Isolation of E. multilocularis metacestodes and in vitro cultivation
E. multilocularis metacestodes were maintained in BALB/c mice by intraperitoneal (i.p.) injection of E. multilocularis protoscoleces. Approximately 6–8 months after infection, the mice were euthanized. Metacestodes dissected from the mouse peritoneal cavity were cut into small tissue blocks of 0.5cm3 around and washed three times with sterile phosphate-buffered saline (PBS). After then, these blocks of metacestodes were cultured under four different conditions: semi-anaerobic, anaerobic, 1% O2 and normoxic condition. For semi-anaerobic culture, three tissue blocks were transferred into a sterile 15ml centrifuge tube which was filled with Dulbecco’s modified Eagle medium (DMEM) (HyClone,Fisher Scientific International Inc.,America) and supplemented with 10% heat-inactivated fetal bovine serum (FBS; ExCell Bio, Saiguo Biotech Co., LTD, Guangzhou, China), 1% penicillin-streptomycin and 10µg of ciprofloxacin/ml (Solarbio Science & Technology Co., Ltd, Beijing, China). The tube was tightly closed and incubated at 37℃ with 5% CO2. For other conditions, every three tissue blocks were placed in each 25-cm2 cell culture flask containing 20 ml of DMEM medium (supplemented as described above). Anaerobic culture were 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 is provided by 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 a period of four weeks with medium changes occurring every 7 days. The follow-up parameters recorded were the production, growth, and proliferation of daughter vesicles.
Then, daughter vesicles derived from E. multilocularis metacestodes were transferred to 6-well plates, one vesicle per well, and cultured for 8 days in three different conditions: anaerobic, 1% O2 and normoxic condition. All vesicles were photographed at day 0 and day 8 under a light microscope (EVOS FL, ThermoFisher Scientific) for size measurement.
Isolation of primary cells of E. multilocularis and in vitro cultivation
Primary cells of E. multilocularis metacestodes were isolated from in vitro grown daughter vesicles as follows. First, vesicles of a minimum of 3 mm in diameter were picked manually from axenic cultures and transferred into a 15 ml tube using a 3 ml pasteur pipette. These vesicles were gentlely washed twice with PBS to remove the serum-containing medium followed by adding 8 volumes of 0.25% (w/v) trypsin/EDTA solution (Solarbio Science & Technology Co., Ltd, Beijing, China). Then repeatedly blow and beat the vesicles with a blue 1000 µl pipette for physical destruction until the ghost pellet became white under visual observation. The tube was incubated for 5 min at 37°C. After serum-containing medium was used to terminate the digestion, centrifuge the tube for 5 min at 1,000 g and the supernatant was discarded. Precipitates were resuspended with 10 ml PBS and sieved through a 70 µm cell strainer (Corning inc.,USA) to remove the lamellar fragment. Distribute the collected fluid containing primary cells and centrifuge at 1,000 g for 5min. Decant the supernatant and resuspend the primary cell pellets carefully with a 1000-µl pipette in 3 ml fresh DMEM medium and counted. The primary cells can now be seeded at 1×106 per well into 6-well cell plate that have been preloaded with 1500µL DMEM medium formulated as before. The plate was incubated anaerobically at 37 ℃ for 5 days using the 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 1µg/ml [8]. The drug powder were resuspended in 40µl of dimethyl sulfoxide (DMSO) / 20 ml culture medium. Control cultures contained 40µl of DMSO / 20 ml culture medium.
Carboxyfluorescein succinimidyl ester (CFSE) staining
To examine the viability of vesicles and proliferation of primary cells of E. multilocularis metacestodes, CFSE staining was used. The vesicles and primary cells were stained with CFSE (Invitrogen) at a final concentration of 5 mM for 15 min at room temperature (RT). After washing twice with PBS, 4,6-diamino-2-phenyl indole (DAPI, Solarbio Science & Technology Co., Ltd, Beijing, China) was added to stain cell nuclei. Then samples were observed under a fluorescent microscope (EVOS FL, ThermoFisher Scientific). In order to examine the proliferation potential of the in vitro cultured primary cells, the CFSE labeled primary cells were incubated under anaerobic condition for 5 days. Since CFSE signal is diluted with each cell division, divided cells exhibited weak CFSE fluorescence were collectively called “CFSE low”, while undivided cells showed str-ong CFSE fluorescence were termed “CFSE high”.
Animal infection with daughter vesicles of E. multilocularis metacestodes
All animal studies were approved by the Experimental Animal Ethics Committee of Ningxia Medical University. The female BALB/C mice (6–8 weeks) were purchased from Animal Center of Ningxia Medical University, and maintained in a specific pathogen-free facility. For in vivo daughter vesicles of E. multilocularis growth assay, the small budding vesicles were transplanted into the peritoneal cavity of normal BALB/c mice. Of note, the in vitro cultured vesicles were easily burst when touched. In order to protect these vesicles from destruction during the transplantation procedure, we immobilized the vesicles in 1% low-melting-point agarose gel. Briefly, three vesicles with the diameter of 1 to 2 millimeter were placed in a round-bottom 96-well plate and then removed the excess liquid by pipette. 1% 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. After that, the cooled agarose solution was added to vesicles-contained 96-well plate (200µl/well) and left at RT for 5 minutes for the agarose to set. As such, microvesicles of E. multilocularis were embeded in agarose gel blocks, we called it “gel jelly”. Each “gel jelly” was surgically implanted into the abdominal cavity of the mouse. After 3 months, mice were sacrificed to examine the parasitic masses in abdominal cavity.
Statistical analysis
The differences in mean vesicle volumes among groups were assessed by 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.). The * indicates a significant difference with the P < 0.05, whereas P > 0.05 was considered not significant (ns).