An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Summary

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Abstract

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase Ι) with tissue, filtration through a 70 µm cell strainer, differential ultracentrifugation, and filtration through a 0.22 µm membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Introduction

Small extracellular vesicles (sEVs) are approximately 30 nm to 150 nm in diameter and are secreted by various cells¹. They can communicate with tissue cells and regulate the local or distant microenvironment by transporting important biological molecules such as lipids, proteins, DNA, and RNA to various organs, tissues, cells, and intracellular parts. Thus, they can also change the behavior of recipient cells^2,3. The isolation and purification of specific sEVs is an essential prerequisite to studying their biological behavior during the development and course of disease. Differential ultracentrifugation-regarded as the gold standard-is commonly used to separate sEVs from the tissues in which they normally reside⁴. Tissue debris, cell debris, large vesicles, and apoptotic bodies can be removed by this technique, leaving only the sEVs.

Collagenase D and DNase I have been shown not to affect the molecular characteristics of cells or vesicles, with the properties of both enzymes contributing to the release of vesicles in the extracellular matrix ⁵. These enzymes have been used to extract sEVs from human metastatic melanoma tissue, colon cancer tissue, and colonic mucosal tissue^5,6,7. However, the concentration and digestion time of collagenase D and DNase I in these methods differ, leading to inconsistent conclusions. To avoid the coprecipitation of other subtypes of sEVs, researchers have removed larger extracellular vesicles (0.1 µm or 0.2 µm in diameter) by filtration and/or differential centrifugation⁸. Depending on the source tissue, different methods of isolation and purification may be required^9,10.

Using the traditional differential ultracentrifugation method to extract sEVs from liver tissue results in a layer of white matter on the surface of the supernatant, without any way of determining its properties. In a previous study¹¹, this layer of white matter was found to affect the purity of the sEVs. Although the particle number and protein concentration of samples isolated by the traditional method were higher than those of the current method, the coefficient of variation was large, possibly because many pollutants can lead to poor repeatability of the results. That is, using detergent (i.e., detecting the solubility of particles in 1% Triton X-100), we found that the purity of sEVs obtained by this method was greater. Hence, we use this method to isolate and purify sEVs derived from colorectal cancer tissue for proteomic research.

At present, the research on sEVs in liver cancer is focused mainly on serum, plasma, and the cell culture's supernatant^12,13,14. However, sEVs derived from liver cancer tissue can more accurately reflect the physiologic pathology and the surrounding microenvironment of liver cancer and effectively avoid the degradation and pollution of other EVs^15,16. With the use of differential ultracentrifugation, this method can enrich the yield and obtain high-quality sEVs, providing an important basis for the further study of liver cancer. This method enables the liver cancer tissue to be separated by sharp separation and to be dissociated by collagenase D and DNase I. Then, the cellular debris, large vesicles, and apoptotic bodies are further removed by filtration and differential ultracentrifugation. Finally, the sEVs are isolated and purified for later studies.

Protocol

Human liver cancer tissue was collected from patients diagnosed with hepatic malignancy at the First Affiliated Hospital of Gannan Medical University. All patients signed an informed consent form, and the collection of human tissue samples was approved by the ethics committee of the First Affiliated Hospital of Gannan Medical University. See the Table of Materials for details related to all materials, equipment, and software used in this protocol.

1. Preparation

1. Place the transference decoloring shaker in the incubator and set the temperature at 37 °C. See Figure 1 for all the other needed equipment.
2. Clean the scalpel and forceps by spraying them with 75% alcohol.
3. Prepare the digestive solution by measuring 6 mg of collagenase D (4 mg/mL) and 24 µL of DNase Ι (80 U/mL) and adding them to 1.5 mL of RPMI-1640 basic medium. Mix by gentle inversion until the additives are completely dissolved.
4. In advance, moisten 70 µm and 0.22 µm cell strainers with 1x phosphate-buffered saline (PBS).
5. Place a 100 mm sterile cell culture dish on the icebox to hold the liver tissues after defrosting.
6. Within 15 min after hepatocellular carcinoma tissue isolation, rinse off the blood on the surface of the tissue block with PBS, transfer the tissue into a sterile frozen tube, and store it at −80 °C until the experiment for not more than 2 weeks.

2. Tissue dissociation

1. Remove the tissue sample from the −80 °C freezer and place approximately 400 mg of the tissue-cut into 2 mm x 2 mm x 2 mm fragments-into the 10 cm cell culture dish on the icebox.
2. Move the tissue into a 6-well plate with 1.5 mL of the digestive solution; then, place the plate on the transference decoloring shaker (20 rpm/min) to incubate for 20 min at 37 °C for complete dissociation of the liver cancer tissue and release of the sEVs.
3. Remove the 6-well plate from the incubator and place it on the icebox. Add 80 µL of phosphatase inhibitor and 200 µL of complete protease inhibitor solution to stop the digestion.
4. Transfer the digestive solution to the 70 µm cell strainer and filter it slowly to remove any large tissue debris. Collect the filtrate and place it in a 2 mL centrifuge tube.

3. Differential ultracentrifugation

NOTE: Perform all the centrifugation steps at 4 °C.

1. Centrifuge the filtrate at 500 × g for 10 min and collect the supernatant in a new 2 mL centrifuge tube. Most of the small tissue debris can then be removed.
2. Centrifuge the supernatant at 3,000 × g for 20 min to remove cell debris. Carefully transfer the supernatant to a new centrifuge tube until the tip of the pipette is about to touch the white substance on the surface.
3. Centrifuge the remaining liquid at 3,000 × g for 3 min; then, aspirate the supernatant to facilitate the collection of vesicles. To ensure the purity of the sEVs, make sure that the white substance is not aspirated.
4. Centrifuge the collected supernatant at 12,000 × g for 20 min and transfer 900 µL of the supernatant to a 4.7 mL ultracentrifuge tube. Centrifuge the remaining liquid at 12,000 × g for 3 min and then collect and mix both supernatants into the same ultracentrifuge tube. Fill the tube completely with PBS.
5. Centrifuge the supernatants at 100,000 × g for 60 min. Discard the supernatant and resuspend the pellet by filling the ultracentrifuge tube with PBS and centrifuging again at 100,000 × g for 60 min. Resuspend the pellet with 50 µL of PBS.
6. Aspirate the sEV suspension using a 1 mL sterile syringe and filter it through a 0.22 µm membrane filter. Collect the filtrate in a 600 µL centrifuge tube and store it at −80 °C for further analysis.

4. Evaluation of enrichment quality

1. Observe the morphology of the sEVs under a transmission electron microscope.
   1. Then, drop 10 µL of the sEV sample onto a copper net, incubate at room temperature for 10 min, and clean it with sterile distilled water, using absorbent paper to remove excess liquid.
   2. Drop 10 µL of 2% uranyl acetate onto the copper net for negative staining for 1 min. Use filter paper to absorb the liquid on the surface of the copper net and dry it for 2 min under an incandescent lamp.
   3. Observe the copper mesh under a transmission electron microscope and image at 80 kV.
2. Use a nanoparticle flow cytometer to determine the size distribution and purity of the sEVs.
   1. Before the machine is started, prepare tubes containing 200 µL of quality control, 200 µL of silica nanospheres, 2 x 200 µL of ultrapure water, 2 x 200 µL of cleaning solution, and 200 µL of PBS.
      NOTE: Before testing the sEV samples, it is necessary to carry out quality control on the instrument and test the particle size standard (the size distribution must be calculated using standard curves generated with several nanomicrospheres of different diameters [68-155 nm] under the same detection condition). QC beads and silica nanospheres were diluted 100x with ultrapure water in a total volume of 200 µL.
   2. Check the volume of the cleaning solution (>10 mL) and note the difference between the levels of the sheath liquid and the waste liquid (20-30 cm).
   3. Turn off the main power supply of the instrument; after 20 s, turn on the computer and wait for the beeps indicating that the instrument is properly connected to run the software.
   4. Click on Start Up to turn on the camera, laser, and air pump.
   5. Click on Sheath Flow-Start Up, and place ultrapure water on the loading platform. After 4 min (instrument's countdown), click on Sample-Boosting | Sample-Unload.
   6. After 30 s, place the blank tube on the loading platform and click on Sample-Boosting | Sample-Unload. Then, place the tube holding the ultrapure water on the loading platform, and, simultaneously, click on Sample-Boosting | Sheath Flow-Purge.
   7. Click on Manual Operation and place the particle concentration tube on the loading platform. Then, click on Sample-Boosting for 1 min and simultaneously select 250 nm Std FL SiNPs quality control in "SAMP. Inf."
   8. Click on Sample-Sampling and turn the detector on by clicking on SPCM.
      NOTE: At this point, the real-time signal waveform can be seen.
   9. Click on Auto Sampling and enter 1.0 into Sampling SET to fix the pressure at 1 KPa. Click on the toolbar and select Large Signal.
   10. Adjust the horizontal position of the laser and set the laser to 2 µm; then, click on L or R to make sure that the signal is strong and uniform.
   11. Click on Time to Record to collect the data, which will automatically jump to Buffer when finished. Then, save the data in a Naf File and click on Sample-Unload.
   12. Place the cleaning solution tube on the loading platform, click on Sample-Boosting, and after 1 min, click on Sample-Unload. Use ultrapure water to remove the residual cleaning solution from the capillary tip.
   13. Place the particle-size standard tube on the loading platform and click on Sample-Boosting for 1 min.
   14. Select 68-155 nm Std FL SiNPs quality control in "SAMP. Inf" and click on Sample-Sampling. Then, click on the toolbar and select Small Signal.
   15. Click on Time to Record to collect the data, which will automatically jump to Buffer when finished. Then, save the data in a Naf File and click on Sample-Unload.
   16. Place the cleaning solution tube on the loading platform, click on Sample-Boosting, and click on Sample-Unload after 1 min. Use ultrapure water to remove the residual cleaning solution from the capillary tip.
   17. Place the PBS tube on the loading platform; click on Sample-Boosting for 1 min.
   18. Select 68-155 nm Std FL SiNPs quality control in "SAMP. Inf" and click on Sample-Sampling. Then, click on the toolbar and select Small Signal.
   19. Click on Time to Record to collect data, which will automatically jump to Buffer when finished. Save the data in a Naf File and click on Sample-Unload.
   20. Place the cleaning solution tube on the loading platform, click on Sample-Boosting and Sample-Unload after 1 min. Use ultrapure water to remove residual cleaning solution from the capillary tip.
   21. Check the sample information as described earlier in steps 4.2.17-4.2.20; change the PBS (step 4.2.17) to the sEV sample and analyze the data.
3. Further identify the sEVs by western blot.
   1. Determine the protein concentrations of the sEVs and tissues by using the BCA protein quantification kit according to the manufacturer's instructions.
   2. Calculate the loading volume of sEVs (e.g., for 5 µg of protein per well) based on these quantitative results. Add loading buffer in a 1:4 ratio and vortex the mixture.
   3. After denaturation in a 100 °C metal bath (dry thermostat) for 10 min, separate the protein on a 12% polyacrylamide gel at 60 V for 80 min.
   4. Transfer the gel onto a 0.22 µm polyvinylidene difluoride membrane at 220 mA for 2 h using transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol).
   5. Block the membrane with 5% nonfat dry milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature and incubate with primary antibody (rabbit monoclonal anti-human CD9 antibody, rabbit monoclonal anti-human CD63 antibody, rabbit monoclonal anti-human TSG101 antibody, mouse monoclonal anti-human GM130 antibody) overnight at 4 °C. Dilute the primary antibodies in 5% nonfat milk at 1:1,000.
   6. Following three washes with TBST, incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h.
   7. Detect the HRP-linked antibody using ECL blotting substrate, and then collect and analyze the images.

Representative Results

sEVs from human liver cancer tissues have played a crucial role in the diagnosis, treatment, and prognosis of patients with liver cancer. This method used common laboratory instruments to isolate and purify sEVs derived from liver cancer tissues; this may provide methodologic support for the study of sEVs. Figure 2 illustrates the general process of enriching sEVs from liver cancer tissues. sEVs in the intercellular spaces of tissues are fully released through tissue cutting and enzymatic hydrolysis. Further, the digestive solution is filtered through 70 µm filters to remove large tissue fragments. Moreover, differential centrifugation steps were performed (at 500 × g, 3,000 × g, and 12,000 × g) to remove small tissue debris, cell debris, large vesicles, and apoptotic bodies (Figure 3A-C).

To obtain high-purity sEVs, two differential centrifugation steps (at 3,000 × g and 12,000 × g) were repeated for 3 min until the white substance on the surface was removed. The collected supernatant was ultracentrifuged at 100,000 × g for 60 min to remove soluble substances, and the pellets were then washed by being resuspending in PBS (Figure 3D,E). After centrifugation at 100,000 × g for 60 min, the pellet was resuspended in 50 µL of PBS. Finally, the sample was filtered through a 0.22 µm membrane and collected in a 600 µL centrifuge tube (Figure 3F) to remove the interference of gelatinous substances and improve the purity of the sEVs.

The purified sEVs were examined under a transmission electron microscope, which revealed the presence of sEVs with a cup-shaped morphology (Figure 4A,B). The particle size ranged from 40 nm to 200 nm, and the average particle size was 89 nm (Figure 5A,B). We assessed the quality of the vesicles by the ratio of the vesicles in all the particles based on the solubility of the particles in 1% Triton X-100. The purity of sEVs was calculated as (1 - C2/C1) × 100%, where C1 and C2 represent the particle numbers detected 1 h before and after Triton X-100 treatment, respectively¹¹. These data showed that the purity of the enriched sEVs was 68.32% (Figure 5C-E). After extracting the sEVs from liver cancer tissue, the samples were treated with PBS and 1% Triton X-100 to evaluate the purity of the sEVs. After treatment, the sEVs were labeled with FITC CD9 and CD9⁺ particles for detection in the nanoparticle flow cytometer. It was found that the number of CD9⁺ particles in the test group decreased (Supplemental Figure S1). The protein markers of sEVs-such as CD63, CD9, and TSG101-were detected by western blot. GM130 was used as the negative control marker of sEVs¹², which was found in tissue cells but not in the sEVs (Figure 6). The protein markers of sEVs derived from cells and tissues-CD63, ALIX, and TSG101-were detected by western blot. GM130 was used as the negative control marker of sEVs (Supplemental Figure 2).

Figure 1: Protocol requirements. (A) A 100 mm cell culture dish, a scalpel, and tweezers for tissue slicing, (B) a 6-well plate, (C) a 70 µm cell strainer, a 0.22 µm membrane filter, and a beaker for cleaning the cell strainer, (D) a 1 mL sterile syringe, and (E) a 4.7 mL ultracentrifuge tube. Please click here to view a larger version of this figure.

Figure 2: Process of enriching small extracellular vesicles from liver cancer tissues. The frozen tissues were weighed, sliced with a scalpel, and incubated with RPMI-1640 medium, collagenase D, and DNase Ι for 20 min at 37 °C. Small extracellular vesicles were enriched by further filtration and differential ultracentrifugation. Please click here to view a larger version of this figure.

Figure 3: Differential ultracentrifugation. (A) After centrifugation at 500 × g for 10 min, a large amount of tissue debris (yellow pellet) and supernatant (pink liquid) can be observed. (B) After centrifugation at 3,000 × g for 20 min, residual tissue debris and a large amount of cell debris were separated from the supernatant. (C) Centrifugation at 12,000 × g for 20 min eliminated the contamination of large-vesicle apoptotic bodies. (D) The pellets were collected through centrifuging at 100,000 × g for 60 min. (E) The centrifugation was repeated at 100,000 × g for 60 min to wash the small vesicles and remove soluble impurities with phosphate-buffered saline. (F) The filtrate was obtained after filtration of the liver cancer tissue-derived small extracellular vesicles through 0.22 µm filters. Please click here to view a larger version of this figure.

Figure 4: Transmission electron microscopy. The shape of the small extracellular vesicles was observed under a transmission electron microscope. Scale bar = (A) 100 nm, (B) 200 nm. Please click here to view a larger version of this figure.

Figure 5: Flow cytometry for sEVs. Representative SSC burst traces of sEVs (A) before and (B) after 1% Triton X-100 treatment for 1 h on ice. (C) Representative SSC distribution histograms of the sEVs before (1,801 events) and (D) after (91 events) Triton X-100 treatment derived from data collected over 1 h each. (E) Purity measurement for sEVs using 1% Triton X-100. The purity of the sEVs was calculated as (1 - C2/C1) × 100%, where C1 and C2 represent the particle number detected 1 h before and after Triton X-100 treatment, respectively. Abbreviations: sEVs = small extracellular vesicles; SSC = side scatter. Please click here to view a larger version of this figure.

Figure 6: Western blot. Western blot was used for the analysis of GM130, TSG101, CD63, and CD9 in the cell extracts and the sEVs. Abbreviation: sEVs = small extracellular vesicles. Please click here to view a larger version of this figure.

Supplemental Figure S1: Detection of FITC CD9. The percentages of CD9-positive sEVs after (A) PBS and (B) 1% Triton X-100 treatment for 1 h were detected by flow cytometry. Abbreviations: sEVs = small extracellular vesicles; FITC = fluorescein isothiocyanate; PBS = phosphate-buffered saline. Please click here to download this File.

Supplemental Figure S2: Western blot used for the analysis of GM130, TSG101, CD63, and ALIX in cell-derived and tissue-derived sEVs. Abbreviation: sEVs = small extracellular vesicles. Please click here to download this File.

Supplemental Figure 3: Coomassie blue staining. Coomassie blue staining of the total proteins from the cell extracts and cultured explant-derived sEVs and fresh and frozen tissue-derived sEVs. Abbreviation: sEVs = small extracellular vesicles. Please click here to download this File.

Supplemental Table S1: Particle to protein ratio of sEVs. Particle to protein ratio compared purity and yield after and before the centrifugation step and filtration step. Please click here to download this File.

Supplemental Table S2: Relationship between tissue size and enzyme concentration. Comparison of the relationship between different enzyme concentrations and tissue block size using particle to protein ratio and particle number of sEVs. Please click here to download this File.

Discussion

This protocol describes a repeatable method for extracting sEVs from liver cancer tissue. High-quality sEVs are obtained by sharp tissue isolation, treatment with digestive enzymes, differential ultracentrifugation, and 0.22 µm filter membrane filtration and purification. For downstream analysis, it is extremely important to ensure the high purity of the sEVs. In the process of differential centrifugation, a layer of white substances (unknown composition) will appear on the surface of the supernatant. As this layer would contaminate the sEVs, we removed it as much as possible by repeated centrifugation (3,000 × g and 12,000 × g) and filtration through a 0.22 µm membrane (Supplemental Table S1). Due to the interference of this white layer, we risked losing sEVs while aspirating the supernatant. Therefore, it is important to proceed carefully through multiple centrifugations and collect the supernatant as carefully as possible without touching the white substance.

This protocol can purify high-quality liver tissue-derived sEVs, but it has some limitations. In these experiments, the volume of tissue blocks is usually ~300-500 mg. The number of sEVs isolated from extremely small tissue blocks was too low to allow for subsequent studies. Considering the influence of the amount of digestive fluid on the yield of tissue-derived sEVs, tissue blocks that are too large should be divided into several parts for extraction, or the amount of digestive enzymes should be increased to ensure that tissue-derived sEVs can be fully obtained. In addition, the method of isolation of sEVs must be adapted to the complexity of the tissue sample. We isolated sEVs from liver tissue and colorectal cancer tissue using this method. However, we have not performed this protocol on other tissues, and these methods may need to be optimized for the isolation of other tissues, for example, by using different types and concentrations of enzymes.

Throughout these procedures, it is very important to obtain high-quality sEVs, and this depends largely on the separation method, the concentration of digestive enzymes, the incubation time, and the proper storage of tissues. In a previous study¹¹, it was determined that the optimal combination of dosage and incubation time of collagenase D and DNase I used to separate sEVs from liver cancer tissues was 4 mg/mL of collagenase D, 80 U/mL of DNase I, and 20 min of incubation time(Supplemental Table S2). We also compared the quality of sEVs from fresh tissue versus frozen tissue and found that the sEVs isolated from fresh tissue had higher total amounts of protein (Supplemental Figure 3). However, the purity of the two types of sEVs was not significantly different through the membrane-breaking experiment (treatment with 1% Triton X-100), indicating that this method is also applicable to fresh liver cancer tissue. The particle concentration and protein content of sEVs obtained by this method were 3.28 × 10¹⁰ ± 0.84 × 10¹⁰ particles/100 mg of tissue and 11.62 µg ± 1.94 µg protein/100 mg of tissue, respectively.However, it is difficult to obtain sEVs from fresh tissue in batches in routine clinical work; therefore, this method can provide a reliable and efficient method for the large-scale purification of sEVs in frozen liver cancer tissue.

In summary, this protocol describes an optimized enrichment method for sEVs, using differential ultracentrifugation and filtration to obtain high-purity sEVs. These tissue sEVs are suitable for downstream analysis, such as the characterization of biomolecular composition, and other studies aimed at characterizing their physiologic or pathophysiologic roles or potential applications as disease markers.

Materials

Name	Company	Catalog Number	Comments
0.22 µm Membrane Filter Unit	Millex	SLGPR33RB
1 mL Sterile syringe	Hubei Xianming Medical Instrument Company	YL01329
2% Uranyl Acetate	Electron Microscopy Sciences	22400-2
4.7 mL Centrifuge Tube	Beckman Coulter	361621
6-well Cell cuture plate	LABSELECT	11110
50 mL Beaker	Tianjin Kangyiheng Experimental Instrument Sales Company	CF2100800
70 µm Cell strainer	Biosharp	BS-70-XBS
100 mm Cell culture dish	CELL TER	CS016-0128
600 µL Centrifuge tube	Axygen	MCT060C
BCA protein quantification kit	Thermo Fisher	RJ240544
Beckman Coulter Optima-Max-TL	Beckman	A95761
BioRad Mini trans-blot	Bio-Rad	1703930
BioRad Mini-Protean	Bio-Rad	1645050
CD63 Antibody	Abcam	ab134045
CD9 Antibody	Abcam	ab263019
Centrifuge 5430R	Eppendorf	5428HQ527333
Cleaning Solution	NanoFCM	C1801
Collagenase D	Roche	11088866001
Copper net	Henan Zhongjingkeyi Technology Company	DJZCM-15-N1
Dry Thermostat	Hangzhou allsheng instruments company	AS-01030-00
FITC Anti Human CD9 Antibody	Elabscience	E-AB-F1086C
Glycine	Solarbio	G8200
Goat horseradish peroxidase (HRP)-coupled secondary anti-mouse antibody	Proteintech	SA00001-1
Goat horseradish peroxidase (HRP)-coupled secondary anti-rabbit antibody	Proteintech	SA00001-2
Methanol	Shanghai Zhenxing Chemical Company
Nanoparticle flow cytometer	NanoFCM INC	FNAN30E20112368
Phosphatase inhibitors(PhosSTOP)	Roche	4906845001
Phosphate Buffered Saline(PBS)	Servicebio	G4202
Polyvinylidene Difluoride Membrane	Solarbio	ISEQ00010
QC Beads	NanoFCM	QS2502
RPMI-1640 basic medium	Biological Industries	C11875500BT
Scalpel	Guangzhou Kehua Trading Company	NN-0623-1
Silica Nanospheres	NanoFCM	S16M-Exo
Transference Decoloring Shaker TS-8	Kylin-Bell	E0018
Transmission Electron Microscope	Thermo Scientific	Talos L120C
Tris	Solarbio	T8060
TSG101 Antibody	Proteintech	28283-1-AP
Tweezer	Guangzhou Lige Technology Company	LG01-105-4X