In this work we described preparation of the novel cell lines expressing human prion protein on PrP0/0-background. Prepared cell lines originated from the Nagasaki mice (PrP0/0) and showed a fibroblast phenotype. The expression level of human prion protein in the developed cell lines was comparable to the physiological expression measured in GT1-7 cells. A great advantage of the prepared cell lines was their short doubling time that allowed obtaining of a large amount of cells for the proteomic experiments. Newly established cell lines open a broad spectrum of applications in the prion research. Besides the study of physiological function of the prion protein or its interactome, the new cell lines could be successfully employed as a unique tool for the better understanding of key events in the pathogenesis of prion diseases.