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

Abstract

To provide a method for enriching extracellular vesicles and / or cell-free DNA with high efficiency and low cost.SOLUTION: Provided is a method for enriching extracellular vesicles and / or cell-free DNA contained in a liquid sample. The method comprises the steps of: (i) preparing the liquid sample containing the extracellular vesicles and / or cell-free DNA; (ii) adding protamine sulfate or spermidine to the sample to obtain an aggregate of the extracellular vesicles and / or cell-free DNA with protamine sulfate or spermidine; and (iii) collecting the agglomerate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for concentrating extracellular vesicles and / or cell-free DNA. [Background technology]

[0002] Traditionally, biopsies have been performed to diagnose diseases by taking a part of the lesion from the living body, but in recent years, liquid biopsies using biological fluid samples have attracted 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.

[0003] 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 there are still 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. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Meddeb,R.et al, Clin.Chem., Vol.65, No.5, pp.623-633(2019) [Non-Patent Document 2] Yang,D.et al, Theranostics, Vol.10, No.8, pp.3684-3707(2020) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made with the aim of solving the problems of the prior art and providing a method for extracting extracellular vesicles and / or cell-free DNA from a biological fluid sample with high yield and low cost using simple procedures. [Means for solving the problem]

[0006] As a result of intensive research, the inventors have found that extracellular vesicles and cell-free DNA in a biological fluid sample can be effectively concentrated by adding protamine sulfate or spermidine to the biological fluid sample.

[0007] 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.

[0008] The liquid sample is preferably a biological fluid sample.

[0009] The method may further comprise the step of: (iv) digesting the aggregates with a protease after step (iii).

[0010] According to one embodiment, the present invention also provides a kit for concentrating extracellular vesicles and / or cell-free DNA, comprising protamine sulfate or spermidine.

[0011] For the extraction of cell-free DNA, the kit preferably further comprises a protease. Effect of the Invention

[0012] 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, enabling extraction of extracellular vesicles and / or cell-free DNA with high efficiency and low cost. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows an electrophoretic image of lipoproteins contained in the supernatant and pellet lysate (exosome extract) obtained from a plasma sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in the present specification.

[0015] 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.

[0016] In the method of this embodiment, a liquid sample containing extracellular vesicles and / or cell-free DNA is prepared and used.

[0017] "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.

[0018] 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 derived therefrom, or established cell lines. 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.

[0019] 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, a culture supernatant obtained by culturing cells, etc. 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.

[0020] 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 adding protamine sulfate or spermidine to the liquid sample, it is preferable to incubate the liquid sample at room temperature for a certain period of time (for example, 10 to 30 minutes).

[0021] 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 include, for example, salts such as sodium chloride, chelating agents such as EDTA, buffers such as PBS, tetraethylene glycol dimethyl ether, etc.

[0022] Then, the 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 × g for 10 to 30 minutes.

[0023] The method of the present embodiment may further include a step of digesting the aggregates with a protease thereafter. Digesting the aggregates may improve the extraction efficiency, particularly of cfDNA. The protease in the present 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 the present 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. Furthermore, the digestion conditions can be appropriately changed depending on the type of protease used. For example, in the case of proteinase K, incubation may be performed at room temperature to 70° C. for 10 to 30 minutes.

[0024] 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.

[0025] The method of this embodiment can concentrate EVs and / or cfDNA in a sample simply by adding protamine sulfate or spermidine, enabling the extraction of EVs and / or cfDNA with high efficiency and low cost.

[0026] 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 the same as those defined in the first embodiment.

[0027] In addition to the above configuration, the kit of this embodiment may further include a protease. The "protease" in the embodiment is the same as that defined in the first embodiment. In addition, the kit of this embodiment may further include a buffer and other reagents used for extracting EVs and / or cfDNA, and an instruction manual describing the procedure for extracting EVs and / or cfDNA. EXAMPLES

[0028] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0029] <1. Extraction of cfDNA> (1) Preparation of liquid samples containing cfDNA As liquid samples containing cfDNA, plasma samples, urine samples, and culture supernatant samples were prepared by the following procedure. Plasma samples: 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 centrifuged at 13,000 rpm (16,060 x g) for 15 minutes to obtain the supernatant used as the plasma sample. Urine samples: Urine samples from five healthy subjects were centrifuged at 2,000×g for 15 minutes. The supernatant was collected and centrifuged at 13,000 rpm for 15 minutes. The supernatant was used as a urine sample. Culture supernatant sample: Human chronic myeloid leukemia cell line K562 (obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) was cultured in RPMI1640 (Fujifilm Wako Pure Chemical, 189-02025) supplemented with 10% FCS (Fujifilm Wako Pure Chemical, 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 centrifuged at 13,000 rpm for 15 minutes to obtain the culture supernatant sample.

[0030] (2) Extraction of cfDNA from plasma samples (2-1) Extraction of cfDNA using protamine sulfate To 1 mL of plasma sample (n=10), 1 / 20 volume of 5 M NaCl (Fujifilm Wako Pure Chemical Industries, Ltd., 191-01665) (50 μL) was added and mixed, and 3 / 20 volume of 1% protamine sulfate (Fujifilm Wako Pure Chemical Industries, Ltd., 160-05193) (150 μL) was added and mixed. After leaving it at room temperature for 10 minutes, it was centrifuged at 13,000 rpm for 10 minutes and the supernatant was removed. The pellet was dissolved by adding 200 μL of PBS, and left at room temperature for 30 minutes with or without adding 40 μL of 20 mg / mL proteinase K (ProK) (Roche Diagnostics, Ltd., 3115836001). Then, cfDNA was extracted using a regular nucleic acid extraction kit, High Pure PCR Template Preparation Kit (Roche Diagnostics, Ltd., 1796828). cfDNA was eluted using 50 μL of Elution Buffer.

[0031] (2-2) Extraction of cfDNA using spermidine 1 mL of plasma sample (n=10) was mixed with 1 / 10 volume of 5M NaCl (100 μL), and then mixed with 1 / 10 volume 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 the mixture was further left at room temperature for 10 minutes. Then, the mixture 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 the mixture was left at room temperature for 30 minutes. Then, cfDNA was extracted using a normal nucleic acid extraction kit, the High Pure PCR Template Preparation Kit. cfDNA was eluted with 50 μL of Elution Buffer.

[0032] As a control, cfDNA was extracted from the same volume (1 mL) of plasma samples (n = 10) using the cobas cfDNA Sample Preparation Kit (Roche Diagnostics, 07247737190), a dedicated kit for cfDNA extraction.

[0033] 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® 96 System (Roche Diagnostics) for reaction and analysis.

[0034] [Reaction solution composition (10 μL)] ·cfDNA solution 1μL 2x FastStart Essential DNA Green Master (Roche Diagnostics, 06402712001) 5μL Primer Premix 1μL Distilled water remaining

[0035] [Reaction conditions] Step 1 (1 cycle): 95℃ 10 minutes Steps 2-3 (40 cycles): 95℃ 10 seconds, 62℃ 30 seconds

[0036] 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 can extract cfDNA efficiently at low cost, and that cfDNA can be extracted with high efficiency by further performing ProK treatment.

[0037] Table 1. Extraction of cfDNA from plasma samples [Table 1]

[0038] (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.

[0039] The results are shown in Table 2. It was confirmed that the method using protamine sulfate and ProK enabled cfDNA extraction with higher efficiency than the method using a commercially available cfDNA extraction kit.

[0040] Table 2. Extraction of cfDNA from urine samples [Table 2]

[0041] (4) Concentration of cfDNA in the sample Instead of the 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 using the same procedure as in 1(2-1) above, to obtain 50 μL of cfDNA eluate.

[0042] The results are shown in Tables 3 and 4. For both urine and culture supernatant samples, the yield of cfDNA increased in a starting amount-dependent manner. This result demonstrated that the method using protamine sulfate and ProK can enrich cfDNA with high efficiency.

[0043] Table 3. Enrichment of cfDNA in urine samples [Table 3]

[0044] Table 4. Enrichment of cfDNA in culture supernatant samples [Table 4]

[0045] <2. Extraction of exosomes> (1) Extraction of exosomes from exosome-containing biofluid samples The COLO201 suspension human colon cancer cell line (obtained from the JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) was cultured in RPMI1640 supplemented with 10% FCS at 37°C until confluent. The cells were harvested, washed twice with PBS(-), and then cultured in RPMI1640 without FCS for 48 hours. The culture medium was harvested and centrifuged at 2,000×g for 15 minutes, and the harvested supernatant was filtered through a sterile syringe filter with a pore size of 0.22 μm (Hawach Scientific, SLPES2522S). Total Exosome Isolation Reagent (from cell culture media) (Thermo Fisher Scientific, 4478359) was added to the filtered supernatant and incubated overnight at 4°C. The cells were then 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 a Qubit Protein Assay Kit (Thermo Fisher Scientific, Q33211), and an exosome solution with a protein concentration of 10 μg / mL was obtained using PBS(-).

[0046] RNaseA (10U) was added to 475μL of the plasma sample and urine sample (n=3 each) prepared in 1(1) above, and the mixture was incubated at 37℃ for 30 minutes, after which 25μL of the above exosome solution was added. 1 / 20 volume of 5M NaCl was added to the obtained exosome sample solution, and mixed, and then 3 / 20 volume of 1% protamine sulfate was added and mixed; alternatively, 1 / 10 volume of 5M NaCl was added to the obtained exosome sample solution, 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, and the supernatant was removed. 150μL 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 commercially available exosome extraction reagents, Total Exosome Isolation Kit (from plasma) (Thermo Fisher Scientific, 4484450) or Total Exosome Isolation Kit (from urine) (Thermo Fisher Scientific, 4484452).

[0047] The efficiency of exosome extraction was evaluated by quantifying miRNA purified from the exosome extract. Specifically, miRNA was purified using the High Pure miRNA Isolation Kit (Roche Diagnostics, 5080576001) and eluted with 50 μL of Elution Buffer. Then, cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, 0489703001). The stem-loop RT primer (5'-GTCAGAGGAGGTGCAGGGTCCGAGGTATTCGCACCTCCTCTGACTCAACA-3': sequence number 1) was used as the primer. The composition of the reaction solution was as described in the manual attached to the kit (1 / 2 scale). The reverse transcription reaction was performed under the following conditions.

[0048] [Reaction conditions] Step 1 (1 cycle): 16℃ 30 minutes Steps 2-4 (60 cycles): 30℃ 30 seconds, 42℃ 30 seconds, 50℃ 1 second Step 5 (1 cycle): 85℃, 5 minutes

[0049] The resulting reaction solution was diluted 5-fold with TE buffer to prepare a cDNA solution. Real-time PCR for miR-21 was then performed under the following conditions. Reactions and analysis were performed using the LightCycler® 96 System (Roche Diagnostics), and the Cp value (mean ± SD) was calculated.

[0050] [Reaction solution composition (10 μL)] ·cDNA solution 2.5μL 2x FastStart Essential DNA Probes Master (Roche Diagnostics, 6402682001) 5μL 10μM forward primer (5'-GCCTGCTAGCTTATCAGACTGATG-3': sequence number 2) 0.2μL 10μM Reverse primer (5'-GTGCAGGGTCCGAGGT-3': sequence number 3) 0.2μL 10μM Universal Probe Library Probe #82 (Roche Diagnostics, 04689054001) 0.2μL Distilled water remaining

[0051] [Reaction conditions] Step 1 (1 cycle): 95℃ 10 minutes Steps 2-3 (40 cycles): 95℃ 10 seconds, 60℃ 30 seconds

[0052] The results are shown in Tables 5 and 6. It was shown that when either protamine sulfate or spermidine was used, exosomes could be extracted with efficiency equal to or higher than that of commercially available exosome extraction reagents.

[0053] Table 5. Cp values ​​of miR-21 extracted from plasma samples spiked with exosomes [Table 5]

[0054] Table 6. Cp values ​​of miR-21 extracted from urine samples spiked with exosomes [Table 6]

[0055] (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 the Cp value (mean ± SD, n = 3) for miR-21 was calculated.

[0056] 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. This result demonstrated that either the method using protamine sulfate or spermidine can concentrate exosomes with high efficiency.

[0057] Table 7. Exosome enrichment in urine samples [Table 7]

[0058] Table 8. Exosome enrichment in culture supernatant samples [Table 8]

[0059] (3) Contamination of lipoproteins during 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.

[0060] 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 mixed, and then 3 / 20 volume of 1% protamine sulfate was added and mixed; 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 leaving it at room temperature for 10 minutes, it was centrifuged at 13,000 rpm for 30 minutes and the supernatant was collected. 500 μL of PBS was added to the pellet to obtain a pellet solution (exosome extract). As a control, a supernatant and a pellet solution were obtained from the same amount of the same sample using a commercially available exosome extraction reagent, Total Exosome Isolation Kit (from plasma). The supernatant and pellet solution were subjected to Quick Gel LIPO (Helena Laboratory, J715) and electrophoresis was performed using Eparizer 2 (Helena Laboratory). The electrophoretic results were captured as images, and the band intensities were quantified using ImageJ (the analysis was performed in triplicate, and the mean value ± SD was calculated).

[0061] 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.

[0062] Table 9. Lipoprotein contamination of exosome extracts [Table 9]

Claims

1. 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 agglomerate; (iii) after step (ii), digesting the aggregates with a protease; The method includes:

2. The method of claim 1 , wherein the liquid sample is a biological fluid sample.

3. A kit for concentrating cell-free DNA comprising protamine sulfate or spermidine sulfate and a protease.

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