A device for separating and purifying exosomes from cell supernatant

Abstract

A cell supernatant exosome separation and purification device includes a main structure and accessories. The main structure includes a mobile phase tube, a stationary phase tube, a pressure regulating component, and a stationary phase packing material. The mobile phase tube is used to hold cell supernatant samples. The pressure regulating component is connected to the mobile phase tube and used to regulate the pressure inside the mobile phase tube. The stationary phase tube is located below the mobile phase tube and is connected to the mobile phase tube by a sealing module. The stationary phase packing material fills the stationary phase tube. The accessories include an exosome collection tube for collecting exosomes that have detached from the stationary phase tube. This invention separates molecules of different sizes from cell supernatant samples by utilizing the specific pore size on the surface of the stationary phase packing material, achieving the separation and purification of exosomes in pretreated samples. This improves the separation and purification efficiency of exosomes while ensuring the purity and biological activity of the exosomes, belonging to the field of exosome separation and purification technology.

Description

Technical Field

[0001] This utility model relates to the field of exosome separation and purification technology, specifically to a device for separating and purifying exosomes from cell supernatant. Background Technology

[0002] Exosomes are extracellular vesicles enclosed in a phospholipid bilayer. Cells actively secrete them into the extracellular environment through a series of complex processes, including endocytosis, fusion, and release. Their size ranges from 30 to 150 nm. Exosomes contain bioactive substances such as nucleic acids, proteins, and lipids, and can act as "information carriers" to deliver rich biological information to other cells, thereby achieving multi-faceted and multi-dimensional alterations in cell state and function.

[0003] Currently, common methods for exosome extraction include ultracentrifugation, density gradient centrifugation, polymer sedimentation, and immunomagnetic bead extraction. Ultracentrifugation utilizes the difference in sedimentation rates of particles of different sizes in the supernatant, separating dead cells and cell debris by controlling centrifugal force and time to obtain exosomes. However, this method results in significant exosome loss and low purity. Density gradient centrifugation extracts exosomes based on the density differences of the centrifugation medium; this method is cumbersome and time-consuming. Polymer sedimentation uses polymers such as polyethylene glycol to adjust the solubility of exosomes, causing them to settle and aggregate. This method is simple to operate and has high extraction efficiency, but the extracted exosomes contain a large amount of polymer that is difficult to remove. Immunomagnetic bead extraction uses magnetic beads coated with exosome-related markers to specifically bind to exosomes before elution and separation. This method yields highly specific exosomes, but their biological activity is low. Therefore, a cell supernatant exosome separation and purification device that can balance separation and purification efficiency with separation purity and biological activity is needed. Utility Model Content

[0004] In view of the technical problems existing in the prior art, the purpose of this utility model is to provide a cell supernatant exosome separation and purification device that has high separation efficiency while ensuring the purity and biological activity of exosomes.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a cell supernatant exosome separation and purification device, comprising a main structure and accessories; the main structure includes a mobile phase tube, a stationary phase tube, a pressure regulating component, and a stationary phase filler; the mobile phase tube is used to hold cell supernatant samples; the pressure regulating component is connected to the mobile phase tube and used to regulate the pressure inside the mobile phase tube; the stationary phase tube is located below the mobile phase tube; the stationary phase tube and the mobile phase tube are connected by a sealing module; and the stationary phase filler is filled inside the stationary phase tube; the accessories include an exosome collection tube, which is used to collect exosomes that have detached from the stationary phase tube.

[0006] As a preferred option, the pressure regulating component is an air pump with a pressure regulating valve, and the pressure regulating valve is a digital display pressure regulating valve.

[0007] As a preferred embodiment, the top of the mobile phase tube has a sample feeding port, and the pressure regulating component is detachably connected to the sample feeding port.

[0008] As a preferred embodiment, the cell supernatant exosome separation and purification device also includes a waste liquid collection tube, which is used to collect the buffer solution and macromolecules eluted from the cell supernatant sample.

[0009] As an alternative, the accessories also include a bracket for mounting the main structure, exosome collection tube, and waste liquid collection tube.

[0010] As a preferred embodiment, the cell supernatant exosome separation and purification device also includes a filter module, which is installed between the mobile phase tube and the stationary phase tube and is used to filter microorganisms.

[0011] As a preferred embodiment, the filter module comprises a filter membrane with a pore size of 0.22 μm.

[0012] As a preferred embodiment, the main structure also includes a fixed phase pipe cover, which is installed at the bottom end of the fixed phase pipe and has a sealing knob structure.

[0013] As a preferred option, the stationary phase packing material is spherical porous particles with a pore size of 30-150 nm and a particle diameter of 5-10 μm.

[0014] The separation principle of the above device is as follows: when the cell supernatant sample flows through the stationary phase packing, large molecules (impurities) cannot enter the internal pores of the stationary phase packing particles and thus elute out earliest, while small molecules (exosomes) penetrate into the interior through specific pores on the surface of the stationary phase packing particles, making the flow path longer and thus elute out later.

[0015] In summary, this utility model has the following advantages:

[0016] (1) This invention can effectively perform physical sieving through the specific pore size of the stationary phase packing material, preferentially retaining exosomes, while effectively separating large-sized cell debris, apoptotic bodies, protein aggregates and small molecule impurities (such as free proteins and metabolites), significantly improving product purity, and effectively maintaining the integrity and biological activity of exosomes.

[0017] (2) The present invention is equipped with a pressure regulating component, which allows the operator to control the optimal flow rate point according to the sample characteristics and specific needs, so as to achieve the optimal balance between separation speed and purity / recovery rate.

[0018] (3) This utility model integrates a filter module, which effectively intercepts all particles and microorganisms larger than 0.22μm, ensuring the cleanliness of the liquid entering the stationary phase tube. This not only extends the service life of the stationary phase packing and the entire device, but also further improves the purity of the product. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall structure of this utility model.

[0020] Figure 2 This is a schematic diagram illustrating the internal structure and working principle of the stationary phase filler of this utility model.

[0021] Figure 3 This is a schematic diagram illustrating the method of using this utility model.

[0022] Among them, 1 is the flowing phase tube, 2 is the pressure regulating valve, 3 is the fixed phase tube, 4 is the fixed phase tube cover, 5 is the fixed phase filler, 6 is the sealing module, 7 is the filter module, 8 is the waste liquid collection tube, 9 is the exosome collection tube, and 10 is the support. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] Example 1

[0025] like Figure 1 As shown, a cell supernatant exosome separation and purification device includes a main structure and accessories. The main structure includes a mobile phase tube 1, a stationary phase tube 3, a pressure regulating component, and a stationary phase packing material 5. The mobile phase tube is used to hold cell supernatant samples. The pressure regulating component is connected to the mobile phase tube and used to regulate the pressure inside the mobile phase tube. The stationary phase tube is located below the mobile phase tube. The stationary phase tube and the mobile phase tube are connected by a sealing module 6. The stationary phase packing material is filled inside the stationary phase tube. The accessories include an exosome collection tube 9, which is used to collect small molecule substances, i.e., exosomes, that have been released from the stationary phase tube.

[0026] The top end of the stationary phase tube is connected to the bottom end of the flowing phase tube via a sealing module, and their interiors are interconnected. The sealing module can be an existing grooved connector with a sealing ring or other structures. The bottom end of the stationary phase tube is a conical outlet.

[0027] Specifically, the pressure regulating component is an air pump with a pressure regulating valve 2, which is a digital display pressure regulating valve. The pressure regulating component can adopt existing technology, and the output pressure can be adjusted by the digital display pressure regulating valve to make the liquid in the mobile phase tube reach a suitable flow rate, thereby realizing the control of the exosome separation and purification rate. When in use, the flow rate of the liquid in the mobile phase tube is in the range of 2.5-5 mL / min.

[0028] Specifically, the mobile phase tube has a sample loading port at its top, and the pressure regulating component is detachably connected to the sample loading port. The outlet of the pressure regulating component is connected to the sample loading port in a conventional detachable manner. If a sample needs to be added during operation, the pressure regulating component can be removed, the sample can be added using an electric pipette, and then the pressure regulating component can be reconnected.

[0029] Specifically, the cell supernatant exosome separation and purification device also includes a waste liquid collection tube 8, which is used to collect the buffer solution and macromolecules eluted from the cell supernatant sample.

[0030] Specifically, the accessories also include a bracket 10, which is used to install the main structure, exosome collection tube, and waste liquid collection tube. The main structure, exosome collection tube, waste liquid collection tube, and bracket can be connected by conventional plug-in, hanging, resting, or other easily disassembled installation methods.

[0031] Specifically, the cell supernatant exosome separation and purification device also includes a filter module 7, which is installed between the mobile phase tube and the stationary phase tube. The filter module is used to filter microorganisms. The filter module can be snapped onto the sealing module, and the liquid in the mobile phase tube enters the stationary phase tube under pressure through the filter module.

[0032] Specifically, the filter module includes a filter membrane with a pore size of 0.22 μm. The filter membrane can remove microorganisms such as bacteria and mycoplasma.

[0033] Specifically, the main structure also includes a fixed phase tube cover 4, which is installed at the bottom of the fixed phase tube and has a sealing knob structure. This knob-based sealing prevents contamination of the inside of the fixed phase tube during storage.

[0034] Specifically, the stationary phase packing consists of spherical porous particles with a pore size of 30-150 nm and a particle diameter of 5-10 μm. For example... Figure 2 As shown, the above-mentioned spherical porous particles have through-holes with a specific pore size, which can be used to separate molecules of different sizes in cell supernatant samples. The material used is an existing porous particle material, such as a material whose main components are silica and polyacrylamide.

[0035] The above-mentioned cell supernatant exosome separation and purification device can be used according to the following steps:

[0036] (1) Place the exosome separation and purification device at room temperature for 30 minutes to allow it to reach the working temperature range of 20-25℃. Then fix the main body of the device, the waste liquid collection tube and the exosome collection tube on the support for easy subsequent operation.

[0037] (2) Open the fixed phase tube cap and add 100ml of high-temperature sterilized phosphate buffer to the mobile phase tube. Control the flow rate of the liquid in the mobile phase tube through the digital display pressure regulating valve to complete the airtightness check and equilibrium pretreatment of the separation and purification device.

[0038] (3) Pre-treat the cell supernatant sample, collect 500ml of cell supernatant, aliquot it into 50ml centrifuge tubes, centrifuge at 500×g 4℃ for 10min to remove dead cells and transfer the supernatant to 50ml centrifuge tubes; centrifuge at 2000×g 4℃ for 10min to remove cell debris and transfer the supernatant to 50ml centrifuge tubes; centrifuge at 10000×g 4℃ for 30min to remove large vesicles and apoptotic bodies and transfer the supernatant to ultrafiltration tubes; centrifuge at 4000×g 4℃ and concentrate the supernatant using ultrafiltration tubes for 30min to finally obtain 5ml of pre-treated cell supernatant sample.

[0039] (4) Add 5 ml of pretreated cell supernatant sample to the mobile phase tube. When all the sample has entered the stationary phase tube, add 50 ml of high-temperature sterilized phosphate buffer to the mobile phase tube. First, use the waste liquid collection tube to collect 35 ml of waste liquid, and then use the exosome collection tube to collect 20 ml of exosome suspension.

[0040] (5) After completing the separation and purification of exosomes, add 150 ml of 75% ethanol solution to the mobile phase tube, disinfect and clean the separation and purification device, and store it in an environment of 4-8℃.

[0041] like Figure 3 As shown, the specific separation process is as follows (including but not limited to the following state nodes):

[0042] T1. After adding 5 ml of cell supernatant sample to the mobile phase tube, the sample will enter the stationary phase tube under the action of the pressure regulating valve. At this time, macromolecules (impurities) and small molecules (exosomes) are mixed and distributed on the top of the stationary phase packing.

[0043] T2. Add 50 ml of high-temperature sterilized phosphate buffer to the mobile phase tube. Under the action of the pressure regulating valve, the mobile phase begins to elute and separate. At this time, macromolecules (impurities) are distributed in the lower part of the stationary phase packing, while small molecules (exosomes) are distributed in the middle and upper part of the stationary phase packing.

[0044] T3. When the cell supernatant sample flows through the stationary phase packing, macromolecular substances (impurities) are the first to elute and flow out. Collect 35 ml of waste liquid using a waste liquid collection tube.

[0045] T4. When the cell supernatant sample flows through the stationary phase packing, small molecules (exosomes) will elute later. Use exosome collection tube 9 to collect 20 ml of exosome suspension.

[0046] The above embodiments are preferred embodiments of the utility model, but the implementation of the utility model is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the utility model shall be considered equivalent substitutions and shall be included within the protection scope of the utility model.

Claims

1. A device for isolating and purifying cell supernatant exosomes, characterized by: Including the main structure and accessories; The main structure includes a mobile phase tube, a stationary phase tube, a pressure regulating component, and a stationary phase packing material. The mobile phase tube is used to hold cell supernatant samples. The pressure regulating component is connected to the mobile phase tube and is used to regulate the pressure inside the mobile phase tube. The stationary phase tube is located below the mobile phase tube. The stationary phase tube and the mobile phase tube are connected by a sealing module. The stationary phase packing material is filled inside the stationary phase tube. The accessories include an exosome collection tube, which is used to collect exosomes that have detached from the stationary phase tube.

2. The cell supernatant exosome isolation and purification device of claim 1, wherein: The pressure regulating component is an air pump with a pressure regulating valve, which is a digital display pressure regulating valve.

3. The device for isolating and purifying cell supernatant exosomes according to claim 1, characterized in that: The top of the mobile phase tube has a sample loading port, and the pressure regulating component is detachably connected to the sample loading port.

4. The device for isolating and purifying cell supernatant exosomes according to claim 1, characterized in that: It also includes waste liquid collection tubes, which are used to collect buffer and macromolecules eluted from cell supernatant samples.

5. The cell supernatant exosome isolation and purification device of claim 4, wherein: The accessories also include a bracket for mounting the main structure, exosome collection tube, and waste liquid collection tube.

6. The cell supernatant exosome isolation and purification device of claim 1, wherein: It also includes a filter module, which is installed between the mobile phase tube and the stationary phase tube and is used to filter microorganisms.

7. The cell supernatant exosome isolation and purification device of claim 6, wherein: The filter module includes a filter membrane with a pore size of 0.22 μm.

8. A cell supernatant exosome separation and purification device according to claim 3, characterized in that: The main structure also includes a fixed phase pipe cover, which is installed at the bottom of the fixed phase pipe and has a sealing knob structure.

9. The cell supernatant exosome isolation and purification device of claim 1, wherein: The stationary phase packing consists of spherical porous particles with a pore size of 30-150 nm and a particle diameter of 5-10 μm.

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