In Vitro Maintenance of Clonorchis sinensis Adult Worms

Abstract

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Locke's solution, RPMI-1640, DMEM, and IMDM media, and in 1×Locke's solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Locke's solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Locke's solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Locke's solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

Production and Deformation of Clonorchis sinensis Eggs during In Vitro Maintenance

“…The collected metacercariae were preserved in cold (4°C) 1× PBS with antibiotics until use. The metacercariae were introduced to male New Zealand white rabbits and adult C. sinensis worms were recovered as described in our previous study [3].…”

“…The fresh worms were distributed in a 6-well culture plate by 10 worms per culture well with 3 mL of test solution or media as described in the previous study [3]. All of the experiment procedures were performed in triplicates.…”

“…The worms were cultivated in a humidified incubator at 37°C in the presence of 5% CO 2 . Some of this experiment procedure was shared with the in vitro cultivation of C. sinensis adults [3].…”

“…Recently adult worms of C. sinensis were in vitro maintained long in broth media [3]. During the cultivation, the worms passed many eggs.…”

Structure-based Discovery of Potential Inhibitors Targeting Sodium-bile Acid Co-transporter of Carcinogenic Liver Fluke Clonorchis Sinensis

“…Notably, bile acids trigger physiological stimuli; however, these metabolites can have detrimental effects on C. sinensis survival, as evidence suggests that the accumulated bile acids can cause toxicity to the worm's tissues and cells [18,19]. Recent studies reported that bile components can be toxic or detrimental to C. sinensis survival [10,20,21]. Under in vitro conditions, higher concentration (> 0.005%) of bile and lithocholic acid signi cantly decreased the worm survival [10,20,21].…”