EXOSOME PURIFICATION METHOD AND INTEGRATED DEVICE THEREOF

Abstract

A method exosome purification and characterization is contemplated, comprising the steps of secondary two-stage tangential ultrafiltration to produce an extracted solution, pretreatment of the extracted solution for characterization, characterization of the extracted solution to detect particle size and concentration, and freeze-drying of the extracted solution. An exosome purification integrated device is also contemplated. Through the disclosed methods and devices, exosomes may be better purified and characterized in a manner that results in high practical value to overcome the problems associated with conventional exosome purification processes on the market today, including tedious purification processes, long durations, and high costs.

Claims

1. A method of exosome purification and characterization, comprising the steps of: S1) introducing an extraction solution containing at least one exosome targeted for extraction into a first-stage tangential flow ultrafiltration device having a first-stage ultrafiltration membrane to obtain from the permeate a first-stage liquid permeate, delivering the first-stage liquid permeate into a second-stage tangential flow ultrafiltration device having a second-stage ultrafiltration membrane to obtain from the retentate an enriched liquid solution, wherein the pore size of the first-stage ultrafiltration membrane of the first-stage tangential flow ultrafiltration device is greater than the pore size of the second-stage ultrafiltration membrane of the second-stage tangential flow ultrafiltration device, and wherein the pore size of the second-stage ultrafiltration membrane of the second-stage tangential flow ultrafiltration device is less than the diameter of the at least one exosome targeted for extraction such thus the exosome targeted for extraction can pass through the first-stage ultrafiltration membrane and cannot pass through the second-stage ultrafiltration membrane; S2) introducing the enriched liquid solution into a fast protein liquid chromatograph to obtain an eluent solution; S3) mixing the eluent solution obtained in step S2 with a phosphate buffered solution to obtain a mixed solution, and injecting the mixed solution into a nanoparticle tracking analyzer to determine particle size and concentration data associated with the enriched liquid solution; and S4) freeze drying the enriched liquid solution obtained in step S1 to obtain a freeze-dried powder permeate.

2. The exosome purification and characterization method according to claim 1, wherein in step S2, the pore size of the first-stage ultrafiltration membrane is greater than or equal to 0.18 μm, and the pore size of the second-stage ultrafiltration membrane is less than or equal to 0.03 μm.

3. The exosome purification and characterization method according to claim 1, wherein step S4 is performed via adding an excipient into the enriched liquid solution, bottling the enriched liquid solution containing the excipient, and then transferring the bottled enriched liquid solution containing the excipient into a vacuum freeze dryer to obtain the freeze-dried powder.

4. The exosome purification and characterization method according to claim 1, wherein in step S2, the fast protein liquid chromatograph is configured to chromatographically treat the enriched liquid solution using a chromatographic column employing an affinity column, the fast protein liquid chromatograph utilizing an equilibrium liquid comprising 0.06M Tris-HCl and 0.5 M NaCl having a PH of 7.9, an eluant comprising 0.06 M Tris-HCl, 0.5 M NaCl, and 0.6 M imidazole having a PH of 7.9, and a flow rate for equilibrating four column beds and eluting one column bed of 4 mL/min.

5. The exosome purification and characterization method according to claim 1, wherein in step S3, the volume ratio of the eluent solution to the phosphate buffered solution is 6:4.

6. The exosome purification and characterization method according to claim 1, wherein in step S1, the extraction solution containing at least one exosome targeted for extraction comprises one or more of: cell supernatant, blood, urine, saliva, amniotic fluid, urine, and seminal fluid.

7. The exosome purification and characterization method according to claim 1, wherein step S3 further comprises a step of comparing the determined particle size and concentration data to an acceptable particle size and concentration range, and if it is detecting that the determined particle size and concentration data is not within the acceptable particle size and concentration range, performing further tangential ultrafiltration treatment upon the enriched liquid solution, wherein the acceptable particle size and concentration range comprises a concentration greater than or equal to 109/ml, and a mean particle size range of between 30 nm to 150 nm.

8. An integrated device for exosome purification and characterization, the integrated device comprising: a tangential flow ultrafiltration system comprising a first-stage tangential flow ultrafiltration device having a first-stage ultrafiltration membrane, and a second-stage tangential flow ultrafiltration device having a second-stage ultrafiltration membrane and a second-stage outlet, the first-stage tangential flow ultrafiltration device and the second-stage tangential flow ultrafiltration device being in fluid communication with one another, the first-stage ultrafiltration membrane having a greater pore size than the pore size of the second-stage ultrafiltration membrane, a fast protein liquid chromatography system having a fast protein liquid chromatography inlet and a fast protein liquid chromatography outlet; a nanoparticle tracking analysis system; and a peristaltic pump for transferring liquid through the integrated device; wherein the second-stage outlet is connected to the fast protein liquid chromatography inlet, and the fast protein liquid chromatography outlet is connected to the nanoparticle tracking analysis system.

9. The integrated device for exosome purification and characterization according to claim 8, wherein the first-stage tangential flow ultrafiltration device includes a first-stage inlet and a first-stage outlet, the second-stage tangential flow ultrafiltration device includes a second-stage inlet, and the first-stage outlet is in fluid communication with the second-stage inlet.

10. The integrated device for exosome purification and characterization according to claim 9, wherein the nanoparticle tracking analysis system is in fluid communication with the first-stage inlet.

11. The integrated device for exosome purification and characterization according to claim 8, wherein the fast protein liquid chromatography system is connected to a freeze-drying refrigeration system.

12. The integrated device for exosome purification and characterization according to claim 11, wherein the freeze-drying refrigeration system comprises a mechanical transfer device, a freeze dryer, and a low-temperature storage space, the mechanical transfer device being operative to transfer a product frozen by the freeze dryer to the low-temperature storage space.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a structure diagram of an exemplary exosome purification integrated device according to the present disclosure;

[0028] FIG. 2 is a diagram of a working principle of an exemplary embodiment of the method of the present disclosure; and

[0029] FIG. 3 is a diagram of a working process of an exemplary embodiment of the method of the present disclosure.

[0030] Common reference numerals are used throughout the drawings and detailed description to indicate the same elements, and are as follows:

[0031] 1—tangential flow ultrafiltration system; 11—first-stage tangential flow ultrafiltration device; 111—first-stage inlet; 112—first-stage outlet; 12—second-stage tangential flow ultrafiltration device; 121—second-stage inlet; 122—second-stage outlet; 2—fast protein liquid chromatography system; 3—nanoparticle tracking analysis system; 4—freeze dryer; 5—mechanical transfer device; 6—low-temperature storage space.

DETAILED DESCRIPTION

[0032] To assist in rendering the objectives, features and advantages of the present disclosure more clear and understandable, the following specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings.

Embodiment 1

[0033] The embodiment provides an exosome purification integrated device, which specifically comprises a tangential flow ultrafiltration system 1, a fast protein liquid chromatography system 2, a nanoparticle tracking analysis system 3 and a peristaltic pump for transferring liquid through the system, which may be communicated through a pipeline; the peristaltic pump is used for transferring a flow of liquid through the device; by adding extraction solution containing the exosome into the device, the extraction solution sequentially flows through the tangential flow ultrafiltration system 1, the fast protein liquid chromatography system 2 and the nanoparticle tracking analysis system 3 to complete the purification of the exosome.

[0034] The tangential flow ultrafiltration system 1 comprises a first-stage tangential flow ultrafiltration device 11 and a second-stage tangential flow ultrafiltration device 12 which are in communication with each other; the first-stage tangential flow ultrafiltration device 11 is provided with a first-stage inlet 111 and a first-stage outlet 112; the second-stage tangential flow ultrafiltration device 12 is provided with a second-stage inlet 121 and a second-stage outlet 122, and the first-stage outlet 112 is communicated with the second-stage inlet 121, thus the introduced extraction solution firstly enters the first-stage tangential flow ultrafiltration device 11 for ultrafiltration, and then flows into the second-stage tangential flow ultrafiltration device 12 for repeated ultrafiltration;

[0035] An ultrafiltration membrane is arranged in each of the first-stage tangential flow ultrafiltration device 11 and the second-stage tangential flow ultrafiltration device 12, and the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device 11 is greater than the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device 12. The pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device 12 is less than the diameter of the exosome in the introduced extraction solution;

[0036] The second-stage tangential flow ultrafiltration device 12 is connected to an inlet of the fast protein liquid chromatography system 2, and an outlet of the fast protein liquid chromatography system 2 is connected to the nanoparticle tracking analysis system 3;

[0037] The fast protein liquid chromatography system 2 is further connected to a freeze-drying refrigeration system, the freeze-drying refrigeration system comprises a mechanical transfer device 5, a freeze dryer 4, and a low-temperature storage space 6; and the mechanical transfer device 5 is used to move a product frozen by the freeze dryer 4 to the low-temperature storage space 6.

Embodiment 2

[0038] The embodiment provides an exosome purification method, comprising the following steps:

[0039] S1. secondary tangential ultrafiltration: introducing an extraction solution containing the exosome into a first-stage tangential flow ultrafiltration device, recovering the ultrafiltered liquid through a first-stage ultrafiltration membrane to obtain from the permeate a first-stage liquid permeate, then enabling the first-stage liquid permeate to flow through the second-stage tangential flow ultrafiltration device continuously, retaining the exosome within the retentate as the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome, and recovering the retentate to obtain enriched liquid;

[0040] It is contemplated that the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is greater than the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome. More particularly, it is contemplated that the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is 0.18 μm, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is 0.03 μm.

[0041] S2. Enriched liquid pretreatment: conducting vacuum filtration or ultrasonic degassing treatment on the buffered solution mobile phase for 15 minutes to remove bubbles; firstly transferring an inlet pipe of the fast protein liquid chromatograph to a deionized water pump from 20% ethanol protection liquid for flushing, and then transferring to a buffered solution pump for flushing, with a flow rate of 0.7 mL/min and pump pressure alarm of 0.2 MPa;

[0042] Introducing the enriched liquid into the fast protein liquid chromatograph for chromatographic treatment to obtain eluent solution;

[0043] In the described embodiment, the fast protein liquid chromatograph is configured to chromatographically treat the enriched liquid solution using a chromatographic column employing an affinity column, the fast protein liquid chromatograph utilizing an equilibrium liquid comprising 0.06M Tris-HCl and 0.5 M NaCl having a PH of 7.9, an eluant comprising 0.06 M Tris-HCl, 0.5 M NaCl, and 0.6 M imidazole having a PH of 7.9, and a flow rate for equilibrating four column beds and eluting one column bed of 4 mL/min.

[0044] S3. Exosome particle size and concentration detection: flushing a detection window of the nanoparticle tracking analyzer with 1 mL of alcohol and 1 mL of pure water successively, then mixing the eluent solution obtained in the step S2 with a phosphate buffered solution in a volume ratio of 6:4, injecting the mixed solution into the nanoparticle tracking analyzer for analyzing and detecting the particle size and the concentration, and feeding back data results, detecting whether the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm; if the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm, entering step S4, or returning to the step S2 again for secondary tangential filtering treatment.

[0045] S4. Freeze-drying: conducting freeze-drying treatment on the enriched liquid or eluent solution to obtain freeze-dried powder, thus completing the purification of the exosome. The freeze-drying refrigeration system is composed of a freeze dryer, a mechanical arm, and a low-temperature storage. The enriched liquid or eluent solution is freeze-dried in the freeze dryer, then is sub-packaged into vials, and then is transferred to the low-temperature storage by the mechanical arm. The low-temperature storage is accompanied with a full alarm system.

Embodiment 3

[0046] The embodiment provides an exosome purification method, comprising the following steps:

[0047] S1. secondary tangential ultrafiltration: introducing an extraction solution containing the exosome into a first-stage tangential flow ultrafiltration device, recovering the ultrafiltered liquid through a first-stage ultrafiltration membrane to obtain from the permeate a first-stage liquid permeate, then enabling the first-stage liquid permeate to flow through the second-stage tangential flow ultrafiltration device continuously, retaining the exosome within the retentate as the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome, and recovering the retentate to obtain enriched liquid;

[0048] The pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is greater than the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome. It is expressly contemplated that the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device may be 0.19 μm, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device may be 0.02 μm.

[0049] S2. Enriched liquid pretreatment: conducting vacuum filtration or ultrasonic degassing treatment on the buffered solution mobile phase for 15 minutes to remove bubbles; firstly transferring an inlet pipe of the fast protein liquid chromatograph to a deionized water pump from 20% ethanol protection liquid for flushing, and then transferring to a buffered solution pump for flushing, with a flow rate of 0.7 mL/min, and pump pressure alarm of 0.2 MPa;

[0050] The enriched liquid in introduced into the fast protein liquid chromatograph for chromatographic treatment to obtain an eluent solution;

[0051] In the described embodiment, the fast protein liquid chromatograph is configured to chromatographically treat the enriched liquid solution using a chromatographic column employing an affinity column, the fast protein liquid chromatograph utilizing an equilibrium liquid comprising 0.06M Tris-HCl and 0.5 M NaCl having a PH of 7.9, an eluant comprising 0.06 M Tris-HCl, 0.5 M NaCl, and 0.6 M imidazole having a PH of 7.9, and a flow rate for equilibrating four column beds and eluting one column bed of 4 mL/min.

[0052] S3. Exosome particle size and concentration detection: flushing a detection window of the nanoparticle tracking analyzer with 1 mL of alcohol and 1 mL of pure water successively, then mixing the eluent solution obtained in the step S2 with a phosphate buffered solution in a volume ratio of 6:4, injecting the mixed solution into the nanoparticle tracking analyzer for particle size and concentration analysis and detection, and feeding back data results, detecting whether the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm; if the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm, entering step S4, or if not, returning to the step S2 again for secondary tangential filtering treatment;

[0053] S4. Freeze-drying: conducting freeze-drying treatment on the enriched liquid to obtain freeze-dried powder, thus completing the purification of the exosome. The freeze-drying refrigeration system is composed of a freeze dryer, a mechanical arm, and a low-temperature storage. The enriched liquid or eluent solution is freeze-dried in the freeze dryer, then is sub-packaged into vials, and then is transferred to the low-temperature storage by the mechanical arm. The low-temperature storage is accompanied with a full alarm system.

Embodiment 4

[0054] The embodiment provides an exosome purification method, comprising the following steps:

[0055] S1. secondary tangential ultrafiltration: introducing an extraction solution containing the exosome into a first-stage tangential flow ultrafiltration device, recovering the ultrafiltered liquid through a first-stage ultrafiltration membrane to obtain from the permeate a first-stage liquid permeate, then enabling the first-stage liquid permeate to flow through the second-stage tangential flow ultrafiltration device continuously, retaining the exosome within the retentate as the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome, and recovering the retentate to obtain enriched liquid;

[0056] The pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device is greater than the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device is less than the diameter of the exosome. It is expressly contemplated that the pore size of the ultrafiltration membrane in the first-stage tangential flow ultrafiltration device may be 0.20 μm, and the pore size of the ultrafiltration membrane in the second-stage tangential flow ultrafiltration device may be 0.01 μm.

[0057] S2. Enriched liquid pretreatment: conducting vacuum filtration or ultrasonic degassing treatment on the buffered solution mobile phase for 15 minutes to remove bubbles; firstly transferring an inlet pipe of the fast protein liquid chromatograph to a deionized water pump from 20% ethanol protection liquid for flushing, and then transferring to a buffered solution pump for flushing, with a flow rate of 0.7 mL/min, and pump pressure alarm of 0.2 MPa;

[0058] The enriched liquid is introduced into the fast protein liquid chromatograph for chromatographic treatment to obtain an eluent solution;

[0059] In the described embodiment, the fast protein liquid chromatograph is configured to chromatographically treat the enriched liquid solution using a chromatographic column employing an affinity column, the fast protein liquid chromatograph utilizing an equilibrium liquid comprising 0.06M Tris-HCl and 0.5 M NaCl having a PH of 7.9, an eluant comprising 0.06 M Tris-HCl, 0.5 M NaCl, and 0.6 M imidazole having a PH of 7.9, and a flow rate for equilibrating four column beds and eluting one column bed of 4 mL/min S3.

[0060] Exosome particle size and concentration detection: flushing a detection window of the nanoparticle tracking analyzer with 1 mL of alcohol and 1 mL of pure water successively, then mixing the eluent solution obtained in the step S2 with a phosphate buffered solution in a volume ratio of 6:4, injecting the mixed solution into the nanoparticle tracking analyzer for particle size and concentration analysis and detection, and feeding back data results, detecting whether the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm; if the concentration range of the exosome reaches 109/ml and the particle ranges from 30 nm to 150 nm, entering step S4, or returning to the step S2 again for secondary tangential filtering treatment;

[0061] S4. Freeze-drying: conducting freeze-drying treatment on the enriched liquid to obtain freeze-dried powder, thus completing the purification of the exosome. The freeze-drying refrigeration system is composed of a freeze dryer, a mechanical arm, and a low-temperature storage. The enriched liquid or eluent solution is freeze-dried in the freeze dryer, then is sub-packaged into vials, and then is transferred to the low-temperature storage by the mechanical arm. The low-temperature storage is accompanied with a full alarm system.

[0062] Although the present disclosure is disclosed as above, the scope of protection of the present disclosure is not limited thereto. Various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present disclosure, all of which should fall within the scope of protection of the present disclosure.