CN113832112A - Mixing method and mixing system for preparing lentivirus - Google Patents

Mixing method and mixing system for preparing lentivirus Download PDF

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CN113832112A
CN113832112A CN202010587853.5A CN202010587853A CN113832112A CN 113832112 A CN113832112 A CN 113832112A CN 202010587853 A CN202010587853 A CN 202010587853A CN 113832112 A CN113832112 A CN 113832112A
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黄一
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Chongqing Precision Biotech Co ltd
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Chongqing Precision Biotech Co ltd
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Abstract

The invention belongs to the field of lentivirus packaging, and particularly relates to a mixing method and a mixing system for preparing lentiviruses. The mixing method for preparing the lentivirus comprises the steps of mixing or reacting a transfection reagent in a mixing container, pumping a culture medium in a cell culture container into the mixing container through a pump and a cell culture container communicating vessel to be mixed with the transfection reagent to obtain a final mixed solution, and pumping the final mixed solution into the cell culture container through the pump and the cell culture container communicating vessel to carry out cell culture; the mixing system for preparing the lentivirus comprises a reagent unit A, a reagent unit B, a mixing unit C, a pump D, a conduit, a cell culture container communicating vessel E, a cell culture unit F, a purification unit G and a numerical control unit H; the mixing method and the mixing system for preparing the lentivirus, provided by the invention, simplify the preparation steps of preparing the lentivirus and provide a lentivirus preparation system which is more closed and is not easily interfered by the external environment.

Description

Mixing method and mixing system for preparing lentivirus
Technical Field
The invention belongs to the field of lentivirus packaging, and particularly relates to a mixing method and a mixing system for preparing lentiviruses.
Background
Lentivirus (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I). A distinction is made between retroviral vectors in general, which have the ability to infect both dividing and non-dividing cells.
Lentivirus is a genus of the retroviridae family, comprising 8 viruses capable of infecting humans and vertebrates, primarily infected cells, dominated by lymphocytes and macrophages, which ultimately affect the individual. A significant feature of lentiviral infections is that the infected individual experiences a latent period of up to several years, mostly before the typical clinical symptoms appear, and then slowly develops, and these pathogens are therefore called lentiviruses. For example, Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia (EIA), Feline Immunodeficiency Virus (FIV) are lentiviruses.
The research of lentiviral vectors has progressed rapidly and is very intensive. The vector can effectively integrate the exogenous gene onto the host chromosome, thereby achieving persistent expression. Can effectively infect various types of cells such as neuron cells, liver cells, myocardial cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capacity, thereby achieving good gene therapy effect, developing clinical research, and having ideal effect, thereby having wide application prospect.
The calcium phosphate method for preparing lentivirus is a common method, and comprises the steps of mixing deionized water, calcium chloride and plasmids in a container, slowly adding HBS solution inwards, uniformly mixing the mixture in a magnetic stirring mode to form a calcium phosphate-DNA (deoxyribonucleic acid) containing compound, adding the transfection compound into a cell factory, and uniformly mixing an internal culture medium and the transfection compound by shaking the cell factory. Because the process of forming the calcium phosphate-DNA compound by mixing the transfection reagent is an open environment (in a biological safety cabinet, a container is open), when raw materials such as plasmids and the like are added, microorganisms are easily introduced, and the pollution risk exists. The transfection compound needs to be transferred into a cell factory and uniformly mixed with a culture medium, and the other way is that the transfection mixed solution is directly added into the cell factory, the purpose of uniformly mixing the transfection compound and the culture medium is achieved by slowly shaking the cell factory, because the cell factory has a special structure, the transfection compound and the culture medium may have the condition of nonuniform mixing, each layer of transfection compound in the cell factory is unevenly distributed, meanwhile, because the production cells used for preparing the lentivirus are HEK293T cells, the adherence is not strong, and the shaking of the mixed transfection compound and the culture medium may cause the cells to fall off, thereby affecting the transfection efficiency of the plasmids. Another way is to add the transfection complex into a simple fluid exchange device, then connect the cell factory with the device, pump the culture medium in the cell factory into the device through a peristaltic pump, mix the culture medium with the transfection complex uniformly, and then pump the culture medium back into the cell factory. The disadvantage of this approach is that the process of transferring transfection reagents to the fluid exchange device is also an open environment (inside the biosafety cabinet) with a certain risk of contamination.
The utility model discloses a chinese utility model patent (CN201822035919.0) discloses a blender convenient to pack lentivirus in batches, and the blender body is equipped with two stock solution cavitys, is A liquid receiver bottle and B liquid receiver bottle respectively, is equipped with the connectivity between A liquid receiver bottle and the B liquid receiver bottle, the connectivity on be equipped with controlling means, the connectivity be the hose, A liquid receiver bottle one side is equipped with fixing device, B liquid receiver bottle bottom is equipped with vibrating structure, B liquid receiver bottle in be equipped with the syringe needle, B liquid receiver bottle top is equipped with the needle inlet, syringe needle one end coupling hose, the syringe needle passes through the needle inlet and gets into B liquid receiver bottle, the syringe needle other end is the liquid outlet, the liquid outlet setting is in B liquid receiver bottle bottom, the liquid outlet slope sets up. The method has the advantages that the labor intensity and time for operation when the solution A and the solution B are mixed during packaging the lentivirus are saved, and the method is also suitable for packaging the lentivirus in batches; in addition, the processing is simple, the device can be used repeatedly, the cost is low, and the popularization is convenient.
However, the mixer disclosed in the above utility model can be used only for mixing the solution A and the solution B for packaging lentivirus, and does not provide a process for introducing the mixture into a culture vessel; the liquid storage bottle of the liquid B is not provided with a liquid outlet, when the liquid A and the liquid B are mixed, the cover can be opened only when the mixed liquid is transferred into the culture container, and the mixed liquid is taken out, so that the pollution is easily caused; the mixed type liquid storage bottle is provided with two liquid storage bottles, so that personnel needs to observe and process the two liquid storage bottles, and the labor is also required to be expended; the liquid storage bottle A is connected with a pressurizing device, and the liquid storage bottle B is provided with a pressure discharge port, so that the liquid storage bottle B is easily filled with the liquid to extrude the liquid.
Disclosure of Invention
One of the objects of the present invention is to provide a mixing method which simplifies the process of preparing viruses, simplifies the steps of mixing reagents to form a transfection reagent and mixing the transfection reagent with a culture medium to be completed in the same device.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a mixing method for preparing lentivirus comprises the steps of mixing or reacting transfection reagents in a mixing container, pumping culture medium in a cell culture container into the mixing container through a pump and a cell culture container communicating device to be mixed with the transfection reagents to obtain final mixed liquid, and pumping the final mixed liquid into the cell culture container through the pump and the cell culture container communicating device to be cultured.
Further, the mixing container consists of a bottle body (1) and a fermentation tank feeding bottle adapter (3); the fermentation tank feed supplement bottle adapter (3) is connected with a CPC male connector, a CPC female plug (4), an air filter (5), a luer female connector, a male plug/needle head filter (6) through a silica gel tube; a transfer pipe of the fermentation tank feeding bottle adapter (3) connected with the CPC male connector and the female plug (4) is connected with a countersunk head (2) through a silicone tube and is arranged at the bottom of the bottle body (1); a switching tube of the fermentation tank feeding bottle adapter (3) which is connected with the luer female connector and the male plug/needle head filter (6) is connected with a silicone tube and is arranged in the bottle body (1), and the dropping position of liquid is controlled by connecting silicone tubes with different lengths; a magnetic stirrer (7) is arranged in the bottle body (1); the cell culture container communicating vessel and the mixing container are connected through a CPC joint of the CPC male joint, the female plug (4) and the CPC female joint (12).
Further, the cell factory communicating vessel comprises a silicone tube (11), a CPC female joint, a CPC male plug (8), a three-way conversion head (9), a CPC female joint (12) and an air filter (5); female joint of a CPC (12) is connected to silicone tube (11) one end, female joint of CPC (12) other end is connected one air cleaner (5), a tee bend conversion head (9) or female joint of CPC and the public end cap of CPC (8) are connected to silicone tube (11) other end, through tee bend conversion head (9) are connected female joint of CPC and the public end cap of CPC (8) and/or tee bend conversion head (9) will cell culture container linker realizes that a plurality of cell culture containers connect.
Further, the pump is a peristaltic pump.
Specifically, the method comprises the following steps:
s1: culturing transfected cells:
digesting cells by enzyme, inoculating the cells into a cell culture container for culture, and achieving 50-90% of confluency;
s2: preparing and mixing a transfection reagent:
preparing a required reagent, and introducing the reagent into the mixing container through a luer female connector and a male plug/needle filter (6) of the mixing container for mixing to obtain a transfection reagent;
s3: mixing a culture medium and a transfection reagent in a cell culture container through a pump and a cell culture container communicating vessel, and pumping the mixture into the cell culture container for culture;
s4: separating and purifying the supernatant.
Specifically, the cell culture container is a cell factory or a cell culture bottle with a CPC connector.
It is another object of the invention to provide a hybrid system adapted to the method.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a mixing system for the preparation of lentiviruses, said system comprising a mixing unit C, a cell culture unit F, a pump D, a conduit and a cell culture container communicator E; the mixing unit C is connected with the pump D through the conduit; the pump D is connected with the cell culture unit F through the cell culture container communicating vessel E; the cell culture container communicating vessel E consists of a conduit and a tee pipe fitting.
Furthermore, a flow meter and an electromagnetic valve are arranged at the feed inlet in the mixing unit C and the connection port of the cell culture container communicating vessel E close to the cell culture unit F.
Further, the mixing system also comprises a reagent unit A and a reagent unit B; the reagent unit A and the reagent unit B are respectively connected with the mixing unit C through a conduit; the reagent unit A is used for preparing a PEI reagent or a calcium chloride or a plasmid reagent; the reagent unit B is used to formulate a plasmid, a 1 × HBS reagent or a 2 × HBS reagent.
Further, the mixing system further comprises a purification unit G; the purification unit G is connected with the cell culture unit F through a conduit and is used for separating and purifying supernatant.
Further, the mixing system also comprises a numerical control unit H; and the numerical control unit H is used for receiving and processing the data transmitted by the flowmeter and controlling the on-off of the electromagnetic valve and the pump D.
The invention has the beneficial effects that:
1. the invention provides a mixing method for preparing lentivirus, which simplifies the flow of virus preparation, and simplifies the steps of mixing reagents to form a transfection reagent and the steps of mixing the transfection reagent and a culture medium to be finished in the same device.
2. The present invention provides a mixing system for the production of lentiviruses, which can be considered as a closed system, reducing the risk of contamination while allowing more thorough mixing of transfection reagents and culture media.
3. The mixing method and the mixing system for preparing the lentivirus, provided by the invention, can ensure that the transfection reagent and the culture medium can be more fully mixed, and simultaneously, the method steps are simplified, the labor cost is saved, and the pollution risk is reduced.
Drawings
FIG. 1: a schematic diagram of a cell culture container connector;
FIG. 2: a front view of the mixing vessel;
FIG. 3: schematic diagram of a hybrid system.
In the figure, 1 is a bottle body; 2 is a countersunk head; 3 is a feeding bottle adapter of the fermentation tank; 4, a CPC male connector and a female plug; 5 is an air filter; 6 is a luer female connector and a male plug/needle filter; 7 is a magnetic stirrer; 8 is a CPC female joint and a CPC male plug; 9 is a three-way conversion head; and 10 is a flow stopping clamp.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Example one
The cell factory communicating vessel comprises a silicone tube 11, a CPC female joint, a CPC male plug 8, a three-way conversion head 9, a CPC female joint 12 and an air filter 5; 11 one end of silicone tube connects a female joint of CPC 12, female joint of CPC 12 other end is connected one air cleaner 5, 11 other ends of silicone tube connect a tee bend conversion head 9 or female joint of CPC and the public end cap of CPC 8, through tee bend conversion head 9 connects female joint of CPC and the public end cap of CPC 8 and/or tee bend conversion head 9 will cell culture container linker realizes that a plurality of cell culture containers connect.
The mixing container consists of a bottle body 1 and a fermentation tank feeding bottle adapter 3; the fermentation tank feeding bottle adapter 3 is connected with a CPC male connector, a CPC female plug 4, an air filter 5, a luer female connector, a male plug/needle filter 6 through a silicone tube; the adapter tube of the fermentation tank feeding bottle adapter 3 connected with the CPC male connector and the female plug 4 is connected with a countersunk head 2 through a silicone tube and is arranged at the bottom of the bottle body 1; the adapter tube of the fermentation tank feeding bottle adapter 3 which is connected with the luer female connector and the male plug/needle filter 6 is connected with a silicone tube and is arranged in the bottle body 1, and the liquid dropping position is controlled by connecting the silicone tubes with different lengths; a magnetic stirrer 7 is arranged in the bottle body 1; the cell culture container communicating vessel and the mixing container are connected through the CPC male connector, the female plug 4 and the CPC connector of the CPC female connector 12.
The bottle body 1 is a 1-20L borosilicate glass reagent bottle.
The air filter 5 is a 0.22 μm air filter.
And the silicone tube connected with the switching tube of the fermentation tank feeding bottle adapter 3 which is connected with the luer female connector and the male plug/needle filter 6 is connected with a sunk head.
The countersunk head is made of tetrafluoro material.
The luer female connector and the needle filter in the male plug/needle filter 6 are 0.22 μm needle filters.
The middle of the cell culture container communicating vessel and the mixing container is connected with a peristaltic pump through a silicone tube 11.
Example two
The mixing container body 1 can be used at 2L, 5L, 10L, 20L as required, the upper end of the hole cover is connected with a 0.22 μm air filter 5, a luer female connector and male/needle filter 6, a CPC male connector and female plug 4, and the mixing container body is sterilized at 121 ℃ for use.
The luer female connector and the male plug/needle filter 6 are connected with a 0.22 mu m needle filter, water, plasmid and calcium chloride are added through the needle filter, and a magnetic stirrer is used for stirring uniformly. The transfection reagent was added through a needle filter with stirring by a magnetic stirrer and mixed well to form a calcium phosphate-DNA precipitate (the mixing method for the PEI method for lentivirus preparation is the same as above).
2 10-layer cell factories or culture flasks can be processed simultaneously by the mixing vessel and cell culture vessel communicating vessels. Can be according to biological safety cabinet size and in-service use demand, increase the quantity of handling cell factory, can highest simultaneous operation 8 10 layers cell factory with cell factory, connect mixing container and cell culture container communicating vessel through the CPC, through the peristaltic pump with cell factory in culture medium pump to the reaction flask of device in, magnetic stirrers is with culture medium and the transfection reagent homogeneous mixing who mixes well simultaneously, again with the mixed culture medium pump in the reaction flask in the cell factory. After the cell factory is flatly placed, the connection with the communication device is disconnected, and the CPC joint of the cell factory is connected with the air filter head and then is placed into the incubator. And (3) replacing a fresh culture medium in a cell factory after 3-6 h of transfection, and collecting lentivirus feed liquid 48h after transfection.
EXAMPLE III
Taking the example of operating 2 10-layer cell factories
(1) The 2L transfection reaction flask and the cell factory communicating vessel are used after being sterilized, and the number of the CPC female joint and the CPC male plug 8 at the tail end of the cell factory communicating vessel and the size of the transfection mixing container are adjusted according to the quantity of the cell factory to be processed.
(2) Inoculating the lentivirus in a cell factory according to proper density 2 days before packaging the lentivirus, changing the liquid of the cell factory before transfection, and adding 1-2L of corresponding culture medium into each 10-layer cell factory. The cell factory needs to be customized with a CPC connector to the factory or to purchase a conversion cover with the CPC connector to be arranged on the cell factory.
(3) When transfection is started, the transfection mixing container is placed on a magnetic stirrer, the luer female connector and the male plug/needle filter 6 connector are unscrewed, a 0.22 mu M needle filter is arranged, and a proper amount of packaging plasmid, skeleton plasmid and 2.5M CaCl are added according to the total area of the culture container2Solution, H2And O. And opening the magnetic stirrer, uniformly stirring, closing the magnetic stirrer, and standing for 5-10 min.
(4) The magnetic stirrer was turned on and the speed was adjusted to form a vortex in the bottle. The HBS solution is added at constant speed through the luer female connector and the male plug/needle filter 6. And after the HBS feeding is finished, closing the magnetic stirrer, and standing and incubating for 5-8 min to fully form a transfection compound.
(5) And opening a male CPC joint and a female CPC joint at the position of a female plug 4 on the transfection mixing container, and connecting the male joint with the female joint at the position of a cell culture container communicating vessel 12. And (3) moving the cell factory into a biological safety cabinet, connecting the female heads of the communicating vessel CPC female joint and the CPC male plug 8 of the cell culture container with the CPC male head pre-installed in the cell factory, and closing the flow stopping clamp 10. The cell factory was slowly turned over 90 °, and after being placed on its side, the liquid level position was marked with a marker. After all the cell factories were connected, the 11 silicone tubes were connected to the peristaltic pumps.
(6) And opening the flow stopping clamp 10, opening the magnetic stirrer to adjust the rotating speed to form vortex, opening the peristaltic pump, pumping the culture medium in the cell factory into the mixing container, and fully and uniformly mixing the culture medium with the transfection reagent. When the decrease of the liquid in the cell factory is observed, the liquid level in a certain cell factory is much lower than that in other cell factories, and the flow stop clip 10 is closed. And (5) closing the peristaltic pump when the liquid in the reaction bottle reaches 80% -90%. When the liquid level in the cell factory reaches the same height, the peristaltic pump is opened to adjust the direction of the liquid to be pumped, and the mixed liquid in the mixing container is pumped into the cell factory. The liquid level in each cell factory is observed, and if the liquid level in any cell factory is higher than that in other cell factories, the flow stop clip 10 is closed when the liquid level rises to the mark line. And (4) stopping the peristaltic pump when the liquid in the reaction bottle is completely pumped into the cell factory. Opening all the cell factory flow stopping clamps 10, closing the flow stopping clamps 10 after the liquid level in the cell factory reaches the same horizontal level, and slowly turning the cell factory to place the cell factory with the front side facing upwards. The cell factory is disconnected from the cell culture container communicating vessel and the cell factory is closed by using an air filter head pre-loaded with a female head or directly using a CPC female head.
(7) The cell factory was placed at 37 ℃ in 5% CO2Culturing in an incubator under the conditions, and collecting virus supernatant.
The mixing method for preparing lentivirus by PEI transfection method is the same as above.
Example four
A mixing system for preparing lentivirus, wherein the system comprises a mixing unit C, a cell culture unit F, a pump D, a conduit and a cell culture container communicating vessel E; the mixing unit C is connected with the pump D through the conduit; the pump D is connected with the cell culture unit F through the cell culture container communicating vessel E; the cell culture container communicating vessel E consists of a conduit and a tee pipe fitting.
And a flow meter and an electromagnetic valve are arranged at the feed inlet in the mixing unit C and the connection port of the cell culture container communicating vessel E close to the cell culture unit F. The mixing system also includes a reagent unit a and a reagent unit B; the reagent unit A and the reagent unit B are respectively connected with the mixing unit C through a conduit; the reagent unit A is used for preparing a PEI reagent or a calcium chloride or a plasmid reagent; the reagent unit B is used to formulate a plasmid, a 1 × HBS reagent or a 2 × HBS reagent. The mixing system further comprises a purification unit G; the purification unit G is connected with the cell culture unit F through a conduit and is used for separating and purifying supernatant. The mixing system also comprises a numerical control unit H; and the numerical control unit H is used for receiving and processing the data transmitted by the flowmeter and controlling the on-off of the electromagnetic valve.
After the reagents for preparing the lentiviruses are prepared in the reagent unit A and the reagent unit B, the reagents are guided into the mixing unit C through the guide pipe D to be mixed or reacted to obtain a transfection reagent, and an electromagnetic valve and a flowmeter are respectively arranged at a feed inlet of the mixing unit C which is connected with the reagent unit A and the reagent unit B to realize automatic liquid mixing; pumping the culture medium in the cell culture unit F into a mixing unit C through a peristaltic pump D and a cell culture container communicating vessel E to be mixed with the transfection reagent, pumping the final mixed liquid in the mixing unit C into each cell culture unit F through the peristaltic pump D and the cell culture container communicating vessel E after the complete mixing, and realizing automatic and accurate liquid mixing by arranging a flow meter and an electromagnetic valve at the position of the cell culture container communicating vessel E, which is close to the connection of the cell culture units F; and connecting the electromagnetic valve, the flowmeter and the peristaltic pump D with a numerical control unit H to realize data monitoring and automatic preparation.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (10)

1. A mixing method for preparing lentivirus is characterized in that transfection reagents are mixed or reacted in a mixing container, a culture medium in a cell culture container is pumped into the mixing container through a pump and a cell culture container communicating device to be mixed with the transfection reagents to obtain final mixed liquid, and the final mixed liquid is pumped into the cell culture container through the pump and the cell culture container communicating device to be cultured.
2. The mixing method according to claim 1, wherein the mixing vessel is composed of a bottle body (1) and a fermenter fill bottle adapter (3); the fermentation tank feed supplement bottle adapter (3) is connected with a CPC male connector, a CPC female plug (4), an air filter (5), a luer female connector, a male plug/needle head filter (6) through a silica gel tube; a transfer pipe of the fermentation tank feeding bottle adapter (3) connected with the CPC male connector and the female plug (4) is connected with a countersunk head (2) through a silicone tube and is arranged at the bottom of the bottle body (1); a switching tube of the fermentation tank feeding bottle adapter (3) which is connected with the luer female connector and the male plug/needle head filter (6) is connected with a silicone tube and is arranged in the bottle body (1), and the dropping position of liquid is controlled by connecting silicone tubes with different lengths; a magnetic stirrer (7) is arranged in the bottle body (1); the cell culture container communicating vessel and the mixing container are connected through a CPC joint of the CPC male joint, the female plug (4) and the CPC female joint (12).
3. Mixing method according to claim 1, wherein the cell factory connectors comprise silicone tubing (11), CPC female and male plugs (8), three-way adapter (9), CPC female connector (12), air filter (5); female joint of a CPC (12) is connected to silicone tube (11) one end, female joint of CPC (12) other end is connected one air cleaner (5), a tee bend conversion head (9) or female joint of CPC and the public end cap of CPC (8) are connected to silicone tube (11) other end, through tee bend conversion head (9) are connected female joint of CPC and the public end cap of CPC (8) and/or tee bend conversion head (9) will cell culture container linker realizes that a plurality of cell culture containers connect.
4. The mixing method according to claim 1, wherein a pump is connected between the cell culture vessel connector and the mixing vessel via a silicone tube (11); the pump is a peristaltic pump.
5. The mixing method according to any one of claims 1 to 4, characterized in that it comprises the following steps:
s1: culturing transfected cells:
digesting cells by enzyme, inoculating the cells into a cell culture container for culture, and achieving 50-90% of confluency;
s2: preparing and mixing a transfection reagent:
preparing a required reagent, and introducing the reagent into the mixing container through a luer female connector and a male plug/needle filter (6) of the mixing container for mixing to obtain a transfection reagent;
s3: mixing a culture medium and a transfection reagent in a cell culture container through a pump and a cell culture container communicating vessel, and pumping the mixture into the cell culture container for culture;
s4: separating and purifying the supernatant.
6. A mixing system for the preparation of lentiviruses, said system comprising a mixing unit C, a cell culture unit F, a pump D, a conduit and a cell culture container communicating vessel E; the mixing unit C is connected with the pump D through the conduit; the pump D is connected with the cell culture unit F through the cell culture container communicating vessel E; the cell culture container communicating vessel E consists of a conduit and a tee pipe fitting.
7. The mixing system of claim 6, wherein the feed port of the mixing unit C and the connection port of the cell culture container connector E near the cell culture unit F are provided with a flow meter and a solenoid valve.
8. The mixing system of claim 6, further comprising reagent unit A and reagent unit B; the reagent unit A and the reagent unit B are respectively connected with the mixing unit C through a conduit; the reagent unit A is used for preparing a PEI reagent or a calcium chloride or a plasmid reagent; the reagent unit B is used to formulate a plasmid, a 1 × HBS reagent or a 2 × HBS reagent.
9. The mixing system as recited in claim 6, further comprising a purification unit G; the purification unit G is connected with the cell culture unit F through a conduit and is used for separating and purifying supernatant.
10. The mixing system as set forth in claim 6, further comprising a numerical control unit H; and the numerical control unit H is used for receiving and processing the data transmitted by the flowmeter and controlling the on-off of the electromagnetic valve and the pump D.
CN202010587853.5A 2020-06-24 2020-06-24 Mixing method and mixing system for preparing lentivirus Pending CN113832112A (en)

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Application publication date: 20211224