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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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 hy...

Dyskusje

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 ...

Materiały

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