An apparatus for purifying exosomes and applications thereof

Abstract

This invention relates to the construction and application of an exosome purification device. The purification device utilizes the differences in size and charge between exosomes and other substances, combining the tangential flow of the sample within the device with the dual effects of membrane sieving and an electric field to remove contaminants and achieve high-purity exosome enrichment. The device includes a pre-filtration section and an exosome purification section. This device is simple to operate, can handle large sample volumes, has a short processing time, is less prone to membrane clogging by contaminants, and achieves high purity exosome separation with minimal structural damage. It can meet the separation and purification needs of exosomes from different sources and can be further applied to research on exosome proteomics, drug loading, activity, and biological functions.

Description

Technical Field

[0001] This invention relates to an apparatus for purifying exosomes, which can purify exosomes from biological samples such as plasma, urine, and cell culture medium. Background Technology

[0002] Exosomes are vesicles with a phospholipid bilayer structure secreted by cells, ranging in size from 30 to 200 nm, and are widely distributed in plasma, urine, and cell culture media. They carry a wealth of important protein information and participate in many crucial biological processes, including intercellular communication and immune responses. Due to their small size, origin from the organism itself, low toxicity, and lack of immunogenicity, they hold great promise for use as drug delivery vehicles for disease treatment. [1] However, the isolation of exosomes from different sources is often hampered by interference from co-separated proteins, resulting in low isolation purity. This hinders their subsequent biological applications and significantly interferes with the proteomics analysis of the exosomes themselves. Therefore, achieving high-purity isolation of exosomes from different samples is essential.

[0003] Currently, ultracentrifugation is the gold standard for the isolation of exosomes. [2] Exosomes are obtained by sedimentation at high speeds using the density difference between exosomes and interfering substances. However, high centrifugation speeds can damage exosomes, resulting in low recovery rates and long processing times. Furthermore, polymer-based sedimentation methods are simpler to operate. [3] While large quantities of exosomes can be recovered, the recovered components contain significant amounts of polymers and co-precipitated proteins, preventing direct in vivo application of the obtained exosomes. Antibody-based affinity enrichment methods can recover exosomes from samples with high specificity. [4] However, the separation of exosomes is closely related to protein expression on the surface of exosomes, and antibodies are expensive, making them impractical for large-scale exosome preparation. Exosome separation based on membrane sieving can achieve direct enrichment of exosomes on the membrane. [5] However, the continuous accumulation of pollutants on the membrane can easily cause membrane blockage, thereby affecting the separation efficiency and purity of exosomes.

[0004] Therefore, to non-destructively enrich large quantities of high-purity exosomes from samples, we developed an exosome purification device. This device utilizes the tangential movement of the sample on the filter membrane surface, leveraging the size sieving effect of the membrane to remove contaminants and effectively intercept exosomes. The tangential movement of the fluid does not easily cause membrane pore blockage. Simultaneously, an electric field is applied to both sides of the filter membrane inside the device, utilizing the difference in charge between the exosome surface and interfering substances to further remove interfering substances. Thus, higher purity exosomes can be obtained, and large-scale exosome preparation can be achieved. The obtained exosome samples can be directly used for proteomics analysis and subsequent biological applications.

[0005] References

[0006] [1].Dongbin Yang,et al.Theranostics.2020,10,3684-3707.

[0007] [2].Kyung Ju Ryu,et al.Ann Lab Med.2020,40,253-258.

[0008] [3]. Yejing Weng, et al. Analyst. 2016, 141, 4640–4646.

[0009] [4].Yoon-Tae Kang,et al.Small.2019,1903600(1-14).

[0010] [5].Fei Liu,et al.ACS Nano.2017,11,10712-10723. Summary of the Invention

[0011] The purpose of this invention is to provide a device for the non-destructive separation of high-purity exosomes from complex samples. Achieving this purpose requires simultaneous control of various components in the pre-filtration section and the exosome purification section of the device, and adjustment of the parameters of each section to achieve optimal exosome separation.

[0012] Therefore, the present invention adopts the following technical solution:

[0013] An apparatus for exosome purification includes a pre-filtration section and an exosome purification section;

[0014] The pre-filtration section includes an ultrafiltration cup, which is a sealed container. A filter membrane is horizontally arranged in the middle of the ultrafiltration cup. The four edges of the filter membrane are sealed to the inner wall of the ultrafiltration cup. The filter membrane divides the ultrafiltration cup into two non-communicating chambers. A gas interface is provided at the upper part of the upper chamber in the ultrafiltration cup. The gas interface is connected to a gas source through a pipeline.

[0015] The exosome purification section includes a sample storage container, a peristaltic pump (06), and an osmosis cell;

[0016] The permeation tank is a closed container. A filter membrane is horizontally arranged in the middle of the permeation tank. The four edges of the filter membrane are sealed to the inner wall of the permeation tank. The filter membrane divides the permeation tank into two non-communicating chambers. A plate-shaped electrode is arranged at the top and bottom of the permeation tank. The two plate-shaped electrodes are parallel, spaced apart and opposite to each other. The two plate-shaped electrodes are connected to an external power source through wires.

[0017] The lower chamber of the ultrafiltration cup is connected to the sample storage container via a pipeline; the inlet of the peristaltic pump is connected to the sample storage container via a pipeline, and the outlet of the peristaltic pump is connected to the upper chamber of the permeation cell via a pipeline; the chamber containing the material to be collected in the permeation cell (which can be the upper or lower chamber) is equipped with a material outlet, which is connected to the sample storage container via a pipeline; the chamber in the permeation cell without the material to be collected (which can be the lower or upper chamber) is connected to the waste liquid collection bottle via a pipeline.

[0018] The ultrafiltration cup is placed on a magnetic stirrer, and a stir bar is placed in the chamber inside the ultrafiltration cup. The stirrer drives the stir bar to move. A liquid material feeding port is provided at the top of the upper chamber, and a sealing cap is provided on the feeding port for opening and closing the feeding port.

[0019] The permeation tank is a group of 2-4 permeation tanks connected in series by pipelines. The first-stage permeation tank is connected to the outlet of the peristaltic pump, and the last-stage permeation tank is connected to the sample storage container.

[0020] That is, the chamber containing the sample to be collected in the previous stage permeation cell is connected to the upper chamber of the next stage permeation cell through a pipeline, and the chamber containing the sample to be collected in the last stage is connected to the sample storage container through a pipeline.

[0021] The chamber of the infiltration tank without any material to be collected is connected to a waste liquid collection bottle via a pipeline.

[0022] A valve or a valve and a pressure gauge are provided at the material outlet of the chamber containing the material to be collected in the permeation tank.

[0023] The filter membrane is one or more of inorganic alumina, polycarbonate, polytetrafluoroethylene or cellulose membrane, and a sieve plate for supporting the filter membrane is provided below the filter membrane.

[0024] Two plate-shaped electrodes on the permeation tank are connected to an external DC power supply via wires, forming an electric field vertical to the filter membrane within the permeation tank; a voltage measuring device is installed between the two plate-shaped electrodes.

[0025] The constructed device is used for the enrichment of exosomes in one or more liquids, such as animal or human plasma, animal or human cell culture medium, or animal or human urine samples.

[0026] 1) The pre-filtration section is used to remove particulate matter larger than exosomes from the sample; the exosome purification section is used to remove contaminants from the sample and thus purify the exosomes.

[0027] The upper part of the ultrafiltration cup in the pre-filtration section is connected to an external nitrogen gas supply. A filter membrane with a pore size of 200-400 nm is horizontally clamped in the middle of the ultrafiltration cup, which is placed on a stirrer. The liquid sample to be processed is placed above the filter membrane in the upper part of the ultrafiltration cup. The pressure generated by the nitrogen gas flow in the ultrafiltration cup achieves filtration of the sample from the membrane to the bottom of the membrane. The filtered sample is then processed in the exosome purification section. The nitrogen gas pressure introduced into the pre-filtration section is adjusted to 0.1-15 MPa through the interface.

[0028] 2) The exosome purification section: the filter membrane in the permeation cell has a pore size of 10-300 nm;

[0029] The pre-filtered sample is fed into the membrane of the permeate tank through the pipeline at a rate of 200 μL / min to 200 mL / min by adjusting the flow rate of the peristaltic pump in the pump control section. The pressure in the entire permeate tank is adjusted to 0.002-0.2 MPa by adjusting the valves in the pressure control section connected in series after the permeate tank. The flow rate ratio between the upper and lower parts of the permeate tank is between 1:0 and 0:1.

[0030] At the same time, the voltage applied to both sides of the permeation tank can be selectively adjusted between 0-100V;

[0031] After repeated filtration of the sample in the purification section of the device, phosphate-buffered saline (PBS) was added to the sample storage container to wash the membrane in the system, and finally the exosomes were collected in the sample storage container.

[0032] The recovered exosomes can be concentrated by volume using a filter membrane with a molecular weight cutoff of 10 kDa.

[0033] Purified exosomes can be used for research in proteomics, drug delivery, activity, and biological function.

[0034] This device is simple to operate, can process large amounts of samples, has a short processing time, is not prone to membrane clogging by contaminants, and produces high purity exosomes with minimal structural damage, thus meeting the separation and purification needs of exosomes from different sources.

[0035] This invention has the following characteristics:

[0036] (1) The device of the present invention removes pollutants by combining the tangential flow of fluid on the filter membrane with an electric field, thereby purifying exosomes.

[0037] (2) The entire device is easy to install, safe to operate, easy to replace, and not easily damaged.

[0038] (3) The device can process a variety of samples, with a large sample processing capacity and short processing time.

[0039] (4) The integrity of the exosomes enriched by the device is not damaged and no other interfering substances are introduced. Attached Figure Description

[0040] Figure 1 Schematic diagram of exosome purification device (In the figure: 01: nitrogen flow, 02: ultrafiltration cup, 03, 09, 14: first, second, and third filter membranes, 04: magnetic stirrer, 05: sample storage container, 06: peristaltic pump, 07, 13: first and last stage permeation cells, 08, 15: first and second electrodes, 10, 16: first and second voltmeters, 11, 17: first and second pressure gauges, 12, 18: first and second valves, 19: waste liquid).

[0041] Figure 2 Size range of purified plasma exosomes.

[0042] Figure 3 Size range of purified urinary exosomes.

[0043] Figure 4 Size range of purified cell culture exosomes.

[0044] Figure 5 LC-MS analysis of purified urinary exosome proteome.

[0045] Figure 6 : LC-MS analysis of purified plasma exosomal proteome. Detailed Implementation

[0046] The exosome purification device consists of a pre-filtration section and an exosome purification section connected in series. The pre-filtration section specifically includes: a magnetic stirrer (04), an ultrafiltration cup (02) with a volume of 10-500 mL, a membrane (03) of one of the following: inorganic alumina, polytetrafluoroethylene, polycarbonate, or cellulose with a pore size of 200-400 nm and an inner diameter of 1-7 cm, and an external nitrogen gas path (01). The exosome purification section includes a pump control section, a permeation tank section, a pressure control section, and a voltage control section. The components are effectively connected in series to ensure the normal operation of the entire system. The ultrafiltration cup contains a membrane (09, 14) of one of the following: inorganic alumina, polytetrafluoroethylene, polycarbonate, or cellulose with a pore size of 200-400 nm and an inner diameter of 1-7 cm. The sample is added to the ultrafiltration cup (02), and under the stirring of the stirrer (04) and the action of the external nitrogen gas flow (01), the sample is filtered from the top to the bottom of the membrane, and large particles of interfering substances are intercepted on the membrane. The nitrogen pressure introduced into the pre-filtration section is 0.1-15 MPa to achieve pre-filtration of samples with a volume of 10-500 mL within 5 min-2 h.

[0047] The pre-filtered sample is introduced into the exosome purification section. The exosome purification section is divided into the following combinations:

[0048] i. A peristaltic pump (06), a single first-stage permeation cell (07) with a membrane (09) having an pore size of 10-50 nm and an inner diameter of 10-60 mm, a first valve (12) for controlling pressure and its first pressure gauge (11) connected in series.

[0049] ii. A peristaltic pump (06), a single first-stage permeation cell (07) with a membrane (09) having an aperture size of 10-50 nm and an inner diameter of 10-60 mm, a first electrode plate (08) added to both sides of the permeation cell, a first voltmeter (10), a first valve (12) for controlling pressure and its first pressure gauge (11) connected in series.

[0050] iii. A peristaltic pump (06) with two first-stage and last-stage permeation cells (07, 13) of membranes (09, 14) with pore sizes of 10-300 nm and inner diameters of 10-60 mm, and first and second pressure valves (12, 18) and their first and second pressure gauges (11, 17) connected in series to control pressure.

[0051] iv. A peristaltic pump (06) with two first-stage and last-stage permeation cells (07, 13) of second and third filter membranes (09, 14) with pore sizes of 10-300 nm and inner diameters of 10-60 mm, first and second electrode plates (08, 15) sandwiched on both sides of each permeation cell, first and second voltmeters (10, 16), first and second pressure valves (12, 18) for controlling pressure, and first and second pressure gauges (11, 17) connected in series.

[0052] To ensure normal operation of the device, the following parameters are adjusted for the different combinations described above. The flow rate of the peristaltic pump (06) in the pump control section is adjusted to allow the sample to enter the second and third filter membranes (09, 14) of the first and last stage permeation cells (07, 13) at a rate of 200 μL / min to 200 mL / min. Simultaneously, the first and second valves (12, 18) of the pressure control section connected in series after the permeation cells are adjusted to achieve a pressure of 0.002-0.2 MPa on the first and second pressure gauges (11, 17). At this point, the flow rate ratio of the liquid through the upper and lower parts of the first and last stage permeation cells (07, 13) is between 1:0 and 0:1. Simultaneously, the first and second voltages (10, 16) on both sides of the permeation cells are adjusted to between 0 and 100 V. The sample was purified into exosomes under the above parameter adjustment. Finally, 0-100 mL of phosphate buffer (PBS) was added for circulation, and the final concentrated volume was 5-50 mL. This part of the sample was recovered as the exosome sample. The recovered exosomes can be concentrated by using a filter membrane with a molecular weight cutoff of 10 kDa for convenient subsequent applications.

[0053] The constructed device was used to isolate exosomes from 1-50 mL of plasma, 10-500 mL of cell culture medium, and 10-500 mL of urine samples.

[0054] Example 1

[0055] Exosome purification device for purifying plasma exosomes

[0056] Plasma samples stored at -80℃ were thawed at room temperature and then centrifuged at 16000g for 20 minutes at 4℃ to remove insoluble matter. 10mL of plasma was then diluted with PBS to 50mL for subsequent exosome purification. The plasma sample was added to a 100mL ultrafiltration vessel containing an inorganic alumina (AAO) membrane with a pore size of 200nm and an inner diameter of 4.4cm. A stirrer was turned on, and nitrogen gas was connected to maintain a pressure of 1MPa for pre-filtration within 20 minutes. The pre-filtered sample was collected in a beaker. The exosome purification process involved a peristaltic pump and a single permeation cell with an inorganic alumina membrane having a pore size of 20nm and an inner diameter of 44mm. Pressure control valves and gauges were connected in series to ensure a tight seal. One end of the tubing was connected to the beaker containing the pre-filtered sample, and the other end was connected to the inlet of the upper part of the permeation cell. Turn on the peristaltic pump and adjust the total flow rate of the sample into the system to 10 mL / min. Simultaneously, adjust the valve downstream of the permeation cell so that the pressure gauge reads 0.008 MPa. At this point, the flow rate ratio of the liquid through the upper and lower parts of the permeation cell is 2:1. The liquid exiting the lower part of the permeation cell is the filtered waste liquid, while the sample flowing out from the upper part of the permeation cell is returned to the beaker containing the sample for continuous circulation and filtration. When approximately 15 mL of sample remains after filtration, add 50 mL of PBS to the system and circulate again. The final filtered volume is 5 mL. To verify the purification effect of the device, 100 μL of the recovered exosome sample was diluted to 1 mL with PBS, and the size distribution of the sample was measured using an NTA (NanoSight 300). The results are as follows. Figure 2 As shown, the sample sizes range from 49.5 nm to 169.5 nm, with an average size of 116 nm, which is within the theoretical size range of exosomes.

[0057] Example 2

[0058] Urinary exosome purification device for purification

[0059] A 100mL urine sample stored at -80℃ was thawed at room temperature and then centrifuged at 4000g for 20 minutes at 4℃ to remove insoluble matter. The sample was then added to a 200mL ultrafiltration vessel containing a polycarbonate membrane with a pore size of 220nm and an inner diameter of 6cm. A stirrer was turned on, and nitrogen gas was connected to maintain a pressure of 5MPa to achieve pre-filtration of the sample within 15 minutes. The pre-filtered sample was then directly introduced into the exosome purification section via tubing. The exosome purification section consists of a peristaltic pump and two permeation cells: a polycarbonate membrane with a pore size of 200nm and an inner diameter of 30mm (the first permeation cell) and a polycarbonate membrane with a pore size of 30nm and an inner diameter of 30mm (the second permeation cell). Pressure control valves and pressure gauges are connected in series. After the sample enters the exosome purification section, the peristaltic pump is turned on, and the total flow rate of the sample introduction system is adjusted to 20 mL / min. Simultaneously, the valve downstream of the permeation cell is adjusted so that the pressure gauge reads 0.01 MPa. At this point, the flow rate ratio of the liquid through the upper and lower parts of the first permeation cell is 1:1. The liquid exiting the lower part of the permeation cell is connected to the inlet of the upper part of the second permeation cell via a pipeline. The liquid exiting the upper part of the permeation cell is recirculated back to the beaker; the portion that cannot permeate through the first membrane is waste liquid. For the sample entering the second permeation cell, the flow rate ratio of the upper and lower parts is controlled to 5:1 by adjusting the valve. The upper portion is recirculated back to the beaker, and the lower portion is waste liquid. This continuous circulation is maintained, controlling the final filtered volume to 10 mL.

[0060] To verify the purification effect of the device, 50 μL of the recovered exosome sample was diluted to 1 mL with PBS, and the size distribution of the sample was measured using NTA (NanoSight 300). The results are as follows. Figure 3 As shown, the average sample size is 163.1 nm, which is within the theoretical size range of exosomes.

[0061] Example 3

[0062] Exosome purification device for purifying cell culture medium exosomes

[0063] Take 200 mL of cell culture medium secreted during HeLa cell culture, centrifuge at 300 g for 5 min at 4℃, then centrifuge at 4000 g for 20 min to remove insoluble matter. Use the supernatant for tangential flow filtration to separate and purify exosomes from the culture medium. Add the sample to a 500 mL ultrafiltration vessel containing a cellulose membrane with a pore size of 200 nm and an inner diameter of 5 cm. Turn on the stirrer and connect an external nitrogen gas supply, maintaining a pressure of 9 MPa, to pre-filter the sample within 30 min. The pre-filtered sample is then directly connected to the exosome purification unit via tubing. The exosome purification unit consists of a peristaltic pump, two permeation cells with inorganic alumina membranes having a pore size of 20 nm and an inner diameter of 44 mm, a voltage control system, and a pressure valve and pressure gauge connected in series to ensure good system sealing. The pre-filtered sample was directly introduced into the inlet of the upper part of the first permeation cell via a peristaltic pump at a total flow rate of 40 mL / min. The pressure valve was adjusted to maintain a flow rate ratio of 20:1 between the upper and lower parts of the first permeation cell. Positive and negative electrodes were applied at both ends, with a voltage of 70V. The upper part was connected to the inlet of the second permeation cell, and the lower part was the permeate waste liquid. Negative and positive electrodes were applied to the upper and lower ends of the second permeation cell, with a voltage of 40V. The air valve at the end of the second permeation cell was adjusted to maintain a flow rate ratio of 20:1 between the upper and lower parts. The upper effluent was recycled back to the first permeation cell, and the lower part was waste liquid. After continuous processing, the final filtered volume was 15 mL. The volume was concentrated to 1 mL using a 10 kDa filter membrane. 5 μL of the recovered exosome sample was diluted to 1 mL with PBS, and the particle size distribution was measured using NTA. The results are as follows: Figure 4 As shown, the average size is 82.9 nm.

[0064] Example 4

[0065] The exosome purification device is used to purify urinary exosomes for subsequent proteomics research.

[0066] Urine samples stored at -20℃ were thawed at room temperature and then centrifuged at 4000g for 30 minutes at 4℃ to remove insoluble matter. 50mL of urine was then added to a 500mL ultrafiltration vessel containing a 200nm pore size and 4.4cm inner diameter inorganic alumina (AAO) membrane. A stirrer was turned on, and nitrogen gas was connected to a pressure of 5MPa to achieve pre-filtration of the sample within 10 minutes. The pre-filtered sample was collected in a beaker. The exosome purification process involved a peristaltic pump, a single permeation cell with a 20nm pore size and 44mm inner diameter inorganic alumina membrane, electrodes, a voltmeter, a pressure control valve, and a pressure gauge connected in series to ensure a tight system. One end of the tubing was connected to the beaker containing the pre-filtered sample, and the other end was connected to the inlet of the upper part of the permeation cell. Positive and negative electrodes were added to the upper and lower parts of the permeation cell, and the voltage was controlled at 36V. Turn on the peristaltic pump and adjust the total flow rate of the sample into the system to 3 mL / min. Simultaneously, adjust the valve downstream of the permeation cell so that the pressure gauge reads 0.003 MPa. At this point, the flow rate ratio of the liquid through the upper and lower parts of the permeation cell is 15:1. The liquid exiting the lower part of the permeation cell is the filtered waste liquid, while the sample flowing out from the upper part of the permeation cell continues to circulate and filter back into the beaker containing the sample. The final filtered volume is 5 mL. The enriched sample was concentrated to a final volume of 500 μL using a 10 kDa filter membrane. 50 μL of the resulting sample was added to 200 μL of 4% SDS and 10 mM DTT, and reacted at 95 °C for 10 min. After cooling to room temperature, 20 mM IAA was added, and the mixture was reacted at 25 °C in the dark for 30 min. The mixture was then filtered through a 30 kDa FASP filter membrane, centrifuged at 15000 g for 40 min, washed three times with 200 μL of 8 M urea each time, and centrifuged at 15000 g for 30 min. Next, it was washed three times with 200 μL of 50 mM ammonium bicarbonate each time, and centrifuged at 15000 g for 30 min. Finally, 100 μL of 50 mM ammonium bicarbonate and 2 μg of trypsin were added, and the mixture was reacted at 37 °C for 16 h. The enzymatically digested peptides were recovered, lyophilized, reconstituted with 0.1% FA, and then analyzed by mass spectrometry. 1100 proteins were identified, and compared with the Exocarta exosome library, 922 exosomal proteins were found. Mass spectra are shown below. Figure 5 .

[0067] Example 5

[0068] The exosome purification device was used to purify plasma oosomes for subsequent proteomics studies. 1 mL of plasma was diluted with PBS to 7 mL and added to a 10 mL ultrafiltration vessel containing a 200 nm pore size polycarbonate membrane. The stirrer was turned on, and nitrogen gas was connected to a pressure of 1 MPa to achieve pre-filtration of the sample within 5 minutes. The pre-filtered sample was collected in a beaker. The exosome purification section consisted of a peristaltic pump, a single permeation cell with a 20 nm pore size and 44 mm inner diameter inorganic alumina membrane, a voltmeter, and a pressure valve connected in series with a pressure gauge to ensure good system sealing. One end of the tubing was connected to the beaker containing the pre-filtered sample, and the other end was connected to the inlet of the upper part of the permeation cell. Positive and negative electrodes were added to the upper and lower parts of the permeation cell, and the voltage was controlled at 80 V. Turn on the peristaltic pump and adjust the total flow rate of the sample into the system to 2 mL / min. Simultaneously, adjust the valve downstream of the permeation cell so that the pressure gauge reads 0.01 MPa. At this point, the flow rate ratio of the liquid through the upper and lower parts of the permeation cell is 20:1. The liquid exiting the lower part of the permeation cell is the filtered waste liquid, while the sample flowing out from the upper part of the permeation cell continues to circulate and filter back into the beaker containing the sample. After final filtration, add 20 mL of PBS to wash the tubing, ensuring a final concentrated volume of 5 mL. Concentrate the enriched sample using a 10 kDa filter membrane to a final volume of 1 mL. Add 2% SDS and 10mM TCEP to a final concentration, react at 56℃ for 30 min, then allow to cool to room temperature. Add 20mM IAA and react in the dark for 35 min. Filter through a FASP membrane with a molecular weight cutoff of 10kDa, centrifuge at 14000g for 60 min, wash three times with 200μL of 8M urea each time, centrifuge at 16000g for 30 min, wash three times with 200μL of 50mM ammonium bicarbonate each time, centrifuge at 15000g for 30 min, finally add 50μL of 25mM ammonium bicarbonate and 1μg of trypsin, react at 37℃ for 14 h. Recover the enzymatically digested peptides, lyophilize, reconstitute with 0.1% FA, and then analyze by mass spectrometry. 350 proteins were identified; compared with the Exocarta exosome library, 190 exosomal proteins were found. See the mass spectra below. Figure 6 .

Claims

1. An apparatus for exosome purification, comprising: (i) a pre-filtration part, (ii) an exosome purification part; The pre-filtration part comprises an ultrafiltration cup (02), which is a closed container, a filter membrane (03) is arranged transversely in the middle of the ultrafiltration cup, the four peripheral edges of the filter membrane are in close connection with the inner wall surface of the ultrafiltration cup, and the filter membrane separates the ultrafiltration cup into two cavities that are not in communication with each other; a gas interface is arranged at the upper part of the upper cavity in the ultrafiltration cup, and the gas interface is connected with a gas source through a pipeline (01); The exosome purification part comprises a sample storage container (05), a peristaltic pump (06) and a permeation cell; The permeation cell is a closed container, a filter membrane is arranged transversely in the middle of the permeation cell, the four peripheral edges of the filter membrane are in close connection with the inner wall surface of the permeation cell, and the filter membrane separates the permeation cell into two cavities that are not in communication with each other; a plate-shaped electrode is arranged at the top and the bottom of the permeation cell respectively, the two plate-shaped electrodes are arranged in parallel, spaced and opposite to each other; the two plate-shaped electrodes are connected with an external power source through wires; The lower cavity of the ultrafiltration cup (02) is connected with the sample storage container (05) through a pipeline; the inlet of the peristaltic pump (06) is connected with the sample storage container (05) in communication through a pipeline, the outlet of the peristaltic pump (06) is connected with the upper cavity in the permeation cell in communication through a pipeline; the cavity where the to-be-collected substance in the permeation cell is located is provided with a material outlet, the material outlet is connected with the sample storage container in communication through a pipeline, and the cavity without the to-be-collected substance in the permeation cell is connected with a waste liquid (19) collecting bottle through a pipeline; The cavity is the lower cavity or the upper cavity; The exosome purification device is used for enrichment of exosomes in a liquid; 1) The pre-filtration part is used for removing contaminant particles with a size larger than that of exosomes in a sample; the exosome purification part is used for removing contaminants in the sample and then purifying the exosomes; The upper part of the ultrafiltration cup in the pre-filtration part is externally connected with nitrogen gas (01); a filter membrane (03) with a pore size of 200-400 nm is transversely clamped in the middle of the ultrafiltration cup, the ultrafiltration cup (02) is placed on a stirrer (04), a liquid sample to be treated is placed above the filter membrane on the upper part of the ultrafiltration cup, and filtration of the sample from the membrane to the bottom of the membrane is realized through the pressure formed by the nitrogen gas flow (01) on the ultrafiltration cup, and the filtered sample is subjected to treatment by the exosome purification part; the nitrogen gas pressure introduced by the pre-filtration part through the adjusting interface is 0.1-15 MPa; 2) The pore size of the filter membrane in the permeation cell of the exosome purification part is 10-20 nm; The pre-filtered sample is adjusted by the flow rate of the peristaltic pump (06) in the pump control part so that the sample enters the membrane of the permeation cell through the pipeline at a speed of 200 μL / min to 200 mL / min; the pressure in the whole permeation cell is adjusted to 0.002-0.2 MPa by adjusting the valve of the pressure control part connected in series behind the permeation cell, and the flow rate ratio of the upper and lower parts of the permeation cell is between 1:0 and 0:1; The voltage applied to the two sides of the permeation cell is adjusted to be between 36 V and 100 V; After the sample is repeatedly filtered in the purification part of the whole device, phosphate buffered saline (PBS) is added to the sample storage container for cleaning of the membrane in the system, and finally the exosomes are collected in the sample storage container. The filter membrane is one or more of inorganic alumina, polycarbonate, polytetrafluoroethylene or cellulose membrane; Two plate-shaped electrodes on the permeation cell are connected to an external direct current power source through wires to form an electric field perpendicular to the filter membrane in the permeation cell; a voltage measuring device is arranged between the two plate-shaped electrodes.

2. The apparatus of claim 1, wherein: The ultrafiltration cup (02) is placed on a magnetic stirrer (04), and a stirring rod is placed in the chamber of the ultrafiltration cup (02), and the stirring rod is driven to move by the stirrer; a liquid material feeding port is arranged at the top of the upper chamber, and a sealing cover for opening and closing the feeding port is arranged on the feeding port.

3. The apparatus of claim 1, wherein: A sieve plate for supporting the filter membrane is arranged below the filter membrane.

4. The apparatus of claim 1, wherein: The permeation cell is a group of 2-4 permeation cells connected in sequence through pipelines, the first stage permeation cell (07) is connected to the outlet of the peristaltic pump, and the last stage permeation cell (13) is connected to the sample storage container (05); The chamber containing the collected material of the previous stage permeation cell is connected to the upper chamber of the next stage permeation cell through pipelines, and the chamber containing the collected material of the last stage is connected to the sample storage container through pipelines. The chamber of the permeation cell without collected material is connected to the waste liquid collection bottle through pipelines.

5. The apparatus of claim 1 or 4, wherein: A valve is arranged at the material outlet of the outlet of the chamber containing the collected material of the permeation cell, or a valve and a pressure gauge are arranged.

6. The apparatus of claim 1, wherein: The recovered exosomes are concentrated in volume by using a filter membrane with a molecular weight cut-off of 10 kDa.

7. The apparatus of claim 6, wherein: The liquid sample to be treated is one of animal or human plasma, animal or human cell culture solution, and animal or human urine sample containing exosomes. The recovered exosomes are concentrated in volume by using a filter membrane with a molecular weight cut-off of 10 kDa. The liquid sample to be treated is one of animal or human plasma, animal or human cell culture solution, and animal or human urine sample containing exosomes.

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