P values of all assays in this study.

The absence of an established in vitro platform is a major obstacle in research on parasitic flatworms, including Fasciola flukes. Fasciola flukes cause zoonotic infections that primarily affect the liver and the bile ducts. Infected juveniles can cause severe liver damage in animals, occasionally leading to sudden death. Although resistance to the only drug for the acute liver stage has been reported worldwide, the search for new drugs has been unsuccessful owing to the critical limitations of previous in vitro cultures. Previous studies have been unable to reproduce liver-stage development in vitro, hindering research on this stage. This study aimed to provide a novel in vitro research platform using a laboratory strain of Fasciola hepatica/gigantica hybrid. Juveniles derived from the livers of mice at 7 and 11 days post-infection (dpi) survived for nearly 100 days in the basic medium consisting of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 50% fetal bovine serum. Bovine red blood cells (RBC) and sex-inducing substances (SIS) that induce sexualization in a free-living flatworm (planarian) were supplemented to examine their effects on the developmental processes in the liver stage, including growth, body shape change, and reproductive development. SIS induced all three processes, although the last was incomplete, suggesting that the sex-inducing ability of SIS is conserved between free-living and parasitic flatworms. However, RBC was somewhat toxic and less effective than SIS for both growth and reproductive development and could not alter body shape. Furthermore, the combined effects of the two supplements were not observed. In this study, the reproducibility of the development was carefully confirmed, and it was shown that a single SIS supplementation is currently the best condition and more closely mimics liver-stage development. This study provides a preliminary but outstanding in vitro research platform for liver-stage juveniles and will facilitate further drug development.