CN219239646U

CN219239646U - High-purity exosome separator

Info

Publication number: CN219239646U
Application number: CN202222715359.XU
Authority: CN
Inventors: 叶永清; 黄能平; 王力; 左鹏举; 仇宇光
Original assignee: Beijing Sany Hematopoietic Technology Co ltd
Current assignee: Beijing Sany Hematopoietic Technology Co ltd
Priority date: 2022-10-16
Filing date: 2022-10-16
Publication date: 2023-06-23
Anticipated expiration: 2032-10-16

Abstract

The utility model discloses a high-purity exosome separation device which comprises a device I and a device II, wherein the device I sorts particles according to the aperture size, the device II is a temperature-sensitive exosome separation device, the aperture size of the device I is 150nm, and an S-shaped glass microtubule is arranged on the inner wall of a temperature-sensitive cell separation tube main body of the device II. The inner wall of the pipe is modified by a silane coupling agent APTES, a layer of gelatin is uniformly coated on the inner part of the pipe by a layer-by-layer self-assembly technology, the surface of the gelatin is modified by streptavidin, and simultaneously, the biotinylated antibody is connected with the streptavidin on the surface of the gelatin, so that the accurate sorting of the exosomes is facilitated, the gelatin is changed into a liquid state from a solid state at a temperature of 37 ℃, the exosomes connected to the surface of the gelatin can be completely dissociated, the pipe is flushed by liquid, the shed exosomes can flow out from a right outlet and be collected, and the high-purity intact exosomes can be obtained by a separation method combining physical size and exosome surface markers.

Description

High-purity exosome separator

Technical Field

The utility model relates to the technical field of exosome separation, and belongs to the field of biological substance separation and purification.

Background

Exosomes are becoming important as important communication media between cells in recent years, and their application prospects in basic research, clinical diagnosis and treatment are being continuously excavated, and how to obtain high-purity exosomes is a primary problem faced by basic research and clinical application of exosomes, and the current common exosome purification method comprises: 1. the gradient centrifugation method is simple, but the prior work and temporary complexity are relatively time-consuming, and the obtained exosome has low content and low purity; 2. the immunomagnetic bead method has short time consumption, does not need ultracentrifugation, but has influence on the biological activity of exosomes, and has high separation and purification cost; in addition, PEG precipitation methods and ultracentrifugation methods have problems such as low purity. Aiming at the problems, the novel design is carried out on the basis of the original separation and purification of exosomes, and a high-purity exosome separation device is developed.

Disclosure of Invention

The present utility model is directed to a high purity exosome separation device, which solves the problems set forth in the background art.

In order to achieve the above purpose, the present utility model provides the following technical solutions: the utility model provides a high purity exosome extraction element, includes device I (1) and device II (2), device I relies on aperture size to select separately exosome granule, device II is temperature-sensitive exosome separator.

The device I comprises a U-shaped pipeline, the pipeline is provided with an inlet (3) and an outlet (4), and the U-shaped pipeline is provided with a small hole (7) with the aperture of 150nm. An S-shaped glass microtubule (5) is arranged in the temperature-sensitive exosome separation tube of the device II (2). The diameter of the S-shaped glass microtube (5) is 100um, and the total length of the pipeline of the S-shaped glass microtube (5) is 1m. The temperature-sensitive exosome separating tube of device II (2), its characterized in that: the formation of the temperature-sensitive characteristic comprises the following steps:

step one:

after the inner wall of the S-shaped glass microtube (5) is modified by a silane coupling agent APTES, a layer of gelatin is uniformly coated on the inner part of the pipeline by a layer-by-layer self-assembly (layer) technology;

step two:

streptavidin attached to the gelatin surface using biotinylated antibodies was then used to isolate exosomes;

step three:

the exosome-containing suspension after the primary screening by the device I flows through a device II S-type glass microtubule (5);

step four:

after the temperature-sensitive exosome separating tube main body (2) separates to obtain exosomes, placing the device II in a 37-degree incubator;

step five:

the tubing was flushed with PBS liquid and the sloughed off exosomes were collected for downstream application analysis from the right outlet (6).

Compared with the prior art, the utility model has the beneficial effects that: the high-purity exosome separating device comprises a high-purity exosome separating device,

1. preliminary screening can be carried out on exosomes through a device I, so as to obtain particles with diameters smaller than 150 nm;

2. after the inner wall of the S-shaped glass microtubule is modified by a silane coupling agent APTES, a layer of gelatin is uniformly coated on the inner part of the pipeline by a layer-by-layer self-assembly technology, and the surface of the gelatin is modified by streptavidin, and simultaneously, the biotin antibody is used for connecting with the streptavidin on the surface of the gelatin, so that the separation work of cells is facilitated;

3. gelatin changes from solid state to liquid state at 37 ℃, so that exosomes connected to the surface of gelatin can be completely dissociated, the subsequent flushing of a pipeline by liquid is facilitated, the exosomes which are convenient to fall off can flow out from the right outlet and be collected, and the separation method can keep higher exosome activity and integrity.

Drawings

FIG. 1 is a schematic diagram of the overall front cross-sectional structure of the present utility model;

in the figure: 1. a separation device I; 2. separation device II 3, sample inlet 4, waste liquid outlet 5, S-shaped glass microtube 6, sample outlet 7 and U-shaped pipeline

FIG. 2 is a photograph of an isolated exosome electron microscope;

Detailed Description

The following description of the embodiments of the present utility model will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present utility model, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the utility model without making any inventive effort, are intended to be within the scope of the utility model.

Example 1 formation of temperature-sensitive exosome separation Properties

Referring to fig. 1, the present utility model provides a technical solution: the utility model provides the following technical scheme: the utility model provides a high purity exosome extraction element, includes device I (1) and device II (2), and temperature-sensitive exosome separation pipe of device II (2), its characterized in that: the formation of the temperature-sensitive characteristic comprises the following steps:

step one:

step two:

step three:

step four:

step five:

EXAMPLE 2 rapid isolation of high purity exosomes

The obtained suspension containing exosomes (such as stem cell supernatant) was passed through the device I and the device II at a rate controlled to 0.1ml/min, twice with PBS solution at the same rate, the temperature-sensitive cell separation tube (device II) was placed in a 37 ℃ cell incubator for 20 minutes, and passed through the device II with PBS solution at the same rate, the desired exosomes were harvested, and the number of exosomes was detected by electron microscopy (fig. 2), stored at 4 ℃ or ultra-low temperature for downstream use.

The above is the working process of the whole device, and what is not described in detail in this specification belongs to the prior art known to those skilled in the art. Although embodiments of the present utility model have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the utility model, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The utility model provides a high purity exosome separator, includes device I (1) and device II (2), its characterized in that: the device I (1) is used for sorting exosome particles by means of pore size, the device II (2) is a temperature-sensitive exosome separation device, an S-shaped glass microtubule (5) is arranged in the device II (2), the inner wall of the S-shaped glass microtubule (5) is modified by a silane coupling agent APTES, a layer of gelatin is uniformly coated on the inside of a pipeline through a layer-by-layer self-assembly technology, the surface of the gelatin is modified by using streptavidin, and a biotinylated antibody is connected with the streptavidin on the surface of the gelatin.

2. The high purity exosome separation apparatus according to claim 1, wherein: the device I (1) comprises a U-shaped pipeline, the U-shaped pipeline is provided with an inlet (3) and an outlet (4), the U-shaped pipeline is provided with a small hole (7), and the aperture size of the small hole (7) is 150nm.

3. The high purity exosome separation apparatus according to claim 1, wherein: the diameter of the S-shaped glass microtube (5) is 100um, and the total length of the pipeline of the S-shaped glass microtube (5) is 1m.