A simple, quick, and economical method for in vitro cultivation of Echinococcus multilocularis metacestode and generation of primary cells

Abstract

Abstract Background Alveolar echinococcosis is one of the most lethal parasitic zoonotic diseases. However, the molecular mechanisms through which Echinococcus multilocularis interacts with its host are poorly understood, and only limited treatments are available. Owing to the advantages of cell culture systems in molecular research, numerous attempts have been devoted to establishing primary cell cultures of Echinococcus multilocularis. In this study, we developed a simple, rapid, and economical method that allows Echinococcus multilocularis metacestode tissue blocks to generate daughter vesicles without host feeder cells in a regular medium. Methods We performed anaerobic, hypoxic (1% O2), normoxic, and semianaerobic (in sealed tubes) cultures of Echinococcus multilocularis metacestode tissues to produce daughter vesicles. Then, the daughter vesicles were cultured at three distinct oxygen concentrations (anaerobic, 1% O2, and normoxic) to identify the optimal cultivation conditions. The viability of the daughter vesicles was assayed using carboxyfluorescein succinimidyl ester staining. Vesicles with high vigour were subsequently used to test the antiparasitic effect of albendazole by isolating primary cells and infecting animals. Results After 4 weeks of incubation, we found that Echinococcus multilocularis metacestode tissues that were cultured in sealed tubes produced daughter vesicles. The daughter vesicles were remarkably enlarged under anaerobic conditions after 8 days of culture, whereas vesicles cultured under the other two conditions only showed a mild increase in volume. Our in vitro cultivated vesicles showed strong viability and could be used to test antiparasitic drugs, isolate primary cells, and infect animals. Conclusions In the study, we established a simple, quick, and economical method for the in vitro generation of vesicles from tissue blocks of Echinococcus multilocularis metacestodes in the absence of host feeder cells. Our in vitro cultivated vesicles, showing appropriate viability, are suitable for screening drugs for the treatment of alveolar echinococcosis both in vitro and in vivo.