Name: isolation and characterization of exosomes from skeletal muscle fibroblasts
Uploaded: 2020-05-16T13:30:00
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 fi...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+exosome+isolation+methods+using+culture+supernatant+for+optimum+yield,+purity+and+downstream+applications.">Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.</a> Science Reports. 9 (1), 5335 (2019).</li><li>Ayala-Mar, S., Donoso-Quezada, J., Gallo-Villanueva, R. C., Perez-Gonzalez, V. H., Gonz&#225;lez-Valdez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+challenges+in+the+recovery+and+purification+of+cellular+exosomes.">Recent advances and challenges in the recovery and purification of cellular exosomes.</a> Electrophoresis. 40 (23-24), 3036-3049 (2019).</li><li>Boutagy, N. 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Muscle Dissection in mouse Available from: <a target="_blank" href="https://www.youtube.com/watch?v=Wh0gXfrHaH8">https://www.youtube.com/watch?v=Wh0gXfrHaH8</a> (2016)</li><li>Mas-Bargues, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+oxygen+concentration+in+stem+cell+culture+for+regenerative+medicine.">Relevance of oxygen concentration in stem cell culture for regenerative medicine.</a> International Journal of Molecular Sciences. 20 (5), 1195 (2019).</li><li>Bois, P. R., Grosveld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKHR+(FOX01a)+is+required+for+myotube+fusion+of+primary+mouse+myoblasts.">FKHR (FOX01a) is required for myotube fusion of primary mouse myoblasts.</a> The EMBO Journal. 22 (5), 1147-1157 (2003).</li><li>Machida, S., Spangenburg, E. E., Booth, F. 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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 f...

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&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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>O2/CO2 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...

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 ...

The advantage of using differential centrifugation for this protocol is that it is easily scalable up to liters and therefore, ideal for isolating exosomes from primary fibroblasts. Purified exosomes can be obtained from different cell types and tissues isolated from various disease models. The exosomes can be used as diagnostic biomarkers.Demonstrating that procedure will be Diantha van de Vlekkert, a scientist from the d'Azzo Laboratory To begin, weigh the mouse muscles and then transfer them to a 10-centimeter dish. Working under a biosafety hood, mince the muscles with scalpels to produce a fine paste. Transfer the finely minced muscle paste to a 50-milliliter tube, and add digestion solution using 3.5 times the volume per milligram of tissue.Incubate for 45 minutes at 37 degrees Celsius. To aid in complete dissociation, mix the suspension thoroughly with a 5-milliliter pipette every 10 minutes. To inactivate the digestion solution, add 20 milliliters of DMEM Complete to the cell suspension.Pipette through a 70-micrometer nylon cell strainer placed on a 50-milliliter tube, and then wash the cell strainer with an additional 5 milliliters of DMEM Complete. Centrifuge the cell suspension at 300g for 10 minutes at room temperature. Carefully remove the supernatant and re-suspend the pellet in 10 milliliters of DMEM Complete.Seed the cells into a 10-centimeter dish, then place the dish in the incubator at 37 degrees Celsius, 5%carbon dioxide, and 3%oxygen. The skeletal muscle fibroblast cultures need to be maintained at a low oxygen level to ensure physiological light conditions. The oxygen level in skeletal muscle is approximately 2.5%After the cells reach 100%confluence, culture them for an additional day, then rinse them with PBS and add one milliliter of trypsinization solution.After incubating the cells for 10 minutes, stop the trypsinization by adding 10 milliliters of DMEM Complete. Transfer the cell suspension to a 50-milliliter tube, and then centrifuge the cells at 300g for 10 minutes at room temperature. Then re-suspend the pellet in 20 milliliters of DMEM complete and seed the cells into a 15-centimeter dish.Collect the conditioned medium from the cultured fibroblasts in a 15-milliliter tube. Centrifuge the conditioned medium at 300g for 10 minutes to pellet the live cells. Transfer the supernatant to a new 50-milliliter tube and pellet the dead cells by centrifuging the tube at 2, 000g for 10 minutes.Next, transfer the supernatant to a 38.5-milliliter polypropylene tube and centrifuge the tube at 10, 000g for 30 minutes to remove organelles, apoptotic bodies, and membrane fragments. To pellet the exosomes, transfer the supernatant to a 38.5-milliliter ultraclear tube, and ultracentrifuge it at 100, 000g for 1.5 hours. Carefully discard the supernatant, leaving approximately one milliliter of conditioned medium.Wash the exosome pellet and re-suspend it in up to 30 milliliters of ice cold PBS. Centrifuge the exosomes again at 100, 000g for 1.5 hours. To complete the purification, remove the supernatant by pipetting, being careful not to disturb the exosomal pellet.The exosome pellet step can be loose and easily detached. Therefore, the removal of the PBS supernatant should be done very carefully with the aid of a 200-microliter pipette. Re-suspend the pellet in ice-cold PBS using 25 microliters per 15-centimeter dish of fibroblasts.Lastly, measure the protein concentration using the BCA Protein Assay Kit and a microplate reader at 562 nanometers. This protocol is a highly reproducible and consistent method for purifying exosomes. Transmission electron microscopy shows the relatively uniform sizes of these exosomes.The immunoblot of exosome lysates shows a canonical pattern of protein expression and the absence of LDH and calnexin protein contaminants. Exosomes were separated by a sucrose density gradient, and individual fractions were probed on a western blot with antibodies against Alix, CD9, and CD81. Exosomes consistently sedimented in fractions three through six.The isolated and concentrated bioactive exosomes can be used for electron microscopy and mass spectrometry analyses, as well as for in-vitro and in-vivo uptake experiments for functional studies. This protocol is suitable for the isolation of exosomes from low secretory cells and can be applied to explore the involvement of exosomes in human fibrotic diseases.

Research

JoVE Journal

Biology

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