Our protocol can be used to produce high quality exosome-enriched extracellular vesicles from embryonic stem cells that express the immune stimulatory factor, GM-CSF. Exosome enriched extracellular vesicles carrying GM-CSF, have the potential to serve a cell-free immune regulatory vesicles that can modulate the immune response. Researchers with the basic molecular and cellular biology training, Should be able to easily as could this protocol, but anyone performing this protocol for the first time should follow the directions closely.
Since our protocol is complicated, visualizing the intricate details of each step will help other researchers To generate exosome free FBS, ultracentrifuge the desired volume of FBS and collect the exosome free supernatant. Before plating the ESD three cells, coat 15 centimeter tissue culture dishes with 0.1%Gelatin at room temperature for 30 minutes. Remove Gelatin by aspiration and culture the ESD three cells without feeder layer cells in ESD three cell culture medium at 37 degrees Celsius in 5%Carbon dioxide humidified incubator until the cells reach 90%confluency.
Wash the almost confluent cultures with five milliliters of 0.05%trypsin per dish followed by a five minute incubation at 37 degrees Celsius in fresh trypsin. At the end of the incubation pull the detached cells in a centrifuge tube and inactivate the trypsin with five milliliters of fresh culture medium. Sediment the cells by centrifugation and re-suspend the pallets in fresh medium for counting For passaging, plate five times 10 to the sixth of the ESD three cells in 15 milliliters of cell culture medium onto new Gelatin coated plates for three days of culture before sub-culturing the cells To collect the cell culture supernatant for the isolation of exosome enriched extracellular vesicles plate one times 10 to the seventh ESD three cells in 15 milliliters of cell culture medium per new Gelatin coated plate for three days prior to collecting the cell culture supernatants For exosome enriched extra cellular vesicle isolation, First sediment the large cell fragments within the supernatants collected from 72 hour cultured ESD three cells by centrifugation.
After collecting the supernatant, Ultracentrifuge the samples and discard the supernatants. Gently rinse each pallette two times with one milliliter of PBS per wash to remove any residual culture supernatant and quantify the exosome enriched extracellular vesicle protein content with a Bicinchoninic acid assay, according to the manufacturer's instruction. Then re-suspend the exosome enriched extracellular vesicles in PBS at a six micrograms per micro meter concentration for storage at minus 80 degrees Celsius.
To visualize the exosome enriched extracellular vesicles by transmission electron microscopy fix three to five micrograms per milliliter of the extracellular vesicles with the final concentration of 2%electron microscope grid paraformaldehyde at room temperature for two hours. At the end of the incubation, load 10 microliters of the fixed samples onto Copper Grids with carbon support film for one minute before draining the grids with filter paper, stay in the grids with an appropriate staining solution, according to the manufacturer's protocol and use tweezers to transfer the grids to a piece of filter paper, then use the transmission electron microscope with a 50, 000 times magnification to acquire electron microscopy images, according to the standard protocols. To prepare Whole Cell Extracts, collect ESD three cells from culture as demonstrated, and re-suspend the harvested cells in PBS for counting.
After a second centrifugation, re-suspend the cells out of five times 10 to the third cells per microliter of SDS page loading buffer containing 0.5%SDS concentration and Sonicate the samples for 10 seconds on a Sonicator with a 10%amplitude. Then heat the samples at 100 degrees Celsius for five minutes. To prepare lysates for the exosome enriched extracellular vesicles, Re-suspend the exosome enriched extracellular vesicles in SDS page loading buffer containing 0.5%SDS at a 1.2 micrograms per microliter concentration and Sonicate the samples for 10 seconds as demonstrated then heat the samples at 100 degrees Celsius for five minutes.
For Western Blot Analysis, load 10 microliter wholesale extract and exosome enriched extracellular vesicle lysate samples into individual Wells of a best stress page gel. At the end of the run, transfer the proteins onto PVDF membranes and incubate the membranes with the appropriate primary and secondary antibodies of interest, then detect the protein expression using an enhanced chemiluminescence detection kit, According to the manufacturer's instructions. To evaluate the amount of GM-CSF within exosome enriched extracellular vesicles, use a kit Murine GM-CSF to coat an ELISA Plate with anti GM-CSF capture antibody next, treat 0.6 micrograms of exosome enriched extracellular vesicles samples with 100 microliters of PBS alone, or PBS plus 0.05%tween 20 at room temperature for 30 minutes.
At the end of the incubation, add the treated samples to individual Wells of the prepared ELISA Plate for a one hour incubation at room temperature followed by washing of the appropriate corresponding Wells with PBS alone, or PBS plus 0.05%tween 20. After the wash, add the detection antibody to the samples for a one hour incubation at room temperature followed by a wash with PBS alone or PBS plus 0.05%tween 20 as demonstrated, Then add Avidin Horseradish Peroxidase to the samples for a 30 minute incubation at room temperature, followed by a wash and measuring the absorbance in each well on a Microplate Reader at 450 nanometers. The GFP fluorescence intensity of a GM-CSF expressing ESD three cell line or an ESD three cell line expressing an empty vector is much higher than that of their parental counterparts.
ELISA reveals that ESD three cells expressing GM-CSF produce markedly higher levels of GMCSF in their cell culture supernatant than do empty vector control cells. Furthermore, the amount of GM-CSF generated by GM-CSF expressing ESD three cells is similar to that produced by STO fibroblasts expressing GM-CSF. Transmission electron microscopy of extracellular vesicles isolated from vector transduced and GM-CSF transduced cell cultures reveal vesicles of different sizes that fall within the expected 30 to 100 nanometer diameter range.
The expression of exosomal markers, including CD81, Annexin V and flotillin-1 is markedly enhanced in extracellular vesicles, isolated from ESD three cells compared to corresponding wholesale extracts by Western blot analysis. Importantly, the presence of other sub-cellular compartment markers in ESD three derived extracellular vesicles was not detected including the endoplasmic reticulum marker, Protein Disulfide Isomerase, the mitochondrial markers, cytochrome C and Oxphos complex IV-subunit IV and the cytosolic marker GAPDH. After washing with 0.05%tween 20, the background GM-CSF levels detected in the control Extracellular vesicles was significantly reduced.
In contrast, GM-CSF levels in the extracellular vesicles of GM-CSF expressing cells was significantly increased by tween 20. Acquiring high quality exosome enriched extracellular vesicles carrying GMCSF is the most important step for the success of this protocol. Researchers can perform additional experiments, such as studies in GM-CSF dependent cell lines and animals to determine whether exosome enriched extracellular vesicles caring GM-CSF can function as cell-free immune regulatory vesicles.
These exosome enriched extracellular vesicles can then be used to explore how modulation of the immune response affects different disease conditions.
