Claims (10)
1. Postupak za kultivaciju epitelijalnih matičnih stanica ili izoliranog tkivnog fragmenta koji se sastoji iz spomenutih epitelijalnih matičnih stanica, postupak obuhvaća
pružanje vanstanične matrice;
inkubaciju spomenutih epitelijalnih matičnih stanica ili spomenutog izoliranog tkivnog fragmenta koji se sastoji iz spomenutih epitelijalnih matičnih stanica s vanstaničnom matricom;
kultivaciju epitelijalnih matičnih stanica ili izoliranog tkivnog fragmenta u prisustvu medija stanične kulture koji se sastoji iz osnovnog medija za životinjske ili ljudske stanice u koje se dodaje:
Noggin;
između 5 i 500 ngram/ml mitogenog faktora rasta izabranog između epidermalnog faktora rasta (EGF), transformirajućeg faktora rasta-alfa, faktora rasta fibroblasta (FGF), neutrofnog faktora porijeklom iz mozga i faktora rasta keratinocita (KGF); i
Wnt agonist izabran između R-spondin 1, R-spondin 2, R-spondin 3 i R-spondin 4.1. The procedure for the cultivation of epithelial stem cells or an isolated tissue fragment consisting of said epithelial stem cells, the procedure includes
providing extracellular matrix;
incubation of said epithelial stem cells or said isolated tissue fragment consisting of said epithelial stem cells with an extracellular matrix;
the cultivation of epithelial stem cells or an isolated tissue fragment in the presence of a cell culture medium consisting of a basic medium for animal or human cells to which is added:
Noggin;
between 5 and 500 ng/ml of a mitogenic growth factor selected from epidermal growth factor (EGF), transforming growth factor-alpha, fibroblast growth factor (FGF), brain-derived neutrophil factor and keratinocyte growth factor (KGF); and
Wnt agonist selected from R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.
2. Postupak prema zahtjevu 1, gdje spomenuti medij kulture dalje sadrži
Rock (Rho-kinaza) inhibitor, opcionalno izabran između Y-27632, Fasudil, i H-1152; i/ili
notch agonista.2. The method according to claim 1, wherein said culture medium further contains
Rock (Rho-kinase) inhibitor, optionally selected from Y-27632, Fasudil, and H-1152; and/or
notch agonists.
3. Postupak prema zahtjevu 1 ili zahtjevu 2, gdje je postupak za kultivaciju organoida, gdje se organoid sastoji iz epitelijalnih matičnih stanica.3. The method according to claim 1 or claim 2, where the method is for the cultivation of an organoid, where the organoid consists of epithelial stem cells.
4. Postupak za dobivanje pankreasnog organoida kultivacijom pankreasnih epitelijalnih matičnih stanica, ili tkivnih fragmenata koji se sastoje iz spomenutih epitelijalnih matičnih stanica, upotrebom postupka prema zahtjevu 1 ili 2, pri čemu korak kultivacije obuhvaća
- kultivaciju spomenutih pankreasnih epitelijalnih matičnih stanica ili tkivnih fragmenata koji se sastoje iz spomenutih epitelijalnih matičnih stanica u kontaktu s vanstaničnom matricom u prvom koraku u mediju koji sadrži Noggin, EGF i KGF ili FGF, i R-spondin 1 kao Wnt agonist, dopunjen s B27, N2, i/ili N-Acetilcisteinom, i opcionalno dalje sadrži gastrin, eksendin i nikotinamid, i
- nakon toga u drugom koraku u mediju koji se sastoji iz EGF i KGF ili FGF, i R-spondin 1 kao Wnt agonist, dopunjen sa B27, N2, i/ili N-Acetilcisteinom.4. A method for obtaining a pancreatic organoid by cultivating pancreatic epithelial stem cells, or tissue fragments consisting of said epithelial stem cells, using the method according to claim 1 or 2, wherein the cultivation step includes
- cultivation of said pancreatic epithelial stem cells or tissue fragments consisting of said epithelial stem cells in contact with the extracellular matrix in the first step in a medium containing Noggin, EGF and KGF or FGF, and R-spondin 1 as a Wnt agonist, supplemented with B27 , N2, and/or N-Acetylcysteine, and optionally further contains gastrin, exendin and nicotinamide, and
- after that in the second step in a medium consisting of EGF and KGF or FGF, and R-spondin 1 as a Wnt agonist, supplemented with B27, N2, and/or N-Acetylcysteine.
5. Postupak za dobivanje želučanog organoida kultivacijom želučanih epitelijalnih matičnih stanica, ili tkivnih fragmenata koji sadrže spomenute epitelijalne matične stanice, upotrebom postupka prema bilo kojem od patentnih zahtjeva 1 do 3, gdje korak kultivacije obuhvaća
- uzgoj spomenutih želučanih epitelijalnih matičnih stanica ili tkivnih fragmenata koji obuhvaćaju spomenute želučane epitelijalne matičnestanice u kontaktu s vanstaničnom matricom u prvom koraku u prvom mediju koji se sastoji iz Noggin, EGF i FGF10, Rspondin 1 i Wnt-3a kao Wnt agonista, i dalje sadrži B27, N2, i/ili N-Acetilcistein, i
- nakon toga u drugom koraku u mediju pri čemu je medij identičan s prvim medijem osim toga što je koncentracija Wnt-3a smanjena u drugom koraku u usporedbi s koncentracijom Wnt-3a koja je prisutna u prvom koraku.5. Method for obtaining a gastric organoid by cultivating gastric epithelial stem cells, or tissue fragments containing said epithelial stem cells, using the method according to any of claims 1 to 3, where the cultivation step comprises
- cultivation of said gastric epithelial stem cells or tissue fragments comprising said gastric epithelial stem cells in contact with the extracellular matrix in the first step in the first medium consisting of Noggin, EGF and FGF10, Rspondin 1 and Wnt-3a as a Wnt agonist, further containing B27, N2, and/or N-Acetylcysteine, and
- thereafter in a second step in the medium, wherein the medium is identical to the first medium except that the concentration of Wnt-3a is reduced in the second step compared to the concentration of Wnt-3a present in the first step.
6. Postupak prema bilo kojem od patentnih zahtjeva 1 do 5, pri čemu vanstanična matrica sadrži kolagen i laminin.6. The method according to any one of claims 1 to 5, wherein the extracellular matrix contains collagen and laminin.
7. Trodimenzionalni tkivni organoid, gdje organoid čuva prisustvo epitelijalnih matičnih stanica koje zadržavaju prisustvo nediferenciranog fenotipa i svojstava samo-obnavljanja, pri čemu su ne-epitelijalne stanice odsutne iz spomenutog organoida, i pri čemu se spomenuti organoid dobiva pomoću postupka koji je definiran u bilo kojem od patentnih zahtjeva definiranim od 1 do 6.7. A three-dimensional tissue organoid, wherein the organoid preserves the presence of epithelial stem cells that retain the presence of an undifferentiated phenotype and self-renewal properties, wherein non-epithelial cells are absent from said organoid, and wherein said organoid is obtained by a method defined in any which of the patent claims defined from 1 to 6.
8. Kolekcija više od 10 organoida, kao što je definirano u zahtjevu 7.8. A collection of more than 10 organoids, as defined in claim 7.
9. Upotreba organoida prema zahtjevu 7, ili kolekcija organoida u zahtjevu 8, u in vitro skriningu (provjeravanju) pri otkrivanju lijeka ili testu toksičnosti.9. Use of an organoid according to claim 7, or a collection of organoids according to claim 8, in an in vitro drug discovery screening or toxicity test.
10. Organoid prema zahtjevu 7, ili kolekcija organoida prema zahtjevu 8, za upotrebu u regenerativnoj medicini.10. An organoid according to claim 7, or a collection of organoids according to claim 8, for use in regenerative medicine.