The advantage of using differential centrifugation for this protocol is that it is easily scalable up to liters and therefore, ideal for isolating exosomes from primary fibroblasts. Purified exosomes can be obtained from different cell types and tissues isolated from various disease models. The exosomes can be used as diagnostic biomarkers.
Demonstrating that procedure will be Diantha van de Vlekkert, a scientist from the d'Azzo Laboratory To begin, weigh the mouse muscles and then transfer them to a 10-centimeter dish. Working under a biosafety hood, mince the muscles with scalpels to produce a fine paste. Transfer the finely minced muscle paste to a 50-milliliter tube, and add digestion solution using 3.5 times the volume per milligram of tissue.
Incubate for 45 minutes at 37 degrees Celsius. To aid in complete dissociation, mix the suspension thoroughly with a 5-milliliter pipette every 10 minutes. To inactivate the digestion solution, add 20 milliliters of DMEM Complete to the cell suspension.
Pipette through a 70-micrometer nylon cell strainer placed on a 50-milliliter tube, and then wash the cell strainer with an additional 5 milliliters of DMEM Complete. Centrifuge the cell suspension at 300g for 10 minutes at room temperature. Carefully remove the supernatant and re-suspend the pellet in 10 milliliters of DMEM Complete.
Seed the cells into a 10-centimeter dish, then place the dish in the incubator at 37 degrees Celsius, 5%carbon dioxide, and 3%oxygen. The skeletal muscle fibroblast cultures need to be maintained at a low oxygen level to ensure physiological light conditions. The oxygen level in skeletal muscle is approximately 2.5%After the cells reach 100%confluence, culture them for an additional day, then rinse them with PBS and add one milliliter of trypsinization solution.
After incubating the cells for 10 minutes, stop the trypsinization by adding 10 milliliters of DMEM Complete. Transfer the cell suspension to a 50-milliliter tube, and then centrifuge the cells at 300g for 10 minutes at room temperature. Then re-suspend the pellet in 20 milliliters of DMEM complete and seed the cells into a 15-centimeter dish.
Collect the conditioned medium from the cultured fibroblasts in a 15-milliliter tube. Centrifuge the conditioned medium at 300g for 10 minutes to pellet the live cells. Transfer the supernatant to a new 50-milliliter tube and pellet the dead cells by centrifuging the tube at 2, 000g for 10 minutes.
Next, transfer the supernatant to a 38.5-milliliter polypropylene tube and centrifuge the tube at 10, 000g for 30 minutes to remove organelles, apoptotic bodies, and membrane fragments. To pellet the exosomes, transfer the supernatant to a 38.5-milliliter ultraclear tube, and ultracentrifuge it at 100, 000g for 1.5 hours. Carefully discard the supernatant, leaving approximately one milliliter of conditioned medium.
Wash the exosome pellet and re-suspend it in up to 30 milliliters of ice cold PBS. Centrifuge the exosomes again at 100, 000g for 1.5 hours. To complete the purification, remove the supernatant by pipetting, being careful not to disturb the exosomal pellet.
The exosome pellet step can be loose and easily detached. Therefore, the removal of the PBS supernatant should be done very carefully with the aid of a 200-microliter pipette. Re-suspend the pellet in ice-cold PBS using 25 microliters per 15-centimeter dish of fibroblasts.
Lastly, measure the protein concentration using the BCA Protein Assay Kit and a microplate reader at 562 nanometers. This protocol is a highly reproducible and consistent method for purifying exosomes. Transmission electron microscopy shows the relatively uniform sizes of these exosomes.
The immunoblot of exosome lysates shows a canonical pattern of protein expression and the absence of LDH and calnexin protein contaminants. Exosomes were separated by a sucrose density gradient, and individual fractions were probed on a western blot with antibodies against Alix, CD9, and CD81. Exosomes consistently sedimented in fractions three through six.
The isolated and concentrated bioactive exosomes can be used for electron microscopy and mass spectrometry analyses, as well as for in-vitro and in-vivo uptake experiments for functional studies. This protocol is suitable for the isolation of exosomes from low secretory cells and can be applied to explore the involvement of exosomes in human fibrotic diseases.
