Tissue-derived exosomes has attracted increasing attention due to their ability to accurately reflect tissue specificity and the microenvironment. Our research focused on figuring out the basic and the translational roles of spleen-derived exosomes in cardiovascular disease. In this study, we develop a practical protocol for isolating exosomes from mice spleen tissues, providing a reproducible technique for subsequent identification analysis and functional studies.
We use a Type I collagenase for digestion, followed by filtration through differential ultracentrifugation to yield high-quality spleen exosome. In contrast that to previous reports, using tissue homogenization to obtain tissue suspension, our approach preserves the membrane integrity of cells in tissues. This technique could be particularly useful in studying the role of exosome in immune responses.
Given the spleen critical function in the immune system, future studies could adapt this protocol to human tissues, enabling the extraction of clinically relevant exosome for diagnostic and therapeutic purpose. To begin, pre-cool the high-speed and ultra-high-speed centrifuges to four degrees Celsius and set the temperature of the desktop shaker to 37 degrees Celsius. Prepare the cell culture dishes, having a diameter of 100 millimeters.
Then, arrange 50 milliliters sterile centrifuge tubes, ice, and sterile cell strainers with a diameter of 70 micrometers. Spray 75%alcohol to sterilize the scissors and forceps and heat them at a high temperature to sterilize further. Then, place the spleen tissue in a sterile 100 millimeter Petri dish on ice and wash off the surface blood with cold 1X PBS.
Thoroughly dry the spleen with sterile gauze before weighing the tissues. Next, using a sterile transfer pipette, add one milliliter of PBS to a 70 micrometer cell strainer to moisten it. Finally, prepare 0.1%Type I collagenase in 1X PBS with a vortex mixer to obtain the digestion buffer.
To begin, prepare the reagents and the sterile tools for tissue processing. With scissors, chop the spleen tissue into small, uniform pieces in a culture dish on ice and transfer them to a 50 milliliter centrifuge tube using a sterile transfer pipette. Then, add the digestion buffer to the tube based on the weight of the spleen tissue, and shake the tube at a 45 degree angle on a horizontal shaker set to 37 degrees Celsius.
Stop the digestion when the tissue chunks have dispersed and most of the pieces have lost their shape. Transfer the digested mixture through a 70 micrometer cell strainer at room temperature to remove fibrous and larger tissue debris. Collect the filtrate in a new 50 milliliter centrifuge tube.
Centrifuge the filtrate at 500 G for 10 minutes at four degrees Celsius. Then, transfer the supernatant to a new tube and repeat the centrifugation step at appropriate speeds. After ultracentrifugation, discard the supernatant and wash the pellet with PBS.
Ultracentrifuge again at 120, 000 G for two hours at four degrees Celsius to isolate the exosomes. Finally, dissolve the isolated exosomes in 200 microliters of sterile PBS per spleen and store them in a two milliliter centrifuge tube at minus 80 degrees Celsius. Transmission electron microscopy images revealed the presence of cup-shaped vesicles with a lipid bilayer, characteristic of exosomes.
Exosome sizes ranged from 30 to 150 nanometers with peak concentration at 60 nanometers as shown by the size distribution graph. Western blot analysis confirmed the presence of exosomal markers, TSG101 and CD9, while GM130 and negative control was not detected.
