Method for exosome separation and extraction by stacked centrifugal filtration

Abstract

A method for exosome separation and extraction by stacked centrifugal filtration. It is used in molecular biology and clinical examination and comprises an exosome separation and extraction kit consisting of the stacked centrifugal filtration device, an incubation buffer and a protease K. The sample to be tested is incubated at room temperature using the incubation buffer and an appropriate amount of protease K, followed by centrifugation in a centrifuge matching the stacked centrifugal filtration device. After mixing thoroughly, the retained liquid in the ultrafiltration tube is collected to obtain the exosomes. The method needs no large experimental equipments except for a centrifuge, which has a low cost and which is convenient and fast, with short operation time and the possibility of carrying out parallel operations with a large number of samples. The high purity exosomes obtained by the method can meet the demand of large-scale clinical applications.

Claims

1. A method for exosome separation and extraction, comprising: 1) adding an incubation buffer into a sample to be tested, the volume of the incubation buffer being 0.1-5 times that of the sample, and adding an amount of protease K to make the final concentration of protease K be 0.2 mg/mL-2.0 mg/mL at the same time, mixing and incubating at room temperature; 2) transferring all liquid in step 1) into a filter tube of a stacked centrifugal filtration device for exosome separation and extraction, positioning the stacked centrifugal filtration device in a centrifuge for centrifugation, and collecting the retentate in an ultrafiltration tube to obtain the exosomes; wherein the stacked centrifugal filtration device for exosome separation and extraction comprises: a filter tube comprising a filter membrane at the bottom of the filter tube, a primary support edge provided with a first air passage at a top tube opening of the filter tube, and a primary filter tube cavity within the filter tube, an ultrafiltration tube arranged outside of the filter tube comprising: an ultrafiltration membrane at the bottom of the ultrafiltration tube, a secondary support edge provided with a second air passage at a top tube opening of the ultrafiltration tube, and a secondary filter tube cavity within the ultrafiltration tube and below the filter tube, a collecting tube arranged outside of the ultrafiltration tube comprising: a collecting tube cavity within the collecting tube and below the ultrafiltration tube, and a cap provided at the top of the collecting tube, when the stacked centrifugal filtration device is capped, the primary support edge is pressed on the secondary support edge, the secondary support edge is pressed on a top tube opening of the collecting tube, and the primary filter tube cavity and the secondary filter tube cavity are connected to the atmosphere through the first and second air passages; and wherein: both left and right sides of the ultrafiltration tube wall are planar and inclining towards a middle part; a secondary filter tube cavity with a V-shape cross section is formed by the two planar walls; and at least one window for the ultrafiltration membrane is arranged on one or both sides of the two planar walls.

2. The method according to claim 1, wherein the incubation buffer comprises: DPBS, 60-240 mM Tris, and 60-180 mM EDTA, and a pH of the incubation buffer is 7-7.5.

3. The method according to claim 1, wherein, for the centrifugation in step 2), a centrifugal force does not exceed 5,000×g, and the centrifugation lasts for 10-30 min.

4. The method according to claim 1, wherein a pore size of the filter membrane is not greater than 1 μm.

5. The method according to claim 1, wherein a molecular weight range of the ultrafiltration membrane is from 30 to 100 kDa.

6. The method according to claim 1, wherein: an ultrafiltration membrane assembly is nested in the ultrafiltration membrane window; the ultrafiltration membrane assembly includes an ultrafiltration membrane fixing plate with the ultrafiltration membrane attached on an inner side of the ultrafiltration membrane fixing plate; and several outflowing holes are arranged at a lower end of the ultrafiltration membrane fixing plate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the stacked centrifugal filtration device of the invention;

(2) FIG. 2 shows the purity testing results of the exosomes.

(3) FIG. 3 shows the purity testing results of the exosomes.

(4) FIG. 4 shows an electron microscope photo of the exosomes.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(5) The reagent formulations used in the invention comprise:

(6) DPBS: KCl 200 mg/L; KH.sub.2PO.sub.4 200 mg/L; NaCl 8 g/L; Na.sub.2HPO.sub.4.Math.7H.sub.2O 2.16 g/L;

(7) Incubation buffer: DPBS+60-240 mM TrisHCl+60-180 mM EDTA, pH=7.2;

(8) Protease K stock solution: 5-40 mg/mL protease K aqueous solution.

(9) Reagents used in the examples are prepared according to the formulations.

(10) The method of the invention comprises the following steps:

(11) 1) adding the incubation buffer into the sample to be tested with 0.1-5 times in volume, and adding protease K stock solution with 0.005-0.4 times in volume of the sample, mixing thoroughly and incubating at room temperature for 8-15 min;

(12) 2) transferring the mixture in step 1) into the filter tube of the stacked centrifugal filtration device, followed by centrifugation; and the retentate containing purified exosomes is thoroughly mixed and collected in the ultrafiltration tube.

(13) Preferably, if an increased purity of the exosomes is required, DBPS may be added into the ultrafiltration tube for centrifugation at 3,000×g for 10 min to wash and remove certain soluble proteins; one more wash is allowed.

(14) Preferably, an exosome solution, which can be used for subsequent treatment, is obtained after well mixing and collection of the retentate.

(15) The invention is further illustrated in combination with detailed embodiments below. However, the invention is not limited to these embodiments.

Example 1. A Stacked Centrifugal Filtration Device for Exosome Separation and Extraction

(16) An embodiment of the invention provides a stacked centrifugal filtration device for exosome separation and extraction (as shown in FIG. 1), comprising:

(17) a filter tube 1 comprising a filter membrane 11 at the bottom, a first support edge 13 provided with an air passage at the top tube opening, and a first filter tube cavity 12 within the filter tube,

(18) an ultrafiltration tube 2 arranged outside of the filter tube 1, comprising an ultrafiltration membrane 21 at the bottom, a second support edge 23 provided with an air passage at the top tube opening, and a second filter tube cavity 22 within the ultrafiltration tube 2 and below the filter tube 1, and

(19) a waste liquid collecting tube 3 arranged outside of the ultrafiltration tube 2, which matches the centrifuge device, comprising a waste liquid collecting tube cavity 31 within the waste liquid collecting tube 3 and below the ultrafiltration tube 2, and a cap provided at the top tube opening of the waste liquid collecting tube 3 through threaded connection or snap fitting connection,

(20) when it is capped, the first support edge 13 may be pressed on the second support edge 23, and the second support edge 23 may be pressed on the top tube opening of the waste liquid collecting tube 3, and meanwhile, the first filter tube cavity 12 and the second filter tube cavity 22 are in communication with the external environment through the air passages.

(21) Both the left and right sides of the ultrafiltration tube wall 2 below the filter tube 1 are planar and inclining towards the middle part. A second filter tube cavity 22 with a V-shape cross section is formed between the two planar tube walls. Scale is provided on the outside wall of the second filter tube cavity 22. An ultrafiltration membrane window is arranged on at least one of the planar tube walls. An ultrafiltration membrane assembly is nested in the ultrafiltration membrane window. The ultrafiltration membrane assembly includes an ultrafiltration membrane fixing plate and an ultrafiltration membrane 21 attached on the inner side of the ultrafiltration membrane fixing plate. Several outflowing holes are arranged at the lower end of the ultrafiltration membrane fixing plate.

(22) Preferably, the pore size of the filter membrane is not greater than 1 μm.

(23) Preferably, the pore size of the filter membrane is not greater than 0.8 μm.

(24) Preferably, the pore size of the filter membrane is 0.22 μm.

(25) The common pore size of the filter membrane is 0.1-100 μm. Common interferents in serum are high abundance proteins and microvesicles (MVs). After the pretreatment under buffer solution and protease K of the invention, a large proportion of high abundance proteins is digested into fragments by protease K. The diameter of a MV is about 200-1,000 nm. The interference from most MVs can be eliminated with a 0.22 μm filter membrane.

(26) Preferably, the ultrafiltration membrane is an ultrafiltration membrane with a molecular weight range of 30-100 kDa for particles.

(27) The MVs are retained in the filter tube by the filter membrane. The main contaminations entering the second filter tube cavity are polypeptide fragments resulted from enzyme digestion of high abundance proteins by protease K. In the invention, most of the polypeptide fragments after treatment are linear molecules with a molecular weight of 5-10 kDa. Therefore, an ultrafiltration membrane with a range molecular weight of greater than 30 kDa is chosen to filter out the polypeptide fragments. At the same time, since the pore size of the ultrafiltration membrane within this range is much smaller than the diameter of the exosomes, the exosomes are retained in the second filter tube cavity.

(28) Using the stacked centrifugal filtration device for exosome separation and extraction of the invention, when the serum sample to be tested is added into the filter tube, the serum sample flows through the filter membrane 11 at the bottom of the filter tube 1 under centrifugal force. A common filter membrane 11 is a micropore filter membrane with a pore size not greater than 1 μm, preferably 0.22 μm. Most MVs are retained in the filter tube 1. The exosomes, soluble proteins, enzymatic hydrolyzed fragments of high abundance proteins enter the ultrafiltration tube 2 through this filter membrane. Due to the ultrafiltration membrane 21 at the lower part of the tube wall of the ultrafiltration tube 2 and the second filter tube cavity 22 at the bottom, during centrifugation, soluble fractions, enzymatic hydrolyzed fragments of high abundance proteins and smaller particles permeate through the ultrafiltration membrane 21 on the side wall and enter the waste liquid collecting tube 3. Due to the second filter tube cavity 22 at the bottom of the ultrafiltration tube 2, part of the liquid is retained during the centrifugation process, and the exosomes are trapped in this liquid. After centrifugation, the ultrafiltration tube 2 is taken out to mix the liquid well in the retaining cavity with a pipette. The retained liquid is collected as the final product, exosome solution.

Example 2. A Kit for Exosome Separation and Extraction

(29) The kit includes the following components:

(30) 1) a stacked centrifugal filtration device as described in example 1.

(31) 2) an incubation buffer, comprising the following components: DPBS, 60-240 mM TrisHCl buffer, 60-180 mM EDTA, and its pH is 7-7.5.

(32) 3) a protease K stock solution with a concentration of 5-40 mg/mL.

(33) 4) DPBS: KCl 200 mg/L; KH.sub.2PO.sub.4 200 mg/L; NaCl 8 g/L; Na.sub.2HPO.sub.4.Math.7H.sub.2O 2.16 g/L.

Example 3. A Method for Exosome Separation and Extraction

(34) Exosomes are separated and extracted using the kit of Example 2.

(35) The following steps are performed:

(36) 1) adding the incubation buffer with a volume 0.5 times of the sample to be tested, and adding protease K stock solution with a volume of 0.05 times, mixing thoroughly and incubating at room temperature for 8-15 min;

(37) 2) transferring the sample treated in step 1) into the filter tube of the stacked centrifugal filtration device (as shown in FIG. 1) to carry out centrifugation, then mix thoroughly, and collect the retained liquid in the ultrafiltration tube.

(38) Preferably, if an increased purity of the exosomes is required, DBPS may be added into the ultrafiltration tube to carry out centrifugation at 3,000×g for 10 min to wash and remove certain soluble proteins; one more wash is allowed.

(39) Preferably, the retained liquid is mixed well and collected as the exosome solution which can be used for subsequent treatment.

(40) The method of the invention and the extraction method of prior art or the commercial reagent extraction method are compared below.

(41) Comparative Experiment Between the Method of the Invention and Commercial Reagent Extraction Method

(42) Currently, reagents for exosome extraction are mainly ExoQuick series from SBI and Total Exosome Isolation Reagent series from ThermoFisher. Both methods work with similar principle and have an equivalent extraction purity. Here compared is the extraction effect of the product suitable for serum with reagents from ThermoFisher vs. the present invention (Example 3) on a same serum sample.

(43) The commercial reagent extraction method includes the following steps:

(44) (1) drawing 200 μL serum into a new EP tube;

(45) (2) adding 40 μL Total Exosome Isolation Reagent (from Serum) and mixing thoroughly;

(46) (3) incubating at 4° C. for 30 min;

(47) (4) taking out the above mixture for centrifugation at 10,000×g for 10 min;

(48) (5) removing the supernatant, and adding 20 μL DPBS to resuspend the precipitation thus obtaining exosome solution.

(49) The purity of the exosomes obtained above and the exosomes obtained in Example 3 are determined and the results are given in FIG. 2.

(50) In FIG. 2, A is the result of exosomes extracted with commercial reagents, while B is the purity result of exosomes extracted with the method of the invention. As shown in FIG. 2, the purity of the exosomes extracted with the method of the invention is equivalent to that of commercial reagents. At the same time, the invention has a shorter operation time than that with commercial reagents and has advantages in cost.

(51) Comparative Experiment of Extraction Systems

(52) Exosome extraction effects of exosome kits with minimum component concentration, maximum component concentration and concentration of the invention are compared.

(53) The specific extraction method is as follows:

(54) Incubation buffer of the invention (DPBS+90 mM TrisHCl+90 mM EDTA, pH=7.2); high concentration incubation buffer (DPBS+250 mM TrisHCl+200 mM EDTA, pH=7.2) and low concentration incubation buffer (DPBS+50 mM TrisHCl+50 mM EDTA, pH=7.2) are prepared respectively.

(55) Protease K stock solution (20 mg/mL), low concentration protease K stock solution (5 mg/mL) and high concentration protease K stock solution (40 mg/mL) are prepared.

(56) 200 μL serum is added into each of 3 new EP tubes. According to different concentrations of reagents added, the experiment is divided into the following three testing groups for comparison, wherein: in the high concentration group, 100 μL high concentration incubation buffer and 10 μL high concentration protease K stock solution are used; in the low concentration group, 100 μL low concentration incubation buffer and 10 μL low concentration protease K stock solution are used; and in the invention group, 100 μL incubation buffer and 10 μL protease K stock solution of the invention are used.

(57) The above mixtures are incubated at room temperature for 10 min. Then the mixtures are transferred into the filter tube of the stacked centrifugal filtration device for centrifugation at 3,000×g for 10 min. Then, the liquid retained in the ultrafiltration tube is collected for determination of the purity of the exosomes. The purity testing results are given in FIG. 3.

(58) As shown by the exosome purity determination results (FIG. 3), the exosome concentration of the invention group is equivalent to that of the low concentration group, with 2.73×10.sup.8 particles/mL and 2.04×10.sup.8 particles/mL, respectively. However, the particle diameters of the exosomes obtained by the invention are mainly (>80%) within the range of below 200 nm, while only about 60% of the products obtained by the low concentration group have a particle diameter below 200 nm with more large particles present.

(59) At the same time, the particle diameter distribution of the invention is equivalent to that of the high concentration group. However, in terms of extracted exosome concentration, the total extracted particle concentration of the high concentration group (1.20×10.sup.8 particles/mL) is only about 50% of that of the invention (2.73×10.sup.8 particles/mL).

(60) In conclusion, the exosomes obtained with the buffer and protease K at working concentrations according to the invention are the best.

(61) TEM Measurement of Exosomes

(62) For the exosomes extracted in Example 3 of the invention from the plasma sample, the retentate is washed twice with 2 mL DPBS, and then centrifuged to collect the retained liquid in the ultrafiltration tube. The retained liquid is immobilized on a copper net for staining, followed by measurement using TEM. The result is shown in FIG. 4.

(63) As shown in FIG. 4, the exosomes extracted from plasma by the invention have an integrated envelope structure with a diameter between 30-150 nm, which is in consistent with the features reported by literatures.

(64) The above embodiments are preferred embodiments of the invention. The invention is not limited by the above examples. Various changes, modifications, substitutes, combinations and simplifications made without departing from the scope of the invention are considered as equivalent substitutes of the invention and are within the protection scope of the invention.