KR102444577B1 - apparatus for extracting extracellular vesicles - Google Patents

Abstract

Nano-vesicles extraction device according to an embodiment of the present invention is an inflow passage through which the mixed fluid containing the nano-vesicles is introduced; a first device in which a first inlet connected to the inflow passage and a first outlet connected to the first circulation passage are formed on an upper surface thereof, and a second inlet and a second outlet are formed on a lower surface thereof; a first membrane provided inside the first device and filtering the mixed fluid introduced into the first device; A third inlet connected to the second outlet and a third inlet connected to the second outlet are formed on an upper surface of the first device, and a third outlet connected to the second circulation passage is formed on the lower surface, and a fourth outlet is formed on the lower surface of the first device. a second device on which an addition is formed; a second membrane provided inside the second device and filtering the mixed fluid introduced into the second device; a first switching valve provided between the inflow passage and the first inlet and connecting the first circulation passage to the first inlet; and a second switching valve provided between the second outlet and the third inlet and connecting the second outlet to the extraction passage. includes

Description

Nano-vesicle extraction device {apparatus for extracting extracellular vesicles}

The present invention relates to a nano-vesicle extraction apparatus, and to a nano-vesicle extraction apparatus capable of rapidly extracting nano-vesicles from a mixed fluid containing nano-vesicles with high purity.

Nano-vesicles (Extracellular vesicles, EVs) are vesicles released by cells to the outside, and include microvesicles (MVs), apoptotic bodies, exosomes, and the like.

These nano-endoplasmic reticulum can be distinguished from each other according to their size and role, but they function as a transport medium for information exchange between cells. Nanoendoplasmic reticulum is derived from cells and acts to stimulate or inhibit the metabolism of other cells.

The nanovesicles reflect the approximate properties and state of the parent cells that release them. The substances that the nano-endoplasmic reticulum moves from the parent cell to other cells are bio-derived substances such as proteins, fats, metabolites, and nucleic acids (DNA/RNA), and these bio-derived substances are accepted It is transmitted to the cell (recipient cell) and performs the role of intercellular signal transduction.

Exosomes are one of the nano-endoplasmic reticulum, and among them, are the smallest vesicular particles with a diameter of about 30 ~ 200 nm, and serve to transport bio-derived substances including proteins. Exosomes have the same genetic and protein information as the parent cells, are structurally stable because they are packaged in a lipid membrane, and exist in abundant amounts in the blood to act as biomarkers.

If a nanovesicle extraction device capable of extracting nanovesicles such as exosomes that can act as such a biomarker is developed, it makes it possible to diagnose diseases from exosomes that have the same genetic and protein information as the parental cells, so a simple structure It is necessary to develop a nano-vesicle extraction device capable of extracting high-purity nano-ER.

Korea Unexamined Patent Publication No. 10-2018-0081354 (Korea University Industry-Academic Cooperation Foundation) 2018.07.16

The problem to be solved by the present invention is to provide a nano-vesicle extraction apparatus capable of rapidly extracting nano-vesicles with high purity from a mixed fluid containing nano-vesicles.

The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Nano-vesicle extraction apparatus according to an embodiment of the present invention for solving the above problems is an inflow passage through which the mixed fluid containing the nano-vesicles is introduced; a first device in which a first inlet connected to the inflow passage and a first outlet connected to the first circulation passage are formed on an upper surface thereof, and a second inlet and a second outlet are formed on a lower surface thereof; a first membrane provided inside the first device and filtering the mixed fluid introduced into the first device; A third inlet connected to the second outlet and a third inlet connected to the second outlet are formed on an upper surface of the first device, and a third outlet connected to the second circulation passage is formed on the lower surface, and a fourth outlet is formed on the lower surface of the first device. a second device on which an addition is formed; a second membrane provided inside the second device and filtering the mixed fluid introduced into the second device; a first switching valve provided between the inflow passage and the first inlet and connecting the first circulation passage to the first inlet; and a second switching valve provided between the second outlet and the third inlet and connecting the second outlet to the extraction passage. includes

In addition, the nano-endoplasmic reticulum may be implemented as an exosome.

In addition, the diameter of the pores of the first membrane may be 200 nm or less, and the diameter of the pores of the second membrane may be less than 30 nm.

In addition, the first membrane may permeate the nano-vesicles of the mixed fluid introduced into the first inlet, and non-permeate the first material of a predetermined size.

In addition, the nano-vesicles that have passed through the first membrane may flow to the second outlet.

In addition, the first material may be implemented as cell debris (cell debris).

In addition, the second membrane may be non-permeable to the nano-vesicles of the mixed fluid introduced into the third inlet, and to transmit a second material of a predetermined size.

In addition, the second material passing through the second membrane may flow to the fourth outlet.

In addition, the second material may be implemented as a serum protein.

In addition, the first device may include: a first upper cover in which the first inlet and the first outlet are formed, a first chamber is formed therein, and the first membrane is disposed under the first chamber; and the first upper cover disposed under the first upper cover, the second inlet portion and the second outlet portion are formed, a second chamber is formed therein, and the first membrane is disposed above the second chamber. 1Lower cover; may include.

The second device may include: a second upper cover having the third inlet and the third outlet formed therein, a third chamber formed therein, and the second membrane disposed under the third chamber; and a second lower cover disposed under the second upper cover, the fourth outlet portion is formed, a fourth chamber is formed therein, and the second membrane is disposed above the fourth chamber; may include.

In addition, the nano-vesicles may be collected in the second chamber and the third chamber.

In addition, a first circulation pump may be provided in the first circulation passage.

In addition, a second circulation pump may be provided in the second circulation passage.

The details of other embodiments are included in the detailed description and drawings.

According to the nano-vesicle extraction apparatus according to an embodiment of the present invention, impurities such as cell debris and serum proteins are filtered from the mixed fluid with a simple structure, and nano-vesicles containing exosomes of high purity can be extracted.

The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those of ordinary skill in the art from the description of the claims.

1 is a perspective view of a nano-vesicle extraction device according to an embodiment of the present invention.
Figure 2 is a schematic diagram of a nano-vesicle extraction device according to an embodiment of the present invention.
Figure 3 is a view showing embodiments of the nano-vesicle extraction apparatus according to an embodiment of the present invention.
4 is a DF mode (a), sTFF mode (b), dTFF mode (c) when operating in the second chamber 15 and the third chamber 34 The content of the nano-vesicles of the mixed fluid flow (intensity) is a graph for
5 is a SEM photograph of the nano-vesicles of the mixed fluid flowing in the second chamber 15 and the third chamber 34 during operation in DF mode (a), sTFF mode (b), and dTFF mode (c).
6 is a graph showing the serum protein concentration and the nano-vesicle concentration for each filtration step of the mixed fluid when operating in the dTFF mode.
7 is a graph showing the ratio of the number of exosome particles in each filtration step of the mixed fluid when operating in the dTFF mode.

In describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. Even if the terms are the same, if the indicated parts are different, it is to be said in advance that the reference numerals do not match.

In addition, the terms described below are terms set in consideration of functions in the present invention, which may vary according to the intentions or customs of operators such as experimenters and measurers, so the definitions should be made based on the content throughout this specification.

In this specification, terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component. and/or includes a combination of a plurality of related listed items or any of a plurality of related listed items.

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

In addition, when a part "includes" a certain component, this means that other components may be further included, rather than excluding other components, unless otherwise stated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.

1 is a perspective view of a nano-vesicle extraction device according to an embodiment of the present invention, Figure 2 is a schematic view of a nano-vesicle extraction device according to an embodiment of the present invention.

1 to 2, the nano-vesicle extraction apparatus according to an embodiment of the present invention is an inflow passage (i) through which the mixed fluid containing the nano-vesicles is introduced, the first connected to the inflow passage (i) on the upper surface A first outlet 11b connected to the first circulation passage S1 is formed in the inlet 11a and the first inlet 11a, and a second inlet 12a and a second outlet 11a are formed on the lower surface thereof. The first device 10, in which 12b) is formed, is provided in the first device 10 and filters the mixed fluid introduced into the first device 10. The first membrane 20, the first device ( 10), a third inlet 31a connected to the second outlet 12b on the upper surface and a third outlet connected to the second inlet 12a by a second circulation passage S2 31b is formed, the second device 30 having a fourth outlet 32b on its lower surface is provided inside the second device 30, and the mixed fluid introduced into the second device 30 is removed. The second membrane 40 for filtering, provided between the inflow passage (i) and the first inlet (11a), the first switching valve (S1) connecting the first inlet (11a) V1), and a second switching valve (V2) provided between the second outlet (12b) and the third inlet (31a) and connecting the second outlet (12b) to the extraction passage (e). .

Nano-vesicles (Extracellular vesicles, EVs) are vesicles that cells release to the outside of the cell as described above in the prior art, microvesicles (MVs), apoptotic bodies, exosomes, etc. include

In general, the diameter of microvesicles (MVs) is 50 ~ 1000 nm, the apoptotic body (apoptotic body) diameter of 50 ~ 5000 nm, the diameter of the exosomes can be formed in the 30 ~ 200 nm.

Hereinafter, the nano-endoplasmic reticulum according to an embodiment of the present invention is implemented as an exosome, but the embodiment is not limited thereto. When the nano-vesicles are implemented as exosomes according to an embodiment of the present invention, the diameter of the nano-vesicles may be implemented in a range of 30 to 200 nm.

The mixed fluid may be composed of distilled water, a culture medium, a phosphate buffer, or a mixture thereof. The mixed fluid contains nano-vesicles. The mixed fluid may include one or more microvesicles, apoptotic bodies, exosomes, serum proteins with a diameter smaller than the exosomes, cell debris larger than the exosomes, proteins and minerals contained in the culture medium.

The mixed fluid flows into the inflow passage (i). The mixed fluid may contain nano-vesicles as described above.

The first device 10 is connected to the inflow passage (i). In this case, a first switching valve V1 may be provided between the inflow passage i and the first inlet 11a. This will be described later.

In the first device 10, the mixed fluid introduced into the inflow passage (i) is filtered. The first device 10 flows the mixed fluid filtered in the first device 10 to a second device 30 to be described later.

The first device 10 has a first inlet portion 11a and a first outlet portion 11b formed on the upper surface. The first inlet 11a and the first outlet 11b may be formed to penetrate through the upper surface of the first device 10 . The first inlet 11a and the first outlet 11b may be connected to the first chamber 14 formed in the first device 10 .

The first inlet (11a) is connected to the inlet (i). In this case, the first inlet (11a) may be connected to the inlet (i) and the tube. Hereinafter, each flow path, an inlet part, an outlet part, and each switching valve to be described later will be described as being connected by a tube.

The mixed fluid is introduced into the first inlet (11a). A first switching valve V1 may be provided between the inflow passage i and the first inlet 11a.

The first outlet 11b may be connected to the first inlet 11a. In this case, the first outlet 11b may be connected to the first inlet 11a through the first circulation passage S1. At this time, the first circulation passage (S1) and the first inlet (11a) may be connected to the first switching valve (V1). This will be described later.

The first device 10 has a second inlet portion 12a and a second outlet portion 12b formed on a lower surface thereof. The second inlet portion 12a and the second outlet portion 12b may be formed to penetrate through the lower surface of the first device 10 . The second inlet 12a and the second outlet 12b may be connected to the second chamber 15 formed in the first device 10 .

The mixed fluid filtered by the first device 10 flows out of the second outlet 12b. The second outlet 12b may be connected to a third inlet 31a of the second device 30 to be described later. In addition, the second outlet (12b) may be connected to the extraction passage (e). In this case, the second outlet 12b and the third inlet 31a may be connected to the second switching valve V2. This will be described later.

The mixed fluid filtered by the second device 30 is introduced into the second inlet 12a. In this case, the second inlet 12a may be connected to the third outlet 31b of the second device 30 . In this case, the second inlet 12a may be connected to the third outlet 31b through the second circulation passage S2. This will be described later.

The first membrane 20 is provided inside the first device 10 . The first membrane 20 may be made of various materials. The first membrane 20 according to an embodiment of the present invention may be formed of a plastic material, preferably a PC (polycarbonate) material, but the embodiment is not limited thereto.

The first membrane 20 is replaceable after use. That is, when the first device 10 is used for a long time, deposits may be generated on the surface of the first membrane 20 and the filtration efficiency may be reduced. In this case, the first membrane 20 may be replaced.

The first membrane 20 filters the mixed fluid introduced into the first device 10 . The first membrane 20 may filter the mixed fluid introduced into the first device 10 through the first inlet 11a. In this case, the first membrane 20 may permeate or impermeate the materials contained in the mixed fluid according to the size of the diameter of the pore.

Here, the first membrane 20 according to an embodiment of the present invention may permeate the nano-vesicles of the mixed fluid introduced into the first inlet 11a, and may non-permeate the first material of a predetermined size.

The first membrane 20 permeates the nano-vesicles. That is, the diameter of the pore (pore) of the first membrane 20 may be formed to a size that can pass through the nano-endoplasmic reticulum is carried out to 200 nm or less. In this case, exosomes having a diameter of 200 nm or less, microvesicles, and serum proteins are transmitted through the pores of the first membrane 20 .

The first membrane 20 is non-permeable to the first material having a predetermined size. Here, the non-permeable first material of a predetermined size may be implemented as cell debris exceeding 200 nm in diameter among the materials contained in the mixed fluid.

That is, cell fragments contained in the mixed fluid and nano-vesicles such as apoptotic bodies and microvesicles exceeding 200 nm in diameter do not pass through the pores of the first membrane 20 and are non-permeable. Hereinafter, a non-permeable material that does not pass through the first membrane 20 is defined as a first material.

In this case, the nano-endoplasmic reticulum that has passed through the first membrane 20 flows to the second outlet 12b. Accordingly, the mixed fluid flowing from the second outlet 12b to the second device 30 may include nano-vesicles and not contain cell debris.

The first device 10 may be implemented in various ways. Here, in the first device 10 according to an embodiment of the present invention, a first inlet portion 11a and a first outlet portion 11b are formed, a first chamber 14 is formed therein, and the first A first upper cover 11 in which the first membrane 20 is disposed, a second inlet 12a and a second outlet 12b are formed in the lower portion of the chamber 14, and a second chamber 15 is formed therein. ) is formed, the first lower cover 12 in which the first membrane 20 is disposed above the second chamber 15, and the first upper cover 11 are disposed in the first chamber 14 and and a first seal 13 sealing an edge between the second chambers 15 .

The first upper cover 11 forms an exterior of the first device 10 . The first upper cover 11 is disposed on the first device 10 . A first inlet portion 11a and a first outlet portion 11b may be formed through the first upper cover 11 .

A first chamber 14 is formed inside the first upper cover 11 . The first chamber 14 may be formed by being depressed in the first upper cover 11 . The first chamber 14 is connected to the first inlet portion 11a and the first outlet portion 11b. In this case, the mixed fluid flows in the first chamber 14 .

The first membrane 20 is disposed under the first chamber 14 . The mixed fluid flowing in the first chamber 14 comes into contact with the first membrane 20 . In this case, as described above, the first membrane 20 filters the mixed fluid. The mixed fluid is filtered by the first membrane 20 in the direction of gravity by its own weight. At this time, the first membrane 20 may permeate the nano-vesicles of the mixed fluid, and may impermeate the cell debris.

The first lower cover 12 forms an exterior of the first device 10 . The first lower cover 12 is disposed under the first upper cover 12 . A second inlet portion 12a and a second outlet portion 12b may be formed through the first lower cover 12 .

A second chamber 15 is formed inside the first lower cover 12 . The second chamber 15 may be formed by being depressed in the first lower cover 12 . The first membrane is disposed above the second chamber 15 . In this case, the mixed fluid transmitted through the first membrane 20 in the first chamber 14 may flow into the second chamber 15 .

The second chamber 15 is connected to the second outlet 12b and the second inlet 12a. In this case, the mixed fluid flowing in the second chamber 15 may flow to the second outlet 12b. Also, the mixed fluid flowing through the second inlet 12a may flow into the second chamber 15 .

The first sealing 13 may be disposed on the first upper cover 11 . The first seal 13 may be formed of a rubber material or a polymer elastic material. The first seal 13 may seal an edge between the first chamber 14 and the second chamber 15 . Accordingly, in the first seal 13 , the mixed fluid flowing in the first chamber 14 and the second chamber 15 flows out between the first chamber 14 and the second chamber 15 to the first upper It is possible to prevent leakage through the gap between the cover 11 and the first lower cover 12 .

The first switching valve V1 may be provided between the inflow passage i and the first inlet 11a. In addition, the first switching valve (V1) may be provided between the first circulation passage (S1) and the first inlet (11a).

Here, the first switching valve V1 according to an embodiment of the present invention may be implemented as a three-way valve. In this case, the first switching valve V1 may connect the first circulation passage S1 to the first inlet 11a.

That is, the first switching valve (V1) connects the inflow passage (i) to the first inlet (11a) and blocks the first circulation passage (S1), or connects the first circulation passage (S1) to the first inlet ( 11a) and block the inflow path (i).

Here, the first circulation pump (P1) may be provided in the first circulation passage (S1). The first circulation pump (P1) flows the mixed fluid discharged from the first outlet (11b) to the first circulation passage (S1). At this time, the mixed fluid flowing into the first circulation passage (S1) may be re-introduced into the first inlet (11a) through the first switching valve (V1).

Accordingly, after the mixed fluid is filtered in the first membrane 20 through the first inlet 11a and the first chamber 14, the first outlet 11b, the first circulation pump P1, and the first Since the circulation passage (S1), the first switching valve (V1), the first inlet (11a) is circulated and re-filtered in the first membrane (20), the filtration efficiency is improved, and the purity of the nano-vesicles is improved (purity) be able to do

The second device 30 is stacked under the first device 10 . The second device 30 is connected to the first device 10 . The second device 30 may be connected to the first device 10 by a tube.

In the second device 30 , the mixed fluid flowing in the first device 10 is filtered. The second device 30 flows the mixed fluid filtered in the second device 30 to the outlet flow path o.

The second device 30 has a third inlet portion 31a and a third outlet portion 31b formed on its upper surface. The third inlet 31a and the third outlet 31b may be formed to penetrate through the upper surface of the second device 30 . The third inlet 31a and the third outlet 31b may be connected to the third chamber 34 formed in the second device 30 .

The third inlet 31a is connected to the second outlet 12b. In this case, the mixed fluid that has passed through the first membrane 20 flows into the third inlet 31a. A second switching valve V2 may be provided between the second outlet 12b and the third inlet 31a. This will be described later.

The mixed fluid filtered by the second device 30 flows out of the third outlet 31b. The third outlet 31b may be connected to the second inlet 12a of the first device 10 . In this case, the third outlet 31b may be connected to the second inlet 12a through the second circulation passage S2. This will be described later.

A fourth outlet 32b is formed on the lower surface of the second device 30 . The fourth outlet 32b may be formed to penetrate through the lower surface of the second device 30 . The fourth outlet 32b may be connected to the outlet passage o. The mixed fluid filtered by the second device 30 flows out into the outflow passage o.

The second membrane 40 is provided inside the second device 30 . The second membrane 40 may be made of various materials. The first membrane 20 according to an embodiment of the present invention may be formed of a plastic material, preferably a PC (polycarbonate) material, but the embodiment is not limited thereto.

The second membrane 40 is replaceable after use. That is, when the second device 30 is used for a long time, the filtration efficiency may be reduced due to the formation of deposits on the surface of the second membrane 40 . In this case, the second membrane 40 may be replaced.

The second membrane 40 filters the mixed fluid introduced into the second device 30 . The second membrane 40 may filter the mixed fluid introduced into the second device 30 through the third inlet 31a. In this case, the second membrane 40 may permeate or impermeate the materials contained in the mixed fluid according to the size of the diameter of the pore.

Here, the second membrane 40 according to an embodiment of the present invention may impermeate the nano-vesicles of the mixed fluid introduced into the third inlet 31a, and transmit a second material of a predetermined size.

The second membrane 40 is non-permeable to the nano-vesicles. That is, the diameter of the pore (pore) of the second membrane 40 is implemented to be less than 200 nm may be formed in a size that the nano-vesicles cannot pass through. In this case, in this case, the exosomes and microvesicles having a diameter of 30 nm or more do not pass through the pores of the second membrane 40 and thus are non-permeable.

The second membrane 40 transmits a second material having a predetermined size. Here, the permeated second material having a predetermined size includes a serum protein having a diameter of less than 30 nm among materials contained in the mixed fluid.

That is, serum proteins and other minerals having a diameter of less than 30 nm pass through the pores of the second membrane 40 . Hereinafter, a material passing through the second membrane 40 is defined as a second material.

In this case, the second material passing through the second membrane 40 flows to the fourth outlet 32b. Accordingly, the mixed fluid flowing out from the fourth outlet 32b to the outflow path o may contain serum protein, which is the second material, and may not contain the nano-endoplasmic reticulum.

The second device 30 may be implemented in various ways. Here, in the second device 30 according to an embodiment of the present invention, a third inlet 31a and a third outlet 31b are formed, a third chamber 34 is formed therein, and the third A second upper cover 31 in which the second membrane 40 is disposed and a fourth outlet 32b are formed under the chamber 34, and a fourth chamber 35 is formed therein, and the fourth chamber The second lower cover 32 , in which the second membrane 40 is disposed on the upper part of the 35 , and the second upper cover 31 , are disposed between the third chamber 34 and the fourth chamber 35 . and a second seal 33 sealing the rim.

The second upper cover 31 forms an exterior of the second device 30 . The second upper cover 31 is disposed on the second device 30 . In this case, the second upper cover 31 is disposed under the first lower cover 12 . A third inlet 31a and a third outlet 31b may be formed through the second upper cover 31 .

A third chamber 34 is formed inside the second upper cover 31 . The third chamber 34 may be formed by being depressed in the second upper cover 31 . The third chamber 34 is connected to the third inlet 31a and the third outlet 31b. In this case, the mixed fluid flows in the third chamber 34 . At this time, the mixed fluid flowing in the third chamber 34 may include nano-vesicles, and may not include cell debris, which is the first material.

A second membrane 40 is disposed below the third chamber 34 . The mixed fluid flowing in the third chamber 34 comes into contact with the second membrane 40 . In this case, as described above, the second membrane 40 filters the mixed fluid. The mixed fluid is filtered by the second membrane 40 in the direction of gravity by its own weight. At this time, the second membrane 40 may not permeate the nano-vesicles of the mixed fluid and permeate the serum protein.

The second lower cover 32 forms an exterior of the second device 30 . The second lower cover 32 is disposed under the second upper cover 31 . The second lower cover 32 may be formed through a fourth outlet 32b.

A fourth chamber 35 is formed inside the second lower cover 32 . The fourth chamber 35 may be formed by being depressed in the second lower cover 32 . The second membrane 40 is disposed above the fourth chamber 35 . In this case, the mixed fluid transmitted through the second membrane 40 in the third chamber 34 may flow into the fourth chamber 35 .

The fourth chamber 35 is connected to the fourth outlet 32b. In this case, the mixed fluid flowing in the fourth chamber 35 may flow to the fourth outlet 32b. The mixed fluid flowing to the fourth outlet 32b flows out into the outlet passage o.

Here, the mixed fluid flowing into the outflow path (o) may contain the second material, serum protein, and may not contain the nano-vesicles. That is, since the nano-vesicles are non-permeable in the second membrane 40 and the serum protein, which is the second material, is permeated, the mixed fluid flowing into the fourth chamber 35 does not contain the nano-vesicles, but only the serum protein.

The second sealing 33 may be disposed on the second upper cover 31 . The second seal 33 may be formed of a rubber material or a polymer elastic material. The second seal 33 may seal the edge between the third chamber 34 and the fourth chamber 35 . Accordingly, in the second seal 33 , the mixed fluid flowing in the third chamber 34 and the fourth chamber 35 flows out between the third chamber 34 and the fourth chamber 35 to the second upper It is possible to prevent leakage through the gap between the cover 31 and the second lower cover 32 .

As described above, when the mixed fluid is filtered in the first device 10 and the second device 30 , the nano-vesicles may be collected in the second chamber 15 and the third chamber 34 .

That is, the mixed fluid introduced into the first chamber 14 is transmitted only through the nano-vesicles by the first membrane 20, and cell debris is not permeated, so that the second chamber 15 contains the nano-vesicles. is moved to

In addition, since the mixed fluid flowing from the second chamber 15 to the third chamber 34 is impermeable to the nano-vesicles by the second membrane 40 and only the serum protein is permeated, it is prepared in a state containing the nano-vesicles. It flows in three chambers (34).

In this case, the nano-ER is collected in the second chamber 15 of the first device 10 and the third chamber 34 of the second device 30 . Accordingly, it is possible to extract the mixed fluid containing the nano-vesicles of high purity through the extraction passage (e), as will be described later.

On the other hand, the first switching valve (V1) may be provided between the inflow passage (i) and the first inlet (11a). In addition, the first switching valve (V1) may be provided between the first circulation passage (S1) and the first inlet (11a).

Here, the first switching valve V1 according to an embodiment of the present invention may be implemented as a three-way valve. In this case, the first switching valve V1 may connect the first circulation passage S1 to the first inlet 11a.

That is, the first switching valve (V1) connects the inflow passage (i) to the first inlet (11a) and blocks the first circulation passage (S1), or connects the first circulation passage (S1) to the first inlet ( 11a) and block the inflow path (i).

In this case, the first circulation pump P1 may be provided in the first circulation passage S1. The first circulation pump (P1) flows the mixed fluid discharged from the first outlet (11b) to the first circulation passage (S1). At this time, the mixed fluid flowing into the first circulation passage (S1) may be re-introduced into the first inlet (11a) through the first switching valve (V1).

Accordingly, after the mixed fluid is filtered in the first membrane 20 through the first inlet 11a and the first chamber 14, the first outlet 11b, the first circulation pump P1, and the first Since the circulation passage (S1), the first switching valve (V1), the first inlet (11a) is circulated and re-filtered in the first membrane (20), the filtration efficiency is improved, and the purity of the nano-vesicles is improved (purity) be able to do

Meanwhile, as described above, the third outlet 31b may be connected to the second inlet 12a through the second circulation passage S2. In this case, a second circulation pump P2 may be provided in the second circulation passage S2. The second circulation pump (P2) flows the mixed fluid discharged from the third outlet (31b) to the second circulation passage (S2). At this time, the mixed fluid flowing into the second circulation passage S2 may be re-introduced into the second inlet 12a.

Accordingly, the mixed fluid is filtered in the second membrane 40 and then circulated to the third outlet 31b, the second circulation pump P2, the second circulation passage S2, and the second inlet 12a. Then, it flows to the second outlet 12b together with the mixed fluid filtered in the first membrane 20 and is re-filtered in the second membrane 40, so that the filtration efficiency is improved, the purity of the nano-vesicles be able to improve

Meanwhile, the second switching valve V2 may be provided between the second outlet 12b and the third inlet 31a. In addition, the second switching valve (V2) may be provided between the second outlet (12b) and the extraction passage (e).

Here, the second switching valve V2 according to an embodiment of the present invention may be implemented as a 3-way valve. In this case, the second switching valve V2 may connect the second outlet 12b to the extraction passage e.

At this time, the second switching valve (V2) connects the second outlet (12b) to the extraction passage (e) and blocks the third inlet (31a), or connects the second outlet (12b) to the third inlet ( 31a), and the extraction passage (e) may be blocked.

In this case, as described above, the nano-vesicles collected in the second chamber 15 and the third chamber 34 may be extracted to the outside through the extraction passage (e). Accordingly, it is possible to extract the mixed fluid containing the nano-vesicles of high purity through the extraction passage (e).

In addition, the pore size of the first membrane 20 and/or the second membrane 40 may be different and used. In this case, a material having a desired size may be filtered or extracted using a membrane having a desired pore size.

Figure 3 is a view showing the embodiments of the nano-vesicle extraction apparatus according to an embodiment of the present invention, Figure 3 (a) is the first circulation pump (P1) and the second circulation pump (P2) does not operate DF (direct filtration) mode, Figure 3 (b) is a sTFF (single tangential flow filtration) mode for operating the second circulation pump (P2), Figure 3 (c) is the first circulation pump (P1) and the second It is an operation diagram of the dTFF (double tangential flow filtration) mode operating the circulation pump (P2).

4 is a DF mode (a), sTFF mode (b), dTFF mode (c) when operating in the second chamber 15 and the third chamber 34 The content of the nano-vesicles of the mixed fluid flow (intensity) is a graph for

5 is a SEM photograph of the nano-vesicles of the mixed fluid flowing in the second chamber 15 and the third chamber 34 when operating in DF mode (a), sTFF mode (b), and dTFF mode (c), FIG. 6 is a graph showing the serum protein concentration and nano-vesicle concentration for each filtration step of the mixed fluid when operating in the dTFF mode, and FIG. 7 is a graph showing the ratio of the number of exosome particles in the filtration step of the mixed fluid when operating in the dTFF mode.

Hereinafter, with reference to FIGS. 3 to 7, the operation and effect of the nano-vesicle extraction apparatus of the present invention will be described.

The DF mode (FIG. 3(a)) is an operation mode in which the first circulation pump P1 and the second circulation pump P2 are not operated. Here, C in FIG. 3 denotes cell debris, EVs denotes nano-endoplasmic reticulum, and P denotes serum proteins.

In the DF mode, the mixed fluid is filtered by its own weight and is not circulated to the first device 10 and the second device 30 . In this case, the mixed fluid introduced into the inflow passage (i) flows into the first inlet (11a). The first switching valve V1 connects the inflow passage i to the first inlet 11a.

The mixed fluid is filtered by the first membrane 20 . In this case, the cell debris is impermeable to the first membrane 20 and remains in the first chamber 14 . The nano-ER and serum proteins penetrate the first membrane 20 and flow into the second chamber 15 .

The mixed fluid flowing into the second chamber 15 flows out to the second outlet 12b. In this case, the second switching valve V2 may connect the second outlet 12b to the third inlet 31a and not to the extraction passage e.

The mixed fluid introduced into the third inlet 31a flows into the third chamber 34 . The mixed fluid flowing in the third chamber 34 is filtered by the second membrane 40 . In this case, the nano-endoplasmic reticulum is non-permeable to the second membrane 40 and remains in the second chamber 15 . The serum protein passes through the second membrane 40 and flows into the fourth chamber 35 .

The mixed fluid containing the serum protein flowing into the fourth chamber 35 flows out to the fourth outlet 32b.

Then, as described above, the second switching valve (V2) operates to extract the nano-vesicles collected in the second chamber 15 and the third chamber 34 to the outside through the extraction passage (e).

At this time, when the mixed fluid collected in the second chamber 15 and the third chamber 34 is analyzed, as shown in FIG. It appears that the second material smaller in size and the first material larger than the exosome size are also found.

In this case, it is analyzed that the cell debris having a size of 200 nm or more is pressed into the first membrane 20 in the second chamber 15 by the action of its own weight, and has passed through the pores of the first membrane 20 . When observing the SEM (electron microscope) picture as shown in (a) of FIG. 5, this It is observed to accumulate after passing through the pores.

The sTFF mode (FIG. 3 (b)) is an operation mode in which the first circulation pump P1 does not operate and only the second circulation pump P2 operates. In the sTFF mode, the mixed fluid is filtered by its own weight and the mixed fluid of the second device 30 is circulated to the second circulation passage S2.

In this case, the mixed fluid introduced into the inflow passage (i) flows into the first inlet (11a). The first switching valve V1 connects the inflow passage i to the first inlet 11a.

The mixed fluid is filtered by the first membrane 20 . In this case, the cell debris is impermeable to the first membrane 20 and remains in the first chamber 14 . The nano-ER and serum proteins penetrate the first membrane 20 and flow into the second chamber 15 .

The mixed fluid flowing into the second chamber 15 flows out to the second outlet 12b. In this case, the second switching valve V2 may connect the second outlet 12b to the third inlet 31a and not to the extraction passage e.

The mixed fluid introduced into the third inlet 31a flows into the third chamber 34 . The mixed fluid flowing in the third chamber 34 is filtered by the second membrane 40 . In this case, the nano-endoplasmic reticulum is non-permeable to the second membrane 40 and remains in the second chamber 15 . The serum protein passes through the second membrane 40 and flows into the fourth chamber 35 .

The mixed fluid containing the serum protein flowing into the fourth chamber 35 flows out to the fourth outlet 32b.

In addition, the mixed fluid flowing into the third chamber 34 may flow into the second circulation passage S2 and may be introduced into the second inlet 12a and re-introduced into the second chamber 15 .

In this case, the mixed fluid flows to the second outlet 12b together with the mixed fluid filtered in the first membrane 20 and is re-filtered in the second membrane 40 .

Then, as described above, the second switching valve (V2) operates to extract the nano-vesicles collected in the second chamber 15 and the third chamber 34 to the outside through the extraction passage (e).

At this time, when the mixed fluid collected in the second chamber 15 and the third chamber 34 is analyzed, as shown in (b) of FIG. 4 , the first material larger than the exosome size is not found. When observing the SEM (electron microscope) photograph as shown in FIG. 5B, turbulence flow occurs in the second chamber 15 by the second circulation passage S2, and Since the pressure action is reduced, it is observed that the cell debris does not pass through the pores of the first membrane 20 because the cell debris is not momentarily compressed.

Accordingly, the mixed fluid containing the nano-vesicles of high purity present in the second chamber 15 and the third chamber 34 can be extracted to the outside through the extraction passage (e).

The dTFF mode (FIG. 3(c)) is an operation mode in which both the first circulation pump P1 and the second circulation pump P2 are operated. In the dTFF mode, the mixed fluid is filtered by its own weight, and the mixed fluid of the first device 10 and the second device 30 is circulated to the first circulation passage S1 and the second circulation passage S2.

In this case, the mixed fluid introduced into the inflow passage (i) flows into the first inlet (11a). The first switching valve V1 connects the inflow passage i to the first inlet 11a.

The mixed fluid is filtered by the first membrane 20 . In this case, the cell debris is impermeable to the first membrane 20 and remains in the first chamber 14 . The nano-ER and serum proteins penetrate the first membrane 20 and flow into the second chamber 15 .

At this time, the mixed fluid remaining in the first chamber 14 flows into the first circulation passage S1 by the first circulation pump P1. In this case, the first switching valve V1 may connect the first circulation passage S1 to the first inlet 11a and block the inflow passage i.

Accordingly, after the mixed fluid is filtered in the first membrane 20 through the first inlet 11a and the first chamber 14, the first outlet 11b, the first circulation pump P1, and the first It is circulated through the circulation passage S1, the first switching valve V1, and the first inlet 11a, and is re-filtered in the first membrane 20.

The mixed fluid flowing into the second chamber 15 flows out to the second outlet 12b. In this case, the second switching valve V2 may connect the second outlet 12b to the third inlet 31a and not to the extraction passage e.

The mixed fluid introduced into the third inlet 31a flows into the third chamber 34 . The mixed fluid flowing in the third chamber 34 is filtered by the second membrane 40 . In this case, the nano-endoplasmic reticulum is non-permeable to the second membrane 40 and remains in the second chamber 15 . The serum protein passes through the second membrane 40 and flows into the fourth chamber 35 .

The mixed fluid containing the serum protein flowing into the fourth chamber 35 flows out to the fourth outlet 32b.

In addition, the mixed fluid flowing into the third chamber 34 flows into the second circulation passage S2 by the second circulation pump P2 and flows into the second inlet 12a, the second chamber 15 . can be reintroduced into

In this case, the mixed fluid flows to the second outlet 12b together with the mixed fluid filtered in the first membrane 20 and is re-filtered in the second membrane 40 .

Then, as described above, the second switching valve (V2) operates to extract the nano-vesicles collected in the second chamber 15 and the third chamber 34 to the outside through the extraction passage (e).

At this time, when the mixed fluid collected in the second chamber 15 and the third chamber 34 is analyzed, the first material larger than the exosome size is not found as shown in FIG. It appears that the content of the endoplasmic reticulum (intensity) is greatly improved. This is shown in Fig. 5 (c), when observing the SEM (electron microscope) picture as shown, the degree of integration of the nano-vesicles appears to be greatly increased.

In dTFF mode operation, referring to FIGS. 6 and 7 , the unfiltered mixed fluid (PF), which is the state before the inflow of the inflow passage (i), for each filtration time point of the mixed fluid, the mixed fluid in the first chamber 14 (C1), the second When the mixed fluid (C2) in the second chamber (15) and the third chamber (34) and the mixed fluid (C3) in the fourth chamber (35) are observed separately by BCA (bicinchoninic acid protein assay), a protein measurement method, serum protein (P) is the largest in C1 after filtration starts, except for PF, which is in the unfiltered mixed fluid state, and is finally filtered and a large amount is discharged from C3. Nano-vesicles (EVs) appear to have the highest content in C2.

In this case, the nano-ER content ratio to serum protein (EVs/protein, %) was 0.2% in PF, 5.44% in C1, 23.98% in C2, and 0.07% in C3, and the nano-ER in the second chamber (15) and the second chamber (15). It appears that the largest amount is collected in the mixed fluid of the 3 chamber (34).

In this case, the content of nano-ER is very low in the mixed fluid flowing out into the outflow path (o), which indicates that the filtration of the nano-ER is effectively performed, and the second chamber 15 and the third chamber 34 corresponding to C2 have high purity Since it appears that the mixed fluid containing the nano-vesicles is present, it is possible to extract high-purity nano-vesicles through the extraction passage (e).

Above, those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The scope of the present invention is indicated by the claims described below rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. should be interpreted

10: first device 20: first membrane
30: second device 40: second membrane

Claims (14)

an inflow passage through which the mixed fluid containing the nano-vesicles is introduced; a first device in which a first inlet connected to the inflow passage and a first outlet connected to the first circulation passage are formed on an upper surface thereof, and a second inlet and a second outlet are formed on a lower surface thereof; a first membrane provided inside the first device and filtering the mixed fluid introduced into the first device; A third inlet connected to the second outlet and a third inlet connected to the second outlet are formed on an upper surface of the first device, and a third outlet connected to the second circulation passage is formed on the lower surface, and a fourth outlet is formed on the lower surface of the first device. a second device on which an addition is formed; a second membrane provided inside the second device and filtering the mixed fluid introduced into the second device; a first switching valve provided between the inflow passage and the first inlet and connecting the first circulation passage to the first inlet; and a second switching valve provided between the second outlet and the third inlet and connecting the second outlet to the extraction passage. including,
The first device may include: a first upper cover in which the first inlet and the first outlet are formed, a first chamber is formed therein, and the first membrane is disposed under the first chamber; a first disposed under the first upper cover, the second inlet portion and the second outlet portion are formed, a second chamber is formed therein, and the first membrane is disposed above the second chamber lower cover; and a first seal disposed on the first upper cover to seal an edge between the first chamber and the second chamber. including,
A first circulation pump is provided in the first circulation passage, and a second circulation pump is provided in the second circulation passage,
The first membrane permeates the nano-vesicles of the mixed fluid introduced into the first inlet, and non-permeates cell debris of a predetermined size,
The second membrane impermeates the nano-vesicles of the mixed fluid introduced into the third inlet, and permeates serum proteins of a predetermined size,
The serum protein that has passed through the second membrane flows to the fourth outlet, and the nano-vesicles that have passed through the first membrane flow into the second outlet.
According to claim 1,
The nano-endoplasmic reticulum is a nano-vesicle extraction device that is an exosome.
According to claim 1,
The diameter of the pores of the first membrane is 200 nm or less, and the diameter of the pores of the second membrane is less than 30 nm.
delete delete delete delete delete delete delete According to claim 1,
The second device is
a second upper cover in which the third inlet and the third outlet are formed, a third chamber is formed therein, and the second membrane is disposed under the third chamber; and
a second lower cover disposed under the second upper cover, the fourth outlet portion is formed, a fourth chamber is formed therein, and the second membrane is disposed above the fourth chamber;
Nano-vesicle extraction device comprising a.
12. The method of claim 11,
The nano-vesicles is a nano-vesicle extraction device that is collected in the second chamber and the third chamber.
delete delete

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