Exosome separation and purification device

Abstract

The utility model relates to an exosome separation and purification device. The exosome separation and purification device comprises a sample input component, a sample output component and a separation component, wherein the sample input component is used for receiving to-be-separated extracellular vesicle samples; the viscoelastic fluid processing assembly is connected with the sample input assembly and comprises a fluid channel for conveying an extracellular vesicle sample and a filling area for filling viscoelastic fluid; and the separating and collecting assembly is used for receiving the exosome obtained by separating the extracellular vesicle sample. Compared with the prior art, the device has the advantages that the exosome can be efficiently and accurately separated and purified through a simple operation process, the yield of the exosome is improved, and the like.

Description

Technical Field

[0001] The utility model relates to the technical field of exosome separation, and in particular to an exosome separation and purification device. Background Art

[0002] Extracellular vesicles are a general term for various membrane-containing vesicle structures released by cells, which are widely present in body fluids such as blood, saliva, and urine. Extracellular vesicles include different subgroups such as exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles play a vital role in the life activities of cells. Exosomes are relatively small extracellular vesicles with a diameter of about 50-150nm. They carry the genetic information and proteins of the mother cells and play an important role in intercellular communication. They are potential diagnostic biomarkers and therapeutic carriers for a variety of diseases, including cancer, infectious diseases, and neurodegenerative diseases. Although exosomes are present in large quantities in body fluids such as blood, urine, synovial fluid, and saliva, the presence of several other types of vesicles in biological fluids often affects the accurate analysis of exosomes.

[0003] In order to better analyze the rich information carried by exosomes, it is necessary to accurately separate and purify exosomes from a wide variety of extracellular vesicles. Due to their tiny size and the complex characteristics of biological fluids, the separation of exosomes faces major challenges. At present, conventional batch technologies for exosome separation mainly include ultracentrifugation, density-based separation, and immunoaffinity capture. These methods are mainly based on the size of exosomes, floating density, and specific marker proteins present on the membrane to achieve separation. However, it consumes a large amount of samples, the operation process is cumbersome, and it needs to rely on expensive instruments and equipment. These shortcomings often lead to low recovery rates of exosomes, easy contamination by other extracellular vesicles, and high costs.

[0004] Patent publication number CN105772116B discloses a system for focusing and separating micro-nano particles and cells based on non-Newtonian effect, including: a working fluid with non-Newtonian fluid effect; a working fluid delivery device for delivering a working fluid with non-Newtonian fluid effect; a microfluidic unit including a microchannel, which is arranged in parallel on the working fluid delivery device, and the microfluidic unit is connected to the working fluid delivery device through a pipeline, and is used to focus or separate the micro-nano particles or cells delivered by the working fluid delivery device in the microchannel; an outlet is arranged at the end of the microfluidic unit, and is used to collect target micro-nano particles or cells; when the outlet is used for focusing, no bifurcation outlet is set; when the outlet is used for separation, the outlet sets the number and width of bifurcation outlets according to the separation characteristics, so that micro-nano particles or cells of different sizes flow into different bifurcation outlets. However, this system only uses non-Newtonian fluids to design the separation of micro-nano particles or cells, and the characteristics of different nanoparticles or cells themselves will greatly affect the separation effect. The characteristics of the separation substance itself are not considered in the device, which may cause the death of the separated cells, low activity, or loss of function of other biological samples. In addition, the separation accuracy cannot be guaranteed without subdivision. Utility Model Content

[0005] The purpose of the present invention is to provide an exosome separation and purification device in order to overcome the defects of the above-mentioned prior art. Through a simple operation process, the exosomes can be separated and purified efficiently and accurately, thereby improving the yield of exosomes.

[0006] The purpose of the utility model can be achieved through the following technical solutions:

[0007] An exosome separation and purification device, comprising:

[0008] a sample input assembly for receiving an extracellular vesicle sample to be isolated;

[0009] a viscoelastic fluid processing component connected to the sample input component, comprising a fluid channel for conveying the extracellular vesicle sample and a filling area filled with viscoelastic fluid;

[0010] and a separation and collection component for receiving the exosomes separated from the extracellular vesicle sample.

[0011] Furthermore, the sample input component includes a sample inlet for inputting the extracellular vesicle sample to be separated, and a buffer inlet for inputting a fluid buffer.

[0012] Furthermore, the sample inlet is connected to a first feeding controller, and the buffer inlet is connected to a second feeding controller. The suspended nanoparticles of different sizes in the extracellular vesicle sample are arranged around the side wall of the fluid channel and controlled by the first feeding controller and the second feeding controller, and driven toward the center line of the fluid channel at different lateral speeds to achieve separation of exosomes in the extracellular vesicle sample. The fluid channel can achieve selective collection of exosomes of different sizes by adjusting parameters such as flow ratio, flow rate, and collection time. The flow characteristics of the fluid, such as inertial force, viscosity, surface tension, etc., are used to achieve the movement and positioning of particles.

[0013] Furthermore, the sample inlet input end is provided with a filter membrane, which can directly separate high-purity extracellular vesicles from cell supernatant and serum.

[0014] Furthermore, a coating is provided on the inner surface of the fluid channel. When no coating is provided, general materials often severely adsorb exosomes. In view of this characteristic, a coating is provided on the inner surface of the fluid channel to reduce the loss of exosomes.

[0015] Furthermore, the sample inlet and the buffer inlet are parallel.

[0016] Furthermore, the sample inlet depth is greater than the buffer inlet depth.

[0017] Furthermore, the sample input component is used to receive an extracellular vesicle sample to be separated, and process the sample to separate exosomes with a diameter of 50 to 150 nm.

[0018] Furthermore, the extracellular vesicle sample to be separated, such as culture medium and serum, is put into the sample input component from the sample inlet after simple pretreatment, such as centrifugation and filtration, to remove impurities and large particles, and at the same time, the fluid buffer is connected from the buffer inlet to allow the liquid mixture to flow to the viscoelastic fluid processing component. The exosomes in the sample are subjected to the elastic lift force related to the particle size in the filling area filled with the viscoelastic fluid, thereby achieving the separation of other extracellular vesicles of different sizes from the exosomes.

[0019] Furthermore, the fluid channel is linear.

[0020] Furthermore, the cross-sectional shape of the fluid channel is circular or rectangular.

[0021] Furthermore, the separation and collection assembly comprises:

[0022] an exosome separation channel connected to the side wall of the fluid channel;

[0023] an exosome collection pool connected to the exosome separation channel;

[0024] and a waste liquid recovery pool connected to the middle of the fluid channel.

[0025] Furthermore, the waste liquid recovery pool is located inside the exosome separation channel, and there is no circulation inside the channel.

[0026] Furthermore, the exosome separation channel is rectangular, parallelogram, rhombus or regular quadrilateral.

[0027] Furthermore, the exosome collection pool is parallel to the waste liquid recovery pool.

[0028] Furthermore, the filter membrane is nanoscale.

[0029] As a novel and simple label-free particle manipulation technology, viscoelastic fluids rely on the particle migration phenomenon caused by the correlation between particle size and elastic lift in viscoelastic media. This technology has been proven to have significant effects in separating tumor cells, blood cells, bacteria, droplets and microspheres. Compared with other separation technologies that rely on electrical and magnetic methods, viscoelastic fluids can achieve continuous manipulation of particles without relying on any externally applied electrical or magnetic fields. When applied to a specific target substance, the physical properties of the species itself need to be considered. Exosomes are newer biological samples than these samples. Conventional fluid materials cannot be directly used for exosomes and need to be re-determined.

[0030] Compared with the prior art, the utility model has the following advantages:

[0031] A separation device designed for extracellular vesicles. Commonly used exosome extraction methods such as density gradient centrifugation require cumbersome preparatory work, complex and time-consuming operations, ultrafiltration is time-consuming and inefficient, and tangential flow separation of exosomes requires expensive equipment. The exosome separation and purification device provided by the utility model can simplify the exosome extraction process. Through a simple operation process, it can efficiently and accurately separate and purify exosomes, improve the yield of exosomes, and effectively separate exosomes of a certain size from cell culture media, thereby achieving efficient and accurate separation of exosomes from other extracellular vesicles, and providing reliable tools and methods for the research and application of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the main body of the exosome separation and purification device shown in Example 1;

[0033] Figure 2 It is a cross-sectional view of the exosome separation and purification device shown in Example 1;

[0034] Figure 3 It is a front view of the exosome separation and purification device shown in Example 1;

[0035] Figure 4 This is a top view of the exosome separation and purification device shown in Example 1.

[0036] Description of the markings in the figure:

[0037] 1-sample input assembly, 11-sample inlet, 12-buffer inlet;

[0038] 2- viscoelastic fluid handling component, 21- fluid channel, 22- filling area;

[0039] 3-separation and collection component, 31-exosome separation channel, 32-exosome collection pool, 33-waste liquid recovery pool. DETAILED DESCRIPTION

[0040] The utility model is described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments are implemented based on the technical solution of the utility model, and provide detailed implementation methods and specific operation processes, but the protection scope of the utility model is not limited to the following embodiments. In the following implementation methods or embodiments, if there is no special description of the functional components or structures, it indicates that they are all conventional components or conventional structures used in the field to achieve the corresponding functions.

[0041] It should be noted that in the description of the present invention, the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0042] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0043] Example 1

[0044] An exosome separation and purification device, such as Figures 1 to 4As shown, including:

[0045] A sample input component 1 for receiving an extracellular vesicle sample to be separated;

[0046] The viscoelastic fluid processing component 2 connected to the sample input component 1 includes a fluid channel 21 for conveying the extracellular vesicle sample and a filling area 22 filled with a viscoelastic fluid. The suspended nanoparticles of different sizes in the extracellular vesicle sample are arranged around the side wall of the fluid channel 21 and driven toward the center line of the fluid channel 21 at different lateral speeds to achieve separation of exosomes in the extracellular vesicle sample.

[0047] and a separation and collection component 3 for receiving the exosomes separated from the extracellular vesicle sample.

[0048] In this embodiment, the sample input component 1 includes a sample inlet 11 for inputting the extracellular vesicle sample to be separated, and a buffer inlet 12 for inputting a fluid buffer. The sample inlet 11 and the buffer inlet 12 are parallel. The depth of the sample inlet 11 is greater than the depth of the buffer inlet 12.

[0049] In this embodiment, the sample inlet 11 is connected to a first feeding controller, and the buffer inlet 12 is connected to a second feeding controller. The suspended nanoparticles of different sizes in the extracellular vesicle sample are arranged around the side wall of the fluid channel 21 and controlled by the first feeding controller and the second feeding controller, and driven toward the center line of the fluid channel 21 at different lateral speeds to achieve the separation of exosomes in the extracellular vesicle sample. The fluid channel can adjust the flow ratio, flow rate, collection time and other parameters through the first feeding controller and the second feeding controller to achieve the selective collection of exosomes of different sizes. The flow characteristics of the fluid, such as inertial force, viscosity, surface tension, etc., are used to achieve the movement and positioning of particles.

[0050] In this embodiment, a filter membrane is provided at the input end of the sample inlet 11, which can directly separate high-purity extracellular vesicles from cell supernatant and serum.

[0051] In this embodiment, a coating is provided on the inner surface of the fluid channel 21. When no coating is provided, general materials often severely adsorb exosomes. In view of this characteristic, a coating is provided on the inner surface of the fluid channel (21) to reduce the loss of exosomes.

[0052] In this embodiment, the sample input component 1 is used to receive the extracellular vesicle sample to be separated, and process the sample to separate exosomes with a diameter of 50 to 150 nm. The extracellular vesicle sample to be separated, such as culture medium and serum, is put into the sample input component 1 from the sample inlet 11 after simple pretreatment, such as centrifugation and filtration, to remove impurities and large particles, and at the same time, the fluid buffer is connected from the buffer inlet 12 to allow the liquid to flow to the viscoelastic fluid processing component 2. The exosomes in the extracellular vesicle sample are subjected to the elastic lift force related to the particle size in the filling area 22 filled with the viscoelastic fluid, thereby achieving the separation of other extracellular vesicles of different sizes from the exosomes.

[0053] In this embodiment, the fluid channel 21 is linear. The cross-sectional shape of the fluid channel 21 is circular. The fluid channel 21 can selectively collect exosomes of different sizes by adjusting parameters such as flow ratio, flow rate, and collection time.

[0054] In this embodiment, the separation and collection assembly 3 comprises:

[0055] An exosome separation channel 31 connected to the side wall of the fluid channel 21;

[0056] An exosome collection pool 32 connected to the exosome separation channel 31;

[0057] And a waste liquid recovery pool 33 connected to the middle of the fluid channel 21.

[0058] In this embodiment, the waste liquid recovery pool 33 is located inside the exosome separation channel 31, and the inside of the waste liquid recovery pool 33 is not circulated. The exosome separation channel 31 is rhombus-shaped. The exosome collection pool 32 is parallel to the waste liquid recovery pool 33. The viscous fluid flows into the waste liquid recovery pool 33.

[0059] In this embodiment, the main material of the sample input component 1, the viscoelastic fluid processing component 2, and the separation and collection component 3 is polydimethylsiloxane.

[0060] In this embodiment, the working process and principle of the exosome separation and purification device are as follows:

[0061] After simple pretreatment, the extracellular vesicle sample is connected to the sample inlet 11 through the filter membrane, and the fluid buffer is connected to the buffer inlet 12. The feed flow ratio of the sample inlet 11 and the buffer inlet 12 can be controlled by the first feed controller and the second feed controller to separate exosomes of different sizes.

[0062] The extracellular vesicle sample and the fluid buffer enter the viscoelastic fluid processing component 2 from the sample input component 1 and are fully mixed in the fluid channel 21, which is composed of a filling area 22 filled with viscoelastic fluid. After the extracellular vesicle sample containing exosomes meets the buffer, the mixture first flows along the side wall of the fluid channel 21 in an aligned manner, and the suspended nanoparticles of different diameters in the extracellular vesicle sample from the sample inlet 11 are arranged around the side wall of the fluid channel 21. The flow characteristics of the fluid, such as inertial force, viscous force, surface tension, etc., are used to achieve the movement and positioning of the particles, and the particles are driven toward the center line of the fluid channel 21 at a lateral speed related to the size controlled by the elastic lift force. Exosomes and other extracellular components are moved to the center line of the fluid channel 21 according to their size, and the larger particles reach the center line last. Large particle waste liquid (including viscous fluid) with a faster centerline migration speed flows out from the middle of the fluid channel 21, flows out along the middle of the fluid channel 21 and enters the waste liquid recovery pool 33; small particle exosomes with a slower migration speed flow out from both sides of the fluid channel 21, pass through the exosome separation channel 31 and then flow into the exosome collection pool 32, thereby realizing label-free continuous separation of nanoparticles of different sizes.

[0063] The above description of the embodiments is to facilitate the understanding and use of the utility model by those skilled in the art. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the utility model is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the utility model without departing from the scope of the utility model should be within the scope of protection of the utility model.

Claims

1. An exosome separation and purification device, characterized in that: include: A sample input component (1) for receiving an extracellular vesicle sample to be separated; A viscoelastic fluid processing component (2) connected to the sample input component (1), comprising a fluid channel (21) for transporting an extracellular vesicle sample and a filling area (22) filled with a viscoelastic fluid; and a separation and collection component (3) for receiving the exosomes separated from the extracellular vesicle sample.

2. The exosome separation and purification device according to claim 1, characterized in that: The sample input component (1) comprises a sample inlet (11) for inputting the extracellular vesicle sample to be separated, and a buffer inlet (12) for inputting a fluid buffer.

3. The exosome separation and purification device according to claim 2, characterized in that: The sample inlet (11) is connected to a first feeding controller, and the buffer inlet (12) is connected to a second feeding controller.

4. The exosome separation and purification device according to claim 2, characterized in that: A filter membrane is arranged at the input end of the sample inlet (11).

5. The exosome separation and purification device according to claim 2, characterized in that: The sample inlet (11) and the buffer inlet (12) are parallel.

6. The exosome separation and purification device according to claim 1, characterized in that: The inner surface of the fluid channel (21) is provided with a coating.

7. The exosome separation and purification device according to claim 1, characterized in that: The cross-sectional shape of the fluid channel (21) is circular or rectangular.

8. The exosome separation and purification device according to claim 1, characterized in that: The separation and collection assembly (3) comprises: an exosome separation channel (31) connected to the side wall of the fluid channel (21); an exosome collection pool (32) connected to the exosome separation channel (31); and a waste liquid recovery pool (33) connected in the middle of the fluid channel (21).

9. The exosome separation and purification device according to claim 8, characterized in that: The waste liquid recovery pool (33) is located inside the exosome separation channel (31), and no fluid circulates inside the channel.

10. The exosome separation and purification device according to claim 8, characterized in that: The exosome collection pool (32) is parallel to the waste liquid recovery pool (33).

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