Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

Summary

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles (SkM-EVs) from rodent muscle tissues through mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It demonstrates consistency and reliability. The isolated SkM-EVs provide insights into muscle homeostasis and diseases, with potential applications as diagnostic biomarkers or therapeutic vehicles.

Abstract

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.

Introduction

Extracellular vesicles (EVs) are nano-sized, membrane-bound particles released by cells into the extracellular environment. These EVs transfer nucleic acids, proteins, and lipids to modulate multiple signaling pathways upon uptake by recipient cells. Specifically, EVs isolated from skeletal muscle (SkM-EVs) have been implicated in muscle degeneration, regeneration, and growth by transferring myogenic regulatory microRNAs or transcription factors^1,2,3,4. As our understanding of SkM-EV subpopulations and characteristics continues to evolve, these vesicles show promise as biomarkers for early diagnostics^5,6 and as therapeutic targets for neuromuscular disorders. This expanding field offers exciting opportunities for both pathological investigation and clinical applications.

Current research on SkM-EVs predominantly focuses on those obtained from cell culture supernatants following ex vivo culture^2,7,8, raising concerns regarding the fidelity of target cell lines after successive passages and the susceptibility of EV characteristics to alteration in artificial environments. This highlights the need to isolate SkM-EVs directly from muscle tissues to avoid ex vivo culture influences and better represent in vivo conditions. Although several approaches for isolating EVs from muscle tissues have been developed^9,10, studies still vary significantly in protocol details, impacting consistency and comparability. A standardized protocol for SkM-EV isolation and characterization is urgently needed to ensure reliability across related studies.

Considering the physiological characteristics of skeletal muscle, we developed a protocol to isolate and purify SkM-EVs from rodent skeletal muscle samples using mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. Through comprehensive analysis using nano-flow cytometry, BCA assay, and Western blot assay, we found that this protocol consistently yields SkM-EVs of high purity and quantity within a limited timeframe. It can be applied to skeletal muscle samples in various conditions, including acute and chronic muscle injuries. Furthermore, with appropriate adjustments, this method can be tailored for muscle tissues from human patients or other animal models. Standardizing the SkM-EV isolation process is essential for ensuring consistency in SkM-EV quality, facilitating comparisons of EV characteristics -- such as yield, size, cargo composition, and function -- and advancing their potential applications as diagnostic biomarkers, as well as enhancing our understanding of their roles in various physiological and pathological processes.

Protocol

This protocol isolates extracellular vesicles (EVs) from skeletal muscle tissue using mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It allows for the isolation of EVs from individual or combined rodent limb muscle groups, including the tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), gastrocnemius (GAS), and quadriceps (QUA). Animal use was approved by the Institutional Animal Care and Use Committee at the University of Georgia, and the study complied with the National Institutes of Health Guidelines for the care and use of animals in research. The animals were housed in the animal facilities within the Department of Animal and Dairy Science at the University of Georgia. Wild-type (C57BL/6J) adult male mice were used in this study, with tissue harvested from mice sacrificed at 3 months of age. Details of the reagents and equipment used are provided in the Table of Materials.

1. Mechanical detachment of muscle groups from the mouse

1. Euthanize the mice with CO₂, followed by cervical dislocation (following institutionally approved protocols). Shave both hindlimbs.
2. Secure the mouse leg in a sterile plate with the anterior side of the leg facing upwards (Figure 1A).
3. Make a small incision (5 mm) in the skin on the lateral side of the mouse ankle using dissection scissors.
4. Insert one blade of the scissors carefully into the incision, positioning it beneath the skin but not in the underlying muscle tissue. Then, cut the skin upwards towards the knee to extend the incision length.
5. Grab the skin along the incision using rough-ended tweezers and expand the opening until the muscle is visible.
6. Locate the TA muscle, which runs from the knee to the ankle on the lateral side of the tibia bone¹¹, and use sharp-end tweezers to grasp and peel off the thin layer of connective tissue covering the muscle (Figure 1B).
7. Near the ankle, identify the two tendons at the end of the TA, one directly connected to the TA muscle and the other, the EDL tendon, which is smaller and located next to the TA tendon on the lateral side. Sever both tendons using dissection scissors.
8. Grab both tendons with rough-ended tweezers. Then, use another pair of rough or sharp-ended tweezers to separate the tendons, thus separating the TA from the EDL (Figure 1C).
9. Lift the TA by the freshly severed tendon with rough-ended tweezers. Then, cut the TA tendon near the knee using dissection scissors, thus removing the muscle. Repeat this process to remove the EDL, and store both muscles in PBS on ice.
10. Release the leg and flip the mouse over so that the posterior face is facing up.
11. With the skin on the leg now partially removed, cut the skin near the ankle using dissection scissors. Then, use rough-ended tweezers to grasp and peel the skin upwards towards the back of the knee, exposing the GAS.
12. Sever the GAS tendon near the ankle using dissection scissors (Figure 1D).
13. Grasp the GAS tendon with rough-ended tweezers and lift it upwards to expose the underside of the GAS.
14. Locate the small SOL tendon on the underside of the GAS close to the knee (where the GAS is still connected). Sever the SOL tendon using dissection scissors, causing the SOL to contract upwards (Figure 1E).
15. Grasp the severed SOL tendon with sharp-ended tweezers and lift it gently, thus detaching the SOL from the underside of the GAS.
16. Sever the end of the SOL where it attaches to the severed end of the GAS using dissection scissors, thus separating the two muscles. Preserve the SOL in PBS on ice.
17. Cut the end of the GAS near the knee and place it in PBS on ice.
18. After removing the four listed muscles below the knee, flip the mouse over so that the anterior face is upwards.
19. If needed, peel the skin further with rough-ended tweezers to expose the QUA. Position the mouse leg at a 90° angle to force the QUA to flex. Sever the tendon closest to the knee using dissection scissors.
    1. Separate the QUA from the femur by cutting between the muscle and bone until the QUA is only connected by the proximal tendon (Figure 1E). Then, sever the proximal tendon and remove the QUA. Place the QUA in PBS on ice.
20. Ensure that after muscle detachments, the tibia and fibula are clearly exposed (Figure 1F).
21. Rinse the muscles several times with PBS to remove contaminants such as fur and blood (Figure 1G). Then, dry the muscles gently with paper towels. Use an analytical balance to measure the muscle weight, which will be used later to calculate the EV collected per gram of muscle.

2. Enzymatic dissociation of detached muscle chunks

1. Gently remove muscle tendons from the muscle sample using sharp-end tweezers, splinter tweezers, scalpels, or scissors (Figure 2A).
2. After tendon removal, cut the muscle longitudinally along the muscle fibers (from tendon to tendon) into 1 mm-thick muscle chunks using scissors or a scalpel to increase the surface area-to-volume ratio (Figure 2B,C).
   NOTE: Try to limit myofiber damage during cutting. For larger muscles such as the TA, GAS, and QUA, make additional cuts compared to smaller muscles like the EDL and SOL to ensure a roughly equal surface area-to-volume ratio across all muscle samples (Figure 2D).
3. Prepare 10 mL of fresh digestive enzyme buffer (DEB) for each mouse and keep it on ice until use.
   1. To prepare DEB, mix 2 mg of collagenase II enzyme per 1 mL of Dulbecco's Modified Eagle's Medium (DMEM) with 1% penicillin-streptomycin solution. Filter the solution through a 0.2 µm filter after mixing.
   2. Ensure the DEB has an enzymatic activity of over 250 units. Vortex the DEB until the solution is thoroughly mixed and the collagenase II enzyme powder is no longer visible.
4. Separate the muscle tissue into aliquots, each containing 20 mg of muscle tissue. Then, add 1 mL of DEB to each aliquot for enzymatic dissociation.
5. Incubate the DEB and muscle mixture on a shaker in the incubator at 37 °C for 12 h at a shaking speed of 100 rotations per minute (rpm) (Figure 2E).

3. Filtration and low-speed centrifugation to enrich SkM-EV

NOTE: Set the refrigerated centrifuge to 4 °C and run it until it reaches the set temperature. Keep all needed tubes and samples on ice until ready for use.

1. Following the enzymatic digestion procedures, ensure no discernible muscle chunks are visible (Figure 3A). Before refrigerated centrifugation, extract 10 µL of solution from the DEB-muscle mixture and transfer it into a 0.2 mL tube for cell viability testing if needed.
2. Prepare and label new empty 1.5 mL microcentrifuge tubes. Set up a 0.45 µm filter and a 5 mL syringe by removing the plunger (do not discard) and screwing the hub into the top end of the 0.45 µm filter. Position the filter so the bottom end is placed into a newly labeled empty tube (Figure 3D). Filter the 1x PBS through a 0.2 µm filter.
3. Centrifuge the mixture for 10 min at 1,000 x g and 4 °C to pellet any debris (Figure 3B). Transfer the supernatant (around 1 mL) into the syringe using a P200 micro-pipette (Figure 3C,E).
4. Reinsert the syringe plunger and slowly push the supernatant through the filter, allowing it to pass through and collect in the newly labeled 1.5 mL microcentrifuge tube (Figure 3F).
5. Remove the plunger again and pipette roughly 200 µL of 1x PBS into the syringe.
6. Reinsert the plunger and slowly push the 200 µL of 1x PBS through the filter (Figure 3G) to remove any remaining supernatant from the filter and into the labeled 1.5 mL microcentrifuge tube.
7. Repeat the filtration process for other tubes, using a new filter for each tissue sample.
8. After filtration, ensure the solution is transparent and devoid of residual muscle chunks (Figure 3H).

4. Differential ultracentrifugation for SkM-EV purification

NOTE: Set the refrigerated centrifuge to 4 °C and run it until it reaches the set temperature. Keep all needed tubes and samples on ice and ready for use.

1. Transfer the filtered solution to new 1.5 mL microtubes (suitable for up to 200,000 x g). Balance all microtubes with an additional 1x PBS to ensure equal volume before placing them into the micro-ultracentrifuge.
2. After ensuring the volume of all tubes is equal, close the tubes and place them into the rotor of the micro-ultracentrifuge to be balanced (Figure 4A).
3. Turn on the micro-ultracentrifuge and set it to run for 1 h at 100,000 x g and 4 °C (Figure 4B).
4. Place the rotor loaded with the tubes into the micro-ultracentrifuge, close the lid, and turn on the vacuum (Figure 4C).
5. Once the vacuum reaches level 2, press the start button and wait until the centrifugation process is complete (Figure 4D).
6. After the micro-ultracentrifuge cycle is complete, click on the vacuum button to repressurize the chamber before removing the rotor and tubes. Keep the tubes on ice and position them upright to avoid disturbing the pellet.
7. Carefully remove the supernatant with a P200 micropipette, avoiding disturbance of the pellet (Figure 4E).
8. Eject the tip, then add 50 µL of 1x PBS into the tube after removing the supernatant using a P200 micropipette. For GAS and QUA samples, add up to 500 µL of 1x PBS to aid dissolution (Figure 4F).
9. Resuspend the pellet by gently pipetting up and down using a P20 micropipette (or P200 micropipette if 500 µL was added) until it is fully broken up and no longer visible.
10. Close the tube and store it at 4 °C for short-term storage (less than one week), -80 °C for long-term storage, or place the tube back on ice if the sample will be immediately characterized using nano-flow cytometry, Western blot, or other assays.

Results

Following this protocol, skeletal muscle extracellular vesicles (SkM-EVs) were successfully extracted from various muscle groups, including the tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), gastrocnemius (GAS), and quadriceps (QUA) from wild-type mice. Subsequent characterization of these SkM-EVs was conducted through nano-flow cytometry, elucidating their distinct features as summarized in Table 1. Remarkably, from a mere 1 g of muscle tissue, an impressive yield of 1.18e14 ± 4....

Discussion

The protocol begins with dissecting hindlimb muscle groups from mice or acquiring postmortem human muscle tissue based on available protocols¹⁶. The following skeletal muscle extracellular vesicles (SkM-EVs) isolation procedures comprise three primary sections: mild enzymatic digestion of the tissue to liberate interstitial SkM-EVs, filtration and low-speed centrifugation, and ultracentrifugation. Finally, various techniques are applied to profile the isolated SkM-EVs.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.6 mL microcentrifuge tubes	ThermoFisher Scientific	05-408-120
1.5 mL micro tube (C)	Eppendorf	5720411009
1.5 mL microcentrifuge tubes	ThermoFisher Scientific	3448	Clear, graduated, nonsterile
BD 5 mL Syringe	Becton Dickinson	14-827-52	luer-lok tip
Cell Counting Slides, Dual Chamber for Cell Counter	Bio-Rad	145-0011
Centrifuge 5430R	Eppendorf	22620667
Cleaning solution	NanoFCM Inc.
Collagenase II	Worthington Biochemical Corporation	LS004176
DPBS (10x) Liquid (-calcium,-magnesium)	Cytiva	SH30378.02	Final used dilution is 1x
DPBS/Modified (1x)	Cytiva	SH30264.02
Dulbecco's Modified Eagle's Medium (DMEM)	Genesee Scientific	25-500
E-POD Pure Water Remote Dispenser	Millipore Sigma	ZRXSP0D01
FA-45-30-11 Microcentrifuge Rotor	Eppendorf	05-401-503
Fisher Vortex Genie 2	ThermoFisher Scientific	12-812
Flow NanoAnalyzer	NanoFCM Inc.
Heratherm OGS60 General Protocol Oven	ThermoFisher Scientific	51028112	Gravity convection, 2.3 cu.ft. 120 V
MemGlow-488 Probe	Cytoskeleton Inc.	MG01-02
Millipak Special Millipore	Millipore Sigma	TANKMPK02
Penicillin-Streptomycin	Gibco	15070063
Pipet-Lite LTS Pipette L-1000XLS+	Rainin	17014382
Pipet-Lite LTS Pipette L-200XLS+	Rainin	17014391
Pipet-Lite LTS Pipette L-20XLS+	Rainin	17014392
Pipette tips ER LTS 1000µL F 768G/8	Rainin	30808038
Pipette tips ER LTS 20µL F 960G/10	Rainin	30807966
Pipette tips ER LTS 200µL F 960G/10	Rainin	30807967
Quality Control beads	NanoFCM Inc.
S110-AT Fixed Angle Rotor	ThermoFisher Scientific	45539	1,10,000 rpm
S16 exo 65-155nm beads	NanoFCM Inc.
Scissors	SPI, USA
Sorvall mX 150+ Micro-Ultracentrifuge	ThermoFisher Scientific	50135641
TC 20 automated cell counter	Bio-Rad	1450102
Trypan Blue Solution	Corning	25-900-CI
Tweezers	SPI, USA
VWR 15mL centrifuge tubes	Avantor	89039-668
Whatman Puradisc 13 Filters	Cytiva	6782-1304	0.45 µm pore size
Wild-type C57BL/6J mice	Jackson Laboratory	stock #000664