡실린&에오신(H&E) 염색을 수행한 결과를 나타낸 것이다. 11 shows the results of hematoxylin & eosin (H&E) staining on gastric tissue excised from mice in Experimental Example 1 and gastric tissue filtered from the Fraction 5 solution.
도 12는 실험예 2에서 분획5 용액로부터 얻어진 세포에 Gif 와 GPR43을 면역 염색한 결과를 나타낸 것이다. 12 shows the results of immunostaining for Gif and GPR43 in cells obtained from the fraction 5 solution in Experimental Example 2.
도 13은 실험예 3에서 분획5 용액로부터 얻어진 세포와 비교예 1에서 분리된 세포에 대하여 다양한 위 상피 세포 마커의 발현 수준을 확인한 결과를 나타낸 것이다.13 shows the results of confirming the expression levels of various gastric epithelial cell markers in cells obtained from the fraction 5 solution in Experimental Example 3 and cells isolated in Comparative Example 1.
도 14는 실험예 3의 실험 모식도를 나타낸 것이다. 14 shows a schematic diagram of the experiment of Experimental Example 3.
도 15는 실험예 3에서 주 세포 특이적으로 RFP 형광 단백질을 발현시킬 수 있는 리포터 마우스(Mist1creEr;R26-tdTomato)의 위로부터 얻어진 분획5 용액에서 침전 및 회수된 세포를 현미경으로 관찰한 사진을 나타낸 것이다. 15 is a micrograph of cells precipitated and recovered from the fraction 5 solution obtained from the stomach of a reporter mouse (Mist1creEr; R26-tdTomato) capable of expressing RFP fluorescent protein in a primary cell-specific manner in Experimental Example 3; will be.
도 16은 실험예 4에서 GF(germ free) 마우스와 SPF(specific pathogen free) 마우스의 위로부터 얻어진 분획1 용액, 분획3 용액 및 분획5 용액에 존재하는 세포에 대하여 다양한 위 상피 세포 마커의 발현 수준을 qRT-PCR로 확인한 결과를 나타낸 것이다. 16 shows expression levels of various gastric epithelial cell markers for cells present in Fraction 1 solution, Fraction 3 solution, and Fraction 5 solution obtained from the stomachs of GF (germ free) mice and SPF (specific pathogen free) mice in Experimental Example 4 It shows the result confirmed by qRT-PCR.
도 17은 실험예 6에서 SPF 마우스와 GF 마우스의 위로부터 얻어진 분획5 용액에서 얻어진 주 세포로부터 분화되는 위 상피 세포를 면역 염색한 결과를 나타낸 것이다. 17 shows the results of immunostaining of gastric epithelial cells differentiated from main cells obtained from the Fraction 5 solution obtained from the stomachs of SPF and GF mice in Experimental Example 6.
도 18은 실시예 2에서 제조된 위 오가노이드를 현미경으로 관찰한 사진을 나타낸 것이다. 18 shows a microscopic photograph of gastric organoids prepared in Example 2.
도 19는 비교예 2에서 제조된 위 오가노이드를 현미경으로 관찰한 사진을 나타낸 것이다. 19 is a microscopic photograph of gastric organoids prepared in Comparative Example 2;
도 20은 실험예 7에서 분획5 용액로부터 얻어진 세포를 제조예 4의 배양 용액 또는 기존의 일반적 배양 용액에서 배양한 뒤 다양한 위 상피 세포 마커의 발현 수준을 분석한 결과를 나타낸 것이다. 20 shows the results of analyzing the expression levels of various gastric epithelial cell markers after culturing the cells obtained from the fraction 5 solution in Experimental Example 7 in the culture solution of Preparation Example 4 or the existing general culture solution.
본 발명은 종래의 위샘에 존재하는 주세포(Chief cell)의 기능과 역할에 대한 연구를 위한 분리된 주세포 배양용 조성물에 관한 것이다. 본 발명은 종래에 사용되는 방법과 달리 위 점막을 외부로 노출시켜, 주세포만을 특이적으로 분리한 주세포 분화시킬 배양용 조성물을 제공하는 것이다. The present invention relates to a composition for culturing isolated chief cells for research on the function and role of chief cells present in conventional gastric glands. Unlike conventional methods, the present invention provides a culture composition for culturing differentiated main cells by specifically isolating only main cells by exposing the gastric mucosa to the outside.
분리된 주세포를 분화시킬 배양용 조성물로 모든 계통, 특히 종래에는 분화시키기 어렵던 벽세포(Parietal cell)까지 분화시키고, 위 점막 오가노이드를 형성시킴으로써, 위장질환 관련 치료 조성물 개발에 중요 발판을 제공할 수 있다.As a culture composition for differentiating the separated main cells, it can differentiate all lineages, especially parietal cells, which have been difficult to differentiate in the past, and form gastric mucosal organoids, thereby providing an important foothold for the development of therapeutic compositions related to gastrointestinal diseases. there is.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
[준비예 1] 기본 용액의 제조[Preparation Example 1] Preparation of Basic Solution
멸균된 3차 증류수에 하기 표 1에 나타낸 조성으로 각 성분을 첨가하여 기본 용액을 제조하였다. A basic solution was prepared by adding each component in the composition shown in Table 1 to sterilized tertiary distilled water.
기본용액basic solution 성분ingredient 구조식constitutional formula 작업 농도
(working concentration)working concentration
(working concentration)
젠타마이신(Gentamicin)Gentamicin 0.1mg/ml0.1 mg/ml
DL-디티오트레이톨(DL-Dithiothreitol)DL-Dithiothreitol HSCH2CH(OH)CH(OH)CH2SHHSCH 2 CH(OH)CH(OH)CH 2 SH 0.5mM0.5 mM
모노나트륨 인산염(Sodium phosphate monobasic)Sodium phosphate monobasic NaH2PO4 NaH 2 PO 4 1mM1 mM
디나트륨 인산염(di-Sodium hydrogen phosphate)di-Sodium hydrogen phosphate Na2HPO4 Na 2 HPO 4 1mM1 mM
탄산수소 나트륨(Sodium bicarbonate) Sodium bicarbonate NaHCO3 NaHC0 3 10mM10 mM
염화 나트륨(Sodium chloride)Sodium chloride NaclNacl 50mM50 mM
염화 칼륨(Potassium chloride) Potassium chloride KClKCl 10mM10 mM
HEPESHEPES C8H18N2O4SC 8 H 18 N 2 O 4 S 50mM50 mM
D-(+)-글루코스(D-(+)-Glucose)D-(+)-Glucose C6H12O6 C 6 H 12 O 6 10mM10 mM
[제조예 1] 제1 세포 분리용 용액의 제조[Preparation Example 1] Preparation of the first cell separation solution
상기 준비예 1에서 제조된 기본 용액에 하기 표 2에 나타낸 성분을 첨가하여 제1 세포 분리용 용액을 제조하였다. 이렇게 제조된 용액은 사용 전 포어 사이즈가 0. 22 μm인 멸균의 셀룰로오스 아세테이트 막(Cellulose acetate membrane) 필터로 여과한 뒤 사용하였다. A first cell separation solution was prepared by adding the components shown in Table 2 to the basic solution prepared in Preparation Example 1. The solution prepared in this way was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 μm before use.
　 성분ingredient 작업 농도
(working concentration)working concentration
(working concentration)
제1 세포 분리용 용액1st Cell Separation Solution EDTAEDTA 2mM / 기본 용액2mM/basic solution
BSABSA 1w/v% / 기본 용액1w/v% / basic solution
프로테이나제 E(Proteinase E)Proteinase E 2.5mg /ml 기본 용액2.5mg/ml basic solution
콜라게나제 1형(Collagenase Type1)Collagenase Type 1 4mg /ml 기본 용액4mg/ml basic solution
[제조예 2] 제2 세포 분리용 용액[Preparation Example 2] Second cell separation solution
상기 준비예 1에서 제조된 기본 용액에 하기 표 3에 나타낸 성분을 첨가하여 제2 세포 분리용 용액을 제조하였다. 이렇게 제조된 용액은 사용 전 포어 사이즈가 0. 22 μm인 멸균의 셀룰로오스 아세테이트 막(Cellulose acetate membrane) 필터로 여과한 뒤 사용하였다.A second cell separation solution was prepared by adding the components shown in Table 3 to the basic solution prepared in Preparation Example 1. The solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 μm before use.
　 성분ingredient 작업 농도
(working concentration)working concentration
(working concentration)
제2 세포 분리용 용액Solution for secondary cell separation EDTAEDTA 2mM / 기본 용액2mM/basic solution
BSABSA 1w/v% / 기본 용액1w/v% / basic solution
[제조예 3] 제3 세포 분리용 용액[Preparation Example 3] Third cell separation solution
상기 준비예 1에서 제조된 기본 용액에 하기 표 4에 나타낸 성분을 첨가하여 제3 세포 분리용 용액을 제조하였다. 이렇게 제조된 용액은 사용 전 포어 사이즈가 0. 22 μm인 멸균의 셀룰로오스 아세테이트 막(Cellulose acetate membrane) 필터로 여과한 뒤 사용하였다.A third cell separation solution was prepared by adding the ingredients shown in Table 4 to the basic solution prepared in Preparation Example 1 above. The solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 μm before use.
　 성분ingredient 구조식constitutional formula 작업 농도
(working concentration)working concentration
(working concentration)
제3 세포 분리용 용액Third cell separation solution 염화 칼슘
(Calcium chloride)calcium chloride
(Calcium chloride) 		Cacl2 Cacl 2 1mM / 기본 용액1 mM / base solution
염화 마그네슘
(Magnesium chloride)magnesium chloride
(Magnesium chloride) 		Mgcl2 Mgcl 2 1.5mM / 기본 용액1.5 mM / base solution
BSABSA 1%One% 1w/v% / 기본 용액1w/v% / basic solution
[제조예 4] 주 세포 배양용 용액[Preparation Example 4] Main cell culture solution
하기 표 5에 나타낸 조성으로 기본 배양 배지에 각 성분을 첨가하여 위의 주 세포 배양용 용액을 제조하였다. 이렇게 제조된 용액은 사용 전 포어 사이즈가 0. 22 μm인 멸균의 셀룰로오스 아세테이트 막(Cellulose acetate membrane) 필터로 여과한 뒤 사용하였다.The above main cell culture solution was prepared by adding each component to the basal culture medium according to the composition shown in Table 5 below. The solution thus prepared was used after filtering with a sterile cellulose acetate membrane filter having a pore size of 0.22 μm before use.
　 인자factor 작업 농도working concentration
주세포
배양 용액main cell
culture solution 		기본 배양 배지Basic culture medium Glutamax 1v/v%Glutamax 1v/v% 37.35v/v%37.35v/v%
HEPES 1mMHEPES 1 mM
Penicillin-Streptomycin 100U/mlPenicillin-Streptomycin 100U/ml
Advanced DMEM/F-12 (Gibco, # 12634028)Advanced DMEM/F-12 (Gibco, # 12634028)
Wnt3a CMWnt3a CM 50v/v%50v/v%
R-spondin CMR-spondin CM 10v/v%10v/v%
mNogginmNoggin 100ng/ml100 ng/ml
B27B27 0.02v/v%0.02v/v%
N-acetyl CysteinN-acetyl Cysteine 1.25mM1.25 mM
hFGF10hFGF10 100ng/ml100 ng/ml
bFGFbFGF 100ng/ml100 ng/ml
mEGFmEGF 25ng/ml25 ng/ml
HGFHGF 200ng/ml200 ng/ml
gastringastrin 1uM1uM
Y-27632Y-27632 10mM10 mM
BSABSA 10w/v%10w/v%
Insulin-Transferrin-Selenium-Sodium PyruvateInsulin-Transferrin-Selenium-Sodium Pyruvate 8ug/ml8ug/ml
HydrocortisoneHydrocortisone 1ug/ml1ug/ml
[실시예 1] 마우스 위로부터의 주 세포의 분리[Example 1] Isolation of principal cells from mouse stomach
도 1은 본 발명에 따라 위 기관에 각 처리 후 주 세포(chief cell), 벽공 세포(pit cell), 벽 세포(parietal cell) 및 협부 세포(isthmus cell)를 분리하는 방법의 모식도를 나타낸 것이다. 구체적인 분리 방법으로는 8주령 C57BL/6 마우스를 CO2챔버를 이용하여 안락사 시킨 뒤 복강을 절개하고, 멸균된 봉합사를 이용하여 식도와 위의 접합 부위를 묶어주었다(도 2). 위를 적출한 뒤 PBS 용액에 침지하여 세척하고(도 3), 위 저 부분(Fundus)을 제외한 전정부(Antrum/Pylorus)를 가위를 이용하여 제거하였다. 멸균된 면봉으로 전위(Forestomach) 부분을 밀어주어 위 점막이 외부로 노출되도록 뒤집어주었다(도 4). 뒤집어진 위를 제2 세포 분리용 용액에 침지하여 세척한 후 절개된 위 저 부분(Fundus)을 멸균된 봉합사로 묶어 주었다(도 5). 전위(Forestomach) 부위에 인슐린 주사기를 이용하여 제1 세포 분리용 용액을 주입하여 위를 부풀렸다(도 6). 이후, 위를 제2 세포 분리용 용액에 침지하여 37도씨 인큐베이터에서 60RPM 속도로 30분간 교반 시킨 후(도 7)(분획1 용액), 30분뒤 얻어진 분획1 용액에서 위를 꺼내어 제3 세포 분리용 용액에 침지하여 동일한 조건으로 30분간 교반 시켰다(분획2 용액). 30분뒤 분획2 용액에서 위를 꺼내어 제3 세포 분리용 용액에 침지하여 60RPM 속도로 30분간 교반 시키고(분획3 용액), 30분뒤 분획3 용액에서 위를 꺼내어 제3 세포 분리용 용액에 침지하고 동일 조건으로 교반시켰다(분획4 용액). 이후 분획4 용액으로부터 위를 꺼내어 제3 세포 분리용 용액에 침지하여 60RPM 속도로 30분간 교반 시키되, 매 5분마다 볼텍스믹서로 30초간 강하게 믹싱하였다(도 8)(분획5 용액). 얻어진 분획5 용액을 100 μm 거름망 필터를 통과시킨 뒤(도 9) 12000RPM으로 원심분리하여 용액에 포함 되어있는 주 세포를 침전시켰다(도 10). 상층액을 제거하고 침전된 주 세포만을 수득하였다. 1 is a schematic diagram of a method for separating chief cells, pit cells, parietal cells, and isthmus cells after each treatment in the gastric organ according to the present invention. As a specific separation method, 8-week-old C57BL/6 mice were euthanized using a CO 2 chamber, the abdominal cavity was incised, and the junction of the esophagus and stomach was tied using a sterilized suture (FIG. 2). After the stomach was removed, it was immersed in a PBS solution and washed (FIG. 3), and the anterior part (Antrum/Pylorus) except for the fundus part was removed using scissors. The forestomach part was pushed with a sterilized cotton swab and turned inside out so that the gastric mucosa was exposed to the outside (FIG. 4). After washing the inverted stomach by immersing it in the second cell isolation solution, the incised fundus was tied with a sterile suture (FIG. 5). The stomach was inflated by injecting the first cell isolation solution into the Forestomach region using an insulin syringe (FIG. 6). Thereafter, the stomach was immersed in the second cell separation solution and stirred at 60 RPM for 30 minutes in an incubator at 37 ° C (Fig. 7) (fraction 1 solution), and after 30 minutes, the stomach was taken out of the obtained fraction 1 solution to separate the third cells. It was immersed in the solution and stirred for 30 minutes under the same conditions (fraction 2 solution). After 30 minutes, take the stomach out of the fraction 2 solution, immerse it in the third cell separation solution, stir at 60 RPM for 30 minutes (fraction 3 solution), and after 30 minutes, take the stomach out of the fraction 3 solution, immerse it in the third cell separation solution, and conditions were stirred (fraction 4 solution). Thereafter, the stomach was taken out of the fraction 4 solution, immersed in the third cell separation solution, stirred at 60 RPM for 30 minutes, and mixed vigorously for 30 seconds with a vortex mixer every 5 minutes (FIG. 8) (fraction 5 solution). The obtained Fraction 5 solution was passed through a 100 μm sieve filter (FIG. 9) and centrifuged at 12000 RPM to precipitate main cells contained in the solution (FIG. 10). The supernatant was removed and only the main precipitated cells were obtained.
[실험예 1][Experimental Example 1]
상기 실시예 1에서 마우스로부터 적출된 위 조직과, 분획5 용액에서 여과된 위 조직에 대하여 헤마톡실린&에오신(H&E) 염색을 수행하여 그 결과를 도 11에 나타내었다. Hematoxylin & eosin (H&E) staining was performed on the gastric tissue excised from the mouse in Example 1 and the gastric tissue filtered from the Fraction 5 solution, and the results are shown in FIG. 11 .
도 11에서 보는 바와 같이, 분획5 용액에서 여과된 위 조직에서는 대부분의 위 상피 세포들이 분리 및 제거된 것을 확인할 수 있었다. As shown in FIG. 11 , it was confirmed that most of the gastric epithelial cells were separated and removed from the gastric tissue filtered in the Fraction 5 solution.
[실험예 2][Experimental Example 2]
상기 실시예 1에서 얻어진 분획5 용액에서 침전 및 회수된 세포를 배양하여 마우스 위 조직의 주 세포 특이적으로 발현되는 단백질로 알려진 Gif 와 GPR43을 면역 염색하여 그 결과를 도 12에 나타내었다. Cells precipitated and recovered from the Fraction 5 solution obtained in Example 1 were cultured and immunostained for Gif and GPR43, which are known to be proteins expressed specifically in the main cells of mouse gastric tissue, and the results are shown in FIG. 12 .
도 12에서 보는 바와 같이, 분리된 세포 전체에서 주 세포 특이적인 단백질인 Gif 와 GPR43가 발현되는 것을 확인할 수 있었다. As shown in FIG. 12, it was confirmed that Gif and GPR43, which are major cell-specific proteins, were expressed in all the isolated cells.
이를 통해 상기 실시예 1에서 얻어진 분획5 용액에서 침전 및 회수된 세포는 위 유래 주 세포인 것을 알 수 있었다. Through this, it was found that the cells precipitated and recovered from the Fraction 5 solution obtained in Example 1 were the main cells derived from the stomach.
[비교예 1][Comparative Example 1]
본 발명에 따른 분리 방법의 위 주 세포 분리 효율이 뛰어남을 증명하기 위하여 이하의 실험을 수행하였다. 상기 실시예 1과 같이 8주령 C57BL/6 마우스를 CO2챔버를 이용하여 안락사 시킨 뒤 복강을 절개하고, 멸균된 봉합사를 이용하여 식도와 위의 접합 부위를 묶어주어 위를 적출한 뒤 PBS 용액에 침지하여 세척하였다. 이후, 적출된 위를 잘게 다진 후(chopping) EDTA로 배양하고 트립신 처리를 통해 단일 세포를 분리하였다. In order to prove that the separation method according to the present invention has excellent separation efficiency of the main cells, the following experiment was performed. As in Example 1, the 8-week-old C57BL/6 mouse was euthanized using a CO 2 chamber, the abdominal cavity was incised, and the junction of the esophagus and stomach was tied using a sterile suture, and the stomach was removed and then placed in a PBS solution. Washed by immersion. Thereafter, the extracted stomach was finely chopped (chopping), cultured with EDTA, and single cells were isolated through trypsin treatment.
[실험예 3][Experimental Example 3]
상기 실시예 1에서 얻어진 분획5 용액에서 침전 및 회수된 세포와 상기 비교예 1에서 분리된 세포로부터 RNA를 추출한 뒤 위 점막을 이루는 각 상피 세포 특이적 마커를 발현하는 지 확인하여 그 결과를 도 13에 나타내었다. 단, 도 13에서 MUC5ac는 선와 세포(foveolar cell)의 마커이고, TFF2는 목 세포(neck cell)의 마커이며, Gif 및 Mist1는 주 세포(chief cell)의 마커이고, H+K+ ATPase는 벽 세포(parietal cell)의 마커이다. RNA was extracted from the cells precipitated and recovered from the Fraction 5 solution obtained in Example 1 and the cells isolated in Comparative Example 1, and then confirmed to express each epithelial cell-specific marker constituting the gastric mucosa, and the results are shown in FIG. 13 shown in However, in FIG. 13, MUC5ac is a foveolar cell marker, TFF2 is a neck cell marker, Gif and Mist1 are chief cell markers, and H+K+ ATPase is a parietal cell marker. (parietal cell) marker.
도 13에서 보는 바와 같이, 실시예 1에서 회수된 세포(#2)에서는 주 세포의 마커(Gif 및 Mist1)만 발현되는 것을 확인할 수 있었으나, 비교예 1에서 분리된 세포(#1)에서는 위 점막을 이루는 상피 세포인 선와 세포(MUC5ac), 목 세포(TFF2), 주 세포(Gif 및 Mist1) 및 벽 세포(H+K+ ATPase)의 마커가 모두 발현되는 것을 확인할 수 있었다. As shown in FIG. 13, it was confirmed that only the main cell markers (Gif and Mist1) were expressed in the cells recovered in Example 1 (#2), but in the cells isolated in Comparative Example 1 (#1), the gastric mucosa It was confirmed that markers of crypt cells (MUC5ac), neck cells (TFF2), principal cells (Gif and Mist1), and parietal cells (H+K+ ATPase), which are epithelial cells that make up the , were all expressed.
이를 통해 본 발명에 따른 분리 방법을 이용하는 경우 위 주 세포만을 높은 효율로 분리할 수 있음을 알 수 있었다. Through this, it was found that only gastric main cells could be separated with high efficiency when using the separation method according to the present invention.
[실험예 4][Experimental Example 4]
도 14는 본 실험의 모식도를 나타낸 것으로, 위에 존재하는 주 세포 특이적으로 RFP 형광 단백질을 발현시킬 수 있는 리포터 마우스(Mist1creEr;R26-tdTomato)에 대하여 타목시펜(Tamoxifen) 5mg을 3회 피하 투여한 뒤, 위를 적출하여 상기 실시예 1과 동일한 방법으로 분획5 용액을 얻고, 이로부터 침전 및 회수된 세포를 현미경으로 관찰하였다. 그 결과는 도 15에 나타내었다. Figure 14 shows a schematic diagram of this experiment, after subcutaneously administering 5 mg of Tamoxifen 3 times to a reporter mouse (Mist1creEr; R26-tdTomato) capable of expressing RFP fluorescent protein specifically in the main cell present in the stomach. , The stomach was removed to obtain a fraction 5 solution in the same manner as in Example 1, and cells precipitated and recovered therefrom were observed under a microscope. The results are shown in FIG. 15 .
도 15에서 보는 바와 같이, 분획5 용액에서 침전 및 회수된 세포들에서 전체적으로 RFP 형광 단백질이 발현되는 것을 확인할 수 있었다. As shown in FIG. 15, it was confirmed that RFP fluorescent protein was expressed as a whole in the cells precipitated and recovered from the fraction 5 solution.
이를 통해 상기 실시예 1의 방법으로 얻어진 분획5 용액에서 침전 및 회수된 세포는 위 유래 주 세포인 것을 알 수 있었다. Through this, it was found that the cells precipitated and recovered from the Fraction 5 solution obtained by the method of Example 1 were the main cells derived from the stomach.
[실험예 5][Experimental Example 5]
GF(germ free) 마우스와 SPF(specific pathogen free) 마우스에 대하여 상기 실시예 1과 동일한 방법을 수행하여 분획1 용액, 분획3 용액 및 분획5 용액을 얻었다. 이후 각 분획 용액에 존재하는 세포에서 mRNA를 추출한 뒤, qRT-PCR로 다양한 위 상피 세포 마커의 발현 수준을 확인하여 그 결과를 도 16에 나타내었다. A fraction 1 solution, a fraction 3 solution, and a fraction 5 solution were obtained in the same manner as in Example 1 for germ free (GF) mice and specific pathogen free (SPF) mice. Thereafter, mRNA was extracted from cells present in each fraction solution, and expression levels of various gastric epithelial cell markers were confirmed by qRT-PCR, and the results are shown in FIG. 16 .
도 16에서 보는 바와 같이, 분획1 용액 및 분획3 용액 유래의 세포들에서는 주 세포 마커와 함께 그 외에 위 상피 세포의 마커가 발현되는 것을 확인할 수 있었으나, 분획5 용액 유래의 세포에서는 주 세포 마커가 강하게 발현되는 것을 확인할 수 있었다. As shown in FIG. 16, it was confirmed that the main cell markers and other gastric epithelial cell markers were expressed in the cells derived from the fraction 1 solution and the fraction 3 solution, but the main cell markers were expressed in the cells derived from the fraction 5 solution. It was confirmed that the expression was strong.
또한, SPF 마우스 유래의 분획5 용액 세포와, GF 마우스 유래의 분획5 용액 세포에서의 증식 세포 마커인 Ki67의 발현 수준을 확인한 결과, GF 마우스 유래의 분획5 용액 세포에서 Ki67가 더 강하게 발현되는 것을 확인할 수 있었는 바, GF 마우스 유래의 분획5 용액 세포의 줄기세포능이 SPF 마우스 유래의 분획5 용액 세포 보다 뛰어난 것을 알 수 있었다. In addition, as a result of confirming the expression level of Ki67, a proliferative cell marker, in Fraction 5 solution cells derived from SPF mice and in Fraction 5 solution cells derived from GF mice, Ki67 was expressed more strongly in Fraction 5 solution cells derived from GF mice. As confirmed, it was found that the stem cell activity of the GF mouse-derived Fraction 5 solution cells was superior to that of the Fraction 5 solution cells derived from SPF mice.
[실험예 6][Experimental Example 6]
상기 실험예 4에서 SPF 마우스와 GF 마우스로부터 얻어진 분획5 용액 유래의 주 세포를 각각 1번의 계대(passage)를 넘기고 7일간 배양한 뒤 이러한 주 세포로부터 분화되는 위 상피 세포를 면역 염색하여 그 결과를 도 17에 나타내었다. The main cells derived from the Fraction 5 solution obtained from the SPF mouse and the GF mouse in Experimental Example 4 were cultured for 7 days after passing one passage each, and immunostaining of gastric epithelial cells differentiated from these main cells was performed. 17.
도 17에서 보는 바와 같이, SPF 마우스 유래의 주 세포 보다 GF 마우스 유래의 주 세포로부터 분화되는 위 상피 세포의 수가 더 많은 것을 확인할 수 있었다. As shown in FIG. 17 , it was confirmed that the number of gastric epithelial cells differentiated from the main cells derived from GF mice was greater than that from the main cells derived from SPF mice.
이를 통해 GF 마우스 유래의 주 세포가 SPF 마우스 유래의 주 세포에 비하여 줄기세포능이 더 뛰어난 것을 알 수 있었다. Through this, it was found that the main cells derived from GF mice had more excellent stem cell activity than the main cells derived from SPF mice.
[실시예 2] 위 오가노이드의 제작[Example 2] Production of gastric organoids
상기 실시예 1에서 분리된 주 세포 펠렛을 RPMI1640 배양액 1㎖에 희석하여 세포 계수 후, 다시 원심 분리하여 세포 결집체를 마트리젤과 혼합하고(15㎕/well), 24웰 플레이트에 분주 후 37℃에서 1시간 배양하였다. 이후 상기 제조예 4의 주 세포 배양 용액을 첨가하고 37℃, 가스 5 부피% CO2 인큐베이터에서 배양하였다. 배양액은 2-3일 간격으로 신선하게 교체하며, 배양 5일 후 다른 상피 세포로의 분화 여부를 관찰하였다. 이렇게 제작된 위 오가노이드를 현미경으로 관찰한 사진을 도 18에 나타내었다. After diluting the main cell pellet isolated in Example 1 in 1 ml of RPMI1640 culture medium, counting the cells, centrifuging again, mixing the cell aggregates with Matrigel (15 μl/well), dispensing into 24-well plates, and then at 37 ° C. Incubated for 1 hour. Then, the main cell culture solution of Preparation Example 4 was added and cultured in an incubator at 37° C., 5 vol% gas CO 2 . The culture medium was freshly replaced every 2-3 days, and differentiation into other epithelial cells was observed after 5 days of culture. A photograph of the prepared gastric organoid under a microscope is shown in FIG. 18 .
도 18에서 보는 바와 같이, 버딩(budding) 형상의 위 오가노이드가 형성된 것을 확인할 수 있었다. As shown in FIG. 18, it was confirmed that organoids having a budding shape were formed.
[비교예 2][Comparative Example 2]
상기 비교예 1에서 분리된 세포 펠렛을 RPMI1640 배양액 1㎖에 희석하여 세포 계수 후, 다시 원심 분리하여 세포 결집체를 마트리젤과 혼합하고, 24웰 플레이트에 분주 후 37℃에서 1시간 배양하였다. 이후 EGF, 가스트린, FGF10, Noggin, Wnt3a, 및 R-spondin 보충된 배양 배지(ENRGFW 배지)를 첨가하여 37℃ CO2 인큐베이터에서 배양하였다. 배양액은 2-3일 간격으로 신선하게 교체하였다. 이렇게 제작된 위 오가노이드를 현미경으로 관찰한 사진을 도 19에 나타내었다.The cell pellet isolated in Comparative Example 1 was diluted in 1 ml of RPMI1640 culture medium, the cells were counted, centrifuged again, the cell aggregates were mixed with Matrigel, and then dispensed into a 24-well plate and incubated at 37 ° C. for 1 hour. Then, a culture medium (ENRGFW medium) supplemented with EGF, gastrin, FGF10, Noggin, Wnt3a, and R-spondin was added and cultured in a 37°C CO 2 incubator. The culture medium was freshly replaced every 2-3 days. A photo of the prepared gastric organoid under a microscope is shown in FIG. 19 .
도 19에서 보는 바와 같이, 원형의 위 오가노이드가 형성된 것을 확인할 수 있었다. As shown in FIG. 19, it was confirmed that circular gastric organoids were formed.
[실험예 7][Experimental Example 7]
상기 실시예 1에서 얻어진 분획5 용액에서 침전 및 회수된 세포를 제조예 4의 주 세포 배양 용액 또는 기존의 일반적인 배양 용액으로 EGF, 가스트린, FGF10, Noggin, Wnt3a, 및 R-spondin 보충된 배양 배지(ENRGFW 배지)에서 에서 37℃, 가스 5 부피% CO2 인큐베이터에서 5일간 배양한 후 실험예 3과 같이 RNA를 추출한 뒤 위 점막을 이루는 각 상피 세포 특이적 마커를 발현하는 지 확인하여 그 결과를 도 20에 나타내었다. Cells precipitated and recovered from the Fraction 5 solution obtained in Example 1 were used as the main cell culture solution of Preparation Example 4 or an existing general culture medium supplemented with EGF, Gastrin, FGF10, Noggin, Wnt3a, and R-spondin ( ENRGFW medium) at 37 ° C, 5 vol% gas CO 2 After culturing for 5 days in an incubator, RNA was extracted as in Experimental Example 3, and then it was confirmed that each epithelial cell-specific marker constituting the gastric mucosa was expressed to help the result 20.
도 20에서 보는 바와 같이, 제조예 4의 주 세포 배양 용액을 이용하여 배양한 경우(#2) 위 점막을 이루는 상피 세포인 선와 세포(MUC5ac), 목 세포(TFF2), 주 세포(Gif 및 Mist1) 및 벽 세포(H+K+ ATPase)의 마커가 모두 발현되는 것을 확인할 수 있었으나, 기존의 일반적인 배양 용액에서 배양한 경우(#1) 벽 세포로의 세포 분화가 일어나지 않은 것을 확인할 수 있었다. As shown in FIG. 20, when cultured using the main cell culture solution of Preparation Example 4 (#2), epithelial cells constituting the gastric mucosa, crypt cells (MUC5ac), neck cells (TFF2), and main cells (Gif and Mist1) ) and parietal cell (H+K+ ATPase) markers were all expressed, but it was confirmed that cell differentiation into parietal cells did not occur when cultured in a conventional general culture solution (#1).
이를 통해 본 발명에 따른 배양 용액을 사용한 경우 위 주 세포로부터 위 점막을 이루는 상피 세포인 선와 세포, 목 세포, 주 세포 및 벽 세포로 효과적인 분화를 유도할 수 있음을 알 수 있었다. From this, it was found that, when the culture solution according to the present invention was used, effective differentiation from gastric principal cells into crypt cells, cervical cells, principal cells, and parietal cells, which are epithelial cells constituting the gastric mucosa, could be induced.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
본 발명은 종래의 위샘에 존재하는 주세포(Chief cell)의 기능과 역할에 대한 연구를 위한 분리된 주세포 배양용 조성물에 관한 것이다. 현재 위샘에 존재하는 주세포의 기능과 역할에 대한 연구가 미미하여, 새로운 분리 방법을 통한 연구의 필요성이 요구되고 있다. 본 발명은 종래에 사용되는 방법과 달리 위 점막을 외부로 노출시켜, 주세포만을 특이적으로 분리한 주세포 분화시킬 배양용 조성물을 제공하는 것이다. 분리된 주세포를 분화시킬 배양용 조성물로 모든 계통, 특히 종래에는 분화시키기 어렵던 벽세포(Parietal cell)까지 분화시키고, 위 점막 오가노이드를 형성시킴으로써, 위장질환 관련 치료 조성물 개발에 치료에 매우 효과적으로 이용될 것으로 기대된다.The present invention relates to a composition for culturing isolated chief cells for research on the functions and roles of chief cells present in conventional gastric glands. Currently, research on the function and role of the main cells present in the gastric gland is insignificant, and the need for research through a new isolation method is required. Unlike conventional methods, the present invention provides a culture composition capable of differentiating main cells by specifically isolating only main cells by exposing the gastric mucosa to the outside. As a culture composition for differentiating the isolated main cells, it differentiates all lineages, especially parietal cells, which were difficult to differentiate in the past, and forms gastric mucosal organoids, which can be used very effectively for treatment in the development of therapeutic compositions related to gastrointestinal diseases. It is expected that

Claims (22)

  1. 소 혈청 알부민(bovine serum albumin; BSA) 및 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(insulin-transferrin-selenium-sodium pyruvate; ITS-A)를 포함하는, 위 세포 배양용 조성물. A composition for culturing stomach cells, comprising bovine serum albumin (BSA) and insulin-transferrin-selenium-sodium pyruvate (ITS-A).
  2. 제1항에 있어서, According to claim 1,
    상기 위 세포는 위 주 세포(chief)인, 위 세포 배양용 조성물. The gastric cells are gastric chief cells, a composition for culturing gastric cells.
  3. 제1항에 있어서, According to claim 1,
    상기 소 혈청 알부민(BSA)의 농도는 1 내지 20 v/v%이고, The concentration of the bovine serum albumin (BSA) is 1 to 20 v / v%,
    상기 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(ITS-A)의 농도는 1 내지 15 ug/ml인, 위 세포 배양용 조성물. The concentration of the insulin-transferrin-selenium-sodium pyruvate (ITS-A) is 1 to 15 ug / ml, the composition for culturing gastric cells.
  4. 제1항에 있어서, According to claim 1,
    상기 위 세포 배양용 조성물은 간세포 성장 인자(hepatocyte growth factor; HGF); Wnt3a 또는 이를 포함하는 조건 배지(conditioned media); R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지; Noggin; B27; N-아세틸 시스테인(N-acetyl cysteine; NAC); 섬유아세포 성장 인자(fibroblast growth factor; FGF); 표피 성장 인자(epidermal growth factor; EGF); 가스트린(gastrin); Y-27632; 및 하이드로코르티손(hydrocortisone)으로 이루어진 군에서 선택된 1종 이상을 더 포함하는, 위 세포 배양용 조성물.The composition for culturing gastric cells includes hepatocyte growth factor (HGF); Wnt3a or conditioned media containing it; R- spondin (R-spondin) or a conditioned medium containing the same; Noggin; B27; N-acetyl cysteine (NAC); fibroblast growth factor (FGF); epidermal growth factor (EGF); gastrin; Y-27632; And hydrocortisone (hydrocortisone) further comprising at least one member selected from the group consisting of, gastric cell culture composition.
  5. 제4항에 있어서, According to claim 4,
    상기 간세포 성장 인자(HGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the hepatocyte growth factor (HGF) is 100 to 1000 ng/ml,
    상기 Wnt3a 또는 이를 포함하는 조건 배지의 농도는 10 내지 80 v/v%이며, The concentration of the Wnt3a or the conditioned medium containing the same is 10 to 80 v / v%,
    상기 R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지의 농도는 0.1 내지 30 v/v%이고, The concentration of the R-spondin or the conditioned medium containing the same is 0.1 to 30 v / v%,
    상기 Noggin의 농도는 10 내지 200 ng/ml이며,The concentration of Noggin is 10 to 200 ng/ml,
    상기 B27의 농도는 0.005 내지 0.1 v/v% 이고, The concentration of B27 is 0.005 to 0.1 v / v%,
    상기 N-아세틸 시스테인(NAC)의 농도는 0.1 내지 5 mM이며, The concentration of N-acetyl cysteine (NAC) is 0.1 to 5 mM,
    상기 섬유아세포 성장 인자(FGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the fibroblast growth factor (FGF) is 100 to 1000 ng/ml,
    상기 표피 성장 인자(EGF)의 농도는 5 내지 100 ng/ml이며, The concentration of the epidermal growth factor (EGF) is 5 to 100 ng/ml,
    상기 가스트린의 농도는 0.01 내지 10 uM이고, The gastrin concentration is 0.01 to 10 uM,
    상기 Y-27632의 농도는 1 내지 50 mM이며, The concentration of Y-27632 is 1 to 50 mM,
    상기 하이드로코르티손의 농도는 0.01 내지 10 ug/ml인, 위 세포 배양용 조성물.The hydrocortisone concentration is 0.01 to 10 ug / ml, the composition for culturing gastric cells.
  6. 제4항에 있어서, According to claim 4,
    상기 섬유아세포 성장 인자(FGF)는 FGF1, FGF2(bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF17, 및 FGF18로 이루어진 군에서 선택된 1종 이상인, 위 세포 배양용 조성물. The fibroblast growth factor (FGF) is at least one member selected from the group consisting of FGF1, FGF2 (bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF17, and FGF18, a composition for culturing gastric cells. .
  7. 제1항에 있어서, According to claim 1,
    상기 위 세포 배양용 조성물은 MEM(Minimum Essential Medium), BME(Basal Medium Eagle), DMEM(Dulbecco's Modified EagleMedium), EMEM(Eagle's minimal essential medium), IMDM(Iscove's Modified Dulbecco's Medium), GMEM(Glas- gow's MEM), F12(Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 배지(Leibovitz's L-15 medium), McCoy's 5 A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM/F-12 및 Advanced RPMI1640로 이루어진 군에서 선택된 1종 이상의 기본 배양 배지를 더 포함하는, 위 세포 배양용 조성물. The gastric cell culture composition is MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified EagleMedium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM) ), F12 (Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5 A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM/F-12, and Advanced RPMI1640, further comprising at least one basal culture medium selected from the group consisting of gastric cell culture composition.
  8. 제7항에 있어서, According to claim 7,
    상기 기본 배양 배지는 글루타맥스(glutamax), 하이드록시에틸피페라진에테인설폰산(Hydroxyethyl piperazine Ethane Sulfonicacid; HEPES) 및 항생제 중 적어도 하나를 더 포함하는, 위 세포 배양용 조성물.Wherein the basal culture medium further comprises at least one of glutamax, hydroxyethyl piperazine ethane sulfonic acid (HEPES) and an antibiotic, a composition for culturing gastric cells.
  9. 소 혈청 알부민(bovine serum albumin; BSA) 및 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(insulin-transferrin-selenium-sodium pyruvate; ITS-A)를 포함하는, 위 주 세포(chief)의 위 점막 세포로의 분화 유도용 조성물. gastric mucosal cells of the chief, including bovine serum albumin (BSA) and insulin-transferrin-selenium-sodium pyruvate (ITS-A) A composition for inducing differentiation of.
  10. 제9항에 있어서, According to claim 9,
    상기 위 점막 세포는 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상인, 분화 유도용 조성물.The gastric mucosal cells are surface mucous cells (mucous foveolar cells), parietal cells (parietal cells) and neck mucous cells (mucus neck cells) at least one selected from the group consisting of, composition for inducing differentiation.
  11. 제9항에 있어서, According to claim 9,
    상기 소 혈청 알부민(BSA)의 농도는 1 내지 20 v/v%이고, The concentration of the bovine serum albumin (BSA) is 1 to 20 v / v%,
    상기 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(ITS-A)의 농도는 1 내지 15 ug/ml인, 분화 유도용 조성물.The concentration of the insulin-transferrin-selenium-sodium pyruvate (ITS-A) is 1 to 15 ug / ml, a composition for inducing differentiation.
  12. 제9항에 있어서, According to claim 9,
    상기 조성물은 간세포 성장 인자(hepatocyte growth factor; HGF); Wnt3a 또는 이를 포함하는 조건 배지(conditioned media); R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지; Noggin; B27; N-아세틸 시스테인(N-acetyl cysteine; NAC); 섬유아세포 성장 인자(fibroblast growth factor; FGF); 표피 성장 인자(epidermal growth factor; EGF); 가스트린(gastrin); Y-27632; 및 하이드로코르티손(hydrocortisone)으로 이루어진 군에서 선택된 1종 이상을 더 포함하는, 분화 유도용 조성물.The composition comprises hepatocyte growth factor (HGF); Wnt3a or conditioned media containing it; R- spondin (R-spondin) or a conditioned medium containing the same; Noggin; B27; N-acetyl cysteine (NAC); fibroblast growth factor (FGF); epidermal growth factor (EGF); gastrin; Y-27632; And hydrocortisone (hydrocortisone) further comprising at least one member selected from the group consisting of, a composition for inducing differentiation.
  13. 제12항에 있어서, According to claim 12,
    상기 간세포 성장 인자(HGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the hepatocyte growth factor (HGF) is 100 to 1000 ng/ml,
    상기 Wnt3a 또는 이를 포함하는 조건 배지의 농도는 10 내지 80 v/v%이며, The concentration of the Wnt3a or the conditioned medium containing the same is 10 to 80 v / v%,
    상기 R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지의 농도는 0.1 내지 30 v/v%이고, The concentration of the R-spondin or the conditioned medium containing the same is 0.1 to 30 v / v%,
    상기 Noggin의 농도는 10 내지 200 ng/ml이며,The concentration of Noggin is 10 to 200 ng/ml,
    상기 B27의 농도는 0.005 내지 0.1 v/v%이고, The concentration of B27 is 0.005 to 0.1 v / v%,
    상기 N-아세틸 시스테인(NAC)의 농도는 0.1 내지 5 mM이며, The concentration of N-acetyl cysteine (NAC) is 0.1 to 5 mM,
    상기 섬유아세포 성장 인자(FGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the fibroblast growth factor (FGF) is 100 to 1000 ng/ml,
    상기 표피 성장 인자(EGF)의 농도는 5 내지 100 ng/ml이며, The concentration of the epidermal growth factor (EGF) is 5 to 100 ng/ml,
    상기 가스트린의 농도는 0.01 내지 10 uM이고, The gastrin concentration is 0.01 to 10 uM,
    상기 Y-27632의 농도는 1 내지 50 mM이며, The concentration of Y-27632 is 1 to 50 mM,
    상기 하이드로코르티손의 농도는 0.01 내지 10 ug/ml인, 분화 유도용 조성물.The concentration of the hydrocortisone is 0.01 to 10 ug / ml, a composition for inducing differentiation.
  14. 소 혈청 알부민(bovine serum albumin; BSA) 및 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(insulin-transferrin-selenium-sodium pyruvate; ITS-A)를 포함하는, 위 오가노이드 배양용 조성물. A composition for culturing gastric organoids comprising bovine serum albumin (BSA) and insulin-transferrin-selenium-sodium pyruvate (ITS-A).
  15. 제14항에 있어서, According to claim 14,
    상기 위 오가노이드는 주 세포(chief cells), 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상을 포함하는, 위 오가노이드 배양용 조성물.The gastric organoid includes at least one selected from the group consisting of chief cells, mucous foveolar cells, parietal cells, and mucus neck cells. A composition for culturing nodules.
  16. 제14항에 있어서, According to claim 14,
    상기 소 혈청 알부민(BSA)의 농도는 1 내지 20 v/v%이고, The concentration of the bovine serum albumin (BSA) is 1 to 20 v / v%,
    상기 인슐린-트랜스퍼린-셀레늄-소디움 피루베이트(ITS-A)의 농도는 1 내지 15 ug/ml인, 위 오가노이드 배양용 조성물.The insulin-transferrin-selenium-sodium pyruvate (ITS-A) concentration is 1 to 15 ug / ml, the composition for culturing gastric organoids.
  17. 제14항에 있어서, According to claim 14,
    상기 조성물은 간세포 성장 인자(hepatocyte growth factor; HGF); Wnt3a 또는 이를 포함하는 조건 배지(conditioned media); R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지; Noggin; B27; N-아세틸 시스테인(N-acetyl cysteine; NAC); 섬유아세포 성장 인자(fibroblast growth factor; FGF); 표피 성장 인자(epidermal growth factor; EGF); 가스트린(gastrin); Y-27632; 및 하이드로코르티손(hydrocortisone)으로 이루어진 군에서 선택된 1종 이상을 더 포함하는, 위 오가노이드 배양용 조성물.The composition comprises hepatocyte growth factor (HGF); Wnt3a or conditioned media containing it; R- spondin (R-spondin) or a conditioned medium containing the same; Noggin; B27; N-acetyl cysteine (NAC); fibroblast growth factor (FGF); epidermal growth factor (EGF); gastrin; Y-27632; And hydrocortisone (hydrocortisone) further comprising at least one member selected from the group consisting of, a composition for culturing gastric organoids.
  18. 제17항에 있어서, According to claim 17,
    상기 간세포 성장 인자(HGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the hepatocyte growth factor (HGF) is 100 to 1000 ng/ml,
    상기 Wnt3a 또는 이를 포함하는 조건 배지의 농도는 10 내지 80 v/v%이며, The concentration of the Wnt3a or the conditioned medium containing the same is 10 to 80 v / v%,
    상기 R-스폰딘(R-spondin) 또는 이를 포함하는 조건 배지의 농도는 0.1 내지 30 v/v%이고, The concentration of the R-spondin or the conditioned medium containing the same is 0.1 to 30 v / v%,
    상기 Noggin의 농도는 10 내지 200 ng/ml이며,The concentration of Noggin is 10 to 200 ng/ml,
    상기 B27의 농도는 0.005 내지 0.1 v/v%이고, The concentration of B27 is 0.005 to 0.1 v / v%,
    상기 N-아세틸 시스테인(NAC)의 농도는 0.1 내지 5 mM이며, The concentration of N-acetyl cysteine (NAC) is 0.1 to 5 mM,
    상기 섬유아세포 성장 인자(FGF)의 농도는 100 내지 1000 ng/ml이고, The concentration of the fibroblast growth factor (FGF) is 100 to 1000 ng/ml,
    상기 표피 성장 인자(EGF)의 농도는 5 내지 100 ng/ml이며, The concentration of the epidermal growth factor (EGF) is 5 to 100 ng/ml,
    상기 가스트린의 농도는 0.01 내지 10 uM이고, The gastrin concentration is 0.01 to 10 uM,
    상기 Y-27632의 농도는 1 내지 50 mM이며, The concentration of Y-27632 is 1 to 50 mM,
    상기 하이드로코르티손의 농도는 0.01 내지 10 ug/ml인, 위 오가노이드 배양용 조성물.The hydrocortisone concentration is 0.01 to 10 ug / ml, the composition for culturing gastric organoids.
  19. 위 주 세포(chief cell)를 마트리겔 및 제14항 내지 제18항 중 어느 한 항의 위 오가노이드 배양용 조성물에 넣고 오가노이드를 형성하는 단계;를 포함하는 위 오가노이드의 제조 방법. A method for producing gastric organoids comprising the step of inserting gastric chief cells into Matrigel and the composition for culturing gastric organoids according to any one of claims 14 to 18 and forming organoids.
  20. 제19항에 있어서,According to claim 19,
    상기 위 오가노이드는 주 세포(chief cells), 표면 점액 세포(mucous foveolar cells), 벽 세포(parietal cell) 및 목 점액 세포(mucus neck cell)로 이루어진 군에서 선택된 1종 이상을 포함하는, 위 오가노이드의 제조 방법.The gastric organoid includes at least one selected from the group consisting of chief cells, mucous foveolar cells, parietal cells, and mucus neck cells. How to make a noid.
  21. 제19항 또는 제20항에 의해 제조된 위 오가노이드. A gastric organoid prepared according to claim 19 or claim 20.
  22. 제21항의 위 오가노이드를 유효성분으로 포함하는 생체 이식용 약학 조성물.A pharmaceutical composition for biotransplantation comprising the gastric organoid of claim 21 as an active ingredient.
PCT/KR2022/008916 2021-06-23 2022-06-23 Composition for culturing stomach cells WO2022270932A1 (en)

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