CN216337716U

CN216337716U - Bioreactor for separating extracellular vesicles

Info

Publication number: CN216337716U
Application number: CN202122996914.6U
Authority: CN
Inventors: 韩顶立; 高山湖; 把小云; 马东雪; 石俊; 龙菲; 杨洋
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-04-19
Anticipated expiration: 2031-12-01

Abstract

The utility model belongs to the technical field of biology, and particularly discloses a bioreactor for separating extracellular vesicles, which comprises a culture container and a collection container, wherein the discharge end of the culture container is connected with the feed end of the collection container and is provided with a switch component for controlling communication or blocking; a filter unit is arranged in the culture container and comprises a filter plate arranged at the lower part of the culture container; the collection container is internally provided with a separation unit which comprises a filter membrane group arranged at the upper part in the collection container. The scheme combines the cell culture process and the vesicle separation and collection process together, reduces the time difference between the cell culture and the vesicle separation and collection, reduces the loss generated in the vesicle recovery process, and can obtain the fresest vesicles in time.

Description

Bioreactor for separating extracellular vesicles

Technical Field

The utility model belongs to the technical field of biology, and particularly relates to a bioreactor for separating extracellular vesicles.

Background

Almost all cells can release vesicles of a Vesicle-like membrane structure coated by a phospholipid bilayer, and the International Society of Extracellular Vesicles (ISEV) defines the vesicles as Extracellular vesicles, which contain various information molecules and play important roles in biological processes such as immune response regulation, antigen presentation, intercellular active molecule transfer, virus and prion transfer, tumor growth, transfer, tumor microenvironment formation and the like.

Extracellular vesicles are a heterogeneous group of vesicles that are available from almost all biological fluids, and their classification is not well defined at present, and ISEVs are divided into 3 subgroups, depending on their diameter: exosomes originate from the endocytic pathway, with diameters of 30-150 nm; microvesicles were released directly from the plasma membrane, with diameters around 100-1000 nm; apoptotic bodies are produced by apoptosis, ranging from about 50nm to 2 μm in diameter, and oncosomes with diameters ranging from 1 to 10 μm released by tumor cells. However, obtaining extracellular vesicles of a particular subpopulation still requires additional purification processes.

At present, methods for separating extracellular vesicles with different diameters are mainly based on size exclusion chromatography and differential centrifugation, and on the one hand, the size exclusion chromatography cannot obtain enough extracellular vesicles, and the differential centrifugation cannot effectively separate a certain subset of extracellular vesicles. In the experiment, the vesicle is continuously transformed at 4 ℃, 37 ℃ and room temperature by a differential centrifugation method which is usually adopted, and some experimental interference factors appearing in the process cannot be controlled; on the other hand, under natural conditions, the yield of vesicles in biological fluids is low, a large amount of manpower, material resources, financial resources and time are required to harvest a certain amount of extracellular vesicles, and long-term sustainable in-vitro culture and collection of extracellular vesicles cannot be achieved.

SUMMERY OF THE UTILITY MODEL

The utility model aims to provide a bioreactor for separating extracellular vesicles, which aims to solve one or more problems in the prior art, such as long whole experiment time period and low efficiency of vesicle separation by culturing cells.

In order to achieve the purpose, the technical scheme of the utility model is as follows:

a bioreactor for separating extracellular vesicles comprises a culture container and a collection container, wherein the discharge end of the culture container is connected with the feed end of the collection container and is provided with a switch assembly for controlling communication or blocking; a filter unit is arranged in the culture container and comprises a filter plate arranged at the lower part of the culture container; the collection container is internally provided with a separation unit which comprises a filter membrane group arranged at the upper part in the collection container.

The principle of the scheme is as follows: the culture vessel is used as the main loading vessel of the bioreactor for culturing cells. During the use, close the ooff valve in advance, make and cut off between culture vessel and the collecting vessel, then add cell culture liquid to culture vessel, start motor simultaneously, the motor drives the stirring subassembly through the pivot and evenly stirs the culture solution, makes cell culture liquid reach better environmental condition, the cell growth of being convenient for.

After the cell culture is finished, the switch valve is opened to enable the culture container to be communicated with the collecting container, the culture solution flows into the collecting container through the switch valve, in the process, the culture solution firstly passes through the filter plate, part of large-particle impurities are filtered out by the filter plate, then the culture solution passes through the filter membrane group, the filter membrane group filters the cell culture solution layer by layer, cells and other particle impurities are filtered out, and finally particles (vesicles) with set sizes are discharged through the discharge end at the bottom of the collecting container to finish the collection of the vesicles.

In the process of cell culture and vesicle separation, cell culture solution can be continuously added into the culture container according to needs, and the culture solution is continuously discharged from the discharge end of the collection container, so that long-term and sustainable cell culture and vesicle obtaining are realized, the test steps are simplified, and the working efficiency is higher.

Meanwhile, the cell culture process and the vesicle separation and collection process are combined together, so that the time difference between the cell culture and the vesicle separation and collection is reduced, the loss generated in the vesicle recovery process is reduced, and the fresest vesicles can be obtained in time.

Drawings

FIG. 1 is a schematic structural diagram of an embodiment;

fig. 2 is a schematic diagram of the first cover in the embodiment.

Reference numerals in the drawings of the specification include: the culture container 1, the accommodating chamber 101, the water bath chamber 102, the collecting container 2, the rotating shaft 3, the motor 4, the brush 5, the filter plate 6, the switch valve 7, the first cover body 8, the second cover body 9 and the filter membrane group 10.

Detailed Description

Various embodiments according to the present invention will be described in detail with reference to the accompanying drawings. Here, it is to be noted that, in the drawings, the same reference numerals are given to constituent parts having substantially the same or similar structures and functions, and repeated description thereof will be omitted. The term "sequentially comprising A, B, C, etc" merely indicates the order of the included elements A, B, C, etc. and does not exclude the possibility of including other elements between a and B and/or between B and C. The description of "first" and its variants is merely for distinguishing the components and does not limit the scope of the utility model, and "first" may be written as "second" and the like without departing from the scope of the utility model.

The drawings in the present specification are schematic views to assist in explaining the concept of the present invention, and schematically show the shapes of respective portions and their mutual relationships.

Hereinafter, preferred embodiments according to the present invention will be described in detail with reference to fig. 1 to 2.

Example 1

The embodiment provides a bioreactor for separating extracellular vesicles, which comprises a culture container 1 and a collection container 2, wherein the discharge end of the culture container 1 is connected with the feed end of the collection container 2 and is controlled to be communicated or blocked by a switch valve 7; a stirring unit and a filtering unit are arranged in the culture container 1, the stirring unit comprises a motor 4, a rotating shaft 3 driven by the motor 4 to rotate and a stirring component arranged on the rotating shaft 3, and the filtering unit comprises a filtering plate 6 arranged below the stirring unit; a separation unit is arranged in the collection container 2 and comprises a filter membrane group 10 arranged at the upper part in the collection container 2.

Referring to fig. 1, the bioreactor is integrally fixed on a support frame. Wherein, the culture container 1 is used as a main loading container of the bioreactor and is used for culturing various biological cells, and the collecting container 2 is used for collecting and discharging vesicles. In this embodiment, the culture container 1 and the collection container 2 are transparent glass containers with a capacity of 2L, both of which are covered with covers, and the covers on the culture container 1 and the collection container 2 have the same structure and are respectively a first cover 8 and a second cover 9. The first cover body 8 is provided with a first sample inlet pipe, the upper end of the first sample inlet pipe is connected with a liquid storage tank (not shown in the figure) storing cell culture liquid, the lower end of the first sample inlet pipe is communicated with the culture container 1, and the bottom of the culture container 1 is provided with a first sample outlet pipe. The collecting container 2 is positioned right below the culture container 1, a second sample inlet pipe is arranged on the second cover body 9, a switch valve 7 is connected between the second sample inlet pipe and the first sample outlet pipe through a flange, the switch valve 7 is used for controlling the second sample inlet pipe to be communicated with or separated from the first sample outlet pipe, and the part can be detached.

The filter unit comprises a filter plate 6, the diameter of the filter plate 6 is slightly smaller than the inner diameter of the culture container 1, so that the filter plate 6 can be placed in the culture container 1, the side edge of the filter plate 6 is attached to the inner wall of the culture container 1, and liquid leakage of the side edge of the filter plate 6 is avoided. The filter plate 6 is placed on the diapire of culture container 1, and in order to facilitate the flow of culture solution, culture container 1 bottom is the funnel-shaped that height all around is low in the middle of, and filter plate 6 just places on the upper limb of funnel-shaped diapire, makes the culture solution after filter plate 6 can directly flow to on the funnel-shaped diapire and continue downward flow along the diapire. In this embodiment, the filter plate 6 is a glass sand core, and the aperture of the filter hole of the glass sand core is 1 μm.

The stirring unit comprises a motor 4, a rotating shaft 3 and a stirring assembly, the motor 4 is fixedly connected to the support frame, the motor 4 is located right above the culture container 1, the upper end of the rotating shaft 3 is coaxially connected with an output shaft of the motor 4 through a coupler, a through hole for the rotating shaft 3 to pass through is formed in the first cover body 8, the rotating shaft 3 passes through the lower end of the through hole and extends to the lower part in the culture container 1, and a bearing assembly can be arranged at the through hole to enable the rotating shaft 3 to be in running fit with the first cover body 8.

In this embodiment, the stirring subassembly includes brush 5 and a plurality of stirring leaf, and specifically, brush 5 includes the horizontal pole of fix with screw on pivot 3, and the thick brush hair that has of horizontal pole downside, brush hair lower extreme and 6 surface contact on the filter plate make brush 5 along with pivot 3 when rotating the brush hair can clear up filter plate 6, avoid impurity pile and block up the filtration pore. As can be seen from the above, the brush 5 has the functions of stirring the culture solution and accelerating the filtration, thereby achieving the effect of dual purposes. In this embodiment, four stirring blades are circumferentially distributed on the rotating shaft 3, and the stirring blades are located above the brush 5.

The separation unit is arranged in the collection container 2, is arranged at the upper part in the collection container 2 and is a size exclusion chromatography component, and specifically, the separation unit comprises a filter membrane group 10, and the filter membrane group 10 is formed by combining an upper layer of filter membrane and a lower layer of filter membrane. In this embodiment, the filter membrane group 10 includes a plurality of filter membranes having different filter pore sizes, and the filter pore sizes of the filter membranes gradually decrease from top to bottom, and specifically, the inside includes two filter membrane layers having filter pore sizes of 1.2 μm, two filter membrane layers having filter pore sizes of 0.8 μm, two filter membrane layers having filter pore sizes of 0.65 μm, two filter membrane layers having filter pore sizes of 0.45 μm, and two filter membrane layers having filter pore sizes of 0.22 μm, respectively, from top to bottom. The middle of each filter membrane is separated by a sieve plate, the sieve plate is a glass flat bottom with a plurality of apertures of 1 mu m, and the edge of the sieve plate is attached to the inner wall of the collection container 2, so that the edge of the sieve plate does not leak liquid. Still be equipped with suction filtration pipe 11 on the lateral wall of collection container 2 bottom, suction filtration pipe 11 external suction filtration pump (not shown in the figure), extract the vesicle to appointed container through the suction filtration pump and store, the suction filtration pump can accelerate the filtration of filter membrane group 10 to the culture solution simultaneously, also can improve the suction dynamics, is convenient for collect the vesicle of capacity. Still be equipped with the discharging pipe on 2 bottoms of collecting vessel, discharging pipe seals in the course of the work, opens discharging pipe discharge exosome after the vesicle obtains to finish

Based on above-mentioned basic structure, during the use, close ooff valve 7 in advance, make and block between culture container 1 and the collecting vessel 2, then add cell culture liquid to culture container 1, start motor 4 simultaneously, motor 4 drives the stirring subassembly through pivot 3 and evenly stirs the culture liquid, makes cell culture liquid reach better environmental condition, the cell growth of being convenient for. In the stirring process, the brush 5 can stir the culture solution as the stirring blade of another form, strengthen the stirring effect, and the brush 5 can also clear up the surface of the filter plate 6 in the stirring process, prevent the filter plate 6 from blocking.

After cell culture is completed, the switch valve 7 is opened to communicate the culture container 1 with the collection container 2, the filtration pump is started simultaneously, culture solution flows into the collection container 2 through the switch valve 7, in the process, the culture solution firstly passes through the filter plate 6, part of large-particle impurities are filtered by the filter plate 6, then the culture solution flows into the collection container 2 again through the filter membrane group 10, the filter membrane group 10 filters the cell culture solution layer by layer, cells and other particle impurities are filtered, and finally particles (vesicles) with set sizes are discharged through the filtration tube 11 at the bottom of the collection container 2, so that collection of the vesicles is completed.

In the process of cell culture and vesicle separation, cell culture solution can be continuously added into the culture container 1 according to needs, and the culture solution is continuously discharged from the discharge end of the collection container 2, so that long-term and sustainable cell culture and vesicle obtaining are realized, the test steps are simplified, and the working efficiency is higher.

Example 2

Referring to fig. 1, this embodiment is an improvement on embodiment 1, specifically, in this embodiment, the culture container 1 and the collection container 2 are both double-glass containers, the inner layers of the culture container 1 and the collection container 2 are both accommodating chambers 101, the interlayer between the inner and outer layers is a water bath chamber 102, and the water bath chamber 102 is externally connected with a constant temperature water bath tank through a water pipe. The circulating constant temperature hot water is continuously injected into the water bath 102 through the constant temperature water bath tank, so that the internal environment of the reaction chamber is kept in a constant temperature state, a better environmental condition is provided, and the growth of cells is facilitated. The constant temperature water bath box is a DC-0506 water bath box of the Shanghai balance instrument, and the water inlet end and the water outlet end of the constant temperature water bath box are respectively communicated with the lower part and the upper part of the water bath room 102.

In some embodiments, the first cover 8 further has a plurality of openings, including a gas outlet, a gas inlet, a sample inlet, and three mounting ports, the gas inlet is externally connected to a nitrogen tank for delivering nitrogen, the gas outlet is externally connected to a gas pump, if an anaerobic condition is required, nitrogen is input into the culture container through the gas inlet, oxygen inside the culture container is discharged through the gas outlet, and both the gas inlet and the gas outlet are connected to a filter with a pore size of 0.22 μm. The sample inlet is provided with the sample inlet pipe. Three installing port is connected with the pH sensor, dissolved oxygen sensor, the carbon dioxide sensor that stretch into in culture container 1 respectively, and processing terminals such as above-mentioned sensor and computer carry out signal connection through connected modes such as communication line, and processing terminal receives the signal that above-mentioned sensor fed back and saves to the high in the clouds (the feedback of being connected of above-mentioned sensor and computer and signal all adopts prior art, no longer has been repeated in this embodiment). The dissolved oxygen sensor is an AMT-D300 intelligent dissolved oxygen sensor, the pH sensor is an AMT-PH300 intelligent pH value sensor, and the carbon dioxide sensor is an AMT-CO2300 intelligent carbon dioxide sensor.

When the water bath device is used, the constant-temperature water bath box is started, the water temperature is set to be 37 ℃, circulating water output by the constant-temperature water bath box enters from the bottom of the water bath room 102 and is discharged from the upper part of the water bath room 102, and water bath circulation is started, so that the internal environment of the accommodating chamber 101 is kept in a constant-temperature state. In the working process, the pH value, the dissolved oxygen amount, the carbon dioxide concentration and other characteristic values of the culture solution are monitored in real time through a computer, and the culture environment of the reaction chamber is ensured.

In some embodiments, the reactor further includes a peristaltic pump, the peristaltic pump includes a first water inlet, a first water outlet, a second water inlet, and a second water outlet, the first water inlet is communicated with the liquid storage tank storing the cell culture fluid through a pipe, the first water outlet is communicated with the first sample inlet pipe, the second water inlet is communicated with the filtration pipe at the lower end of the collection container 2, and the second water outlet is connected with a collection bottle for collecting vesicles. When the culture medium collection bottle is used, the peristaltic pump is started, the infusion and drainage functions are started simultaneously, the flow speed of the infusion and drainage is set, the culture medium is added into the culture container 1 at a constant speed, and the cell culture medium is drained from the collection container 2 to the collection bottle at a constant speed, so that the culture medium is updated and replaced. The peristaltic pump is a basic peristaltic pump, the model of the driver is L100-1E, and the model of the pump head is DG-2 (10 rollers).

The foregoing detailed description of the preferred embodiments of the utility model has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. The bioreactor for separating extracellular vesicles is characterized by comprising a culture container (1) and a collection container (2), wherein the discharge end of the culture container (1) is connected with the feed end of the collection container (2) and is provided with a switch assembly for controlling communication or blocking; a filter unit is arranged in the culture container (1), and the filter unit comprises a filter plate (6) arranged at the lower part of the culture container (1); a separation unit is arranged in the collection container (2), and the separation unit comprises a filter membrane group (10) arranged at the upper part in the collection container (2).

2. A bioreactor for isolating extracellular vesicles according to claim 1, wherein: the culture container is also internally provided with a stirring unit which is positioned above the filter plate (6) and comprises a motor (4), a rotating shaft (3) driven by the motor (4) to rotate and a stirring component arranged on the rotating shaft (3).

3. A bioreactor for isolating extracellular vesicles according to claim 2, wherein: the stirring component comprises a brush (5) fixedly connected to the bottom of the rotating shaft (3), and bristles on the lower portion of the brush (5) are in surface contact with the filter plate (6).

4. A bioreactor for isolating extracellular vesicles according to claim 2 or 3, wherein: the stirring assembly comprises a plurality of stirring blades fixedly connected to the rotating shaft (3).

5. A bioreactor for isolating extracellular vesicles according to claim 4, wherein: every filter membrane of filtration aperture specification all has two, all is equipped with the sieve between two filter membranes of same specification filter aperture.

6. A bioreactor for isolating extracellular vesicles according to claim 1, 2, 3 or 5, wherein: the culture medium collection device is characterized by further comprising a peristaltic pump, wherein the peristaltic pump comprises a first water inlet, a first water outlet, a second water inlet and a second water outlet, the first water inlet and the first water outlet are respectively connected with the feed ends of the culture medium container and the culture container (1), and the second water inlet and the second water outlet are respectively connected with the discharge end of the collection container (2) and the collection bottle.

7. A bioreactor for isolating extracellular vesicles according to claim 1, wherein: the culture container (1) and the separation container are both double-layer glass containers, the inner layer is a containing chamber (101) for containing culture solution, the interlayer between the inner layer and the outer layer is a water bath chamber (102), and the water bath chamber (102) is externally connected with a constant-temperature water bath tank through a pipe fitting.

8. A bioreactor for isolating extracellular vesicles according to claim 1, 2, 3 or 5, wherein: cultivate container (1) top and be equipped with first lid (8), be equipped with gas outlet, air inlet, introduction port and supply the through-hole that pivot (3) passed on first lid (8), still be connected with pH sensor, dissolved oxygen sensor, the carbon dioxide sensor that stretches into in cultivating container (1) on first lid (8).

9. A bioreactor for isolating extracellular vesicles according to claim 1, wherein: the filter plate (6) is a glass sand core.