Name: isolation and characterization of exosomes from skeletal muscle fibroblasts
Uploaded: 2020-05-16T13:30:00.000+00:00
Duration: 6 min 27 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Alessandra d&#8217;Azzo holds the Jewelers for Children (JFC) Endowed Chair in Genetics and Gene Therapy. This work was supported in part by NIH grants R01GM104981, RO1DK095169, and CA021764, the Assisi Foundation of Memphis, and the American Lebanese Syrian Associated Charities.

All procedures in mice were performed according to animal protocols approved by the St. Jude Children&#8217;s Research Hospital Institutional Animal Care and Use Committee and National Institutes of Health guidelines.
1. Preparation of solutions and media
<ol>
	<li>Prepare digestion solution by mixing 15.4 mL of PBS with 2.5 mL of 20 mg/mL collagenase P (5 mg/mL final concentration), 2 mL of 11 U/mL dispase II (1.2 U/mL final concentration), and 100 &#181;L of 1.0 M CaCl2 (5 mM final concentration).</li>
	<li>Prepare 500 mL of primary fibroblasts medium (DMEM complete) by mixing 440 mL of DMEM with 50 mL of FBS (10%), 5.0 mL of pen/strep (100 U/mL and 100 &#181;g/mL, respectively) and 5.0 mL of glutamine supplement (2 mM). Filter-sterilize the medium with a 0.22 &#181;m pore size vacuum filter.</li>
	<li>Prepare exosome-free serum by overnight ultracentrifugation of FBS in 38.5 mL polypropylene tubes at 100,000 x g at 4 &#176;C and transfer the supernatant to a new tube.</li>
	<li>Prepare 500 mL of exosome-free medium by mixing 440 mL of DMEM with 50 mL of exosome-free serum (10%), 5 mL of pen/strep (100 U/mL and 100 &#181;g/mL), and 5 mL of glutamine supplement (2 mM). Filter-sterilize the medium with a 0.22 &#181;m pore size vacuum filter.</li>
	<li>Prepare 0.5 M Tris-HCl (pH 7.4) by dissolving 6.057 g of Tris base in 90 mL of dH2O and adjusting the pH to 7.4 with HCl. Adjust the volume to 100 mL with dH2O.</li>
	<li>Prepare 10 mM Tris-HCl (pH 7.4)/1 mM Mg(Ac)2 solution (sucrose working solution) by mixing 97.9 mL of dH2O with 2 mL of 0.5 M Tris-HCl (pH = 7.4) and 100 &#181;L of 1 M Mg(Ac)2. Add protease inhibitors to the solution just before use and keep the solution on ice.</li>
	<li>Prepare sucrose density gradient solutions on ice according to Table 1. These solutions are sufficient to prepare two sucrose gradient tubes.</li>
	<li>Prepare 100% TCA by adding 40 mL of dH2O to 100 g of TCA powder and mix until fully dissolved. Adjust the final volume with dH2O to 100 mL.</li>
	<li>Prepare 80% ethanol by mixing 20 mL of dH2O with 80 mL of 100% ethanol.</li>
	<li>Prepare western blot running buffer by mixing 1.8 L of dH2O with 200 mL of 10x running buffer.</li>
	<li>Prepare western blot transfer buffer by mixing 1.4 L of dH2O with 200 mL of 10x transfer buffer and 400 mL of methanol. Precool the transfer buffer to 4 &#176;C.</li>
	<li>Prepare 20x Tris-buffered saline (TBS) by dissolving 122 g of Tris base and 180 g of sodium chloride in 850 mL of dH2O (pH 8.0). Adjust the volume to 1 L with dH2O.</li>
	<li>Prepare blocking buffer by dissolving 5 g of nonfat dry milk with 1 mL of 10% Tween-20, 5 mL of 20x TBS, and 94 mL of dH2O.</li>
	<li>Prepare antibody buffer by dissolving 3 g of BSA with 1 mL of 10% Tween-20, 5 mL of 20x TBS, and 94 mL of dH2O.</li>
	<li>Prepare washing buffer by mixing 100 mL of 20x TBS and 20 mL of 10% Tween-20 (10x) with 1.88 L of dH2O.</li>
	<li>Prepare developing solution by mixing 1 volume of luminol/enhance with 1 volume of stable peroxide buffer.</li>
</ol>
2. Dissection of gastrocnemius (GA) mouse muscle15,16
<ol>
	<li>Prepare 50 mL tubes containing 10 mL PBS on ice.</li>
	<li>Euthanize the mice in a CO2 chamber followed by cervical dislocation.</li>
	<li>Remove the gastrocnemius muscle from both legs and transfer them to the tube with PBS on ice. 
	NOTE: Remove the soleus muscle from the GA muscle before transferring the GA muscle to PBS.</li>
</ol>
3. Mouse primary fibroblast isolation and culture
<ol>
	<li>Weigh the muscles and transfer them to a 10 cm dish under a biosafety hood. Mince the muscles with scalpels until it becomes a fine paste.</li>
	<li>Transfer the finely minced muscle paste to a 50 mL tube and add 3.5x volume/mg tissue of digestion solution to the tube and incubate for 45 min at 37 &#176;C. Mix the suspension thoroughly with a 5 mL pipet every 10 min (this will aid in complete dissociation). 
	NOTE: Alternatively, a tissue dissociator can be used for this step.</li>
	<li>Add 20 mL of DMEM complete to the cell suspension to inactivate the digestion solution. Transfer to a 70 &#181;m nylon cell strainer placed on a 50 mL tube. Collect the flow-through and wash the cell strainer with an additional 5 mL of DMEM complete.</li>
	<li>Centrifuge the cell suspension at 300 x g at room temperature (RT) for 10 min and carefully remove the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of DMEM complete and seed the cells into a 10 cm dish.</li>
	<li>Culture the cells at 37 &#176;C, 5% CO2, 3% O2 (passage 0 or P0). 
	NOTE: 1) These cultures need to be maintained at a low oxygen level to ensure physiological-like conditions (O2 level in skeletal muscle is around 2.5%)17. 2) The passage number (e.g., P0) of a cell culture is a record of the number of times the culture has been subcultured. 3) Primary fibroblasts at P0 are routinely cultured until 100% confluency plus 1 day (and only for P0). This is to purge other types of cells and assure that the cells present in the culture are only fibroblasts, depleted of myogenic cells based on microscope examination. The skeletal muscle from an adult animal at 2 months of age contains about 2% myogenic progenitors4. In addition, myogenic progenitors usually do not attach to the uncoated dishes; therefore, they are lost during subculturing18,19,20. Myogenic cells require a different medium (F10) supplemented with 20% FBS and basic fibroblast growth factor (bFGF)18. In DMEM complete, medium fibroblasts have a higher proliferation rate than the myogenic cells, which results in the elimination of these contaminating cells before the first passage.</li>
	<li>Rinse the P0 cells with PBS when 100% confluent plus 1 day. Add 1 mL of trypsinization solution and incubate at 37 &#176;C, 5% CO2, 3% O2 to detach the cells. Stop the enzymatic activity by using 10 mL of DMEM complete.</li>
	<li>Centrifuge the cells at 300 x g for 10 min at RT and resuspend the pellet in 20 mL of DMEM complete and seed into a 15 cm dish (passage 1 or P1). The cells are grown until 80%&#8211;90% confluency and can be expanded until passage 3. 
	NOTE: Depending on the downstream experiments passage 1, passage 2 (P2) or passage 3 (P3) can be used.</li>
</ol>
4. Seeding of cells and collection of conditioned medium
<ol>
	<li>Wash the fibroblasts (P1, P2, or P3) with 10 mL of PBS, add 2.5 mL of trypsinization solution to the cells and incubate at 37 &#176;C, 5% CO2, 3% O2. Stop enzymatic activity by using 10 mL of DMEM complete. 
	NOTE: After passage 4, primary cells are discarded and should not be used for further experiments, as they change characteristics.</li>
	<li>Collect the cells and centrifuge at 300 x g at RT for 10 min.</li>
	<li>Resuspended the cell pellet in 10 mL of exosome-free medium and count the cells.</li>
	<li>Seed the cells at 1.5&#8211;2.0 x 106 cells per dish (15 cm) and incubate at 37 &#176;C, 5% CO2, 3% O2.</li>
	<li>Collect the conditioned medium between 16&#8211;24 h in 50 mL tubes on ice. 
	NOTE: If cells are not 80% confluent, fresh exosomal free medium can be added again and collected after an additional 16&#8211;24 h period. The two collections can be combined for further processing.</li>
</ol>
5. Purification of exosomes using differential and ultra-centrifugation
NOTE: All steps are performed at 4 &#176;C or on ice. Balance the tube with a tube filled with water when needed.
<ol>
	<li>Centrifuge the conditioned medium at 300 x g for 10 min for removal of live cells and transfer the supernatant to a new 50 mL tube.</li>
	<li>Remove dead cells by centrifugation at 2,000 x g for 10 min and transfer the supernatant to a 38.5 mL polypropylene tube.</li>
	<li>Centrifuge the supernatant at 10,000 x g for 30 min for the removal of organelles, apoptotic bodies, and membrane fragments. Transfer the supernatant to a 38.5 mL ultra-clear tube.</li>
	<li>Ultracentrifuge the supernatant at 100,000 x g for 1.5 h to pellet the exosomes.</li>
	<li>Carefully discard the supernatant, leaving approximately 1 mL of conditioned medium, and wash the exosome pellet in a total volume of 30 mL of ice-cold PBS.</li>
	<li>Centrifuge at 100,000 x g for 1.5 h and carefully discard the supernatant by pipetting, being careful not to disturb the exosomal pellet.</li>
	<li>Resuspend the pellet in ice-cold PBS (~25 &#956;L per 15 cm dish) and measure the protein concentration using the BCA protein assay kit and a microplate reader at 562 nm. 
	NOTE: The protein concentration is measured by a 1:2 dilution with 0.1% Triton-X100 in dH2O. The yield of exosomes from a 15 cm dish (20 mL of conditioned medium) of primary fibroblasts at 80% confluency is ~3&#8211;4 &#956;g.</li>
</ol>
6. Characterization of exosomes by sucrose density gradient
<ol>
	<li>Load the sucrose gradient in a 13 mL ultra-clear tube by pipetting (from the bottom up) the following: 1.5 mL of 2.0 M, 2.5 mL of 1.3 M, 2.5 mL of 1.16 M, 2.0 mL of 0.8 M, and 2.0 mL of 0.5 M sucrose solutions.</li>
	<li>Mix 30&#8211;100 &#181;g of exosomes with 0.25 M sucrose solution and adjust the volume to 1 mL.</li>
	<li>Load carefully the exosomes on top of the gradients and ultra-centrifuge the samples for 2.5 h at 100,000 x g and 4 &#176;C.</li>
	<li>Remove the tubes from the ultra-centrifuge and place them on ice and collect 1.0 mL fractions starting from the top of the gradient. Transfer them to 1.7 mL tubes on ice.</li>
	<li>Add 110 &#181;L of 100% TCA to each tube, mix well, and incubate for 10 min at RT.</li>
	<li>Centrifuge for 10 min at 10,000 x g and RT and carefully discard the supernatant.</li>
	<li>Resuspend the pellet in 500 &#181;L of pre-cooled 80% ethanol and leave the samples to wash for 10 min at -20 &#176;C.</li>
	<li>Centrifuge for 10 min at 10,000 x g and 4 &#176;C and discard the supernatant.</li>
	<li>Dry the pellet for 10&#8211;15 min at RT and resuspend the pellet in PBS for in vitro experiments. Measure the protein concentration using BCA protein assay kit or resuspend the pellet directly in 14.5 &#181;L of dH2O, 5 &#181;L of 4x Laemmli buffer, and 0.5 &#181;L of 1 M DTT for western blot analyses.</li>
</ol>
7. Exosome detection by western blot analysis
<ol>
	<li>Mix 5&#8211;10 &#181;g of exosomes from step 5.7 with 5 &#181;L of 4x Laemmli buffer, and 0.5 &#181;L of 1 M DTT, then adjust the final volume to 20 &#181;L with dH2O. Heat all samples, including those in step 6.9, for 5 min at 98 &#176;C.</li>
	<li>Load the samples into a 10% TGX stain-free gel and load the protein ladders according to the manufacturer&#8217;s recommendations. 
	NOTE: Choose the percentage of the gel according to the molecular weight of the protein of interest.</li>
	<li>Run the gel at 100 V for ~1 h in running buffer until the loading dye is at the bottom of the gel. 
	NOTE: The run time may vary according to the equipment used or type and percentage of gel.</li>
	<li>Image the gel. Images of the stain-free gels are used as the loading control for immunoblots.</li>
	<li>Transfer the gel using a PVDF membrane for 1.5 h at 80 V or overnight at 30 V at 4 &#176;C.</li>
	<li>Block the membranes in blocking buffer for 1 h at RT.</li>
	<li>Incubate the membranes for specific antibodies (Table 2) in antibody buffer overnight at 4 &#176;C with agitation.</li>
	<li>Wash the membranes in washing buffer and incubate the membranes with the appropriate secondary antibodies (Table 2) for 1 h at RT with agitation.</li>
	<li>Wash the membranes in washing buffer and image the membranes using develop solution and a camera-based imager. Alternatively, use an X-ray film to develop the membranes.</li>
</ol>

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None of the authors have any conflicts of interest to declare.

A critical step for the successful isolation of exosomes from the culture media, as outlined in this protocol, is the proper establishment and maintenance of primary mouse fibroblast cultures from adult skeletal muscle. These cultures need to be maintained at a low oxygen level to ensure physiological-like conditions (O2 level in skeletal muscle is ~2.5%)15. Primary fibroblasts will change characteristics when passed in culture too many times. Hence, a low passage number is imperative for ideal exosome yield. Purified exosomes should either be used immediately for experimental purposes or kept frozen in aliquots at -80 &#176;C until needed. Another important step in the protocol is the use of sucrose solutions prepared fresh every time. A limitation of the method is that it is time-consuming, because primary fibroblasts do not generally secrete high amounts of exosomes, like other secretory cells. Therefore, large cultures should be employed, which result in large volumes of conditioned media to be processed.
The advantage of differential centrifugation used here is that it represents a scalable approach (up to liters) and is the method of choice to isolate exosomes from primary fibroblasts. An alternative method for larger volumes (up to 100 mL per column) is size exclusion chromatography (SEC). This method is fast and can separate proteins from exosomes. The column does not pellet exosomes, so further concentration or centrifugation steps are required. One of the disadvantages of the SEC method is that the column can be clogged over time and overloaded. Other current methods are based on the use of small volumes of biological fluids (i.e., urine, CSF, serum, plasma) and commercially available kits. Although these kits ensure reproducibility, they are costly and do not always produce a high yield of pure exosomes. The outlined protocol for exosome purification is straightforward and can be applied to different types of cells, whole tissues and organs, or other biological fluids depending on the experimental need.

Exosomes are heterogeneous extracellular vesicles ranging in size from 30&#8211;150 nm. They are established key players in physiological and pathological processes, given their ubiquitous distribution in tissues and organs1,2. Exosomes carry a complex cargo of proteins, lipids, DNA types, and RNA types, which vary according to the type of cells from which they are derived1,2,3. Exosomes are enriched in proteins that have different functions (i.e., tetraspanins, including CD9 and CD63) are responsible for fusion events. For example, heat shock proteins HSP70 and HSP90 are involved in antigen binding and presentation. Additionally, Alix, Tsg101, and flotillin participate in exosome biogenesis and release and are widely used as markers of these nanovesicles2,3,4.
Exosomes also contain a variety of RNAs (i.e., microRNAs, long noncoding RNAs, ribosomal RNAs) that can be transferred to recipient cells, where they influence downstream signaling3. Being enclosed by a single unit membrane, exosome bioactivity depends not only on the cargo of proteins and nucleic acids, but also on lipid components of the limiting membrane1. Exosomal membranes are enriched in phosphatidylserine, phosphatidic acid, cholesterol, sphingomyelin, arachidonic acid, and other fatty acids, all of which can influence exosome stability and membrane topology2,3. As a result of the cargo and lipid arrangement, exosomes initiate signaling pathways in receiving cells and participate in the maintenance of normal tissue physiology1,2,4,5. Under certain pathological conditions (i.e., neurodegeneration, fibrosis, and cancer), they have been shown to trigger and propagate pathological stimuli4,6,7,8,9,10,11.
Owing to their ability to propagate signals to neighboring or distant sites, exosomes have become valuable biomarkers for the diagnosis or prognosis of disease conditions. In addition, exosomes have been used experimentally as vehicles of therapeutic compounds2,12. The potential application of these nanovesicles in the clinic makes the isolation method increasingly important in order to achieve maximum yield, purity, and reproducibility. Different techniques for the isolation of exosomes have been developed and implemented. Generally, exosomes can be isolated from conditioned cell culture media or body fluids by differential centrifugation, size exclusion chromatography, and immune capture (using commercially available kits). Each approach has unique advantages and disadvantages that have been discussed previously1,2,13,14.
The outlined protocol focuses on the 1) isolation and culture of primary fibroblasts from adult mouse gastrocnemius muscle and 2) purification and characterization of exosomes released into the culture medium by these cells. A well-established protocol for the isolation of exosomes from primary fibroblasts for functional studies is currently lacking. Primary fibroblasts do not secrete large amounts of exosomes, making the isolation and purification process challenging. This protocol describes the purification of large amounts of pure exosomes from large culture volumes while maintaining their morphological integrity and functional activity. Purified exosomes obtained from conditioned medium have been used successfully in in vitro uptake experiments to induce specific signaling pathways in recipient cells. They have also been used for comparative proteomic analyses of exosomal cargos from multiple biological samples4.

<table><tbody><tr><td>10 cm dishes</td><td>Midwest Scientific, TPP</td><td>TP93100</td><td></td></tr><tr><td>15 cm dishes</td><td>Midwest Scientific</td><td>TP93150</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific, Pierce</td><td>23225</td><td></td></tr><tr><td>Bovine serum albumin Fraction V</td><td>Roche</td><td>10735094001</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C1016-100G</td><td></td></tr><tr><td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td><td>Eppendorf</td><td>022620659/5427754008</td><td></td></tr><tr><td>Chemidoc MP imaging system</td><td>BioRad</td><td>12003154</td><td></td></tr><tr><td>Collagenase P</td><td>Sigma, Roche</td><td>11 213 857 001</td><td>100 mg</td></tr><tr><td>cOmplete protease inhibitor cocktail</td><td>Millipore/Sigma, Roche</td><td>11697498001</td><td></td></tr><tr><td>Criterion Blotter with plate electrodes</td><td>BioRad</td><td>1704070</td><td></td></tr><tr><td>Criterion TGX stain-free protein gel</td><td>BioRad</td><td>5678034</td><td>10% 18-well, midi-gel</td></tr><tr><td>Criterion vertical electrophoresis cell (midi)</td><td>BioRad</td><td>NC0165100</td><td></td></tr><tr><td>Dispase II</td><td>Sigma, Roche</td><td>04 942 078 001</td><td>neutral protease, grade II</td></tr><tr><td>Dithiothreitol</td><td>Sigma/Millipore, Roche</td><td>10708984001</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modification Eagles Medium</td><td>Corning</td><td>15-013-CV</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Pharmco by Greenfield Global</td><td>111000200</td><td></td></tr><tr><td>Falcon 50 mL conical centrifuge tubes</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10437-028</td><td></td></tr><tr><td>Fluostar Omega multi-mode microplate reader</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>GlutaMAX supplement</td><td>Thermo Fisher Scientific, Gibco</td><td>35050-061</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Immobilon-P Transfer membranes</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Laemmli sample buffer (4x)</td><td>BioRad</td><td>1610747</td><td></td></tr><tr><td>Magnesium acetate solution</td><td>Sigma</td><td>63052-100ml</td><td></td></tr><tr><td>Non-fat dry milk</td><td>LabScientific</td><td>M-0842</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>MC0-18M</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific, Gibco</td><td>15140-122</td><td>10,000 U/ml</td></tr><tr><td>Premium microcentrifuge tubes</td><td>Fisher Scientific, Midwest Scientific</td><td>AVSC1510</td><td>1.7 mL</td></tr><tr><td>Protected disposable scalpels</td><td>Fisher Scientific, Aspen Surgical Bard-Parker</td><td>372610</td><td></td></tr><tr><td>Running buffer</td><td>BioRad</td><td>1610732</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific, Fisher Chemical</td><td>S271-3</td><td></td></tr><tr><td>Stericup Quick release-GP sterile vacuum filtration system</td><td>Millipore</td><td>S2GPU05RE</td><td>500 mL</td></tr><tr><td>Sterile cell strainer (70 mm)</td><td>Fisher Scientific, Fisher brand</td><td>22-363-548</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific, Fisher Chemical</td><td>S5-500</td><td></td></tr><tr><td>SuperSignal west Femto</td><td>Thermo Fisher Scientific</td><td>34096</td><td></td></tr><tr><td>Thin wall Polypropylene tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>Transfer buffer</td><td>BioRad</td><td>16110734</td><td></td></tr><tr><td>Trichloroacetic Acid</td><td>Sigma</td><td>91228-100G</td><td></td></tr><tr><td>Tris base</td><td>BioRad</td><td>1610719</td><td></td></tr><tr><td>Triton-X100 solution</td><td>Sigma</td><td>93443-100mL</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Thermo Fisher Scientific, Gibco</td><td>12604-013</td><td>No phenol red</td></tr><tr><td>Tween-20</td><td>BioRad</td><td>#1610781</td><td></td></tr><tr><td>Ultra-centrifuge Optima XPM</td><td>Beckman Coulter</td><td>A99842</td><td></td></tr><tr><td>Ultra-clear tube (14x89 mm)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Ultra-clear tubes (25x89 mm)</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Water bath Isotemp 220</td><td>Fisher Scientific</td><td>FS220</td><td></td></tr></tbody></table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol is suitable for the purification of exosomes from large volumes of conditioned medium in a cost-effective manner. The procedure is highly reproducible and consistent. Figure 1 shows transmission electron microscopy (TEM) image of exosomes purified from the culture medium of mouse primary fibroblasts. Figure 2 shows the protein expression pattern of canonical exosomal markers, and the absence of cytosolic (LDH) and ER (calnexin) protein contaminants. Figure 3 shows the distribution of canonical exosomal markers (Alix, CD9, and CD81) after sucrose density gradients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61127/61127fig1v2.jpg" /> 
Figure 1: Representative TEM image of exosomes isolated from culture medium of mouse primary fibroblasts.&#160;Shown are the relatively uniform sizes of these nanovesicles. Across black lines denote the diameter of the individual vesicles. Scale bar = 100 nm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61127/61127fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61127/61127fig2v2.jpg" /> 
Figure 2: Immunoblots of exosome lysates probed with antibodies against exosome, cytosolic, and ER markers.&#160;Alix, CD81, CD9, flotillin1, syndecan1, syntenin1 (exosome), cytosolic (LDH), and ER (calnexin) markers. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61127/61127fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61127/61127fig3v2.jpg" /> 
Figure 3: Exosomes separated by a sucrose density gradient.&#160;Individual fractions were probed on a western blot with antibodies against Alix, CD9, and CD81. Based on the fractionation pattern of the markers, exosomes consistently sediment in fractions 3&#8211;6, which correspond to densities of 1.096&#8211;1.140 g/mL. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61127/61127fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Sucrose (g)</td>
			<td>Sucrose working solution (mL)</td>
		</tr>
		<tr>
			<td>2.0 M</td>
			<td>2.74</td>
			<td>4.0</td>
		</tr>
		<tr>
			<td>1.3 M</td>
			<td>2.67</td>
			<td>6.0</td>
		</tr>
		<tr>
			<td>1.16 M</td>
			<td>2.38</td>
			<td>6.0</td>
		</tr>
		<tr>
			<td>0.8 M</td>
			<td>1.37</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>0.5 M</td>
			<td>0.86</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>0.25 M</td>
			<td>0.26</td>
			<td>3.0</td>
		</tr>
	</tbody>
</table>
Table 1: Sucrose density gradient solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Host</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Rat</td>
			<td>BD Biosciences</td>
			<td>553758</td>
		</tr>
		<tr>
			<td>CD81</td>
			<td>Mouse</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-166029</td>
		</tr>
		<tr>
			<td>Alix</td>
			<td>Rabbit</td>
			<td>d&#39;Azzo lab</td>
			<td>Alix</td>
		</tr>
		<tr>
			<td>Flotilin1</td>
			<td>Mouse</td>
			<td>BD Biosciences</td>
			<td>610820</td>
		</tr>
		<tr>
			<td>Syndecan1</td>
			<td>Rabbit</td>
			<td>Life Technologies</td>
			<td>36-2900</td>
		</tr>
		<tr>
			<td>Syntenin1</td>
			<td>Rabbit</td>
			<td>Millipore/Sigma</td>
			<td>AB15272</td>
		</tr>
		<tr>
			<td>LDH</td>
			<td>Goat</td>
			<td>Chemicon</td>
			<td>AB1222</td>
		</tr>
		<tr>
			<td>Calnexin</td>
			<td>Goat</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-6465</td>
		</tr>
		<tr>
			<td>rat-HRP</td>
			<td>Donkey</td>
			<td>Jackson Imm. Res Lab</td>
			<td>112-035-003</td>
		</tr>
		<tr>
			<td>Mouse-HRP</td>
			<td>Goat</td>
			<td>Jackson Imm. Res Lab</td>
			<td>115-035-044</td>
		</tr>
		<tr>
			<td>Rabbit-HRP</td>
			<td>Goat</td>
			<td>Jackson Imm. Res Lab</td>
			<td>111-035-144</td>
		</tr>
	</tbody>
</table>
Table 2: Primary and secondary antibodies.

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

isolation-characterization-exosomes-from-skeletal-muscle

Universidad San Sebastian

Research

JoVE Journal

Biology

3.3K Views.  St. Jude Children's Research Hospital. This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Video: Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Refining Techniques for Murine Spleen-Derived Exosome Isolation

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...