Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.
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.
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 cells2,3. 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 tissue5,6,7. 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 required9,10.
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 supernatant12,13,14. 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 EVs15,16. 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.
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
2. Tissue dissociation
3. Differential ultracentrifugation
NOTE: Perform all the centrifugation steps at 4 °C.
4. Evaluation of enrichment quality
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.
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.
The authors have nothing to disclose.
The authors thank the First Affiliated Hospital of Gannan Medical University for supporting this work. This work was supported by the National Natural Science Foundation of China (grant numbers 82260422).
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 |