阅读说明：本技术 一种便携式培养细胞的外泌体制备、富集、纯化收集系统 (Portable exosome preparation, enrichment, purification collection system of cultured cells ) 是由 崔大祥 彭家伟 梁辉 周诚 于 2021-08-09 设计创作，主要内容包括：本发明涉及一种便携式培养细胞的外泌体制备、富集、纯化收集系统,包括生产系统、浓缩收集系统、循环流体系统。富集收集装置中超滤膜与超滤膜管形成锐角夹角,优选范围为30-5°,设置夹角,使得平行流过浓缩外管的包含外泌体的流体培养基经过超滤膜时,尽可能避免流体培养基滤液垂直经过超滤膜形成“死滤”,不仅起到超滤作用,同时也会冲带走超滤膜表面截留的外泌体,防止后续截留的外泌体在超滤膜上积累造成滤孔的堵塞,起到模拟切向流过滤效果。使后续生产容器中培养细胞产生的外泌体源源不断的在富集装置中富集、纯化,获得高浓度、高纯度的外泌体。并且各组成设备均可灵活拆卸定期更换,防止污染以及长期使用造成的培养物黏附堵塞。(The invention relates to a portable exosome preparation, enrichment, purification and collection system for cultured cells, which comprises a production system, a concentration and collection system and a circulating fluid system. Ultrafiltration membrane and ultrafiltration membrane tube form acute angle contained angle among the enrichment collection device, preferred scope is 30-5, set up the contained angle, when making the fluid medium that contains the exosome of concentrated outer tube of concurrent flow through the ultrafiltration membrane, avoid fluid medium filtrating perpendicular through the ultrafiltration membrane formation "dying to strain" as far as possible, not only play the ultrafiltration effect, also can wash away the exosome of intercepting on the ultrafiltration membrane surface simultaneously, prevent that the subsequent exosome of intercepting from accumulating on the ultrafiltration membrane and causing the jam of filtration pore, play the simulation tangential flow and strain the effect. The exosome source generated by culturing cells in the subsequent production container is continuously enriched and purified in the enrichment device, and the exosome with high concentration and high purity is obtained. And all the components can be flexibly disassembled and replaced regularly, so that the culture is prevented from being adhered and blocked due to pollution and long-term use.)

1. A portable exosome preparation, enrichment, purification collection system for cultured cells, comprising a production vessel (22) and a collection vessel (2), characterised by comprising a production system, an enrichment collection system and a circulating fluid system, wherein,

the production system at least comprises a production container (22) and a stirrer (7), wherein a culture medium (23), a microcarrier (4) required by 3D culture and a cultured cell (3) are filled in the production container (22), and the production container (22) is provided with a filling port (6), a liquid outlet (8) and a liquid return port (24); the production cells (3) are adhered to the surface of the microcarrier (4) to grow, under the stirring action of the stirrer (7), the microcarrier (4) and the culture cells (3) growing on the surface are suspended in a turbulent flow culture medium, and the stimulation of the turbulent flow promotes the massive secretion of the exosome EVs (5);

the enrichment and collection system consists of a detachable ultrafiltration membrane pipe (25), an ultrafiltration membrane (19) arranged in the ultrafiltration membrane pipe (25) and forming an included angle with the wall of the ultrafiltration membrane pipe along the flow direction of a fluid, a first concentration outer pipe, a second concentration outer pipe (28) (29) and a collection container (2), wherein the first concentration outer pipe, the second concentration outer pipe and the collection container are hermetically connected with the ultrafiltration membrane pipe (25), the first concentration outer pipe, the second concentration outer pipe and the collection container are connected into a circulating system through a fluid pipe (1) through a first inlet interface and a second outlet interface (16) and (20) which are connected with two sides, and the ultrafiltration membrane pipe (25) is seamlessly connected with the first concentration outer pipe, the second concentration outer pipe (28) and the concentration outer pipe (29) through a first spiral interface, a second spiral interface (17) and a spiral interface (18) on two sides to form a hollow concentration container; the collecting container (2) is connected to the bottom of a first concentrating outer pipe (28) in the liquid inlet direction;

the circulating fluid system comprises a circulating system consisting of a fluid pipe (1), a filter membrane (9), a pipe fitting connector (14), a peristaltic pump (10) and a three-way valve, EVs (5) produced by the production system are filtered by the filter membrane (9) and then enter the circulating fluid system from a liquid outlet (8) of the production container along with a culture medium (23), microcarriers (4) and culture cells (3) are intercepted in the production container (22) to continuously produce the EVs (5), the culture medium (23) and the produced EVs (5) flow to the enrichment collection system through the liquid outlet (8) by the peristaltic pump (10), meanwhile, the culture medium (26) in the enrichment collection system returns to the production container (22) through a liquid return port (24), and finally the dynamic circulating fluid system with the EVs enriched and the culture medium capable of being recycled is formed.

2. The system for preparing, enriching, purifying and collecting exosomes of portable cultured cells according to claim 1, wherein the production vessel (22) in the production line has a volume of 50 mL to 60L, and is a fermenter, or a glass culture vessel.

3. The system for preparing, enriching and purifying exosomes of portable cultured cells according to claim 1, wherein the pore size of the ultrafiltration membrane (19) is 30-200 nm, so that culture medium (26) in the enrichment collection system can freely pass through to retain EVs in fluid, and dynamic enrichment of EVs is realized.

4. The portable exosome preparation, enrichment, purification and collection system for cultured cells according to claim 1 or 3, characterized in that the ultrafiltration membrane (19) and the ultrafiltration membrane tube (25) form an included angle of 30-5 °, so that when a fluid culture medium (26) containing enriched EVs (27) flowing through the concentration outer tube (28) in parallel passes through the ultrafiltration membrane (19), the fluid culture medium can wash away the EVs (27) retained on the surface of the ultrafiltration membrane (19), thereby preventing the EVs from accumulating on the ultrafiltration membrane (19) to cause the blockage of filter pores, and achieving the effect of simulating tangential flow filtration. So that EVs (5) produced by the cultured cells (3) in the subsequent production vessel (22) can be continuously enriched in the enrichment collection system, and the enrichment collection of EVs produced by any form of cultured cells including 3D cultured cells can be realized.

5. A portable exosome preparation, enrichment, purification collection system for cultured cells according to claim 4, characterised in that the filter membrane (9) is selected with a pore size of 0.22-0.45 μm.

6. The portable exosome preparation, enrichment, purification collection system for cultured cells according to claim 1, characterized in that the agitator (7) is replaceable to meet the optimal growth environment required for EVs production by suspension culture of different types of cultured cells according to adjustment of its size, type of paddle, and rotation speed.

7. The method for the purification and collection of an exosome preparation, enrichment and purification collection system for the portable cultured cells according to any one of claims 1 to 6, characterized in that when the enriched EVs-containing culture medium (26) obtained in the enrichment collection system is re-flowed back to the inside of the production vessel (22) from the liquid return port, the culture medium (23) in the production system is changed to PBS buffer so that the enriched EVs-containing culture medium is continuously diluted and purified until the culture medium is completely replaced, the collection vessel (2) is opened, and the purified EVs are collected.

8. The method for replacing a culture medium in an exosome preparation, enrichment, purification collection system for portable cultured cells according to any one of claims 1 to 7, characterized in that in the circulating fluid system, an inlet (11) and a vertical outlet (13) of a three-way valve are opened, a horizontal outlet (12) of the three-way valve is closed, a new culture medium is added from a filling port (6), and an old culture medium is discharged from the vertical outlet (13) of the three-way valve (13), so that the replacement of the new culture medium and the old culture medium is conveniently realized.

9. The system for preparing, enriching, purifying and collecting exosomes of portable cultured cells according to claim 1, wherein the cultured cells (3) include, but are not limited to, umbilical cord mesenchymal stem cell adherent growth cells.

10. A portable exosome preparation, enrichment, purification collection system for cultured cells according to claim 1, characterised in that the microcarriers (4) are non-cytotoxic micron material meeting the cell adhesive growth characteristics, with a particle size of 20-200 μ ι η.

Technical Field

The invention relates to a portable exosome preparation, enrichment, purification and collection system for cultured cells.

Technical Field

Exosomes (EVs) are spherical membrane vesicles with the particle size of 30-150 nm formed by surrounding lipid bilayers, are subcellular structures secreted by cells, and play an important role in the process of intercellular information transmission. At present, methods for collecting exosomes mainly comprise ultracentrifugation, ultrafiltration, kit extraction and the like. The ultracentrifuge is limited by the fact that the ultracentrifuge is expensive and long in time consumption, and is difficult to popularize and apply. The extraction method of the kit usually utilizes a chemical reagent or a modified micron nano material to capture exosomes for purification, so that the surface structure of the exosomes can be damaged to a certain extent. The ultrafiltration method is to intercept the particles with larger size and pass the substances with small particle size or small molecular weight by utilizing different intercepted molecular weights or particle sizes, thereby achieving the purpose of extraction. At present, commercial ultrafiltration for extracting EVs has two forms of ultrafiltration tangential flow filtration and hollow fiber tubes, but depends on import, is expensive and has small flux, and is difficult to be suitable for actual large-scale production.

Disclosure of Invention

In view of the above disadvantages, the present invention aims to provide a portable exosome preparation, enrichment, purification and collection system for cultured cells, which is suitable for large-scale production.

The invention aims to provide the following scheme for realization: a portable exosome preparation, enrichment, purification collection system for cultured cells, comprising a production vessel and a collection vessel, comprising a production system, an enrichment collection system and a circulating fluid system,

the production system at least comprises a production container and a stirrer, wherein the production container is filled with a culture medium, microcarriers required by 3D culture and cultured cells, and is provided with a filling port, a liquid outlet and a liquid return port; the production cells are adhered to and grow on the surface of the microcarrier, the microcarrier and the culture cells growing on the surface are suspended in a turbulent flow culture medium under the stirring action of a stirrer, and the stimulation of the turbulent flow promotes the massive secretion of the exosome EVs;

the enrichment collection system consists of a detachable ultrafiltration membrane pipe, ultrafiltration membranes arranged in the ultrafiltration membrane pipe and forming an included angle with the wall of the ultrafiltration membrane pipe along the fluid flowing direction, a first concentration outer pipe, a second concentration outer pipe and a collection container, wherein the first concentration outer pipe and the second concentration outer pipe are hermetically connected with the ultrafiltration membrane pipe; the collecting container is connected to the bottom of the first concentrating outer pipe in the liquid inlet direction;

the circulating fluid system comprises a fluid pipe, a filter membrane, a pipe fitting connector, a peristaltic pump, a three-way valve and the like, and the fluid pipe can be connected to the required length through a connector in a sealing manner according to actual requirements; EVs generated in the production system is filtered by a filter membrane and then enters the circulating fluid system from a liquid outlet of the production container along with the culture medium, microcarriers and cultured cells are blocked in the production container to continuously produce the EVs, the culture medium and the produced EVs flow to the enrichment and collection system through the liquid outlet of the production container by the peristaltic pump, meanwhile, the culture medium in the enrichment and collection system flows back to the interior of the production container again through a liquid return port, and finally, the dynamic circulating fluid system with the EVs enriched and the culture medium recycled is formed.

The portable exosome preparation, enrichment, purification and collection system device for the cultured cells can be successfully applied to enrichment, purification and collection of the cultured cell EVs.

On the basis of the scheme, the production container in the production system has the volume of 50 mL-60L, and can be a fermentation tank, a glass culture container common in laboratories and the like.

On the basis of the scheme, the aperture of the ultrafiltration membrane is 30-200 nm so as to meet the requirement that the culture medium in the enrichment and collection system passes through freely to intercept EVs in the fluid and realize the dynamic enrichment of the EVs.

Furthermore, the preferred range of the included angle formed by the ultrafiltration membrane and the ultrafiltration membrane is 30-5 degrees, and a certain included angle is formed, so that when a fluid culture medium which flows through the concentration outer tube in parallel and contains the enriched EVs passes through the ultrafiltration membrane, the situation that filtrate of the fluid culture medium vertically passes through the ultrafiltration membrane to form 'dead filtration' is avoided as much as possible, the ultrafiltration effect is achieved, meanwhile, the EVs intercepted on the surface of the ultrafiltration membrane can be washed away, the subsequent intercepted EVs are prevented from being accumulated on the ultrafiltration membrane to cause filter hole blockage, the effect of simulating tangential flow filtration is achieved, the EVs generated by cultured cells in a subsequent production container are continuously enriched in an enrichment and collection system, and the device can be used for enrichment and collection of the EVs generated by cultured cells in any form including 3D cultured cells.

The enrichment collection system is not only suitable for collecting EVs generated by 3D cultured cells, but also suitable for enriching and collecting EVs generated by any form of cultured cells.

Preferably, the size of the pores of the filter membrane at the liquid outlet is 0.22-0.45 μm, and in practice, filter membranes with different pore sizes can be selected according to the particle sizes of the microcarriers and cultured cells.

On the basis of the scheme, the stirrer is replaceable, so that the size, the type of the stirring paddle and the rotating speed of the stirrer can be adjusted according to requirements to meet the optimal growth environment required by the suspension culture of different types of cultured cells for producing EVs.

The invention provides a purification and collection method of a portable exosome preparation, enrichment and purification collection system for cultured cells, which is characterized in that when an enriched EVs-containing culture medium obtained in the enrichment collection system flows back to the interior of a production container from a liquid return port, the culture medium in the production container is changed into PBS buffer solution, so that the enriched EVs-containing culture medium is continuously diluted and purified until the culture medium is completely replaced, the collection container is opened, and the purified EVs are collected.

In the circulating fluid system, an inlet and a vertical outlet in a three-way valve are opened, a horizontal outlet of the three-way valve is closed, a new culture medium is added from a filling port of a production container, and an old culture medium is discharged from the vertical outlet of the three-way valve, so that the convenient replacement of the new culture medium and the old culture medium is realized.

In the production system of the present invention, the cultured cells include, but are not limited to, adherent growing cells of umbilical cord mesenchymal stem cells.

In the production system, the microcarrier is a non-cytotoxic micron material meeting the cell adhesive growth characteristics, and the particle size is 20-200 microns.

Advantageous effects

Compared with the prior ultracentrifugation method, tangential flow filtration, hollow fiber tube ultrafiltration method, kit extraction and other methods. When the method is used for enriching the exosomes by ultrafiltration, the phenomenon that the filtrate of the fluid culture medium vertically passes through the ultrafiltration membrane to form 'dead filtration' is avoided, so that the ultrafiltration effect is achieved, the exosomes intercepted on the surface of the ultrafiltration membrane are washed away, the blockage of filter holes caused by the accumulation of the subsequently intercepted exosomes on the ultrafiltration membrane is prevented, and the effect of simulating tangential flow filtration is achieved. The method is suitable for generating enrichment, purifying and collecting the exosome of the cultured cells at the same time, and the exosome with high concentration and high purity is obtained. And all the components can be flexibly disassembled and replaced regularly, so that the culture is prevented from being adhered and blocked due to pollution and long-term use.

In conclusion, the system device is suitable for the characteristics of producing exosomes such as sustainability, low cost, high concentration and high purity, and has remarkable economic and social benefits.

Drawings

FIG. 1 is a schematic diagram of the system of the present invention;

FIG. 2 is a schematic structural view of an ultrafiltration enrichment collection device;

FIG. 3 is a transmission electron microscope image of exosomes extracted by the system device of the present invention;

in the production system, the production process includes the steps of,

22-production vessel; 21-electric machine;

3-culturing the cells; 4-microcarriers; 5-Exosomes (EVs);

6-a filling opening; 7-stirrer;

8-a liquid outlet; 9-filter membrane; 24-liquid return port of production system; 23-culture medium;

in the circulating liquid system, the liquid is circulated,

1-fluid pipe; 10-peristaltic pump; 11. 12, 13-three-way valve; 14-pipe connector;

in the enrichment system, the concentration of the carbon dioxide is controlled,

2-a collection vessel; 15-one-way valve;

16-inlet to the enrichment system;

17-liquid inlet with screw interface; 18-spiral interface liquid outlet; 19-ultrafiltration membrane;

20-outlet of the enrichment system;

25-ultrafiltration membrane tube;

26-enrichment of the medium in the system; 27-enriched EVs;

28. 29-concentration outer tube one, two.

Detailed Description

In order to achieve the purpose, the invention adopts the following technical scheme:

example 1

A portable exosome preparation, enrichment, purification collection system for cultured cells, comprising a production system, an enrichment collection system and a circulating fluid system, comprising a production vessel 22, wherein,

the production system comprises a production container 22 and a stirrer 7, wherein a culture medium 23, microcarriers 4 required by 3D culture and cultured cells 3 are filled in the production container 22, and the production container 22 is provided with a filling port 6, a liquid outlet 8 and a liquid return port 24; the production cells 3 are adhered to the surface of the microcarrier 4 to grow, under the stirring action of the stirrer 7, the microcarrier 4 and the culture cells 3 growing on the surface float in a turbulent flow culture medium, and the stimulation of the turbulent flow promotes the massive secretion of the exosome EVs 5;

the enrichment and collection system consists of a detachable ultrafiltration membrane pipe 25, an ultrafiltration membrane 19 which is arranged in the ultrafiltration membrane pipe 25 and forms an included angle with the wall of the ultrafiltration membrane pipe along the fluid flowing direction, a first concentration outer pipe, a second concentration outer pipe 28, 29 which are hermetically connected with the ultrafiltration membrane pipe 25 and a collection container 2, the ultrafiltration membrane pipe 25 is connected into a circulating system through a fluid pipe 1 through inlet and outlet ports 16, 20 which are connected at two sides, and the ultrafiltration membrane pipe 25 is seamlessly connected with the first concentration outer pipe, the second concentration outer pipe 28, 29 through spiral ports I, II 17, 18 at two sides to form a hollow concentration container; the collecting container 2 is connected with the inlet 16 of the enrichment system and the bottom of a first concentrating outer pipe 28 of the section of the liquid inlet 17 of the screw connector;

the circulating fluid system comprises a circulating system consisting of a fluid pipe 1, a filter membrane 9, a pipe fitting connector 14, a peristaltic pump 10 and a three-way valve, wherein EVs5 produced by the production system is filtered by the filter membrane 9 and then enters the circulating fluid system along with a culture medium 23 from a liquid outlet 8 of the production container, the microcarriers 4 and the cultured cells 3 are intercepted in the production container 22 to continuously produce the EVs5, the culture medium 23 and the produced EVs5 are conveyed to the enrichment collection system through the liquid outlet 8 by the peristaltic pump 10, meanwhile, the culture medium 26 in the enrichment collection system returns to the interior of the production container 22 through a liquid return port 24, and finally, the dynamic circulating fluid system with enriched EVs and recyclable culture medium is formed.

In the invention, a certain included angle is formed between the ultrafiltration membrane 19 and the section of the ultrafiltration membrane tube 25 in the enrichment and collection device, the preferable range is 30-5 degrees, and the certain included angle is set, so that when the fluid culture medium 26 which flows through the concentration outer tube in parallel and contains exosomes passes through the ultrafiltration membrane 19, the phenomenon that filtrate of the fluid culture medium 26 vertically passes through the ultrafiltration membrane to form 'dead filtration' is avoided as much as possible, the ultrafiltration effect is achieved, the enriched EVs 27 intercepted on the surface of the ultrafiltration membrane 19 can be washed away, the blockage of filtration pores caused by the accumulation of the subsequently intercepted EVs on the ultrafiltration membrane 19 is prevented, the simulated tangential flow filtration effect is achieved, the EVs5 generated by the cultured cells 3 in the subsequent production container 22 are continuously enriched in the enrichment system device, and then the collected culture medium containing EVs is added into the production container 22 from the filling port 6.

The components of the invention are convenient to disassemble and assemble, and can be disassembled and assembled according to the use requirements.

Application example 1

Preparing, enriching and purifying the EVs by adopting the exosome preparation, enrichment and purification collecting system of the portable cultured cells, wherein the cultured cells are selected to be Mesenchymal Stem Cells (MSC); the microcarrier 4 is of the Cytodex 1 type and comprises the following steps:

the first step is as follows: inside the production vessel 22, cultured cells 3 are seeded to the surface of the microcarrier 4 at a certain concentration by 3D culture technique for culture, and the stirrer 7 is maintained at a stirring speed of 40 rpm for continuous culture for 5D, wherein the maximum volume of the culture medium 23 such as DMEM medium is determined by the cultured cells 3 and the vessel volume, preferably 50 mL-30L.

In the second step, as shown in FIG. 1, opening the three-way valve inlet 11 and outlet 12, closing the three-way valve vertical outlet 13, allowing the culture medium 23 and EVs5 to flow from the outlet 8 to the enrichment collection system under the action of the peristaltic pump 10 and the filter membrane 9, while the microcarriers 4 and the cultured cells 3 are intercepted in the production vessel 22 to continue producing EVs, and at the same time, the culture medium 26 in the enrichment system is returned to the interior of the production vessel 22 through the liquid return port 24, and the collection vessel 2 is opened to collect the EVs 27 enriched in the enrichment system.

In the third step, another identical culture apparatus is prepared, and the culture medium collected in the above-described enrichment system including EVs is fed into the production vessel 22 through the filling port 6, with the difference that: the culture medium 23 was changed to PBS buffer so that the enriched EVs-containing culture medium was continuously diluted and purified until the culture medium was completely replaced, at which time the collection vessel 2 was opened and the EVs containing only PBS liquid was collected. The transmission electron microscope image of exosome extracted by the system device is shown in fig. 3, the EVs obtained by enrichment and purification of the system device has good size and dispersity, and the particle size is about 33 nm.

Application example 2

The system of the invention is adopted to obtain the purified EVs, and comprises the following steps:

the first step is as follows: inside the production vessel 22, cultured cells 3 such as Mesenchymal Stem Cells (MSC) are seeded at a certain concentration on the surface of microcarrier 4Cytodex type 1 for culture using 3D culture technique, 3D static culture is performed while maintaining the stirrer 7 at 0 rpm, and continuous culture is performed for 5D, wherein the maximum volume of vessel medium 23 such as DMEM medium is determined by the cultured cells 3 and the vessel volume, preferably 50 mL to 30L.

And secondly, opening a three-way valve liquid inlet 11 and a horizontal liquid outlet, closing a three-way valve vertical liquid outlet 13, enabling the culture medium 23 and the EVs5 to flow to the enrichment collection system through a liquid outlet 8 under the action of a peristaltic pump 10 and a filter membrane 9, stopping the microcarriers 4 and the cultured cells 3 in a production container 22 to continuously produce the EVs, simultaneously refluxing the culture medium 26 to the inside of the production container 22 again through a liquid return port 24, opening the collection container 2, and collecting the enriched EVs 27 in the liquid culture medium.

In the third step, another identical culture apparatus is prepared, and the culture medium containing EVs collected in the above step is added to the production vessel 22 from the filling port 6, except that the culture medium 23 is changed to PBS buffer so that the enriched culture medium containing EVs is continuously diluted and purified until the culture medium is completely replaced, at which time the collection vessel 2 is opened and EVs containing only PBS liquid is collected. Thus obtaining the EVs with high concentration and high purity.

All the components of the invention can be flexibly disassembled and replaced regularly, thus preventing the culture from being adhered and blocked due to pollution and long-term use.

Application example 3

The difference between the application example and the application example 1 is that MSC with the same concentration is inoculated into a commercial culture dish for 2D cell adherent culture for 5D, then the upper layer culture medium is collected and is put into the system device of the invention through a filling port 6, and the EVs in the culture medium is continuously enriched, purified and collected by PBS.