Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Summary

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Abstract

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Introduction

Exosomes are the smallest subset of EVs, 50–200 nm in diameter, and have multiple biological functions across a diverse array of signaling processes^1,2,3,4,5. They govern cellular and tissue homeostasis primarily by facilitating intercellular communication through cargo molecules such as lipids, proteins, and nucleic acids^6,7,8,9. One critical step in EV research is the isolation process. Differential ultracentrifugation (UC), with or without density gradient centrifugation (DGC), is considered the gold standard approach, but this method carries major limitations, including inefficient EV recovery rates and low scalability^10,11,12, that restrict its best utilization to larger volume samples, such as cell culture supernatant or high exosome production specimens. The advantages and disadvantages of other methods, such as size exclusion by ultrafiltration or chromatography, immunoaffinity isolation by beads or columns, and microfluidics, are well described, and modern supplemental procedures have been developed to overcome and minimize technical limitations in each approach^{11,12,13,14,15}. Others have shown that an ultrafiltration centrifugation (UFC) with a nanoporous membrane in the filter unit is an alternative technique that provides comparable purity to a UC method^16,17,18. This technique could be considered as one of the alternative isolation methods.

Bronchoalveolar lavage fluid (BALF) contains EVs that possess numerous biological functions in various respiratory conditions^19,20,21,22. Studying BALF-derived EVs entails some challenges due to the invasiveness of the bronchoscopy procedure in humans, as well as a limited amount of lavage fluid recovery. In small laboratory animals such as mice, only a few milliliters can be recovered in normal lung conditions, even less in inflamed or fibrotic lungs²³. Consequently, collecting a sufficient amount of BALF for EV isolation by a differential ultracentrifugation for downstream applications may not be feasible. However, isolating correct EV populations is a crucial factor for studying EV biological functions. The delicate balance between efficiency and efficacy continues to be a challenge in well-established EV isolation methods.

In this current study, we demonstrate that a centrifugal ultrafiltration approach, utilizing a 100 kDa molecular weight cut-off (MWCO) nanomembrane filter unit, is suitable for small-volume biological specimen such as BALF. This technique is simple, efficient, and provides high purity and scalability to support the study of BALF-derived EVs.

Protocol

The utilization of animals and all animal procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at Cedars-Sinai Medical Center (CSMC).

1. Murine Bronchoalveolar Lavage Fluid (BALF) Collection and Preparation

1. BALF collection
   1. Euthanize mice with a cocktail of ketamine (300 mg/kg) and xylazine (30 mg/kg) via the intraperitoneal route followed by cervical dislocation.
   2. Insert a 22 G angiocatheter into the trachea. Attach an insulin syringe containing 1 mL (mL) of ice-cold sterile Dulbecco’s phosphate buffer saline (DPBS) and instill 1 mL of DBPS into both lungs through the angiocatheter.
   3. Slowly withdraw the syringe plunger to retrieve BALF and dispense the BALF into a 50-mL conical tube. Keep the BALF on ice.
   4. Repeat steps 1.1.2 and 1.1.3 3x (4x in total in each mouse).
      Note: Approximately 0.8 mL is generally retrieved per milliliter of instillation. Also, the following steps can be performed for individual mice (i.e., 3 mL of BALF), but pooling multiple BALF samples will allow the isolation of a larger batch of EVs for consistency in downstream experiments.
2. BALF preparation
   1. Pool BALF from 25 mice and divide it into two equal sets (~35 mL per aliquot).
   2. Centrifuge the BALF at 400 x g, at 4 °C for 5 min, to remove cells and other cellular debris and collect the supernatant.
   3. Centrifuge the supernatant at 1,500 x g, at 4 °C for 10 min, to remove cell debris. Collect the supernatant and proceed to the EV isolation steps.

2. Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid

NOTE: In this study, two EV isolation techniques, namely UFC and ultracentrifugation with buoyant used to isolate EVs from BALF. The detailed protocol of each method is described below.

1. Ultrafiltration centrifugation (UFC) enrichment method
   NOTE: This method was modified from a previously described protocol¹⁰.
   1. Filter the supernatant from step 1.2.3 through a 0.2 μm sterile syringe filter and keep the filtered BALF on ice.
      NOTE: This is a size exclusion step whereby only vesicles smaller than 200 nm are collected.
   2. Equilibrate the 100 kDa MWCO centrifugal filter unit with sterile DPBS for 10 min. Centrifuge the centrifugal unit at 1,500 x g for 10 min at 4 °C to discard the DPBS.
      CAUTION: Once the membrane in the filter device is equilibrated with DPBS, the membrane must be kept wet at all time until the device is used.
   3. Fill the filter unit with 15 mL of BALF sample from step 2.1.1 and centrifuge at 3,000 x g for 30 min at 4 °C. The flow-through BALF can be discarded or collected into a separate canonical tube and stored at -80 °C for future use.
   4. Repeat step 2.1.3 for the remaining 0.2 µm-filtered BALF.
      Note: It took three repetitions of centrifugations to sufficiently concentrate the BALF EVs from the original starting volume of 35 mL. This resulted in 1–1.5 mL of retentate.
   5. Wash the retentate with 14 mL of sterile DPBS by a gently pipetting repetitively. Centrifuge the filter unit at 3,000 x g, at 4 °C for 30 min, to remove the DPBS and to concentrate the EV retentate.
   6. Collect the concentrated BALF-derived EVs from the filter device by inserting a pipettor into the bottom of the filter device and withdrawing the sample using a side-to-side sweeping motion to ensure total recovery.
   7. Aliquot the BALF-derived EVs and store them at -80 °C for further particle quantification and characterization (see step 3).
2. Ultracentrifugation (UC) with buoyant density gradient centrifugation (DGC)
   NOTE: The following protocol was modified from the previously described protocol²⁴.
   1. Transfer the supernatant from step 2.1.1 into a 37-mL ultracentrifuge tube and centrifuge the sample at 10,000 x g for 30 min at 4 °C using ultracentrifuge. Collect the supernatant and centrifuge at 100,000 x g, at 4 °C for 60 min. While the EV pellets are centrifuged, prepare different concentrations of buoyant density gradient buffers (Table 1) for step 2.2.3.
   2. Discard the supernatant and resuspend the EV pellets in 200 μL of DPBS.
   3. Mix the EV suspension with 300 μL of 50% iodixanol working solution (Table 1) and transfer it to the 15 mL ultracentrifuge tube. On top of the 50% iodixanol-EV mixture suspension, sequentially layer the following buffer solution in the order from the bottom to the top: 30% iodixanol (4.5 mL), 25% of iodixanol (3 mL), 15% iodixanol (2.5 mL), and 5% iodixanol (6 mL). Centrifuge at 100,000 x g, at 4 °C for 230 min.
      NOTE: The buoyant density gradient is based on the percent of iodixanol scaling with the highest concentration (50%) at the bottom to the lowest concentration (5%) at the top. To generate different concentrations of iodixanol, various amounts of homogenization medium (Table 1) were mixed with working solution (50% iodixanol).
   4. Collect the 15% and 25% fraction and dilute them in sterile DPBS to bring up the volume to 15 mL. Transfer them to a new small ultracentrifuge tube and centrifuge at 100,000 x g, at 4 °C for 60 min.
   5. Discard the supernatant and resuspend the EV pellets in 50 μL of sterile DPBS for further characterization.

3. Extracellular Vesicle Quantification

NOTE: The BALF-derived EVs recovery yield is quantitated with two metrics.

1. Nanoparticle tracking analysis (NTA) measurement
   1. Dilute the EV sample at 1:200–1:500 in 1 mL of DPBS and load the sample into an insulin syringe. Attach a sample syringe to a syringe pump and begin to measure the particle numbers and size (see Table of Materials).
   2. Set the camera level at 14 and the detection threshold at 1 for all sample measurements. Five repetitive measurements with 1,500 frames in 30 s were recorded for each sample, with a delay of 20 s between reads. Combine and average the data for final concentration and size reports.
      NOTE: For accurate capturing of all particles, adjust the camera level as appropriate to visualize all particles and use similar settings for all sample measurements in each experiment.
2. Protein quantification
   NOTE: The bicinchoninic acid (BCA) protein assay was used to measure the protein concentration of the BALF-derived EVs.
   1. Solubilize the EV samples in 1x lysis buffer.
   2. Quantify the amount of protein in the BALF-derived EVs per BCA standard protocol using colorimetric detection by measuring the absorbance at 560 nm in a plate reader.

4. Detection of BALF-derived Extracellular Vesicles

NOTE: Commonly known exosome surface marker proteins (TSG101, CD63, CD81, and CD9) were used to verify EV recoveries by SDS-PAGE and immunoblotting and flow cytometry analysis.

1. SDS-PAGE and immunoblotting
   1. Dissolve an equal amount of EV proteins of each sample with a blotting loading buffer (lithium dodecyl sulfate) and 50 mM dithiothreitol (DTT) in a 1.6 mL tube. Heat the samples at 70 °C for 10 min.
   2. Load the samples from step 4.1.1 into a 4–12% Bis-Tris Plus acrylamide gel and run electrophoresis (150 volts, 35 mA) for 35 min.
   3. Transfer proteins to a nitrocellulose membrane using a dry transfer method.
   4. Block the membrane with 5% skimmed milk for 60 min, rocking at room temperature (RT).
   5. Incubate the membrane with an antibody to an EV surface protein marker, Tumor Susceptibility Gene 101 (TSG101), at 1:500 dilution in 5% BSA in Tris-buffered saline Tween-20 (TBST) at 4 °C, rocking overnight.
   6. The next day, wash the membrane 3x, 10 min each wash, in TBST buffer, and incubate it with anti-rabbit IgG, an HRP-linked antibody, at 1:5,000 dilution for 60 min at RT.
   7. Wash the membrane 3x, 10 min each wash, in TBST buffer. Develop the membrane with chemiluminescent HRP antibody detection reagent and image (see Table of Materials for imaging system).
2. Flow cytometry
   1. Dilute BALF-derived EVs in 49 μL of PEB staining buffer (PBS + 5 mM EDTA + 0.5% BSA, filtered through a 0.1 μm syringe filter membrane).
   2. Add each of the following antibodies into each individual sample: 1) rat anti-mouse PE CD63 antibody (100 ng per reaction); 2) rat anti-mouse PE CD81 (500 ng); 3) rat anti-mouse FITC CD9 (200 ng). Incubate at 4 °C, rocking for 60 min in the dark.
   3. Dilute the samples with 450 μL of membrane-filtered PEB staining buffer and subject the samples to flow cytometry analysis (see Table of Materials)²⁵.
   4. Adjust the flow cytometer settings as follows: 1) set all channels on hyper log (hlog); 2) set the trigger on SSC at 4; 3) turn off the secondary trigger. Run the samples in a low-speed setting and acquire at least 10,000 events in each sample.
   5. Perform low cytometry data analysis in each sample using analysis software (see Table of Materials).

Results

We performed EV isolation from mouse BALF using UFC and UC-DGC isolation methods on the same day. The UFC method required approximately 2.5–3 h, whereas the UC-DGC technique required 8 h of processing time. This did not include buffers and reagent preparation time. It should be noted that some other tasks could be performed during the long centrifugation periods. Nevertheless, the entire procedure lasted nearly an entire day for the UC-DGC isolation technique.

Discussion

In the past few decades, scientists have unraveled the significances of EVs in cellular homeostasis. More importantly, EVs play major roles in many disease processes by modulating neighboring and distant cells through their bioactive cargo molecules^{1,21,22,26,27,28,29,}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
Amicon Ultra-15 centrifugal filters Ultracel-100K	Sigma-Millipore, St. Louis, MO	UFC910024
Dulbecco's Phosphate Buffered Saline (DPBS)	Corning Cellgro, Manassas, VA	21-031-CV
Sucrose	Sigma-Millipore, St. Louis, MO	EMD8550
HEPES	Research Products International, Prospect, IL	75277-39-3
EDTA	Corning Cellgro, Manassas, VA	46-034-CI
Sodium Chloride	Sigma-Millipore, St. Louis, MO	S3014-1KG
OptiPrep	Sigma-Millipore, St. Louis, MO	MKCD9753	Density Gradient Medium
Ketamine	VetOne, Boise, ID	13985-702-10
Xylazine	Akorn Animal Health, Lake Forest, IL	59399-110-20
Syringe 1 mL	BD Syringe, Franklin Lakes, NJ	309656
Angiocatheter 20 G	BD Syringe, Franklin Lakes, NJ	381703
Centrifuge tubes 15 mL	VWR, Radnor, PA	89039-666
Centrifuge tubes 50 mL	Corning Cellgro, Manassas, VA	430828
Bicinchonic acid (BCA) protein assay	Pierce, Thermo Fischer Scientific, Rockford, IL	23235
Rabbit anti-mouse TSG101 Antibody	AbCam, Cambridge, MA	AB125011
Rat anti-mouse PE-CD63 Antibody	Biolegend, San Diego, CA	143904
CD81
CD9
Anti-rabbit IgG, HRP-linked antibody	Cell Signaling Technology, Danvers, MA	7074S
4x LDS
10x Reducing agent (Bolt)
10x Lysis buffer (Bolt)	Cell Signaling Technology, Danvers, MA
Bolt 4-12% Bis-Tris Plus acrylamide gel	Invitrogen, Thermo Fisher Scientific, Waltham, MA	NW04120
iBlot 2 Nitrocellulose mini stacks	Invitrogen, Thermo Fisher Scientific, Waltham, MA	IB23002
Chemiluminescent HRP antibody detection reagent HyGLO	Denville Scientific, Holliston, MA	E2400
Ultracentrifuge tubes 17 mL	Beckman Coulter, Pasadena, CA	337986
Ultracentrifuge tubes 38.5 mL	Beckman Coulter, Pasadena, CA	326823
Corning SFCA Syringe Filters 0.2 µm pore	Thermo Fisher Scientific, Waltham, MA	09-754-13
Equipment
Centrifuge	Eppendorf, Hamburg, Germany	-
Ultracentrifuge	Beckman Coulter, Pasadena, CA	-
Nanosight (NS300)	Malvern, Worcestershire, UK	-	To measure particle size distribution and particle concentration
MACSQuant Analyzer 10 flow cytometer	Miltenyi Biotec, Bergisch Gladbach, Germany	-
iBlot Transfer Apparatus	Thermo Fischer Scientific, Waltham, MA	-
Bio-Rad ChemiDoc MP Imaging System	Bio-Rad, Hercules, CA
FlowJo v. 10			Analysis software