Isolation of E. multilocularis metacestodes and in vitro cultivation
E. multilocularis metacestodes were maintained in BALB/c mice via intraperitoneal (i.p.) injection of E. multilocularis protoscoleces. Approximately 6–8 months after infection, the mice were euthanised. Metacestodes dissected from the peritoneal cavity of the mice were cut into small tissue blocks (approximately 0.5 cm3) and washed three times with sterile phosphate-buffered saline (PBS). After then, these blocks of metacestodes were cultured under four different conditions: semianaerobic, anaerobic, 1% O2, and normoxic conditions. For the semianaerobic culture, three tissue blocks were transferred into a sterile 15 mL centrifuge tube that was filled with Dulbecco’s modified Eagle medium (DMEM) (HyClone, Fisher Scientific International Inc.,Logan, Utah, USA) and supplemented with 10% heat-inactivated foetal bovine serum (FBS; ExCell Bio, Saiguo Biotech Co., Ltd., Guangzhou, China), 1% penicillin–streptomycin, and 10 µg ciprofloxacin/mL (Solarbio Science & Technology Co., Ltd, Beijing, China). The tube was tightly closed and incubated at 37 ℃ with 5% CO2. For the other conditions, three tissue blocks were placed in 25 cm2 cell culture flasks containing 20 mL of DMEM (supplemented as described above). Anaerobic culture was performed in a sealed container with an AnaeroPack pouch (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at 37 ℃. A constant hypoxic environment of 1% O2 was provided using an incubator with an O2 sensor and N2 gas supply at 37 ℃ with 5% CO2. The normoxic group was cultured normally. The cultures were maintained for four weeks with medium changes occurring every 7 days. The follow-up parameters recorded included the production, growth, and proliferation of daughter vesicles.
The daughter vesicles derived from E. multilocularis metacestodes were transferred to 6-well plates (one vesicle per well) and cultured for 8 days under three different conditions: anaerobic, 1% O2, and normoxic conditions. All the vesicles were photographed on days 0 and 8 with a light microscope (EVOS FL; Thermo Fisher Scientific) to measure size.
Isolation of primary cells of E. multilocularis and in vitro cultivation
Primary E. multilocularis metacestode cells were isolated from the daughter vesicles grown in vitro as follows: First, vesicles with a minimum diameter of 3 mm were manually selected from axenic cultures and transferred into a 15 mL tube using a 3 mL Pasteur pipette. These vesicles were gently washed twice with PBS to remove the serum-containing medium, which was followed by adding 8 volumes of 0.25% (w/v) trypsin/EDTA solution (Solarbio Science & Technology Co., Ltd, Beijing, China). Then, we repeatedly blew and beat the vesicles with a blue 1000 µL pipette to physically destroy them until the ghost pellet became white, per visual observation. The tubes were incubated for 5 min at 37°C. After the serum-containing medium was used to terminate the digestion, the tube was centrifuged for 5 min at 1,000 ×g, and the supernatant was discarded. The precipitates were resuspended with 10 mL of PBS and sieved through a 70 µm cell strainer (Corning Inc., Corning, New York, USA) to remove the lamellar fragment. The collected fluid containing the primary cells was distributed and centrifuged at 1,000 ×g for 5 min. The supernatant was decanted, and the primary cell pellets were carefully resuspended with a 1,000 µL pipette in 3 mL of fresh DMEM and counted. Primary cells were seeded at 1×106 per well into 6-well cell plates that were preloaded with 1500 µL of DMEM formulated as previously described. The plate was anaerobically incubated at 37 ℃ for 5 days using a sealed AnaeroPack pouch, as described above.
Drug preparation
Albendazole (ABZ, Solarbio Science & Technology Co., Ltd, Beijing, China) solution was prepared according to the recommended dose at 1 µg/mL [8]. The drug powder was resuspended in 40 µL of dimethyl sulphoxide (DMSO) per 20 mL of culture medium. The control cultures contained 40 µL of DMSO per 20 mL of culture medium.
Carboxyfluorescein succinimidyl ester (CFSE) staining
CFSE staining was used to examine the viability of the vesicles and the proliferation of the primary cells of E. multilocularis metacestodes. The vesicles and primary cells were stained with 5 mM CFSE (Thermo Fisher Scientific, Waltham, MA, USA)) for 15 min at room temperature (around 25℃). After washing twice with PBS, 4,6-diamino-2-phenyl indole (DAPI; Solarbio Science & Technology Co., Ltd., Beijing, China) was added to stain the cell nuclei. Samples were observed under a fluorescence microscope (EVOS FL; Thermo Fisher Scientific). To examine the proliferation potential of the primary cells cultured in vitro, the CFSE-labelled primary cells were incubated under anaerobic conditions for five days. Because the CFSE signal is diluted with each cell division, divided cells that exhibited weak CFSE fluorescence were collectively termed “CFSE low”, whereas undivided cells that showed showed strong CFSE fluorescence were termed “CFSE high”.
Animal infection with daughter vesicles of E. multilocularis metacestodes
All animal experiments were approved by the Animal Ethics Committee of Ningxia Medical University. Female BALB/c mice (6–8 weeks old) were purchased from the Animal Center of Ningxia Medical University and maintained in a specific pathogen-free facility. For the in vivo growth assay of daughter vesicles of E. multilocularis, small budding vesicles were transplanted into the peritoneal cavity of normal BALB/c mice. Notably, in vitro-cultured vesicles burst easily when touched. To protect these vesicles from destruction during the transplantation procedure, we immobilised the vesicles in 1% low-melting-point agarose gel. Briefly, three vesicles with diameters of 1–2 mm were placed in a round-bottom 96-well plate, and excess liquid was removed using a pipette. Next, agarose powder in PBS was heated in a microwave oven until fully dissolved. The melted agarose gel was slowly cooled to 37°C in a 37°C water bath. Then, the cooled agarose solution was added to the vesicles-containing 96-well plate (200 µL/well) and left at RT for 5 min for the agarose to set. The microvesicles of E. multilocularis were embedded in agarose gel blocks. Each gel was surgically implanted into the abdominal cavity of the mouse. After 3 months, the mice were sacrificed to examine the parasitic masses in their abdominal cavity.
Statistical analysis
The differences in the mean vesicle volumes among groups were assessed using unpaired t-test for comparisons between two groups and two-way ANOVA for comparisons between more than two groups using GraphPad Prism 8.0 (GraphPad Software Inc.). In this text, an asterisk (*) indicates a significant difference at P < 0.05, whereas P > 0.05 was considered not significant (ns).