CN211522189U - A filtration separator for extracellular vesicle - Google Patents
A filtration separator for extracellular vesicle Download PDFInfo
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- CN211522189U CN211522189U CN202021630112.2U CN202021630112U CN211522189U CN 211522189 U CN211522189 U CN 211522189U CN 202021630112 U CN202021630112 U CN 202021630112U CN 211522189 U CN211522189 U CN 211522189U
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Abstract
The present disclosure describes a filtration separation device for extracellular vesicles, comprising: a sample injector; the first membrane filter comprises a first filter membrane, and the inlet of the first membrane filter is connected to the outlet of the sample injector through a first three-way valve; the inlet of the second membrane filter is connected to the outlet of the first membrane filter through a second three-way valve; a first container connected to an outlet of the second membrane filter through a third three-way valve, the first container being connected to a vacuum pump for pumping gas in the first container; and one or more liquid adding devices connected to at least one of the first, second, and third three-way valves for injecting the balancing liquid into the at least one of the first, second, and third three-way valves. The filtering and separating device can clean the filtering membrane in the membrane filter and collect particulate matters such as extracellular capsules and the like attached to the filtering membrane, and is easy and quick to operate.
Description
Technical Field
The present disclosure relates to a filtration separation device for extracellular vesicles.
Background
Extracellular Vesicles (EV) refer to vesicular bodies of a double-layer membrane structure that are shed from the cell membrane or secreted from the cell, and have a diameter of between 50 nm and 2 mm. The extracellular vesicles widely and stably exist in various body fluids, such as peripheral blood, urine, saliva, cerebrospinal fluid, milk, ascites, amniotic fluid and other body fluids, and carry various biomolecules (including protein, mRNA, miRNA and the like) from cells, and are important tools for carrying out substance transportation, signal transduction and realizing physiological functions of cells. EVs are largely divided into three major classes, depending on their biological origin: exosomes, microvesicles, and apoptotic bodies. Wherein the Exosomes (Exosomes) are extracellular vesicles having a diameter of about 40-150 nm.
The separation and purification of extracellular vesicles are usually achieved by ultracentrifugation, immunomagnetic beads, ultrafiltration, precipitation or kits. For example, CN210856130U discloses a manual extracellular vesicle separation system which employs a chromatographic column packed with a porous particulate packing to filter the filtration liquid. CN108865971A discloses a method and apparatus for separation of exosomes using porous anodic alumina membrane.
Particulate matter such as extracellular vesicles often adheres to the filter in the separation system after use. However, the above extracellular vesicle separation device is difficult to clean the filter and collect the particulate matter therein, which is not favorable for the reuse of the filter.
SUMMERY OF THE UTILITY MODEL
The present disclosure provides a filtration separation device for extracellular vesicles, comprising: a sample injector; the first membrane filter comprises a first filter membrane, and the inlet of the first membrane filter is connected to the outlet of the sample injector through a first three-way valve; the inlet of the second membrane filter is connected to the outlet of the first membrane filter through a second three-way valve; a first container connected to an outlet of the second membrane filter through a third three-way valve, the first container being connected to a vacuum pump for pumping gas in the first container; and one or more liquid adding devices connected to at least one of the first, second, and third three-way valves for injecting the balancing liquid into the at least one of the first, second, and third three-way valves.
The filtering and separating device can inject the balance liquid into the membrane filter according to the requirement so as to clean the filter membrane in the membrane filter and collect the particulate matters such as extracellular capsules attached to the filter membrane, and meanwhile, the operations such as disassembling and reassembling the filtering and separating device are not needed, and the operation is easy and quick.
In some embodiments according to the present disclosure, the outlet of the sample injector is removably connected to a first end of a first three-way valve; the inlet of the first membrane filter is detachably connected with the second end of the first three-way valve; the outlet of the first membrane filter is detachably connected with the first end of the second three-way valve; the inlet of the second membrane filter is detachably connected with the second end of the second three-way valve; the outlet of the second membrane filter is detachably connected with the first end of the third three-way valve; the inlet of the first container is detachably connected with the second end of the third three-way valve; the liquid adding device is connected to at least one of the third end of the first three-way valve, the third end of the second three-way valve and the third end of the third three-way valve.
In some embodiments according to the present disclosure, the priming device comprises one or more of a peristaltic pump, a magnetic gear pump, a plunger pump, a syringe.
In some embodiments according to the present disclosure, the first filter membrane and the second filter membrane are uniform pore size anodized aluminum thin films.
In some embodiments according to the present disclosure, the uniform pore size anodized aluminum oxide film of the first filter membrane has a pore size in the range of 150 nm to 300 nm; the uniform pore size anodic alumina membrane of the second filter membrane has a pore size in the range of 30 nm to 150 nm.
Further, in some embodiments according to the present disclosure, the uniform pore size anodized aluminum thin film of the first filter membrane has a pore size of 300 nm; the uniform-aperture anodic aluminum oxide film of the second filter membrane has an aperture of 30 nm.
In some embodiments according to the present disclosure, the uniform aperture anodized aluminum thin film is convex downward.
In some embodiments according to the present disclosure, a microfiltration membrane covers the outlet of the applicator to filter impurities.
Drawings
Fig. 1 is a schematic illustration of a filtration separation apparatus for extracellular vesicles according to some embodiments of the present disclosure;
fig. 2 is a partially enlarged schematic view of a first three-way valve of the filtering separation device for extracellular vesicles of fig. 1.
Detailed Description
The filtration and separation device for extracellular vesicles according to the present invention will be described in detail with reference to the accompanying drawings and specific examples. Advantages and features of the present invention will become apparent from the following detailed description and claims. It is noted that the drawings are in greatly simplified form and employ non-precise ratios for the purposes of facilitating and distinctly facilitating the description of the embodiments of the present invention.
Referring to fig. 1, in some embodiments according to the present disclosure, a filtration and separation device for extracellular vesicles is disclosed, which includes a sample injector 1, a first membrane filter 3, a second membrane filter 5, a first container 7, a vacuum pump 8, and one or more priming devices 10.
The sample injector 1 is used for storing a liquid to be filtered, such as peripheral blood, urine, saliva, cerebrospinal fluid, milk, ascites, amniotic fluid, or other body fluids. Alternatively, the outlet of the sample applicator 1 may be covered with a microfiltration membrane having a pore size of about 1 μm to filter out platelets and cellular debris and the like that may be contained in the body fluid.
The first membrane filter 3 comprises a first filter membrane 31. The inlet of the first membrane filter 3 is connected to the outlet of the injector 1 via a first three-way valve 2. The first filter 31 may be used for the initial filtration from the sample applicator 1.
The second membrane filter 5 comprises a second filter membrane 51. The inlet of the second membrane filter 51 is connected to the outlet of the first membrane filter 3 by a second three-way valve 5. The first filter membrane 31 may receive the primarily filtered liquid from the first membrane filter 3 and filter it again.
The first container 7 is connected to an outlet of the second membrane filter 5 through a third three-way valve 6, and the first container 7 is connected to a vacuum pump 8 for pumping gas in the first container 7. The first container 7 may be used to receive filtered liquid. The vacuum pump 8 may be connected to the first container 7 by a suction pipe 9, and the vacuum pump 8 may be a manual or electric vacuum pump.
The charging device 10 is connected to at least one of the first, second and third three- way valves 2, 4, 6 for charging at least one of the first, second and third three- way valves 2, 4, 6 with the balancing liquid. Alternatively, the priming device 10 may comprise one or more of a peristaltic pump, a magnetic gear pump, a plunger pump, a syringe.
The filtration and separation device can realize the separation and purification of extracellular vesicles (such as exosomes) with different particle sizes through a double filtration mechanism, can also inject the balance liquid into the membrane filter as required by arranging a three-way valve and a liquid adding device and the like for injecting the balance liquid, so as to clean the filter membrane in the membrane filter, collect particulate matters such as extracellular vesicles attached to the filter membrane, and the like, and meanwhile, the filtration and separation device does not need to be disassembled, reassembled and the like, and the operation is simple and quick.
In particular, in some embodiments according to the present disclosure, the outlet of the sample injector 1 may be detachably connected with the first end 21 of the first three-way valve 2; the inlet of the first membrane filter 3 may be detachably connected with the second end 22 of the first three-way valve 2 (as shown in fig. 2); the outlet of the first membrane filter 3 may be detachably connected with a first end of a second three-way valve 4; the inlet of the second membrane filter 5 may be detachably connected with the second end of the second three-way valve 4; the outlet of the second membrane filter 5 may be detachably connected with a first end of a third three-way valve 6; the inlet of the first container 7 may be detachably connected with the second end of the third three-way valve 6; the charging device 10 may be connected to at least one of the third terminal 23 of the first three-way valve 2, the third terminal of the second three-way valve 4 and the third terminal of the third three-way valve 6.
The above connections may be made by standard luer fittings. Alternatively, the first, second, and third three- way valves 2, 4, and 6 may be the same structure or different structures, respectively. Each end of the first, second and third three- way valves 2, 4, 6 may be provided with a switch to individually close or open the corresponding end.
In some embodiments according to the present disclosure, the pore sizes of the first filter 31 and the second filter 51 may be different from each other, so as to intercept and classify extracellular vesicles (e.g., exosomes) with different particle sizes, respectively, and perform corresponding analysis, research and application after collection.
Alternatively, the filter membranes in the first and second membrane filters 3 and 5 are uniform pore size anodic alumina thin films. The porous anodic alumina membrane is used as an inorganic membrane with a unique honeycomb structure, and can be used for separating and purifying extracellular vesicles, particularly exosomes, due to the advantages of parallel and strictly vertical pore channels to the surface of the membrane, adjustable pore diameter (hundreds of nanometers to a few tenths of nanometers), narrow distribution range and the like.
As an example, the uniform pore diameter anodized aluminum thin film of the first filter membrane 31 has a pore diameter in the range of 150 nm to 300 nm; the uniform pore size anodized aluminum oxide film of the second filter membrane 51 has a pore size in the range of 30 nm to 150 nm. Preferably, the uniform pore diameter anodic alumina membrane of the first filter membrane 31 has a pore diameter of 300 nm; the uniform pore diameter anodized aluminum film of the second filter membrane 51 had a pore diameter of 30 nm.
In some embodiments according to the present disclosure, the uniform pore diameter anodized aluminum oxide film is convex downward, so that when liquid is filtered through the uniform pore diameter anodized aluminum oxide film, impurities are easily accumulated in the central region of the uniform pore diameter anodized aluminum oxide film, and the aluminum oxide film is not completely blocked. The liquid may also pass through the anodized aluminum film at the peripheral portion of the film to avoid complete plugging. Since the outer shells of the first membrane filter 3 and the second membrane filter 5 can be made of transparent materials, most of the blocked places of the membranes in the above scheme are concentrated in the central area, so that users can find the problem to be dealt with in time more easily.
In performing filtration separation using the filtration separation apparatus according to the embodiment of the present disclosure, first, the first ends (upper ends) and the second ends (lower ends) of the first three-way valve 2, the second three-way valve 4, and the third three-way valve 6 are opened, and the third ends (lateral ends) are closed, and the air in the first container 7 is evacuated using the vacuum pump 8, and the vacuum pump 8 may be stopped when the air pressure in the first container 7 is lower than a predetermined value; then, the outlet of the sample injector 1 is opened, and the liquid in the sample injector 1 enters the first membrane filter 3 under the action of suction force and is filtered by the first filter membrane 31, preferably by an anodic alumina membrane with uniform pore size; thereafter, the primarily filtered liquid flows through the second three-way valve 4 and into the second membrane filter 5 and is filtered by the second filter membrane 51, preferably by a uniform pore size anodic alumina membrane; the liquid after the re-filtration passes through the third three-way valve 6 into the first container 7. And because the pore size of the second filter 51 is smaller than that of the first filter 31, extracellular vesicles (such as exosomes) with different particle sizes can be intercepted and classified respectively, and then collected for corresponding analysis, research and application.
When the filtering and separating device according to the embodiment of the present disclosure is cleaned and particulate matter such as extracellular capsules attached to the first filter membrane 31 and the second filter membrane 51 are collected, respectively, one or more charging devices 10 may be connected to at least one of the third terminal 23 of the first three-way valve 2, the third terminal of the second three-way valve 4, and the third terminal of the third three-way valve 6, respectively.
For example, in order to clean the first membrane filter 3 and collect the particulate matter and the like adhering thereto, the user may couple the liquid adding device 10 to the third end 23 of the first three-way valve 2 after the end of filtration, and adjust the first three-way valve 2 and the second three-way valve 4 so that the sample injector 1, the second membrane filter 5 are not communicated with the first membrane filter 3, and the third end of the second three-way valve 4 is open to the outside of the filtration-separation device; the user can then inject the balancing fluid into the first three-way valve 2, which can thereby wash out the particles and the like adhering to the first filter membrane 31, and the washed fluid can be discharged from the third end of the second three-way valve 4.
Preferably, for better cleaning and collection effect, the liquid adding device 10 can be coupled to the third end of the second three-way valve 4, and the first three-way valve 2 and the second three-way valve 4 are adjusted so that the sample injector 1, the second membrane filter 5 are not communicated with the first membrane filter 3, and the third end 23 of the first three-way valve 2 is open to the outside of the filtration and separation device; the user can then inject the balancing fluid into the second three-way valve 4, which thereby flushes the particles, etc. adhering to the first filter membrane 31, and the flushed fluid can be discharged from the third port 23 of the first three-way valve 4. Compare in last clean mode, this clean mode can realize the washing away from up of following of first filter membrane 31, and because most particulate matter etc. are located the upper surface of first filter membrane 31, can realize better cleanness and collection effect.
In the above manner, the discharged liquid can be collected while cleaning the first filter membrane 31, so as to obtain biological materials suspended in the discharged liquid, such as extracellular vesicles or exosomes, and perform corresponding analysis, research and application after collection.
The cleaning of the second filter membrane 51 may be performed in the same or similar manner as the cleaning of the first filter membrane 31. Also, while cleaning the second filter membrane 51, the discharged liquid may be collected, thereby obtaining biological materials, such as extracellular vesicles or exosomes, suspended in the discharged liquid, and performing corresponding analysis, research and application after collection. Due to the difference of the pore sizes of the first filter membrane 31 and the second filter membrane 51, the separation and purification of extracellular vesicles (such as exosomes) with different particle sizes are realized. Moreover, because the filtering membrane is adopted instead of the filtering column, compared with the filtering column, the biological material is more difficult to attach to the filtering membrane, and is convenient for a user to obtain.
For example, in the case where the uniform pore diameter anodized aluminum thin film of the first filter 31 has a pore diameter of 300 nm and the uniform pore diameter anodized aluminum thin film of the second filter 51 has a pore diameter of 30 nm. Extracellular vesicles with a particle size of more than 300 nm can be obtained by collecting extracellular vesicles and the like adhering to the first filter 31, and extracellular vesicles with a particle size of between 30 nm and 300 nm can be obtained by collecting extracellular vesicles and the like adhering to the second filter 51. Whereas extracellular vesicles with a particle size of less than 30 nm can be obtained in the first container 7. And then the extracellular vesicles with various particle sizes can be analyzed, researched and applied correspondingly.
It is also possible to clean the first filter membrane 31 and the second filter membrane 51 simultaneously. And before the above cleaning operation, the respective components of the filtering and separating device may be first disassembled.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The terms "plurality" and "a plurality" in the present disclosure and appended claims refer to two or more than two unless otherwise specified.
It will be apparent to those skilled in the art that various changes and modifications can be made to the disclosed magnetic frame without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. A filtration separation device for extracellular vesicles, comprising:
a sample injector;
the first membrane filter comprises a first filter membrane, and the inlet of the first membrane filter is connected to the outlet of the sample injector through a first three-way valve;
a second membrane filter comprising a second filter membrane, an inlet of the second membrane filter being connected to an outlet of the first membrane filter by a second three-way valve;
a first container connected to an outlet of the second membrane filter through a third three-way valve, the first container being connected to a vacuum pump for pumping gas in the first container; and
one or more liquid adding devices connected to at least one of the first, second, and third three-way valves for injecting a balancing liquid into at least one of the first, second, and third three-way valves.
2. The filtration separation apparatus for extracellular vesicles according to claim 1, wherein:
the outlet of the sample injector is detachably connected with the first end of the first three-way valve;
the inlet of the first membrane filter is detachably connected with the second end of the first three-way valve;
the outlet of the first membrane filter is detachably connected with the first end of the second three-way valve;
the inlet of the second membrane filter is detachably connected with the second end of the second three-way valve;
the outlet of the second membrane filter is detachably connected with the first end of the third three-way valve;
the inlet of the first container is detachably connected with the second end of the third three-way valve;
the charging device is connected to at least one of the third end of the first three-way valve, the third end of the second three-way valve, and the third end of the third three-way valve.
3. The filtration separation apparatus for extracellular vesicles according to claim 1, wherein:
the liquid adding device comprises one or more of a peristaltic pump, a magnetic gear pump, a plunger pump, a syringe pump and an injector.
4. The filtration separation apparatus for extracellular vesicles according to claim 1, wherein:
the first filter membrane and the second filter membrane are uniform-aperture anodic aluminum oxide films.
5. The filtration separation apparatus for extracellular vesicles according to claim 4, wherein:
the aperture of the uniform-aperture anodic aluminum oxide film of the first filter membrane is in the range of 150 nm to 300 nm;
the uniform-aperture anodic aluminum oxide film of the second filter membrane has an aperture in the range of 30 nm to 150 nm.
6. The filtration separation apparatus for extracellular vesicles according to claim 5, wherein:
the aperture of the uniform-aperture anodic aluminum oxide film of the first filter membrane is 300 nm;
the aperture of the uniform-aperture anodic aluminum oxide film of the second filter membrane is 30 nm.
7. The filtration separation apparatus for extracellular vesicles according to claim 4, wherein:
the uniform-aperture anodic aluminum oxide film protrudes downwards.
8. The filtered separation device for extracellular vesicles according to claim 1, further comprising:
a microfiltration membrane covering an outlet of the sample injector to filter impurities.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202021630112.2U CN211522189U (en) | 2020-08-07 | 2020-08-07 | A filtration separator for extracellular vesicle |
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| CN202021630112.2U CN211522189U (en) | 2020-08-07 | 2020-08-07 | A filtration separator for extracellular vesicle |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865971A (en) * | 2018-03-14 | 2018-11-23 | 深圳拓扑精膜科技有限公司 | A kind of separation method and its separator of excretion body |
| CN114984759A (en) * | 2022-05-25 | 2022-09-02 | 上海交通大学 | Device and method for filtering and purifying marine diatom sediment sample |
-
2020
- 2020-08-07 CN CN202021630112.2U patent/CN211522189U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865971A (en) * | 2018-03-14 | 2018-11-23 | 深圳拓扑精膜科技有限公司 | A kind of separation method and its separator of excretion body |
| CN114984759A (en) * | 2022-05-25 | 2022-09-02 | 上海交通大学 | Device and method for filtering and purifying marine diatom sediment sample |
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