Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles in rodent muscle tissues. This extracellular vesicle will provide invaluable insights into the muscle human status disease mechanism with potential application as diagnostic biomarkers and therapeutical vehicles. This standardized protocol will enable the analysis and the comparison of characteristic of skeleton muscle derived EV across different muscle samples, including the yield, size, cargo composition and function.

These results will advance our understanding of their roles in various physiologic and pathological processes, and will also facilitate the application of these EVs as diagnostic biomarkers. Our laboratory will focus on reliably producing high purity and high-yield skeletal muscle interstitial extracellular vesicles in an efficient and time effective manner. We aim to explore their pathological role and therapeutic potential, particularly in the context of neuromuscular disorders.

To begin, secure the euthanized mouse leg in a sterile plate with the anterior side of the leg facing upwards. Using dissection scissors, make a small incision of five millimeters in the skin on the lateral side of the mouse ankle. Insert one blade of the scissors into the incision, positioning it beneath the skin, but above the muscle tissue.

Then, cut the skin upwards towards the knee to extend the incision. Use rough-ended tweezers to grab the skin along the incision and pull it back to widen the opening, until the muscle becomes visible. Now, locate the tibialis anterior, or TA muscle, running from the knee to the ankle, along the lateral side of the tibia bone.

Use sharpened tweezers to grasp and carefully peel off the connective tissue covering the TA muscle. Near the ankle, identify the TA tendon connected to the TA muscle and the smaller EDL tendon, located next to the TA tendon on the lateral side. Use dissection scissors to sever both tendons.

Use rough-ended tweezers to grab both tendons. With another pair of tweezers, carefully separate the tendons, isolating the TA from the EDL. Then, lift the TA muscle by the freshly-severed tendon using rough-ended tweezers.

Use dissection scissors to cut the TA tendon near the knee, removing the muscle. Now, release the leg and flip the mouse so that the posterior side of the leg faces upwards. With part of the skin already removed, use dissection scissors to cut the remaining skin near the ankle.

Grab the cut skin with rough-ended tweezers and peel it upwards towards the back of the knee, exposing the gastrocnemius muscle. Use dissection scissors to sever the gastrocnemius tendon near the ankle. Grasp the gastrocnemius tendon with rough-ended tweezers and lift it upwards to expose the underside of the gastrocnemius muscle.

Then, locate the smaller soleus tendon beneath the gastrocnemius, close to the knee, where the gastrocnemius is still connected. Use dissection scissors to sever the soleus tendon, allowing the soleus muscle to contract upwards. Grasp the severed soleus tendon with sharp-ended tweezers and lift it gently to detach the soleus muscle from the underside of the gastrocnemius.

Use dissection scissors to sever the end of the soleus, where it attaches to the severed end of the gastrocnemius, fully separating the two muscles. Using dissection scissors, cut the end of the gastrocnemius near the knee and place it in PBS on ice. After removing the four muscles below the knee, flip the mouse so that the anterior side faces upwards.

Position the mouse leg at a 90-degree angle to force the quadriceps muscle to flex. Use dissection scissors to sever the tendon closest to the knee. Then, to separate the quadriceps from the femur, cut between the muscle and the bone until only the proximal tendon remains attached.

Sever the proximal tendon using dissection scissors, and remove the quadriceps. Rinse all removed muscles several times with PBS to remove contaminants such as fur and blood. Gently dry the muscles using paper towels.

Begin by gently removing the tendons from the muscle sample using sharp-end tweezers, splinter tweezers, scalpels or scissors. Then, using scissors or a scalpel, cut the muscle longitudinally along the muscle fibers from one tendon to the other, into one-millimeter thick chunks. To prepare digestive enzyme buffer, mix two milligrams of collagenase, two enzyme per milliliter of DMEM with 1%penicillin streptomycin solution.

After mixing, filter the solution through a 0.2 micrometer filter. Separate the muscle tissue into aliquots, each containing 20 milligrams of muscle. Add one milliliter of the prepared buffer to each aliquot for enzymatic dissociation and incubate.

Prepare and label new 1.5 milliliter microcentrifuge tubes. Set up a 0.45 micrometer filter by removing the plunger from a five-milliliter syringe. Screw the syringe hub into the top end of the 0.45 micrometer filter and position the bottom end of the filter into a labeled microcentrifuge tube.

Then, centrifuge the mixture for 10 minutes at 1000 G and four degrees Celsius to pellet debris. Use a P-200 micropipette to transfer the supernatant into the syringe. After reinserting the plunger into the syringe, slowly push the supernatant through the filter, allowing it to collect in the labeled 1.5 milliliter microcentrifuge tube.

Remove the syringe plunger again. Using a P-200 micropipette, add approximately 200 microliters of PBS into the syringe. Reinsert the plunger and slowly push the 200 microliters of PBS through the filter to flush any remaining supernatant into the labeled microcentrifuge tube.

Transfer the filtered solution to new 1.5 milliliter microcentrifuge tubes, suitable for speeds up to 200, 000 G.After closing the tubes, place them in the rotor of the micro ultracentrifuge to ensure they are balanced. Turn on the micro ultracentrifuge and set it to run for one hour at 100, 000 G and four degrees Celsius. Place the loaded rotor with the tubes inside the micro ultracentrifuge.

Close the lid and turn on the vacuum to initiate the process. Once the vacuum reaches level two, press the start button and wait for the centrifugation process to complete. After the centrifugation cycle is complete, click the vacuum button to repressurize the chamber before removing the rotor and tubes.

Carefully remove the supernatant from each tube using a P-200 micropipette. Then, use the same P-200 micropipette to add 50 microliters of PBS to each tube.