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Biology

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

doi: 10.3791/61127 Published: May 16, 2020
Diantha van de Vlekkert1, Xiaohui Qiu1, Ida Annunziata1, Alessandra d'Azzo1

Abstract

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.

Introduction

Exosomes are heterogeneous extracellular vesicles ranging in size from 30–150 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.

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Protocol

All procedures in mice were performed according to animal protocols approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee and National Institutes of Health guidelines.

1. Preparation of solutions and media

  1. 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 µL of 1.0 M CaCl2 (5 mM final concentration).
  2. 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 µg/mL, respectively) and 5.0 mL of glutamine supplement (2 mM). Filter-sterilize the medium with a 0.22 µm pore size vacuum filter.
  3. Prepare exosome-free serum by overnight ultracentrifugation of FBS in 38.5 mL polypropylene tubes at 100,000 x g at 4 °C and transfer the supernatant to a new tube.
  4. 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 µg/mL), and 5 mL of glutamine supplement (2 mM). Filter-sterilize the medium with a 0.22 µm pore size vacuum filter.
  5. 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.
  6. 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 µL of 1 M Mg(Ac)2. Add protease inhibitors to the solution just before use and keep the solution on ice.
  7. Prepare sucrose density gradient solutions on ice according to Table 1. These solutions are sufficient to prepare two sucrose gradient tubes.
  8. 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.
  9. Prepare 80% ethanol by mixing 20 mL of dH2O with 80 mL of 100% ethanol.
  10. Prepare western blot running buffer by mixing 1.8 L of dH2O with 200 mL of 10x running buffer.
  11. 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 °C.
  12. 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.
  13. 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.
  14. 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.
  15. Prepare washing buffer by mixing 100 mL of 20x TBS and 20 mL of 10% Tween-20 (10x) with 1.88 L of dH2O.
  16. Prepare developing solution by mixing 1 volume of luminol/enhance with 1 volume of stable peroxide buffer.

2. Dissection of gastrocnemius (GA) mouse muscle15,16

  1. Prepare 50 mL tubes containing 10 mL PBS on ice.
  2. Euthanize the mice in a CO2 chamber followed by cervical dislocation.
  3. 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.

3. Mouse primary fibroblast isolation and culture

  1. 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.
  2. 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 °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.
  3. Add 20 mL of DMEM complete to the cell suspension to inactivate the digestion solution. Transfer to a 70 µm 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.
  4. Centrifuge the cell suspension at 300 x g at room temperature (RT) for 10 min and carefully remove the supernatant.
  5. Resuspend the pellet in 10 mL of DMEM complete and seed the cells into a 10 cm dish.
  6. Culture the cells at 37 °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.
  7. Rinse the P0 cells with PBS when 100% confluent plus 1 day. Add 1 mL of trypsinization solution and incubate at 37 °C, 5% CO2, 3% O2 to detach the cells. Stop the enzymatic activity by using 10 mL of DMEM complete.
  8. 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%–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.

4. Seeding of cells and collection of conditioned medium

  1. 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 °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.
  2. Collect the cells and centrifuge at 300 x g at RT for 10 min.
  3. Resuspended the cell pellet in 10 mL of exosome-free medium and count the cells.
  4. Seed the cells at 1.5–2.0 x 106 cells per dish (15 cm) and incubate at 37 °C, 5% CO2, 3% O2.
  5. Collect the conditioned medium between 16–24 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 16–24 h period. The two collections can be combined for further processing.

5. Purification of exosomes using differential and ultra-centrifugation

NOTE: All steps are performed at 4 °C or on ice. Balance the tube with a tube filled with water when needed.

  1. 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.
  2. Remove dead cells by centrifugation at 2,000 x g for 10 min and transfer the supernatant to a 38.5 mL polypropylene tube.
  3. 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.
  4. Ultracentrifuge the supernatant at 100,000 x g for 1.5 h to pellet the exosomes.
  5. 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.
  6. 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.
  7. Resuspend the pellet in ice-cold PBS (~25 μL 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 ~3–4 μg.

6. Characterization of exosomes by sucrose density gradient

  1. 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.
  2. Mix 30–100 µg of exosomes with 0.25 M sucrose solution and adjust the volume to 1 mL.
  3. 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 °C.
  4. 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.
  5. Add 110 µL of 100% TCA to each tube, mix well, and incubate for 10 min at RT.
  6. Centrifuge for 10 min at 10,000 x g and RT and carefully discard the supernatant.
  7. Resuspend the pellet in 500 µL of pre-cooled 80% ethanol and leave the samples to wash for 10 min at -20 °C.
  8. Centrifuge for 10 min at 10,000 x g and 4 °C and discard the supernatant.
  9. Dry the pellet for 10–15 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 µL of dH2O, 5 µL of 4x Laemmli buffer, and 0.5 µL of 1 M DTT for western blot analyses.

7. Exosome detection by western blot analysis

  1. Mix 5–10 µg of exosomes from step 5.7 with 5 µL of 4x Laemmli buffer, and 0.5 µL of 1 M DTT, then adjust the final volume to 20 µL with dH2O. Heat all samples, including those in step 6.9, for 5 min at 98 °C.
  2. Load the samples into a 10% TGX stain-free gel and load the protein ladders according to the manufacturer’s recommendations.
    NOTE: Choose the percentage of the gel according to the molecular weight of the protein of interest.
  3. 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.
  4. Image the gel. Images of the stain-free gels are used as the loading control for immunoblots.
  5. Transfer the gel using a PVDF membrane for 1.5 h at 80 V or overnight at 30 V at 4 °C.
  6. Block the membranes in blocking buffer for 1 h at RT.
  7. Incubate the membranes for specific antibodies (Table 2) in antibody buffer overnight at 4 °C with agitation.
  8. Wash the membranes in washing buffer and incubate the membranes with the appropriate secondary antibodies (Table 2) for 1 h at RT with agitation.
  9. 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.

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Representative Results

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.

Figure 1
Figure 1: Representative TEM image of exosomes isolated from culture medium of mouse primary fibroblasts. Shown are the relatively uniform sizes of these nanovesicles. Across black lines denote the diameter of the individual vesicles. Scale bar = 100 nm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Immunoblots of exosome lysates probed with antibodies against exosome, cytosolic, and ER markers. Alix, CD81, CD9, flotillin1, syndecan1, syntenin1 (exosome), cytosolic (LDH), and ER (calnexin) markers. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Exosomes separated by a sucrose density gradient. 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 3–6, which correspond to densities of 1.096–1.140 g/mL. Please click here to view a larger version of this figure.

Sucrose (g) Sucrose working solution (mL)
2.0 M 2.74 4.0
1.3 M 2.67 6.0
1.16 M 2.38 6.0
0.8 M 1.37 5.0
0.5 M 0.86 5.0
0.25 M 0.26 3.0

Table 1: Sucrose density gradient solutions.

Antibody Host Company Catalog number
CD9 Rat BD Biosciences 553758
CD81 Mouse Santa Cruz Biotechnologies sc-166029
Alix Rabbit d'Azzo lab Alix
Flotilin1 Mouse BD Biosciences 610820
Syndecan1 Rabbit Life Technologies 36-2900
Syntenin1 Rabbit Millipore/Sigma AB15272
LDH Goat Chemicon AB1222
Calnexin Goat Santa Cruz Biotechnologies sc-6465
rat-HRP Donkey Jackson Imm. Res Lab 112-035-003
Mouse-HRP Goat Jackson Imm. Res Lab 115-035-044
Rabbit-HRP Goat Jackson Imm. Res Lab 111-035-144

Table 2: Primary and secondary antibodies.

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Discussion

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 °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.

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Disclosures

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

Acknowledgments

Alessandra d’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.

Materials

Name Company Catalog Number Comments
10 cm dishes Midwest Scientific, TPP TP93100
15 cm dishes Midwest Scientific TP93150
BCA protein assay kit Thermo Fisher Scientific, Pierce 23225
Bovine serum albumin Fraction V Roche 10735094001
CaCl2 Sigma C1016-100G
Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11 Eppendorf 022620659/5427754008
Chemidoc MP imaging system BioRad 12003154
Collagenase P Sigma, Roche 11 213 857 001 100 mg
cOmplete protease inhibitor cocktail Millipore/Sigma, Roche 11697498001
Criterion Blotter with plate electrodes BioRad 1704070
Criterion TGX stain-free protein gel BioRad 5678034 10% 18-well, midi-gel
Criterion vertical electrophoresis cell (midi) BioRad NC0165100
Dispase II Sigma, Roche 04 942 078 001 neutral protease, grade II
Dithiothreitol Sigma/Millipore, Roche 10708984001
Dulbecco’s Modification Eagles Medium Corning 15-013-CV
Dulbecco’s Phosphate Buffered Saline Corning 21-031-CV
Ethanol 200 proof Pharmco by Greenfield Global 111000200
Falcon 50 mL conical centrifuge tubes Corning 352070
Fetal Bovine Serum Gibco 10437-028
Fluostar Omega multi-mode microplate reader BMG Labtech
GlutaMAX supplement Thermo Fisher Scientific, Gibco 35050-061
Hydrochloric acid Fisher Scientific A144S-500
Immobilon-P Transfer membranes Millipore IPVH00010
Laemmli sample buffer (4x) BioRad 1610747
Magnesium acetate solution Sigma 63052-100ml
Non-fat dry milk LabScientific M-0842
O2/CO2 incubator Sanyo MC0-18M
Penicillin-Streptomycin Thermo Fisher Scientific, Gibco 15140-122 10,000 U/ml
Premium microcentrifuge tubes Fisher Scientific, Midwest Scientific AVSC1510 1.7 mL
Protected disposable scalpels Fisher Scientific, Aspen Surgical Bard-Parker 372610
Running buffer BioRad 1610732
Sodium Chloride Fisher Scientific, Fisher Chemical S271-3
Stericup Quick release-GP sterile vacuum filtration system Millipore S2GPU05RE 500 mL
Sterile cell strainer (70 mm) Fisher Scientific, Fisher brand 22-363-548
Sucrose Fisher Scientific, Fisher Chemical S5-500
SuperSignal west Femto Thermo Fisher Scientific 34096
Thin wall Polypropylene tubes Beckman Coulter 326823
Transfer buffer BioRad 16110734
Trichloroacetic Acid Sigma 91228-100G
Tris base BioRad 1610719
Triton-X100 solution Sigma 93443-100mL
TrypLE Express Enzyme Thermo Fisher Scientific, Gibco 12604-013 No phenol red
Tween-20 BioRad #1610781
Ultra-centrifuge Optima XPM Beckman Coulter A99842
Ultra-clear tube (14x89 mm) Beckman Coulter 344059
Ultra-clear tubes (25x89 mm) Beckman Coulter 344058
Water bath Isotemp 220 Fisher Scientific FS220

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Cite this Article

van de Vlekkert, D., Qiu, X., Annunziata, I., d'Azzo, A. Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts. J. Vis. Exp. (159), e61127, doi:10.3791/61127 (2020).More

van de Vlekkert, D., Qiu, X., Annunziata, I., d'Azzo, A. Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts. J. Vis. Exp. (159), e61127, doi:10.3791/61127 (2020).

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