CN213376341U - Semi-automatic totally-enclosed cell transfection reagent preparation device - Google Patents

Semi-automatic totally-enclosed cell transfection reagent preparation device Download PDF

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CN213376341U
CN213376341U CN202022024504.0U CN202022024504U CN213376341U CN 213376341 U CN213376341 U CN 213376341U CN 202022024504 U CN202022024504 U CN 202022024504U CN 213376341 U CN213376341 U CN 213376341U
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bottle
transfection reagent
reagent
transfection
calcium chloride
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曹元元
高海
赵云灿
罗易坤
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Shanghai Yaoming Junuo Biomedical Research And Development Co ltd
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Shanghai Yaoming Junuo Biomedical Research And Development Co ltd
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Abstract

The utility model relates to the field of biotechnology, a device is prepared to totally closed cell transfection reagent of semi-automatization is provided, include: transfection reagent prepares bottle, buffer solution storage bottle, plasmid calcium chloride bottle and transfection reagent culture medium mixing bottle, transfection reagent prepares the bottle through the feed liquor pipe with the buffer solution storage bottle with plasmid calcium chloride bottle is connected, transfection reagent prepares the bottle through the transfection reagent drain pipe with the transfection reagent culture medium mixing bottle is connected, transfection reagent prepares the bottle and is provided with control by temperature change magnetic stirrers, the inside turbidity sensor that is provided with liquid feeding shower nozzle and turbidity detector of transfection reagent preparation bottle, the liquid feeding shower nozzle with the feed liquor pipe intercommunication. The utility model provides a device is prepared to totally closed cell transfection reagent of semi-automatization can replace traditional artifical manual operation mode, increases the controllability of operation, improves the reproducibility of experiment, reduces the pollution risk.

Description

Semi-automatic totally-enclosed cell transfection reagent preparation device
Technical Field
The utility model relates to the field of biotechnology, especially, relate to a device is prepared to totally closed cell transfection reagent of semi-automatization.
Background
With the development of scientific research level, exogenous DNA substances are generally introduced into cells by means of specific experimental means, so that researches on functions of DNA and the like are realized or a large amount of specific proteins are prepared. Cell transfection refers to a technique for introducing foreign molecules such as DNA, RNA, etc. into eukaryotic cells, and is a common tool for studying gene expression regulation, mutation analysis, protein production, etc. Many methods for introducing DNA into mammalian cells are available, and among them, calcium phosphate transfection is considered to be an economical and simple method with low toxicity to cells, and is one of the commonly used methods for transfection of mammalian cells. Calcium phosphate transfection is primarily achieved by forming calcium phosphate DNA precipitation complexes and delivering the DNA into cells cultured in vitro. Firstly, DNA is mixed with a calcium chloride reagent, and then a phosphate buffer reagent is dropwise added into the mixed solution, so that DNA-calcium phosphate precipitate is formed, the adhesion of the DNA on the cell surface can be promoted, and the exogenous DNA is taken in through endocytosis of the cell. The size of the formed precipitate has a great influence on the transfection efficiency, and the aggregated DNA-calcium phosphate particles are not favorable for adhesion into cells, which affects the transfection efficiency. The operation is influenced by various factors, the transfection efficiency is very unstable, the repeatability of experimental results among batches is poor, and different laboratories usually need to spend great effort to optimize a transfection system when selecting a calcium phosphate transfection method, which wastes time and labor.
Conventional calcium phosphate transfection reagents are typically formulated by manually mixing the two reagents and transferring the mixture into cells for transfection. In manual operation, the transfection efficiency is influenced by factors such as operator variation, environment temperature change, reagent pH and mixing mode, the problems that the open operation is easy to pollute and the quality of the transfection reagent is difficult to measure exist, and the factors cause the problems that the operation process is difficult to control, the repeatability of the transfection efficiency is poor, the pollution exists and the like.
SUMMERY OF THE UTILITY MODEL
In view of the above shortcomings in the prior art, an object of the present invention is to provide a semi-automatic and fully-enclosed cell transfection reagent preparation device, which is used for solving the problems of the prior art, such as difficulty in controlling the operation process, poor repeatability of transfection efficiency, pollution, etc.
To achieve the above and other related objects, the present invention provides a semi-automatic and fully-enclosed cell transfection reagent preparing device, comprising: transfection reagent prepares bottle, buffer solution storage bottle, plasmid calcium chloride bottle and transfection reagent culture medium mixing bottle, transfection reagent prepares the bottle through the feed liquor pipe with the buffer solution storage bottle with the plasmid calcium chloride bottle is connected, transfection reagent prepares the bottle through the transfection reagent drain pipe with the transfection reagent culture medium mixing bottle is connected, transfection reagent prepares the bottle and is provided with control by temperature change magnetic stirrers, the inside turbidity sensor who is provided with liquid feeding shower nozzle and turbidity detector of bottle is prepared to transfection reagent, the liquid feeding shower nozzle with feed liquor pipe intercommunication, transfection reagent prepares the bottle and still is provided with the air cleaner.
Preferably, the liquid inlet pipe is a Y-shaped pipe, a port of the main part of the liquid inlet pipe is connected with the transfection reagent preparation bottle, and two ports of the branch part of the liquid inlet pipe are respectively connected with the buffer solution storage bottle and the plasmid calcium chloride bottle.
Preferably, two ports of the branch part of the liquid inlet pipe are respectively connected with the buffer solution storage bottle and the plasmid calcium chloride bottle by aseptic welding.
Preferably, the main part of the liquid inlet pipe is provided with a peristaltic pump.
Preferably, the transfection reagent outlet tube is provided with a peristaltic pump.
Preferably, the buffer solution storage bottle, the plasmid calcium chloride bottle and the transfection reagent culture medium mixing bottle are all provided with a temperature-controlled magnetic stirrer.
Preferably, the branch part of the liquid inlet pipe and the transfection reagent liquid outlet pipe are both provided with valves.
Preferably, the liquid feeding spray head is connected with the port of the liquid inlet pipe through a quick sterile connector or a sterile welding mode.
Preferably, the transfection reagent culture medium mixing bottle is connected with the port of the transfection reagent liquid outlet pipe through a quick sterile joint or a sterile welding mode
Preferably, the transfection reagent preparation bottle is connected with a port of the transfection reagent liquid outlet pipe through a quick sterile connector or a sterile welding mode.
As mentioned above, the utility model discloses a totally closed cell transfection reagent of semi-automatization prepares device has following beneficial effect:
the utility model discloses a constancy of temperature before the control by temperature change magnetic stirrers guarantees that reagent mixes and reagent are even, the reagent gets into transfection reagent preparation bottle with the mode of spraying, help forming the transfection reagent of moderate size, control the reagent temperature of transfection reagent preparation in-process, advance kind speed and mixing speed, the controllability of transfection reagent preparation process has been increased, be favorable to improving the reproducibility of preparing reagent batch through fixed parameter, increase the turbidity detector in addition, be used for the turbid detection of transfection reagent, the quality standard whether the testing result can regard as transfection reagent to use, improve production technology's robustness.
Drawings
Fig. 1 is a schematic diagram of a semi-automatic and totally-enclosed cell transfection reagent preparation device according to the present invention.
Description of the element reference numerals
1 temperature control magnetic stirrer
2 buffer solution storage bottle
3 plasmid calcium chloride bottle
4 liquid inlet pipe
5 peristaltic pump
6 air filter
7 transfection reagent preparation bottle
8 turbidity detector
9 transfection reagent drain pipe
10 transfection reagent culture medium mixing bottle
11 liquid feeding spray head
Detailed Description
Please refer to fig. 1, it should be understood that the structure, ratio, size and the like shown in the drawings attached to the present specification are only used for matching with the content disclosed in the specification, so as to be known and read by those skilled in the art, and are not used for limiting the limit conditions that the present invention can be implemented, so that the present invention has no essential significance in the technology, and any structure modification, ratio relationship change or size adjustment should still fall within the scope of the present invention without affecting the function and the achievable purpose of the present invention. Meanwhile, the terms such as "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for convenience of description, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes, and the present invention is also regarded as the scope of the present invention.
As shown in fig. 1, the utility model provides a semi-automatic totally closed cell transfection reagent preparation device, including: transfection reagent prepares bottle 7, buffer solution storage bottle 2, plasmid calcium chloride bottle 3 and transfection reagent culture medium mixing bottle 10, transfection reagent prepares bottle 7 through feed liquor pipe 4 with buffer solution storage bottle 2 with plasmid calcium chloride bottle 3 connects, transfection reagent prepares bottle 7 through transfection reagent drain pipe 9 with transfection reagent culture medium mixing bottle 10 connects, transfection reagent prepares bottle 7 is provided with control by temperature change magnetic stirrer 1, the inside turbidity sensor who is provided with liquid feeding shower nozzle 11 and turbidity detector 8 of transfection reagent prepares bottle 7, liquid feeding shower nozzle 11 with feed liquor pipe 4 intercommunication, transfection reagent prepares bottle 7 still is provided with air cleaner 6.
The utility model is used for cell transfection reagent prepares, and plasmid calcium chloride bottle 3 is used for storing plasmid, 1M calcium chloride and water, and buffer solution storage bottle 2 is used for storing buffer solution, and transfection reagent prepares bottle 7 as the container of preparing transfection reagent and is used for storing the transfection reagent who prepares the completion, and transfection reagent culture medium mixing bottle 10 is as the container of mixing transfection reagent and culture medium and is used for storing the transfection reagent culture medium of preparing the completion. The transfection reagent preparation bottle 7 is connected with the buffer solution storage bottle 2 and the plasmid calcium chloride bottle 3 through the liquid inlet pipe 4, the transfection reagent preparation bottle 7 is connected with the transfection reagent culture medium mixing bottle 10 through the transfection reagent liquid outlet pipe 9, and the connection mode can enable the preparation process of the transfection reagent and the mixing process of the transfection reagent culture medium to be carried out in closed bottles, so that reagent transfer is not required to be opened, and the risk of pollution of the transfection reagent is reduced. The temperature control magnetic stirrer 1 arranged on the transfection reagent preparation bottle 7 can control the reagent temperature and the mixing speed in the transfection reagent preparation process, manual stirring is not needed, the controllability of the transfection reagent preparation process is improved, and the repeatability among prepared reagent batches is favorably improved through fixed parameters. The liquid adding spray head 11 arranged in the transfection reagent preparation bottle 7 can enable the reagent to enter the transfection reagent preparation bottle 7 in a spraying mode, and is beneficial to forming the transfection reagent with a proper size. The inside turbidity sensor that is provided with turbidity detector 8 of transfection reagent preparation bottle 7 can detect the turbidity of transfection reagent, and the testing result can regard as the quality standard whether transfection reagent can use to improve the robustness of production technology, transfection reagent preparation bottle 7 still is provided with air cleaner 6, ensures that transfection reagent preparation bottle internal and external pressure is unanimous, prevents simultaneously that unfiltered outside air from polluting the reagent of transfection reagent preparation bottle 7 inside.
In one embodiment, the liquid inlet pipe 4 is a Y-shaped pipe, a port of a main portion of the liquid inlet pipe 4 is connected to the transfection reagent preparation bottle 7, and two ports of branch portions of the liquid inlet pipe 4 are respectively connected to the buffer solution storage bottle 2 and the plasmid calcium chloride bottle 3. The reagent in the buffer solution storage bottle 2 and/or the reagent in the plasmid calcium chloride bottle 3 can enter the transfection reagent preparation bottle 7 through a port of the main part of the liquid inlet pipe 4, and the liquid inlet pipe 4 is a Y-shaped pipe, so that the connection mode can be simplified.
In one embodiment, two ports of the branch portion of the liquid inlet pipe 4 are respectively connected with the buffer solution storage bottle 2 and the plasmid calcium chloride bottle 3 by aseptic welding. This connection prevents bacteria, viruses or dust at the connection from contaminating the reagents in the buffer storage bottle 2 and the plasmid calcium chloride bottle 3.
In an embodiment, the main portion of the inlet pipe 4 is provided with a peristaltic pump 5. The peristaltic pump 5 can quantitatively pump the reagents in the buffer solution storage bottle 2 and the plasmid calcium chloride bottle 3 and transfer the pumped reagents into the transfection reagent preparation bottle 7.
In one embodiment, the transfection reagent outlet 9 is provided with a peristaltic pump 5. The peristaltic pump 5 can quantitatively extract the transfection reagent in the transfection reagent preparation bottle 7 and deliver the extracted transfection reagent into the transfection reagent culture medium mixing bottle 10.
In one embodiment, the buffer solution storage bottle 2, the plasmid calcium chloride bottle 3, the transfection reagent preparation bottle 7, and the transfection reagent medium mixing bottle 10 are each provided with a temperature-controlled magnetic stirrer 1. The temperature control magnetic stirrer 1 can control the temperature and the mixing speed of the reagent, manual stirring is not needed, the controllability of the preparation process of the transfection reagent is increased, the buffer solution storage bottle 2 and the temperature control magnetic stirrer 1 arranged in the plasmid calcium chloride bottle 3 can respectively stir the reagent in the bottle, so that the reagent in the bottle can be uniformly mixed, the temperature of the reagent in the experimental process is kept constant, the temperature control magnetic stirrer 1 arranged in the transfection reagent preparation bottle 7 can control the mixing of the buffer solution and the plasmid calcium chloride, the temperature of the transfection reagent during mixing and standing can be set and maintained, the temperature control magnetic stirrer 1 arranged in the transfection reagent culture medium mixing bottle 10 can control the mixing of the transfection reagent and the culture medium, and the temperature during mixing can be set and maintained.
In one embodiment, the branch portion of the liquid inlet pipe 4 and the transfection reagent liquid outlet pipe 9 are both provided with valves, the branch portion of the liquid inlet pipe 4 is provided with a valve for controlling the liquid inlet sequence of calcium chloride and buffer liquid, and the liquid outlet pipe is provided with a valve for preventing the solution from mistakenly entering the transfection reagent culture medium mixing bottle due to unbalanced pressure inside and outside the bottle when the calcium chloride and the buffer liquid enter the transfection reagent preparation bottle.
In one embodiment, the liquid feeding nozzle 11 is connected to the port of the liquid feeding pipe 4 by a quick aseptic joint or aseptic welding. This connection prevents bacteria, viruses or dust at the connection from contaminating the reagents in the transfection reagent preparation vial 7. Meanwhile, the liquid adding nozzle 11 adds the buffer solution into the calcium chloride solution in a spraying mode for mixing, so that the solution is rapidly and uniformly distributed, and the transfection reagent with consistent shape is favorably formed.
In one embodiment, the transfection reagent media mixing bottle 10 is connected to the port of the transfection reagent outlet tube 9 by a quick sterile connector or by aseptic welding. This connection prevents bacteria, viruses, or dust at the connection from contaminating the reagents in the transfection reagent media mix bottle 10.
In one embodiment, the transfection reagent preparation bottle 7 is connected to the port of the transfection reagent outlet tube 9 by a quick sterile connector or by aseptic welding. This connection prevents bacteria, viruses, or dust at the connection from contaminating the reagents in the transfection reagent media mix bottle 10.
The following description is provided for illustrative purposes, and other advantages and features of the present invention will become apparent to those skilled in the art from the following detailed description.
Adopt the utility model discloses the step that the device is used for realizing the quick batch cell transfection in laboratory is as follows: plasmid, 1M calcium chloride and water were first added to plasmid calcium chloride bottle 3. According to the scheme shown in figure 1, a buffer solution storage bottle 2, a plasmid calcium chloride bottle 3, a transfection reagent preparation bottle 7, a transfection reagent culture medium mixing bottle 10, a liquid inlet pipe 4, a transfection reagent liquid outlet pipe 9, a liquid adding spray head 11, a turbidity detector 8 and a temperature control magnetic stirrer 1 are connected, wherein plasmid calcium chloride bottle 3 and buffer solution storage bottle 2 are connected with the branching portion of feed liquor pipe 4 with aseptic welding mode, the trunk of feed liquor pipe 4 passes through quick aseptic joint or aseptic welding with the pipe on 7 bottle lids of transfection reagent preparation bottle, turbidity sensor and turbidity detector 8 on 7 bottle lids of transfection reagent preparation bottle are connected, transfection reagent culture medium mixing bottle 10 passes through the pipeline on the bottle lid and is connected with aseptic welding mode transfection reagent drain pipe 9, the pipe on 7 bottle lids of transfection reagent preparation bottle passes through quick aseptic joint or aseptic welding and is connected with the other end of transfection reagent drain pipe 9. After connection, the buffer solution storage bottle 2, the plasmid calcium chloride bottle 3, the transfection reagent preparation bottle 7 and the transfection reagent culture medium mixing bottle 10 are all provided with a temperature control magnetic stirrer 1. The temperature control magnetic stirrer 1 below the plasmid calcium chloride bottle 3, the buffer solution storage bottle 2 and the transfection reagent culture medium mixing bottle 10 is started, the rotating speed is set to be 30rpm, and the temperature is set to be: the plasmid calcium chloride bottle 3 is set at 30 ℃, and the buffer solution storage bottle 2, the transfection reagent preparation bottle 7 and the transfection reagent culture medium mixing bottle 10 are set at 37 ℃. After keeping for 20min, starting the temperature control magnetic stirrer 1 below the transfection reagent preparation bottle 7, setting the rotation speed to be 150rpm, the temperature to be 37 ℃, starting the peristaltic pump 5, setting the pump speed to be 150rpm, starting a valve of a connecting pipeline part of the liquid inlet pipe 4 and the plasmid calcium chloride bottle 3, conveying the reagent in the plasmid calcium chloride bottle 3 to the transfection reagent preparation bottle 7, and closing the valve. And opening a valve of a connecting pipeline part of the liquid inlet pipe 4 and the buffer solution storage bottle 2, and conveying the reagent in the buffer solution storage bottle 2 to the transfection reagent preparation bottle 7. After the reagents in the buffer solution storage bottle 2 are completely conveyed, the rotating speed of the temperature control magnetic stirrer 1 below the transfection reagent preparation bottle 7 is set to be 0 rpm. Standing for 10-15 min, starting a turbidity detector 8, measuring turbidity, and if the turbidity meets the set technological standard, setting the rotating speed of the temperature control magnetic stirrer 1 below the transfection reagent preparation bottle 7 to be 100rpm, and keeping for 1 min. Starting the peristaltic pump 5 between the transfection reagent preparation bottle 7 and the transfection reagent culture medium mixing bottle 10, setting the parameter at 200rpm, conveying the transfection reagent to the transfection reagent culture medium mixing bottle 10, keeping for 5min, completing the mixing of the transfection reagent and the culture medium, and allowing the mixed culture medium to enter the next cell transfection step.
To sum up, the utility model discloses various shortcomings in the prior art have effectively been overcome and high industry value has.
The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (10)

1. A semi-automatic totally-enclosed cell transfection reagent preparation device is characterized by comprising: transfection reagent prepares bottle (7), buffer solution storage bottle (2), plasmid calcium chloride bottle (3) and transfection reagent culture medium mixing bottle (10), transfection reagent prepares bottle (7) through feed liquor pipe (4) with buffer solution storage bottle (2) with plasmid calcium chloride bottle (3) are connected, transfection reagent prepares bottle (7) through transfection reagent drain pipe (9) with transfection reagent culture medium mixing bottle (10) are connected, transfection reagent prepares bottle (7) is provided with control by temperature change magnetic stirrers (1), the inside turbidity sensor that is provided with liquid feeding shower nozzle (11) and turbidity detector (8) of transfection reagent prepares bottle (7), liquid feeding shower nozzle (11) with feed liquor pipe (4) intercommunication, transfection reagent prepares bottle (7) still is provided with air cleaner (6).
2. The apparatus of claim 1, wherein the reagent dispensing device comprises: the liquid inlet pipe (4) is a Y-shaped pipe, a port of a main part of the liquid inlet pipe (4) is connected with the transfection reagent preparation bottle (7), and two ports of a branch part of the liquid inlet pipe (4) are respectively connected with the buffer solution storage bottle (2) and the plasmid calcium chloride bottle (3).
3. The apparatus of claim 2, wherein the reagent dispensing device comprises: two ports of the branch part of the liquid inlet pipe (4) are respectively connected with the buffer solution storage bottle (2) and the plasmid calcium chloride bottle (3) in an aseptic welding mode.
4. The apparatus of claim 2, wherein the reagent dispensing device comprises: the main part of the liquid inlet pipe (4) is provided with a peristaltic pump (5).
5. The apparatus of claim 1, wherein the reagent dispensing device comprises: the transfection reagent liquid outlet pipe (9) is provided with a peristaltic pump.
6. The apparatus of claim 1, wherein the reagent dispensing device comprises: the buffer solution storage bottle (2), the plasmid calcium chloride bottle (3) and the transfection reagent culture medium mixing bottle (10) are all provided with a temperature control magnetic stirrer (1).
7. The apparatus of claim 1, wherein the reagent dispensing device comprises: the branch part of the liquid inlet pipe (4) and the transfection reagent liquid outlet pipe (9) are both provided with valves.
8. The apparatus of claim 1, wherein the reagent dispensing device comprises: the liquid adding nozzle (11) is connected with the port of the liquid inlet pipe (4) through a quick aseptic joint or an aseptic welding mode.
9. The apparatus of claim 1, wherein the reagent dispensing device comprises: the transfection reagent culture medium mixing bottle (10) is connected with the port of the transfection reagent liquid outlet pipe (9) through a quick sterile joint or a sterile welding mode.
10. The apparatus of claim 1, wherein the reagent dispensing device comprises: the transfection reagent preparation bottle (7) is connected with the port of the transfection reagent liquid outlet pipe (9) through a quick sterile joint or a sterile welding mode.
CN202022024504.0U 2020-09-15 2020-09-15 Semi-automatic totally-enclosed cell transfection reagent preparation device Active CN213376341U (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265333A (en) * 2021-06-23 2021-08-17 无锡生基医药科技有限公司 Totally-enclosed, semi-automatically-controlled and modularized plasmid transient transfection system

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265333A (en) * 2021-06-23 2021-08-17 无锡生基医药科技有限公司 Totally-enclosed, semi-automatically-controlled and modularized plasmid transient transfection system

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