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).
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