This procedure addressed the need for simple and reliable mesenchymal stem cell-derived extracellular vesicle collection, and of application in translation of research. Collection of extracellular vesicles by differential centrifugation is simple, readily scalable, and reproducible, which will be useful to the field. Extracellular vesicles indistinct}from indistinct}mesenchymal stem cell is a indistinct}application in modern disease treatments.
I will not be afraid of mistakes. This operation is simple and easy to learn. There are many details in this simple program that can be better presented through video.
To begin, culture the mesenchymal stem cells to more than 90%confluence. Replace the culture medium in each dish with 8 mL of supplemented alpha-MEM. After 48 hours of culture, the cells must be grown evenly to fully collect the culture medium into 50 mL centrifuge tubes.
Then, centrifuge the culture medium at 800 x g for 10 minutes at four degrees Celsius. Transfer the supernatant to 1.5 mL conical tubes to remove the cell fragments and cellular debris. Centrifuge the supernatant at 16, 000 x g for 30 minutes at four degrees Celsius.
Use a pipette to transfer the suspension to ultracentrifuge tubes. Then, centrifuge at 150, 000 x g for two hours at four degrees Celsius. Resuspend the microvesicle pellet in 50 uL of PBS per dish.
Discard the supernatant and collect the residue, which is exosomes. Resuspend the exosomes from seven dishes of cells in 50 uL of PBS. In a 15 mL tube, dilute 1 uL of extracellular vesicles in 1, 499 uL of PBS and mix.
Then, start the compatible software for the tracking analyzer. Fill the sample cell with distilled water and then allow the instrument to start the cell check. Calibrate the instrument with a prepared standard solution.
Ensure that the particle number displayed on the software detection interface is between 50 to 400, preferably around 200, and click okay. Next, rinse the sample cell with 5 mL of distilled water. Before sample analysis, flush the channel with 1 mL of distilled water.
Ensure that the particle number displayed on the software detection interface is less than 10. In a 1.5 mL conical tube, resuspend the extracellular vesicles with 250 uL of PBS. In another 1.5 mL conical tube, prepare the working solution of the PKH26 dye.
Mix the resuspended extracellular vesicles with the working solution. Allow it to stand at room temperature for five minutes and then add 500 uL of EV-depleted FBS to stop the reaction. For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius.
Discard the supernatant. Add 1 mL of PBS to rinse the residue. Centrifuge again, and discard the supernatant to remove the unbound dye.
For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius. Resuspend the residue with 200 uL of PBS. Drop 20 uL of suspension on the slide and observe it under a fluorescence microscope.
Resuspend extracellular vesicles in 200 uL of PBS, and then mix with heparin solution at a 10:1 volume ratio. Place the mouse into the caudal vein imaging system. Press the switch to lift the lever.
With the help of a tail vein illustrator, perform a systematic injection of the extracellular vesicle suspension through the tail vein. Analysis of particle size distribution using NTA demonstrated that the size of exosomes from human mesenchymal stem cells ranged from 40 to 335 nm, with a peak size of about 100 nm. Also, the size of microvesicles ranged from 50 to 445 nm, with a peak size of 150 nm.
Morphological characterization of exosomes showed that they exhibited a typical cup shape. Extracellular vesicles were efficiently labeled by PKH26, which was observed by the gross view as labeled pellets, as well as by fluorescent microscopy. The operator should pay attention during supernatant collection, because cells must be blown evenly to fully collect the released EVs retained in the indistinct}of mesenchymal stem cells.
In order to verify the success of the injection, in vivo imaging of biodistribution of fluorescence-labeled EVs or frozen sections of specific tissue sites of engraftment can be performed.
