JP7625408B2 - Methods for Enriching Extracellular Vesicles and/or Cell-Free DNA - Google Patents

Description

The present invention relates to a method for concentrating extracellular vesicles and/or cell-free DNA.

Traditionally, biopsies have been performed to diagnose diseases by taking a part of a lesion from a living body, but in recent years, liquid biopsies, which use biological fluid samples, have been attracting attention as a less invasive testing method. Analytical targets for liquid biopsies include circulating tumor cells (CTCs), cell-free DNA (cfDNA) such as circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) such as exosomes.

cfDNA can be extracted by conventional nucleic acid extraction methods using spin columns or magnetic beads (Non-Patent Document 1). However, since cfDNA exists only in very small amounts, at the nanogram level, per 1 mL of biological fluid sample, the problem is that a large amount of biological fluid sample is required. In addition, EVs can be extracted by ultracentrifugation or immunoprecipitation using an antibody that recognizes an antigen on the membrane surface (Non-Patent Document 2), but these still have problems with poor recovery efficiency and operability. Many kits for extracting cfDNA and EVs are commercially available, but all of them are relatively expensive. Therefore, it is desirable to establish a method for extracting cfDNA and EVs with high yield and low cost.

Meddeb, R. et al., Clin. Chem. , Vol. 65, No. 5, pp. 623-633 (2019) Yang, D. et al, Theranostics, Vol. 10, No. 8, pp. 3684-3707 (2020)

The present invention aims to solve the problems of the prior art and provide a method for extracting extracellular vesicles and/or cell-free DNA from a biological fluid sample with high yield and low cost using simple operations.

As a result of extensive research, the inventors have found that by adding protamine sulfate or spermidine to a biological fluid sample, extracellular vesicles and cell-free DNA in the biological fluid sample can be effectively concentrated.

That is, according to one embodiment, the present invention provides a method for concentrating extracellular vesicles and/or cell-free DNA contained in a liquid sample, the method comprising the steps of (i) preparing a liquid sample containing extracellular vesicles and/or cell-free DNA, (ii) adding protamine sulfate or spermidine to the sample, thereby obtaining aggregates of the extracellular vesicles and/or cell-free DNA and the protamine sulfate or spermidine, and (iii) recovering the aggregates.

The liquid sample is preferably a biological fluid sample.

The method may further include (iv) digesting the aggregates with a protease after step (iii).

In one embodiment, the present invention provides a kit for concentrating extracellular vesicles and/or cell-free DNA, comprising protamine sulfate or spermidine.

For the extraction of cell-free DNA, it is preferable that the above kit further contains a protease.

According to the method of the present invention, by adding protamine sulfate or spermidine to a biological fluid sample, extracellular vesicles and/or cell-free DNA contained in trace amounts in the biological fluid sample can be concentrated, making it possible to extract extracellular vesicles and/or cell-free DNA with high efficiency and low cost.

FIG. 1 shows an electrophoretic image of lipoproteins contained in the supernatant and pellet solution (exosome extract) obtained from a plasma sample.

The present invention is described in detail below, but is not limited to the embodiments described in this specification.

According to a first embodiment, the present invention provides a method for concentrating extracellular vesicles and/or cell-free DNA contained in a liquid sample, the method comprising the steps of (i) preparing a liquid sample containing extracellular vesicles and/or cell-free DNA, (ii) adding protamine sulfate or spermidine to the sample, thereby obtaining aggregates of the extracellular vesicles and/or cell-free DNA and the protamine sulfate or spermidine, and (iii) recovering the aggregates.

In the method of this embodiment, a liquid sample containing extracellular vesicles and/or cell-free DNA is prepared and used.

"Extracellular vesicles" (hereinafter also referred to as "EVs") refer to lipid bilayer vesicles secreted from cells. EVs are broadly classified into exosomes, microvesicles, and apoptotic bodies based on their size and surface markers, and the EVs in this embodiment may be any of these or a mixture of these. "Cell-free DNA" (hereinafter also referred to as "cfDNA") refers to free DNA fragments released from cells due to cell death or damage.

The extracellular vesicles and cell-free DNA in this embodiment may be secreted from any cell, and may be secreted from either normal or diseased cells. The type of cells from which the EVs and cfDNA are derived is not particularly limited, and may be, for example, dendritic cells, T cells, B cells, nerve cells, stem cells, cancer cells, primary cultured cells or cell lines derived therefrom. Therefore, the cfDNA in this embodiment may be circulating tumor DNA (ctDNA). The organism from which the cells are derived is also not particularly limited, and may be any vertebrate, but is preferably a mammal such as a mouse, rat, rabbit, pig, cow, goat, monkey, or human, and is particularly preferably a human.

The liquid sample in this embodiment is not particularly limited as long as it contains extracellular vesicles and/or cell-free DNA, and may be, for example, a body fluid obtained from a living body or a culture supernatant obtained by culturing cells. Examples of body fluids include, but are not limited to, blood, plasma, serum, saliva, urine, gastric juice, pancreatic juice, bile, sweat, tears, breast milk, nasal secretions, and intestinal fluids.

Next, protamine sulfate or spermidine is added to the liquid sample. This allows for the production of aggregates of extracellular vesicles and/or cell-free DNA with protamine sulfate or spermidine. The final concentration of protamine sulfate added to the liquid sample may be, for example, in the range of 100 μg/ml to 15 mg/ml, and preferably in the range of 600 μg/ml to 6.5 mg/ml. The final concentration of spermidine added to the liquid sample may be, for example, in the range of 0.8 μg/ml to 85 μg/ml, and preferably in the range of 4 μg/ml to 45 μg/ml. After the addition of protamine sulfate or spermidine to the liquid sample, it is preferable to incubate the sample at room temperature for a certain period of time (for example, 10 to 30 minutes).

To stabilize EVs and cfDNA and further improve extraction efficiency, other components may be added before or at the same time as adding protamine sulfate or spermidine, as appropriate. Such other components may include, for example, salts such as sodium chloride, chelating agents such as EDTA, buffers such as PBS, tetraethylene glycol dimethyl ether, etc.

Then, aggregates of extracellular vesicles and/or cell-free DNA with protamine sulfate or spermidine are collected. The aggregates can be collected, for example, by centrifugation at 4°C and 16,000 x g for 10 to 30 minutes.

The method of this embodiment may further include a step of digesting the aggregates with a protease. Digesting the aggregates may improve the extraction efficiency, particularly of cfDNA. The protease in this embodiment is not particularly limited, and may be, for example, a serine protease such as proteinase K, trypsin, or chymotrypsin; a cysteine protease such as cathepsin B, cathepsin L, cathepsin K, or papain; or an aspartic acid protease such as pepsin, chymosin, or cathepsin D, and may be used alone or in combination of two or more selected from these. The protease in this embodiment is preferably proteinase K. The concentration of the protease may be appropriately changed depending on the type of protease, but for example, in the case of proteinase K, it may be in the range of 10 mg/ml to 100 mg/ml, and preferably in the range of 15 mg/ml to 30 mg/ml. Digestion conditions can be changed as appropriate depending on the type of protease, but for example, if proteinase K is used, incubation can be performed at room temperature to 70°C for 10 to 30 minutes.

Extracellular vesicles and/or cell-free DNA aggregates with protamine sulfate or spermidine or their protease digests can be dissolved in a buffer such as PBS and then subjected to a known and established method for extracting EVs/cfDNA. For example, cfDNA can be extracted using a spin column or magnetic beads, and EVs can be extracted by ultracentrifugation or immunoprecipitation using an antibody against a surface marker. Many kits for extracting EVs and/or cfDNA based on these methods are commercially available, and such commercially available products can also be used in this embodiment.

The method of this embodiment can concentrate EVs and/or cfDNA in a sample simply by adding protamine sulfate or spermidine, enabling highly efficient and low-cost extraction of EVs and/or cfDNA.

According to a second embodiment, the present invention provides a kit for concentrating extracellular vesicles and/or cell-free DNA, comprising protamine sulfate or spermidine. In this embodiment, "extracellular vesicles" and "cell-free DNA" are defined as in the first embodiment.

The kit of this embodiment may further include a protease in addition to the above configuration. The "protease" in this embodiment is the same as that defined in the first embodiment. The kit of this embodiment may further include a buffer and other reagents used for extracting EVs and/or cfDNA, and instructions describing the procedure for extracting EVs and/or cfDNA.

The present invention will be further explained with reference to the following examples. Note that the present invention is not limited to these examples.

<1. Extraction of cfDNA>
(1) Preparation of cfDNA-Containing Liquid Samples As cfDNA-containing liquid samples, plasma samples, urine samples, and culture supernatant samples were prepared by the following procedure.
Plasma sample: Blood was collected from five healthy subjects using EDTA2K-containing blood collection tubes (BD Vacutainer blood collection tubes, BD Japan) and centrifuged at 2,000 x g for 15 minutes. The supernatant was collected and further centrifuged at 13,000 rpm (16,060 x g) for 15 minutes to obtain a plasma sample.
Urine samples: Urine samples from five healthy subjects were centrifuged at 2,000×g for 15 minutes. The supernatant was collected and further centrifuged at 13,000 rpm for 15 minutes to obtain a urine sample.
Culture supernatant sample: Human chronic myeloid leukemia cell line K562 (obtained from JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) was cultured in RPMI1640 (Fujifilm Wako Pure Chemical Industries, Ltd., 189-02025) supplemented with 10% FCS (Fujifilm Wako Pure Chemical Industries, Ltd., 556-33865) in a 50 ml flask until it became confluent. The medium was then collected and centrifuged at 2,000 x g for 15 minutes. The supernatant was collected and further centrifuged at 13,000 rpm for 15 minutes to obtain the supernatant as the culture supernatant sample.

(2) Extraction of cfDNA from plasma samples (2-1) Extraction of cfDNA using protamine sulfate 1 mL of plasma sample (n = 10) was mixed with 1/20 volume of 5M NaCl (Fujifilm Wako Pure Chemical Industries, 191-01665) (50 μL), and further mixed with 3/20 volume of 1% protamine sulfate (Fujifilm Wako Pure Chemical Industries, 160-05193) (150 μL). After standing at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 10 minutes and the supernatant was removed. 200 μL of PBS was added to dissolve the pellet, and 40 μL of 20 mg/mL proteinase K (ProK) (Roche Diagnostics, 3115836001) was added or not added, and the mixture was left at room temperature for 30 minutes. Then, cfDNA was extracted using a conventional nucleic acid extraction kit, High Pure PCR Template Preparation Kit (Roche Diagnostics, 1796828). Elution of cfDNA was performed using 50 μL of Elution Buffer.

(2-2) Extraction of cfDNA using spermidine 1 mL of plasma sample (n = 10) was mixed with 1/10 amount of 5M NaCl (100 μL), and further mixed with 1/10 amount of 0.01% spermidine (FUJIFILM Wako Pure Chemical Industries, 191-13831) (100 μL). After standing at room temperature for 10 minutes, 500 μL of tetraethylene glycol dimethyl ether (Tokyo Chemical Industry) was added and mixed, and further stood at room temperature for 10 minutes. Then, it was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was removed. 160 μL of PBS was added to dissolve the pellet, and 80 μL of 20 mg / mL ProK was added or not added, and it was left at room temperature for 30 minutes. Then, cfDNA was extracted using a conventional nucleic acid extraction kit, High Pure PCR Template Preparation Kit. The cfDNA was eluted using 50 μL of Elution Buffer.

As a control, cfDNA was extracted from the same amount (1 mL) of plasma samples (n = 10) using the Cobas cfDNA Sample Preparation Kit (Roche Diagnostics, 07247737190), a dedicated kit for cfDNA extraction.

The extracted cfDNA solution was quantified using the 129 bp Primer Premix included in the KAPA hg DNA Quantification and QC Kit (Roche Diagnostics, KK4966). The concentration of the extracted cfDNA solution (mean ± SD) was determined using the LightCycler (registered trademark) 96 System (Roche Diagnostics) for reaction and analysis.

[Reaction solution composition (10 μL)]
・cfDNA solution 1μL
・2x FastStart Essential DNA Green Master (Roche Diagnostics, 06402712001) 5 μL
・Primer Premix 1μL
・Distilled water remaining

[Reaction conditions]
Step 1 (1 cycle): 95°C 10 minutes Steps 2-3 (40 cycles): 95°C 10 seconds, 62°C 30 seconds

The results are shown in Table 1. It was shown that by agglomerating cfDNA using protamine sulfate, cfDNA can be extracted with the same efficiency as a commercially available cfDNA extraction kit. Furthermore, by digesting the protamine sulfate-cfDNA aggregates with ProK, cfDNA can be extracted with higher efficiency than a commercially available cfDNA extraction kit. Even when spermidine was used, by digesting the spermidine-cfDNA aggregates with ProK, cfDNA can be extracted with higher efficiency than a commercially available cfDNA extraction kit. These results show that the method using protamine sulfate or spermidine is low-cost yet capable of extracting cfDNA efficiently, and that by further performing ProK treatment, cfDNA can be extracted with high efficiency.

Table 1. Extraction of cfDNA from plasma samples

(3) Extraction of cfDNA from urine samples Using 2 mL of urine sample (n=5) instead of 1 mL of plasma sample, the cfDNA extraction efficiency was compared between the method using protamine sulfate and ProK and the method using a commercially available cfDNA extraction kit, following the same procedure as in 1(2-1) above.

The results are shown in Table 2. It was confirmed that the method using protamine sulfate and ProK allows for more efficient extraction of cfDNA than the method using a commercially available kit specifically for cfDNA extraction.

Table 2. Extraction of cfDNA from urine samples

(4) Concentration of cfDNA in samples Instead of 1 mL of plasma sample, 1 mL, 2.5 mL, 5 mL, or 10 mL of urine sample or culture supernatant sample (n=3 each) was used, and cfDNA was extracted using protamine sulfate and ProK in the same manner as in 1(2-1) above to obtain 50 μL of cfDNA eluate.

The results are shown in Tables 3 and 4. In both the urine samples and the culture supernatant samples, the yield of cfDNA increased depending on the starting amount. These results demonstrate that the method using protamine sulfate and ProK can concentrate cfDNA with high efficiency.

Table 3. Enrichment of cfDNA in urine samples

Table 4. Enrichment of cfDNA in culture supernatant samples

2. Extraction of exosomes
(1) Extraction of exosomes from exosome-containing biological fluid samples. The floating human colon cancer cell line COLO201 (obtained from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition) was cultured at 37°C until confluent in RPMI1640 supplemented with 10% FCS. The cells were collected, washed twice with PBS(-), and then cultured in RPMI1640 without FCS for 48 hours. The culture medium was collected and centrifuged at 2,000 x g for 15 minutes, and the collected supernatant was filtered with a sterile syringe filter (Hawach Scientific, SLPES2522S) with a pore size of 0.22 μm. Total Exosome Isolation Reagent (from cell culture media) (Thermo Fisher Scientific, 4478359) was added to the supernatant after filtration and incubated overnight at 4 ° C. Then, the mixture was centrifuged at 13,000 rpm for 60 minutes, the supernatant was removed, and the pellet was suspended in PBS (-). The protein amount of the suspension was measured using Qubit Protein Assay Kit (Thermo Fisher Scientific, Q33211), and an exosome solution was obtained in which the protein concentration was adjusted to 10 μg / mL using PBS (-).

RNase A (10 U) was added to 475 μL of the plasma sample and urine sample (n = 3 each) prepared in 1 (1) above, and the mixture was incubated at 37 ° C for 30 minutes, and then 25 μL of the above exosome solution was added. 1/20 volume of 5 M NaCl was added to the obtained exosome sample solution and mixed, and 3/20 volume of 1% protamine sulfate was added and mixed; or 1/10 volume of 5 M NaCl was added to the obtained exosome sample solution and mixed, and 1/10 volume of 0.01% spermidine was added and mixed. After standing at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was removed. 150 μL of PBS was added to dissolve the pellet, and an exosome extract was obtained. As a control, an exosome extract was prepared from the same amount of the same sample using a commercially available exosome extraction reagent, Total Exosome Isolation Kit (from plasma) (Thermo Fisher Scientific, 4484450) or Total Exosome Isolation Kit (from urine) (Thermo Fisher Scientific, 4484452).

The efficiency of exosome extraction was evaluated by quantifying miRNA purified from the exosome extract. Specifically, miRNA was purified using High Pure miRNA Isolation Kit (Roche Diagnostics, 5080576001) and eluted with 50 μL of Elution Buffer. Then, cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, 0489703001). The stem loop RT primer (5'-GTCAGAGGAGGTGCAGGGTCCGAGGTATTCGCACCTCCTCTGACTCAACA-3': SEQ ID NO: 1) was used as the primer. The composition of the reaction solution was as described in the instructions included with the kit (1/2 scale). The reverse transcription reaction was carried out under the following conditions.

[Reaction conditions]
Step 1 (1 cycle): 16°C 30 minutes; Steps 2-4 (60 cycles): 30°C 30 seconds, 42°C 30 seconds, 50°C 1 second; Step 5 (1 cycle): 85°C, 5 minutes

The resulting reaction solution was diluted 5-fold with TE buffer to prepare a cDNA solution. Next, real-time PCR for miR-21 was performed under the following conditions. Reactions and analysis were performed using a LightCycler (registered trademark) 96 System (Roche Diagnostics), and the Cp value (mean value ± SD) was calculated.

[Reaction solution composition (10 μL)]
・cDNA solution 2.5 μL
2x FastStart Essential DNA Probes Master (Roche Diagnostics, 6402682001) 5 μL
10 μM forward primer (5'-GCCTGCTAGCTTATCAGACTGATG-3': SEQ ID NO: 2) 0.2 μL
10 μM reverse primer (5'-GTGCAGGGTCCGAGGT-3': SEQ ID NO: 3) 0.2 μL
10 μM Universal Probe Library Probe #82 (Roche Diagnostics, 04689054001) 0.2 μL
・Distilled water remaining

[Reaction conditions]
Step 1 (1 cycle): 95°C 10 minutes Steps 2-3 (40 cycles): 95°C 10 seconds, 60°C 30 seconds

The results are shown in Tables 5 and 6. It was shown that whether protamine sulfate or spermidine was used, exosomes could be extracted with efficiency equal to or greater than that of commercially available exosome extraction reagents.

Table 5. Cp values of miR-21 extracted from plasma samples spiked with exosomes

Table 6. Cp values of miR-21 extracted from urine samples spiked with exosomes

(2) Concentration of exosomes in samples Exosomes in the samples were extracted using protamine sulfate or spermidine in the same manner as in 2(1) above, except that 20 μL or 200 μL of the exosome solution prepared in 2(1) above was added to 1 mL or 10 mL of RPMI1640 and used as a sample, and the Cp value (mean ± SD, n = 3) for miR-21 was calculated.

The results are shown in Tables 7 and 8. It was confirmed that the exosome yield increased depending on the starting amount in either the method using protamine sulfate or spermidine. These results demonstrate that either the method using protamine sulfate or spermidine can concentrate exosomes with high efficiency.

Table 7. Exosome enrichment in urine samples

Table 8. Exosome enrichment in culture supernatant samples

(3) Contamination of lipoproteins accompanying exosome extraction Lipoproteins such as LDL and HDL are present in plasma, and miRNAs are also contained in lipoprotein particles. Therefore, in order to perform accurate exosome analysis, it is necessary to eliminate the contamination of lipoproteins during exosome extraction. Therefore, the presence or absence of lipoprotein contamination in the method of the present invention was confirmed.

500 μL of the plasma sample (n=2) prepared in 1(1) above was mixed with 1/20 volume of 5M NaCl (FUJIFILM Wako Pure Chemical Industries, 191-01665), and then mixed with 3/20 volume of 1% protamine sulfate; or 1/10 volume of 5M NaCl was added and mixed, and then 1/10 volume of 0.01% spermidine was added and mixed. After standing at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 30 minutes to collect the supernatant. 500 μL of PBS was added to the pellet to obtain a pellet dissolution solution (exosome extract). As a control, a supernatant and a pellet dissolution solution were obtained from the same sample in the same amount using a commercially available exosome extraction reagent, Total Exosome Isolation Kit (from plasma). The supernatant and pellet solution were applied to Quickgel LIPO (Helena Laboratory, J715) and electrophoresis was performed using Eparizer 2 (Helena Laboratory). The electrophoresis results were obtained as images, and the band intensities were quantified using ImageJ (analysis was performed in triplicate, and the mean value ± SD was calculated).

The results are shown in Figure 1. Lane 1: Supernatant obtained from plasma sample 1 by protamine treatment, lane 2: Pellet solution obtained from plasma sample 1 by protamine treatment, lane 3: Supernatant obtained from plasma sample 2 by protamine treatment, lane 4: Pellet solution obtained from plasma sample 2 by protamine treatment, lane 5: Supernatant obtained from plasma sample 1 by spermidine treatment, lane 6: Pellet solution obtained from plasma sample 1 by spermidine treatment, lane 7: Supernatant obtained from plasma sample 2 by spermidine treatment, lane 8: Pellet solution obtained from plasma sample 2 by spermidine treatment, lane 9: Supernatant obtained from plasma sample 1 by commercial reagent treatment, lane 10: Pellet solution obtained from plasma sample 1 by commercial reagent treatment, lane 11: Supernatant obtained from plasma sample 2 by commercial reagent treatment, lane 12: Pellet solution obtained from plasma sample 2 by commercial reagent treatment, lane 13: Plasma sample 1, and lane 14: Plasma sample 2. The band intensities in Figure 1 are also shown in Table 9. The pellet lysate prepared using protamine sulfate or spermidine contained only trace amounts of lipoproteins, whereas the pellet lysate prepared using a commercially available exosome extraction reagent contained significant amounts of lipoproteins. These results indicate that the method of extracting exosomes using protamine sulfate or spermidine can minimize lipoprotein contamination and prepare highly pure exosomes.

Table 9. Lipoprotein contamination of exosome extracts

Claims (3)

A method for concentrating extracellular vesicles and/or cell-free DNA contained in a liquid sample, comprising:
( i ) adding protamine sulfate or spermidine to a liquid sample containing extracellular vesicles and/or cell-free DNA , thereby obtaining aggregates of the extracellular vesicles and/or cell-free DNA with protamine sulfate or spermidine;
( ii ) recovering the aggregate;
(iii) after step (ii), digesting the aggregates with a protease;
The method includes: The method of claim 1, wherein the liquid sample is a biological fluid sample. A kit for concentrating cell-free DNA comprising protamine sulfate or spermidine sulfate and a protease.