WO2022270932A1 - Composition pour la culture de cellules stomacales - Google Patents

Composition pour la culture de cellules stomacales Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
cells
concentration
composition
gastric
growth factor
Prior art date
Application number
PCT/KR2022/008916
Other languages
English (en)
Korean (ko)
Inventor
남기택
이부현
Original Assignee
연세대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 연세대학교 산학협력단 filed Critical 연세대학교 산학협력단
Publication of WO2022270932A1 publication Critical patent/WO2022270932A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/345—Gastrin; Cholecystokinins [CCK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Physiology (AREA)
  • Nutrition Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Transplantation (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une composition pour la culture de cellules principales isolées à partir de tissus ou d'organes vivants, en particulier de l'estomac, et un procédé de culture de cellules utilisant la composition.
PCT/KR2022/008916 2021-06-23 2022-06-23 Composition pour la culture de cellules stomacales WO2022270932A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020210081690A KR20230000033A (ko) 2021-06-23 2021-06-23 위 세포 배양용 조성물
KR10-2021-0081690 2021-06-23

Publications (1)

Publication Number Publication Date
WO2022270932A1 true WO2022270932A1 (fr) 2022-12-29

Family

ID=84545779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/008916 WO2022270932A1 (fr) 2021-06-23 2022-06-23 Composition pour la culture de cellules stomacales

Country Status (2)

Country Link
KR (1) KR20230000033A (fr)
WO (1) WO2022270932A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220170629A (ko) * 2021-06-23 2022-12-30 연세대학교 산학협력단 조직 또는 기관으로부터 세포 분리용 조성물

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020182728A1 (en) * 2001-03-29 2002-12-05 Vijayakumar Ramiya Method for transdifferentiation of non pancreatic stem cells to the pancreatic differentiation pathway
KR20150050861A (ko) * 2013-11-01 2015-05-11 주식회사 비비에이치씨 중간엽 줄기세포로부터 유도된 만능 줄기세포를 이용하여 연골세포로 분화시키는 방법
KR20150070496A (ko) * 2013-12-16 2015-06-25 건국대학교 산학협력단 돼지 정원줄기세포를 체외 배양하기 위한 배양액 조성물 및 배양방법
KR20210028562A (ko) * 2019-09-04 2021-03-12 한국생명공학연구원 증식 가능한 간 오가노이드 분화용 배지 조성물 및 이를 이용한 간 오가노이드의 제조방법
KR20210051138A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 사람 폐 조직으로부터 2형 폐포 세포 분리 및 계대배양 방법, 및 이를 이용한 폐 오가노이드 제작 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020182728A1 (en) * 2001-03-29 2002-12-05 Vijayakumar Ramiya Method for transdifferentiation of non pancreatic stem cells to the pancreatic differentiation pathway
KR20150050861A (ko) * 2013-11-01 2015-05-11 주식회사 비비에이치씨 중간엽 줄기세포로부터 유도된 만능 줄기세포를 이용하여 연골세포로 분화시키는 방법
KR20150070496A (ko) * 2013-12-16 2015-06-25 건국대학교 산학협력단 돼지 정원줄기세포를 체외 배양하기 위한 배양액 조성물 및 배양방법
KR20210028562A (ko) * 2019-09-04 2021-03-12 한국생명공학연구원 증식 가능한 간 오가노이드 분화용 배지 조성물 및 이를 이용한 간 오가노이드의 제조방법
KR20210051138A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 사람 폐 조직으로부터 2형 폐포 세포 분리 및 계대배양 방법, 및 이를 이용한 폐 오가노이드 제작 방법

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20220170629A (ko) * 2021-06-23 2022-12-30 연세대학교 산학협력단 조직 또는 기관으로부터 세포 분리용 조성물
KR102611109B1 (ko) 2021-06-23 2023-12-07 연세대학교 산학협력단 조직 또는 기관으로부터 세포 분리용 조성물

Also Published As

Publication number Publication date
KR20230000033A (ko) 2023-01-02

Similar Documents

Publication Publication Date Title
WO2022270932A1 (fr) Composition pour la culture de cellules stomacales
WO2019198995A1 (fr) Procédé de conversion à base d'exosomes pour cellules immunitaires
WO2012008733A2 (fr) Cellules souches issues de tissu de placenta primaire et agent thérapeutique contenant celles-ci
WO2019004792A9 (fr) Procédé de préparation et utilisation d'un microsphéroïde de cellule souche cardiaque d'origine humaine
WO2021071289A2 (fr) Composition permettant d'augmenter le caractère souche et son utilisation
WO2019050350A2 (fr) Cellule de type de sertoli dérivée de cellule souche procédé de préparation associé et utilisation associée
WO2024076173A1 (fr) Composition pour induire une différenciation de cellules souches issues du tissu adipeux en cellules de papille dermique, et procédé de différenciation utilisant la composition
WO2016006782A1 (fr) Composition favorisant la stabilité de stockage de cellules souches
WO2012161519A1 (fr) Lignée de cellules souches adultes introduite avec un gène du facteur de croissance hépatocytaire et un gène du facteur de transcription neurogène ayant un motif basique hélice-boucle-hélice et ses utilisations
WO2020067774A1 (fr) Cellules souches mésenchymateuses dérivées de la synoviale et leur utilisation
WO2015023147A1 (fr) Cellule souche mésenchymateuse traitée par un inhibiteur de signal mtor/stat3 ayant une activité immunomodulatrice, et composition pour thérapie cellulaire la comprenant, destinée à prévenir ou traiter des troubles immuns
WO2022065859A1 (fr) Procédé de reprogrammation directe de cellules somatiques en cellules bêta pancréatiques à l'aide de microarn, et composition de différenciation
WO2012133992A1 (fr) Procédé de différenciation de cellules souches mésenchymateuses dérivées de tissus adipeux en chondrocytes
WO2024014721A1 (fr) Composition anticancéreuse comprenant des exosomes dérivés de cellules souches et son procédé de préparation
WO2020209636A1 (fr) Procédé pour induire une reprogrammation directe d'une cellule urinaire dans une cellule progénitrice rénale et composition pharmaceutique comprenant ladite cellule reprogrammée par le même procédé pour prévenir ou traiter une maladie des lésions cellulaires rénales
WO2023090928A1 (fr) Procédé d'isolement, de maintien, de prolifération et de différenciation de cellules monoclonales dérivées de cellules souches épithéliales de glandes salivaires humaines ou de cellules progénitrices et procédé de production de vésicules extracellulaires pour le traitement de maladies des glandes salivaires
WO2021107234A1 (fr) Cellules souches hématopoïétiques à inactivation simultanée de gènes ccr5/cxcr4 personnalisées pour le traitement ou la prévention d'une infection par le vih, et leur procédé de préparation
WO2019117454A1 (fr) Additif de milieu pour transformation cellulaire hautement efficace à l'aide d'un facteur de régulation de stress d'organite cellulaire
WO2015194710A1 (fr) Composition comprenant des cellules souches mésenchymateuses traitées avec un inhibiteur de stat3 en tant que principe actif pour la prévention ou le traitement de maladies immunitaires
WO2022270931A1 (fr) Composition pour isoler des cellules d'un tissu ou d'un organe
WO2019039922A1 (fr) Composition pharmacologique de prévention ou de traitement du lupus, comprenant un sécrétome dérivé de cellules souches mésenchymateuses
WO2023043191A1 (fr) Composition pharmaceutique pour la prévention ou le traitement d'une maladie hépatique comprenant un milieu de culture de cellules souches surexprimant la protéine iap
WO2021118325A1 (fr) Procédé de préparation de cellules souches mésenchymateuses ayant une viabilité améliorée par introduction de virus anticancéreux
WO2022231083A1 (fr) Composition pour la prévention ou le traitement d'une maladie auto-immune, comprenant des mitochondries en tant que principe actif
WO2024043521A1 (fr) Procédé de construction d'organoïde alvéolaire à l'aide de cellules souches pluripotentes induites dérivées de cellules alvéolaires

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22828779

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22828779

Country of ref document: EP

Kind code of ref document: A1