Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

Our research focuses on the characterization of the Echinococcus granulosus extracellular vesicles and their role in parasite-host interactions. We investigate the composition uptake by host dendritic cells and the potential influence on immunomodulation. We demonstrated that E. granulosus EV are taken up by dendritic cells, influencing maturation and antigen presentation. We also identify key immunomodulatory and antigenic proteins in EV cargo, which may influence parasite-host interactions and immune responses.

Our protocol optimizes in vitro parasite culture for enhanced small EV production and ensures high-quality isolation and integrates dendritic cell assays to study immunomodulation. It can be applied as a whole or in parts. Our future research will explore the in vivo effects of E. granulosus EVs on immune responses, their potential as vaccine components, and their impact on dendritic cells, microphages, and these cells' immune models.

[Narrator] To begin, clean the ventral surface of the euthanized mouse with hydatid disease using 70% alcohol. Surgically open the peritoneal cavity to remove the developed metacestodes using scissors and forceps. Then, transfer the metacestode masses to a sterile Petri dish using forceps. Remove the connective tissue using forceps, if needed, to release the cysts from the metacestode masses. Wash the obtained metacestodes with supplemented PBS at four degrees Celsius. Now, prepare the culture medium using all the required components, and mix the solution gently by inversion. Transfer five milliliters of the prepared culture medium into each Leighton tube. Add the parasites to the culture medium and incubate the tubes at 37 degrees Celsius for five days without changing the medium. Collect the parasite culture medium from each Leighton tube and transfer it into a 15-milliliter conical tube. Centrifuge the collected medium at 300g for 10 minutes at four degrees Celsius and transfer the supernatant to a new 15-milliliter conical tube. Now, centrifuge the supernatant at 2,000g for 10 minutes at four degrees Celsius. Transfer the resulting supernatant to a new 1.5-milliliter tube using a pipette. Centrifuge the tube at 10,000g for 30 minutes at four degrees Celsius to remove smaller cell debris. Using a pipette, transfer the supernatant to a tube suitable for the ultracentrifuge rotor. Mark one side of the tube with a marker. And place the tube in the rotor with the marked side facing up. Centrifuge the tubes at 100,000g for one hour at four degrees Celsius. Pour off the supernatant quickly and let the tube rest upside down for one minute. Wash the pellet with at least three milliliters of PBS to remove the contaminating proteins. Resuspend the pellet multiple times using a pipette along all tube faces, focusing on the marked side where the pellet is expected to be. After repeating the ultracentrifugation, resuspend the pellet in 30 microliters of PBS. Transfer the resuspended sample to a 1.5-milliliter tube and freeze the extracellular vesicles at minus 80 degrees Celsius for storage. Spray the euthanized five- to eight-week-old female CF-1 mouse with ethanol before placing it into a tissue culture hood. Place the mouse on a dissecting board in the supine position. Using forceps and dissecting scissors, make a vertical T incision above the urethra and extend it horizontally to the top of the lower extremities. Using forceps, separate the skin along both hind limbs to expose the leg bones and tissues. With the hands, remove the skin of each leg after pushing from the ankle towards the abdomen, pulling the skin to the opposite side, leaving both legs free of skin. Now, carefully remove the femur and tibia using scissors and forceps, ensuring no breakage. Fasten the tip of each bone with forceps. And cut the tendons to remove muscle fascias around the bones. Complete cleaning the muscle tissue with paper napkins. Place each removed bone into a sterile 50-milliliter tube containing two milliliters of supplemented complete RPMI medium to remove debris. After discarding the medium, wash the bones twice with 70% ethanol for five minutes each time. Transfer the washed bones to a sterile Petri dish and cut off the two-bone epiphyses using sharp dissecting scissors to access the bone marrow cells. Using a 25-gauge needle attached to a 20-milliliter syringe containing complete medium, carefully flush the bone marrow cells from each of the four bones into a sterile Petri dish. Then, gently homogenize the medium containing eluded marrow by pipetting to remove bone connective tissue and cell lumps. Transfer the sample to a sterile 50-milliliter conical tube, passing the cells through a sterile 70-micrometer polypropylene cell strainer to remove connective tissue and bone debris. Centrifuge the cells at 450g for seven minutes at four degrees Celsius. Carefully remove and discard the supernatant, ensuring the pellet remains adhered to the tube wall. After incubating the cells for one minute at room temperature, resuspend them in 500 microliters of RBC lysis buffer and neutralize the lysis buffer with three milliliters of complete medium. Centrifuge the cells at 450g for seven minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in five milliliters of complete medium. After filtering the sample as demonstrated, count the cells using a hemocytometer. Add 300 nanograms per milliliter of recombinant murine FMS-related tyrosine kinase three ligand, or FLT3L, to the culture medium. Plate the cells in a multi-well plate at a concentration of one times 10 to the power of six cells per milliliter. Incubate the cells for seven days at 37 degrees Celsius in a humidified atmosphere with 5% carbon dioxide. On day three, remove one milliliter of medium from each well without disturbing the cells, and replace it with one milliliter of pre-warmed fresh complete medium, supplemented with 150 nanograms per milliliter of recombinant murine FLT3L. The extracellular vesicles, or EVs, purified from the Echinococcus granulosus larval stage were primarily small EVs with sizes ranging from 50 to 200 nanometers, confirmed by dynamic light scattering and transmission electron microscopy. Bone marrow-derived dendritic cells demonstrated morphological differentiation over seven days, progressing from small, round hematopoietic cells to stellate-shaped cells with cytoplasmic extensions. After one hour of incubation, the dendritic cells captured fluorescently-labeled small EVs, which co-localized with MHCIII molecules in endosomal-lysosomal compartments, as confirmed by confocal microscopy.