CN211522189U - A filtration separator for extracellular vesicle - Google Patents

Abstract

The present disclosure describes a filtration separation device for extracellular vesicles, comprising: a sample applicator; a first membrane filter including a first filter membrane, the inlet of the first membrane filter being connected to a first three-way valve through a first three-way valve The outlet of the sample injector; the second membrane filter, including the second filter membrane, the inlet of the second membrane filter is connected to the outlet of the first membrane filter through the second three-way valve; the first container, the first container passes through the second three-way valve. A three-way valve is connected to the outlet of the second membrane filter, the first container is connected to a vacuum pump for sucking gas in the first container; and one or more liquid adding devices are connected to the first three-way valve , at least one of the second three-way valve and the third three-way valve for injecting the balance liquid into at least one of the first three-way valve, the second three-way valve and the third three-way valve. The filtration and separation device can clean the filter membrane in the membrane filter and collect particulate matter such as extracellular vesicles attached thereon, and the operation is easy and fast.

Description

A filter separation device for extracellular vesicles

technical field

The present disclosure relates to filtration isolation devices for extracellular vesicles.

Background technique

Extracellular vesicles (EVs) are double-membrane vesicle-like bodies shed from cell membranes or secreted by cells, with diameters ranging from 50 nm to 2 mm. Extracellular vesicles exist widely and stably in various body fluids, such as peripheral blood, urine, saliva, cerebrospinal fluid, milk, ascites, amniotic fluid and other body fluids, and carry various cell-derived biomolecules (including proteins, mRNAs, etc.). , miRNA, etc.), is an important tool for cells to carry out material transport, signal transduction, and achieve physiological functions. According to their biological origin, EVs are mainly divided into three categories: exosomes, microvesicles, and apoptotic bodies. Among them, exosomes are extracellular vesicles with a diameter of about 40-150 nm.

The isolation and purification of extracellular vesicles are usually achieved by methods such as ultracentrifugation, immunomagnetic beads, ultrafiltration, precipitation or kits. For example, CN210856130U discloses a manual extracellular vesicle separation system, which uses a chromatography column filled with porous particle fillers to filter and filter liquid. CN108865971A discloses a method and device for separating exosomes using a porous anodic aluminum oxide film.

After the filter in the separation system is used, particles such as extracellular vesicles are often attached. However, the above extracellular vesicle separation device is difficult to clean the filter and collect the particulate matter, which is not conducive to the reuse of the filter.

Utility model content

The present disclosure provides a filtration and separation device for extracellular vesicles, which includes: a sample applicator; a first membrane filter, including a first filter membrane, the inlet of the first membrane filter is connected through a first three-way valve To the outlet of the sample injector; the second membrane filter, including the second filter membrane, the inlet of the second membrane filter is connected to the outlet of the first membrane filter through the second three-way valve; the first container, the first container passes through A third three-way valve is connected to the outlet of the second membrane filter, the first container is connected to a vacuum pump for sucking gas in the first container; and one or more liquid addition devices are connected to the first three-way at least one of the valve, the second three-way valve and the third three-way valve for injecting the balance liquid into at least one of the first three-way valve, the second three-way valve and the third three-way valve.

The filtration and separation device can inject a balance liquid into the membrane filter as required, so as to clean the filter membrane in the membrane filter and collect the extracellular vesicles and other particles attached to it, and at the same time, it is not necessary to carry out the filtration and separation device. Disassembly, reassembly and other operations, the operation is easy and fast.

In some embodiments according to the present disclosure, the outlet of the sampler is detachably connected to the first end of the first three-way valve; the inlet of the first membrane filter is detachably connected to the second end of the first three-way valve connection; the outlet of the first membrane filter is detachably connected to the first end of the second three-way valve; the inlet of the second membrane filter is detachably connected to the second end of the second three-way valve; the second membrane filter The outlet of the container is detachably connected to the first end of the third three-way valve; the inlet of the first container is detachably connected to the second end of the third three-way valve; the liquid adding device is connected to the third end of the first three-way valve. At least one of the three ends, 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 liquid addition device includes one or more of a peristaltic pump, a magnetic gear pump, a plunger pump, a syringe pump, and 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 oxide membranes.

In some embodiments according to the present disclosure, the pore size of the uniform pore size anodic aluminum oxide film of the first filter membrane is in the range of 150 nm to 300 nm; the pore size of the uniform pore size anodic aluminum oxide film of the second filter membrane is 30 nm to 30 nm. 150 nm range.

Further, in some embodiments according to the present disclosure, the pore size of the uniform pore size anodic aluminum oxide film of the first filter membrane is 300 nm; the pore size of the uniform pore size anodic aluminum oxide film of the second filter membrane is 30 nm.

In some embodiments according to the present disclosure, the uniform pore size anodized aluminum film protrudes downward.

In some embodiments according to the present disclosure, a microfiltration membrane covers the outlet of the sampler to filter impurities.

Description of drawings

1 is a schematic diagram of a filtration isolation device for extracellular vesicles according to some embodiments of the present disclosure;

FIG. 2 is a partially enlarged schematic view of the first three-way valve of the filter separation device for extracellular vesicles of FIG. 1 .

Detailed ways

The filtration and separation device for extracellular vesicles disclosed in the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. The advantages and features of the present invention will become more apparent from the following detailed description and claims. It should be noted that the accompanying drawings are all in a very simplified form and use inaccurate ratios, and are only used for the purpose of assisting in explaining the embodiments of the present invention conveniently and clearly.

Referring to FIG. 1 , in some embodiments according to the present disclosure, a filter separation device for extracellular vesicles is disclosed, which includes a sample applicator 1 , a first membrane filter 3 , a second membrane filter 5 , A first container 7 , a vacuum pump 8 , and one or more liquid addition devices 10 .

The sampler 1 is used to store liquids to be filtered, such as peripheral blood, urine, saliva, cerebrospinal fluid, milk, ascites, amniotic fluid and other body fluids. In addition, the outlet of the sample injector 1 can be covered with a microfiltration membrane, and the pore size of the microfiltration membrane is about 1 μm, so as to filter and remove platelets and cell debris and the like that may be contained in the body fluid.

The first membrane filter 3 includes a first filter membrane 31 . The inlet of the first membrane filter 3 is connected to the outlet of the sample injector 1 through the first three-way valve 2 . The first filter membrane 31 can perform primary filtration from the sample injector 1 .

The second membrane filter 5 includes a second filter membrane 51 . The inlet of the second membrane filter 51 is connected to the outlet of the first membrane filter 3 through the second three-way valve 5 . The first filter membrane 31 can receive the primary filtered liquid from the first membrane filter 3 and filter it again.

The first container 7 is connected to the outlet of the second membrane filter 5 through the third three-way valve 6 , and the first container 7 is connected to a vacuum pump 8 for sucking the gas in the first container 7 . The first container 7 may be used to receive filtered liquid. The vacuum pump 8 can be connected to the first container 7 through the suction pipe 9, and the vacuum pump 8 can be a manual or electric vacuum pump.

The dosing device 10 is connected to at least one of the first three-way valve 2, the second three-way valve 4 and the third three-way valve 6 for supplying the first three-way valve 2, the second three-way valve 4 and the third three-way valve 2. At least one of the three-way valves 6 is injected with a balance liquid. Optionally, the liquid adding device 10 may include one or more of a peristaltic pump, a magnetic gear pump, a plunger pump, a syringe pump, and a syringe.

In addition to realizing the separation and purification of extracellular vesicles (such as exosomes) with different particle sizes through a double filtration mechanism, the filtration and separation device can also be equipped with a three-way valve and a liquid addition device for injecting a balance solution. Etc., balance liquid can be injected into the membrane filter as needed to clean the filter membrane in the membrane filter and collect extracellular vesicles and other particulate matter attached to the filter membrane without disassembling the filter separation device. , reassembly and other operations, the operation is simple and fast.

Specifically, 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 connected with the first three-way valve 2. The second end 22 of the through valve 2 is detachably connected (as shown in Figure 2); the outlet of the first membrane filter 3 can be detachably connected to the first end of the second three-way valve 4; the second membrane filter The inlet of 5 can be detachably connected with the second end of the second three-way valve 4; the outlet of the second membrane filter 5 can be detachably connected with the first end of the third three-way valve 6; The inlet can be detachably connected to the second end of the third three-way valve 6; the liquid addition device 10 can be connected to the third end 23 of the first three-way valve 2, the third end and the third end of the second three-way valve 4 At least one of the third ends of the three-way valve 6 .

The above connections can be made via standard Luer connectors. Optionally, the first three-way valve 2 , the second three-way valve 4 and the third three-way valve 6 may have the same structure or different structures. Each end of the first three-way valve 2 , the second three-way valve 4 and the third three-way valve 6 may be respectively provided with switches to individually close or open the corresponding ends.

In some embodiments according to the present disclosure, the pore sizes of the first filter membrane 31 and the second filter membrane 51 may be different from each other, so as to intercept, classify, and classify extracellular vesicles (eg, exosomes) with different particle sizes, respectively. Corresponding analysis, research and application are carried out after collection.

Alternatively, the filter membranes in the first membrane filter 3 and the second membrane filter 5 are uniform pore size anodized aluminum oxide membranes. As an inorganic membrane with a unique honeycomb structure, the porous anodic alumina thin film can be used for separation and purification due to its pore channels parallel and strictly perpendicular to the membrane surface, adjustable pore size (hundreds of nanometers to tenths of nanometers), and narrow distribution range. Purification of extracellular vesicles, especially isolation and purification of exosomes.

As an example, the pore size of the uniform pore size anodic aluminum oxide film of the first filter membrane 31 is in the range of 150 nm to 300 nm; the pore size of the uniform pore size anodic aluminum oxide film of the second filter film 51 is in the range of 30 nm to 150 nm . Preferably, the pore size of the anodic aluminum oxide film with uniform pore size of the first filter membrane 31 is 300 nm; the pore size of the anodic aluminum oxide film with uniform pore size of the second filter film 51 is 30 nm.

In some embodiments according to the present disclosure, the uniform pore size anodic aluminum oxide film protrudes downward, so that when the liquid is filtered through the uniform pore size anodic aluminum oxide film, impurities are easily gathered in the central area of the uniform pore size anodic aluminum oxide film , the alumina film will not be completely blocked. Liquid can also pass through the membrane at the peripheral portion of the anodized aluminium membrane to avoid complete clogging. Since the casings of the first membrane filter 3 and the second membrane filter 5 can be made of transparent materials, and most of the places where the membranes are blocked in the above solution are concentrated in the central area, it is easier for users to find out and deal with the problem in time.

When performing filtration separation using the filtration separation device according to the embodiment of the present disclosure, first, the first ends (upper ends) and the second ends of the first three-way valve 2 , the second three-way valve 4 , and the third three-way valve 6 are opened. end (lower end), close the third end (lateral end), and use the vacuum pump 8 to evacuate the air in the first container 7, and the vacuum pump 8 can be stopped when the air pressure in the first container 7 is lower than a predetermined value ; Then, open the outlet of the sampler 1, the liquid in the sampler 1 enters the first membrane filter 3 under the action of suction, and is filtered by the first filter membrane 31, preferably by an anodic aluminum oxide membrane with uniform pore size; after , the liquid that has been filtered for the first time flows through the second three-way valve 4 and enters the second membrane filter 5, and is filtered by the second filter membrane 51, preferably by an anodized aluminum oxide membrane with uniform aperture; The third three-way valve 6 enters the first container 7 . And since the pore size of the second filter membrane 51 is smaller than the pore size of the first filter membrane 31, extracellular vesicles (eg exosomes) with different particle sizes can be intercepted and classified, and corresponding analysis can be performed after collection. , research and application.

When cleaning the filtration and separation device according to the embodiment of the present disclosure and respectively collecting particulate matter such as extracellular vesicles attached to the first filter membrane 31 and the second filter membrane 51, one or more liquid addition devices may be added accordingly. The 10 is connected to at least one of the third end 23 of the first three-way valve 2 , the third end of the second three-way valve 4 , and the third end of the third three-way valve 6 .

For example, in order to clean the first membrane filter 3 and collect the particles attached to it, the user can connect the liquid adding device 10 to the third end 23 of the first three-way valve 2 after the filtration is completed, and adjust the first three-way valve 2 . Through valve 2 and second three-way valve 4, so that the sampler 1 and 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 leads to the filter separation device. Then the user can inject the balance liquid into the first three-way valve 2, and the balance liquid can flush out the particles attached to the first filter membrane 31, and the flushed liquid can be removed from the third end of the second three-way valve 4. discharge.

Preferably, in order to achieve better cleaning and collection effects, 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 can be adjusted so that the adding The sample device 1 and 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 leads to the outside of the filter separation device; The balancing liquid is injected into the first filter membrane 31 , and the balancing liquid can flush out the particles and the like attached to the first filter membrane 31 , and the flushed liquid can be discharged from the third end 23 of the first three-way valve 4 . Compared with the previous cleaning method, this cleaning method can realize the scouring of the first filter membrane 31 from bottom to top, and since most of the particles are located on the upper surface of the first filter membrane 31, better cleaning and cleaning can be achieved. Collection effects.

In the above manner, while cleaning the first filter membrane 31, the discharged liquid can be collected, so as to obtain biological materials suspended in the discharged liquid, such as extracellular vesicles or exosomes, and corresponding Analysis, Research and Application.

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 . In addition, while cleaning the second filter membrane 51, the discharged liquid can be collected, so as to obtain biological materials suspended in the discharged liquid, such as extracellular vesicles or exosomes, etc., and perform corresponding analysis after collection, research and application. Due to the difference in the pore size 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 is achieved. Moreover, since the present application uses a filter membrane instead of a filter column, compared with the filter column, the biological material is less easily attached to the filter membrane, which is convenient for users to obtain.

For example, in the case where the pore size of the anodic aluminum oxide film with uniform pore size of the first filter membrane 31 is 300 nm, and the pore size of the anodic aluminum oxide film with uniform pore size of the second filter film 51 is 30 nm. Collecting extracellular vesicles and the like attached to the first filter membrane 31 can obtain extracellular vesicles with a particle size greater than 300 nm, and collecting the extracellular vesicles attached to the second filter membrane 51 can obtain particle diameters of 30 nm and 300 nm. extracellular vesicles between nm. In the first container 7, extracellular vesicles with a particle size of less than 30 nm can be obtained. Afterwards, the corresponding analysis, research and application of extracellular vesicles of various particle sizes can be carried out.

It is also possible to clean the first filter membrane 31 and the second filter membrane 51 at the same time. And before the above cleaning operation, the various components of the filter and separation device can also be disassembled first.

In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. The indicated orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation or a specific orientation. construction and operation, and therefore should not be construed as limiting the invention.

In addition, the technical features involved in the different embodiments of the present invention described below can 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," "the," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will also be understood that the term "and/or" as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

In the present disclosure and the appended claims, "plurality" and "plurality", unless otherwise specified, refer to two or more.

Obviously, those skilled in the art can make various changes and modifications to the magnetic stand disclosed in the present invention without departing from the spirit and scope of the present invention. Thus, provided that these 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 these 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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