Exosome removal culture medium preparation device

Abstract

In order to solve the technical problems that in the exosome production industry, when a cell culture medium is prepared, the exosome removal step of a reagent is tedious, and the reagent is prone to being polluted, the utility model provides an exosome removal culture medium preparation device which comprises a filtering device composed of a shell, a hollow fiber membrane filter, a first peristaltic pump and a second peristaltic pump. A first liquid inlet, a first liquid return opening and a first liquid outlet are formed in the hollow fiber membrane filter; the additive bottle is provided with a second liquid outlet, a second liquid supplementing opening and a second liquid returning opening, and the basic culture medium bottle is provided with a third liquid inlet and a third liquid outlet; an inlet and an outlet of the first peristaltic pump are respectively connected with the second liquid outlet and the first liquid inlet of the additive, an inlet and an outlet of the second peristaltic pump are respectively connected with the third liquid outlet and the second liquid supplementing port, the exosome in the culture medium additive can be quickly removed, the working efficiency is improved, and the pollution risk is reduced.

Description

Technical Field

[0001] The utility model relates to the technical field of exosome-removed culture medium devices, and in particular to an exosome-removed culture medium configuration device. Background Art

[0002] Exosomes are biological structures surrounded by a phospholipid bilayer membrane that are actively released to the outside of cells through a series of processes such as "endocytosis-fusion-excretion". They have a diameter of 30 to 150 nm and can be secreted by almost all types of cells. Exosomes were once considered to be "waste" discharged by cells to the outside of cells, but studies in recent years have shown that exosomes carry important biological molecules from maternal cells, including RNA, proteins, lipids, metabolites and other molecules, and play a vital role in cell signal transduction and cell physiological and pathological activities.

[0003] Currently, exosomes have been widely used in research in the fields of medical testing and disease treatment. For example, stem cell exosome therapy, as a cell-free treatment method, greatly reduces the risks of rejection and ectopic differentiation after allogeneic transplantation, and has achieved significant therapeutic effects in related research on various types of diseases such as trauma repair, ischemic diseases, neurodegenerative diseases, diabetes, and COVID-19.

[0004] The complete culture medium used in cell experiments is composed of a basal culture medium and one or more additives. Common additives include fetal bovine serum, platelet lysate, serum extract, etc., which carry exosomes from maternal cells such as bovine blood cells or human blood cells. If this complete culture medium is used to directly culture stem cells for subsequent extraction of stem cell exosomes, the exosomes in the culture medium and the exosomes secreted by the stem cells will be mixed together and cannot be separated. Therefore, if high-purity stem cell exosomes are to be produced, the exosomes in the complete culture medium need to be removed in advance.

[0005] The conventional method for removing exosomes from culture medium is ultracentrifugation, that is, the prepared complete culture medium is centrifuged with an ultracentrifuge, the supernatant is collected, and the exosomes are removed in the form of precipitation. However, ultracentrifugation is time-consuming, the equipment is expensive, and it is an open operation. The obtained exosome-free culture medium also needs to be filtered and sterilized, which consumes a lot of time and experimental materials. Utility Model Content

[0006] In order to solve the above technical problems, the utility model provides a device for configuring exosome-free culture medium, and the technical solution adopted is as follows:

[0007] A device for preparing exosome-free culture medium, comprising:

[0008] The filtering device is composed of a housing, a hollow fiber membrane filter, a first peristaltic pump and a second peristaltic pump, wherein the hollow fiber membrane filter is provided with a first liquid inlet, a first liquid return port and a first liquid outlet;

[0009] The additive bottle is fixed to one side of the filtering device, the additive bottle is connected to an additive bottle cap, and the additive bottle cap is provided with a second liquid outlet, a second liquid replenishing port and a second liquid return port;

[0010] A basal culture medium bottle, wherein the basal culture medium bottle is provided with a basal culture medium bottle cap, and the basal culture medium bottle cap is provided with a third liquid inlet and a third liquid outlet;

[0011] The inlet and outlet of the first peristaltic pump are connected to the second liquid outlet and the first liquid inlet respectively, the inlet and outlet of the second peristaltic pump are connected to the third liquid outlet and the second liquid replenishment port respectively, the first liquid outlet is connected to the third liquid inlet, and the first liquid return port is connected to the second liquid return port.

[0012] Furthermore, the additive bottle cap and the basal culture medium bottle cap are both provided with air filter membranes.

[0013] Furthermore, a liquid level sensor is provided on the additive bottle.

[0014] Furthermore, the liquid level sensor includes an upper liquid level sensor and a lower liquid level sensor, and the upper liquid level sensor and the lower liquid level sensor are respectively located at the upper part and the lower part of the additive bottle.

[0015] Furthermore, the hollow fiber membrane filter is provided with a syringe interface.

[0016] Furthermore, a plug is provided at the syringe interface.

[0017] The beneficial effects of the utility model are:

[0018] 1. The tangential flow ultrafiltration principle of the hollow fiber membrane filter is used to quickly remove the exosomes in the culture medium additives, which improves the work efficiency. In addition, the hollow fiber membrane ultrafiltration can also filter and sterilize, so the additives used do not need to purchase sterile reagents;

[0019] 2. During filtration, the remaining volume in the additive bottle is detected and the second peristaltic pump is used to automatically replenish and dilute the additive, which can effectively reduce the waste of additives;

[0020] 3. By replacing additive bottles or basal culture medium bottles of different specifications, different volumes of liquid preparation can be achieved, which is easy to operate;

[0021] 4. The overall structure has good sealing performance, which can prevent the entry of exogenous microorganisms during operation and effectively prevent contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the structure of the utility model

[0023] Figure 2 Schematic diagram of the connection structure between the filter device and the additive bottle

[0024] Figure 3 Schematic diagram of the hollow fiber membrane filter structure

[0025] Figure 4 Shell side view

[0026] Figure 5 Top view of the additive bottle cap structure

[0027] Figure 6 Schematic diagram of the basic culture medium bottle structure

[0028] Among them, 1-shell, 101-bin cover, 102-duct groove, 103-upper pipe clamp, 104-lower pipe clamp, 105-fixing clamp;

[0029] 2-hollow fiber membrane filter, 201-first liquid inlet, 202-first liquid return port, 203-first liquid outlet, 204-syringe interface, 205-plug;

[0030] 3- first peristaltic pump;

[0031] 4- second peristaltic pump;

[0032] 5-additive bottle, 501-additive bottle body, 502-additive bottle cap, 503-first air filter membrane, 504-second liquid outlet, 505-second liquid replenishment port, 506-second liquid return port;

[0033] 6-basal culture medium bottle, 601-basal culture medium bottle body, 602-basal culture medium bottle cap, 603-third liquid inlet, 604-third liquid outlet. DETAILED DESCRIPTION

[0034] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0035] In the description of the utility model, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the viewing direction or position relationship, and are only for the convenience of describing the utility model, 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 utility model.

[0036] A device for preparing exosome-free culture medium, such as Figure 1-Figure 6 As shown, it includes a filtering device, an additive bottle 5, and a culture medium bottle 6. The filtering device is composed of a shell 1, a hollow fiber membrane filter 2, a first peristaltic pump 3, and a second peristaltic pump 4.

[0037] Specifically, Figure 1-Figure 4 As shown, a bin cover 101 is provided at the upper end of the front side of the shell 1, and conduit grooves 102 are provided on the left and right sides. The purpose of providing the conduit grooves 102 is to facilitate the passage of pipelines. The overall structure of the shell 1 is closed, and the hollow fiber membrane filter 2 is fixedly connected to the inside of the shell 1 through a fixing frame 105. The left and right sides of the hollow fiber membrane filter 8 are respectively provided with a first liquid return port 202 and a first liquid inlet 201, and the left and right sides of the upper part are respectively provided with a first liquid outlet 203 and a syringe interface 204, and a plug 205 is provided on the syringe interface 204. The first peristaltic pump 3 and the second peristaltic pump 4 are fixedly connected to the front side of the shell 1. In addition, the upper and lower left sides of the shell 1 are respectively fixedly connected with an upper tube clamp 103 and a lower tube clamp 104.

[0038] like Figure 2 and Figure 5 As shown, the additive bottle 5 consists of an additive bottle body 501 and an additive bottle cap 502. The additive bottle body 501 is fixedly connected to the left side of the shell 1 through an upper tube clamp 103 and a lower tube clamp 104, and an upper liquid level sensor and a lower liquid level sensor are respectively provided at the upper tube clamp 103 and the lower tube clamp 104. The remaining liquid volume inside the additive bottle body 501 can be automatically detected by two sets of liquid level sensors; a first air filter membrane 503 is provided on the additive bottle cap 502, and a second liquid outlet 504, a second liquid replenishing port 505 and a second liquid return port 506 are sequentially provided on the additive bottle cap 502 from left to right.

[0039] like Figure 6As shown, the basic culture medium bottle 6 comprises a basic culture medium bottle body 601 and a basic culture medium bottle cap 602. The basic culture medium bottle cap 602 is provided with a third liquid inlet 603 and a third liquid outlet 604. The lower end of the third liquid inlet 603 passes through the basic culture medium bottle cap 602 from top to bottom and is located in the upper part of the basic culture medium bottle body 601. The third liquid outlet 604 is connected to the bottom of the basic culture medium bottle body 601 22. Moreover, a second air filter membrane is also provided on the basic culture medium bottle cap 602. Its function is the same as that of the first air filter membrane on the additive bottle cap 502, which is to balance the internal and external pressure difference and prevent external impurities from entering the bottle.

[0040] like Figure 1 As shown, the connection relationship between the various components is specifically as follows: the inlet of the first peristaltic pump 3 is connected to the second liquid outlet 504, and the outlet is connected to the first liquid inlet 201 through a pipeline; the inlet of the second peristaltic pump 4 is connected to the third liquid outlet 604, and the outlet is connected to the second liquid replenishment port 505; the first liquid return port 202 is connected to the second liquid return port 506; the first liquid outlet 203 is connected to the culture medium inlet pipe 603.

[0041] The working principle of this device is as follows:

[0042] In the initial state, a plug 205 is provided at the syringe interface 204 to keep the overall sealing of the device, and the first peristaltic pump 3 is started. The additive passes through the first peristaltic pump 3 and enters the hollow fiber filter 2 for filtration, and the filtrate passes through the first liquid outlet 203 and the third liquid inlet 603 to enter the basic culture medium bottle 6, while the exosomes in the additive cannot pass through the filter pores of the hollow fiber membrane, and therefore pass through the first liquid return port 202 and the second liquid return port 506 to return to the additive bottle 5; when the additive level drops to the position of the lower liquid level sensor at the lower tube clamp 104, the first peristaltic pump 3 stops working, and the second peristaltic pump 4 is started, and the liquid in the basic culture medium bottle 6 The liquid enters the additive bottle 5 through the third liquid outlet 604 and the second liquid replenishment port 505. When the liquid level in the additive bottle 5 reaches the upper liquid level sensor position at the upper tube clamp 103, the second peristaltic pump 4 stops working and the first peristaltic pump 3 is started. The above-mentioned cycle operation is repeated, and finally the configured exosome complete culture medium is obtained in the basic culture medium bottle 6; at the same time, a small amount of exosome complete culture medium remains in each pipeline and the external space of the hollow fiber membrane filter. If it needs to be collected, the syringe interface 204 can be connected through a syringe to push the remaining exosome complete culture medium in each pipeline and the external space of the hollow fiber membrane filter into the basic culture medium bottle 6.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A device for preparing exosome-free culture medium, characterized in that: include: The filtering device is composed of a housing, a hollow fiber membrane filter, a first peristaltic pump and a second peristaltic pump, wherein the hollow fiber membrane filter is provided with a first liquid inlet, a first liquid return port and a first liquid outlet; The additive bottle is fixed to one side of the filtering device, and an additive bottle cap is provided on the additive bottle. The additive bottle cap is provided with a second liquid outlet, a second liquid replenishing port and a second liquid return port. A basal culture medium bottle, wherein the basal culture medium bottle is provided with a basal culture medium bottle cap, and the basal culture medium bottle cap is provided with a third liquid inlet and a third liquid outlet; In addition, the inlet and outlet of the first peristaltic pump are connected to the second liquid outlet and the first liquid inlet respectively, the inlet and outlet of the second peristaltic pump are connected to the third liquid outlet and the second liquid replenishment port respectively, the first liquid outlet is connected to the third liquid inlet, and the first liquid return port is connected to the second liquid return port.

2. The exosome-removed culture medium configuration device according to claim 1, characterized in that: The additive bottle cap and the basic culture medium bottle cap are both provided with air filter membranes.

3. The exosome-removed culture medium configuration device according to claim 1, characterized in that: The additive bottle is provided with a liquid level sensor.

4. The exosome-removed culture medium configuration device according to claim 3, characterized in that: The liquid level sensor comprises an upper liquid level sensor and a lower liquid level sensor, and the upper liquid level sensor and the lower liquid level sensor are respectively located at the upper part and the lower part of the additive bottle.

5. The exosome-removed culture medium configuration device according to claim 1, characterized in that: The hollow fiber membrane filter is provided with a syringe interface.

6. The exosome-removed culture medium configuration device according to claim 5, characterized in that: A plug is arranged at the syringe interface.

Images