20.500.12556/RUL-184068 Vzpostavitev in karakterizacija mišjih jetrnih organoidov v kupolah Matrigela Establishment and characterization of mouse liver organoids in Matrigel domes Organoidi so pomanjšani in poenostavljeni tridimenzionalni modelni sistemi organov in vitro, ki se samoobnavljajo, samoorganizirajo ter izražajo funkcionalnost, ki je podobna organom in vivo. Prvi cilj magistrske naloge je bil vzpostavitev dveh kultur jetrnih organoidov v kupolah Matrigela. Prvo kulturo smo vzpostavili iz krioprezerviranih jetrnih organoidov iz miši linije C57BL/6, kupljenih v podjetju StemcellTM Technologies (oznaka SC), drugo kulturo pa iz primarnih celic jeter miši (oznaka OI), prav tako linije C57BL/6. Za vsako kulturo smo testirali dve različni gojišči, in sicer komercialno gojišče (oznaka SC) ter v laboratoriju pripravljeno gojišče (oznaka HM). Drugi cilj je bil karakterizacija morfoloških, molekularnih in funkcionalnih lastnosti organoidov, s čimer smo želeli ovrednotiti njihovo podobnost z jetrnim tkivom miši ter uporabnost kot in vitro modela. Z uporabo elektroporacije pa smo želeli oceniti njihovo primernost za uporabo pri elektrokemoterapiji. Uspešno smo vzpostavili tri skupine organoidov, in sicer OI_HM, SC_HM in SC_SC. OI_SC skupine organoidov nismo uspeli vzpostaviti. Vse vzpostavljene kulture so izkazovale podobno dinamiko rasti in med njimi nismo zaznali statistično značilnih razlik. Pri karakterizaciji organoidov smo ugotovili, da smo pri vseh treh skupinah večinoma vzpostavili holangiocitno in ne hepatocitno kulturo organoidov. Vzpostavljeni organoidi so namreč izražali nižjo količino genov, značilnih za hepatocitne organoide (Alb, Cyp1a2, Cyp3a11 in HNF4a) in povišano količino genov, značilnih za holangiocitne organoide (Krt19). Tudi pri histološki analizi s H&E barvanjem je večina organoidov izkazovala morfologijo značilno za holangiocitne organoide. S PAS barvanjem pa smo glikogen potrdili le pri nekaterih organoidih, s čimer smo dodatno dokazali, da je večina vzpostavljenih organoidov holangiocitnih. Ne glede na to, smo vzpostavljene organoide uporabili za poskuse elektroporacije, kjer smo dokazali, da vzpostavljene organoide lahko uspešno elektroporiramo s propidijevim jodidom (PI) in bleomicinom (BLM). Organoids are miniaturized and simplified three-dimensional in vitro model systems of organs that are capable of self-renewal and self-organization and exhibit functionality resembling that of organs in vivo. The first aim of the master’s thesis was to establish two cultures of liver organoids in Matrigel domes. The first culture was established from cryopreserved liver organoids derived from C57BL/6 mice and purchased from Stemcell™ Technologies, designated SC. The second culture was established from primary liver cells isolated from C57BL/6 mice, designated OI. For each culture, two different media were tested: a commercial medium, designated SC, and a laboratory-prepared medium, designated HM. The second aim was to characterize the morphological, molecular and functional properties of the organoids in order to evaluate their similarity to mouse liver tissue and their applicability as an in vitro model. Using electroporation, we also aimed to assess their suitability for use in electrochemotherapy. We successfully established three organoid groups, namely OI_HM, SC_HM and SC_SC. The OI_SC organoid group could not be established. All established cultures exhibited similar growth dynamics, and no statistically significant differences were observed among them. During organoid characterization, we found that in all three groups we had predominantly established cholangiocytic rather than hepatocytic organoid cultures. The established organoids showed lower expression of genes characteristic of hepatocytic organoids (Alb, Cyp1a2, Cyp3a11 and HNF4a) and increased expression of a gene characteristic of cholangiocytic organoids (Krt19). Histological analysis with H&E staining also showed that most organoids displayed morphology characteristic of cholangiocytic organoids. PAS staining confirmed the presence of glycogen only in some organoids, further supporting the conclusion that the majority of the established organoids were cholangiocytic. Nevertheless, the established organoids were used for electroporation experiments, in which we demonstrated that they can be successfully electroporated with propidium iodide (PI) and bleomycin (BLM). organoidi Matrigel vzpostavitev karakterizacija elektroporacija organoids Matrigel establishment characterization electroporation true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-06-25 08:15:47 2026-06-25 08:15:59 2026-06-29 14:46:11 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 280260 282990083 57957.pdf 57957.pdf 1 A517885DA09BF720496C99734971D275 80154ece01a85b08d0d6ea7a9462a4e6292a4bf865044d3748461e21a0191f08 3fc39e35-705d-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236527 Biotehniška fakulteta 0 0 0