Transfection mixture preparation device, gene transfection system and related method

Abstract

The invention provides a transfection mixture preparation device, a gene transfection system and a related method, and relates to the technical field of molecule and cell biology. The transfection mixture preparation device comprises a workbench and a bag body, the bag body is a cuboid-like whole and can be divided into a nucleic acid bag, a reagent bag and a connecting bag according to functions, the two ends of the connecting bag are communicated with the nucleic acid bag and the reagent bag respectively, the nucleic acid bag is used for containing exogenous nucleic acid diluent, and the reagent bag is used for containing transfection reagent diluent. The reagent bag can move to a position higher than the nucleic acid bag under the action of external force, so that transfection reagent diluent in the reagent bag flows into the nucleic acid bag through the connecting bag and is mixed with exogenous nucleic acid diluent, and finally a transfection mixture is obtained. Therefore, the transfection reagent diluent is added into the exogenous nucleic acid diluent in a pouring manner, and the volume of the reactor is not seen in the process, so that the transfection mixture has very short uniform mixing time all the time, and the transfection efficiency is ensured.

Description

Technical Field

[0001] This application relates to the fields of molecular and cell biology, and in particular to an apparatus for preparing transfection mixtures, a gene transfection system, and related methods. Background Technology

[0002] Gene transfection is a technique that introduces exogenous nucleic acids (such as DNA and RNA) into cells, allowing these exogenous nucleic acids to maintain their biological functions within the cells. The gene transfection process involves: diluting the transfection reagent (such as PEI, a cationic polymer) to obtain a transfection reagent diluent; diluting the exogenous nucleic acid to obtain an exogenous nucleic acid diluent; introducing the transfection reagent diluent into the exogenous nucleic acid diluent and mixing them thoroughly to obtain a transfection mixture; introducing the transfection mixture into the cells to be transfected, mixing them thoroughly, and then culturing for a period of time. Typically, the mixing time between the transfection reagent diluent and the exogenous nucleic acid diluent during the preparation of the transfection mixture significantly affects the transfection efficiency of the cells to be transfected; the longer the mixing time, the lower the transfection efficiency.

[0003] In related technologies, for small-scale experiments, a pipette can be used to add the transfection reagent diluent to the exogenous nucleic acid diluent. This process is instantaneous, thus avoiding the low transfection efficiency problem caused by long mixing times. However, in actual gene transfection, the cells to be transfected are usually cultured in a large reactor, which increases the volume of the required transfection mixture. This means that both the transfection reagent diluent and the exogenous nucleic acid diluent are required. In this case, the transfection reagent diluent cannot be added to the exogenous nucleic acid diluent instantaneously as in small-scale experiments, resulting in a longer mixing time and lower transfection efficiency of the transfection mixture. In other words, the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent is affected by the reactor volume; the larger the reactor volume, the longer the mixing time, and the lower the transfection efficiency of the transfection mixture. Summary of the Invention

[0004] This application provides a transfection mixture preparation apparatus, a gene transfection system, and a related method, aiming to solve the problem in related technologies that the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent is severely affected by the volume of the reactor used to culture the cells to be transfected.

[0005] To address the aforementioned drawbacks in related technologies, this application provides a transfection mixture preparation apparatus, comprising a worktable and a bag body. The bag body is functionally divided into three parts: a nucleic acid bag, a reagent bag, and a connecting bag. The two ends of the connecting bag are connected to the nucleic acid bag and the reagent bag, respectively. Both the nucleic acid bag and the reagent bag are placed on the worktable. The nucleic acid bag is used to contain exogenous nucleic acid diluent; the reagent bag is used to contain transfection reagent diluent; the reagent bag is also designed to detach from the worktable under external force and move away from the worktable to pour the transfection reagent diluent into the nucleic acid bag, thereby mixing the transfection reagent diluent with the exogenous nucleic acid diluent to obtain a transfection mixture.

[0006] In some implementations, the nucleic acid bag has an internal nucleic acid port, the reagent bag has an internal reagent port, and the connecting bag has internal connection ports at both ends, which connect to the nucleic acid port and the reagent port, respectively. Specifically, when both the nucleic acid bag and the reagent bag are placed on the worktable, both the nucleic acid port and the reagent port face away from the worktable.

[0007] In some implementations, the transfection mixture preparation apparatus further includes a first motor, a first isolation sleeve, and a first stirring paddle. A first mounting hole communicating with the interior is provided on the nucleic acid bag. The first motor is mounted on a worktable, the first isolation sleeve is disposed within the first mounting hole, and the first stirring paddle is located inside the nucleic acid bag. The first stirring paddle is mounted on the first isolation sleeve and rotatably engages with it. The first motor is used to connect with the first isolation sleeve when the nucleic acid bag is placed on the worktable and to drive the first stirring paddle to rotate using magnetic force. In one implementation, the first mounting hole is located at the center of the bottom of the nucleic acid bag and opposite the nucleic acid port. In another implementation, a hollow first protrusion extending outward from the center of the first isolation sleeve is formed, and a first boss is formed in the center of the first stirring paddle. The first boss is inserted into the first protrusion to allow the first stirring paddle to rotatably engage with the first isolation sleeve. The first motor has a first recess that matches the first protrusion, specifically used to ensure that the first recess mates with the first protrusion when the nucleic acid bag is placed on the worktable.

[0008] In some implementations, the transfection mixture preparation apparatus further includes a second motor, a second isolation sleeve, and a second stirring paddle. A second mounting hole communicating with the interior is provided on the reagent bag. The second motor is mounted on a worktable, the second isolation sleeve is disposed within the second mounting hole, and the second stirring paddle is located inside the reagent bag, mounted on and rotatably engaged with the second isolation sleeve. The second motor is used to connect with the second isolation sleeve when the reagent bag is placed on the worktable and to drive the second stirring paddle to rotate using magnetic force. In one implementation, the second mounting hole is located at the center of the bottom of the reagent bag and opposite the reagent port. In another implementation, a hollow second protrusion extending outward from the middle of the second isolation sleeve is formed, and a second boss is formed in the middle of the second stirring paddle. The second boss is inserted into the second protrusion to allow the second stirring paddle to rotatably engage with the second isolation sleeve. The second motor has a second recess adapted to the second protrusion, specifically used to ensure that the second recess mates with the second protrusion when the reagent bag is placed on the worktable.

[0009] In some implementations, when both the nucleic acid bag and the reagent bag are placed on the worktable, the middle of the connecting bag is higher than both ends. Furthermore, the transfection mixture preparation device also includes a nucleic acid storage tank, a nucleic acid infusion tube, and a nucleic acid infusion pump. The connecting bag also has a nucleic acid orifice connected internally and close to the nucleic acid port. Both the nucleic acid storage tank and the nucleic acid infusion pump are positioned on the worktable. One end of the nucleic acid infusion tube is connected to the connecting bag through the nucleic acid orifice, and the other end is connected to the nucleic acid storage tank. The nucleic acid storage tank is used to store exogenous nucleic acid diluent; the nucleic acid infusion pump is used to pump the exogenous nucleic acid diluent from the nucleic acid storage tank into the nucleic acid bag through the nucleic acid infusion tube after both the nucleic acid bag and the reagent bag are placed on the worktable.

[0010] In some implementations, the transfection mixture preparation device also includes a reagent storage tank, a reagent infusion tubing, and a reagent infusion pump. The connecting bag also has a reagent port connected to the interior and close to the reagent outlet. Both the reagent storage tank and the reagent infusion pump are mounted on a workbench. One end of the reagent infusion tubing is connected to the connecting bag through the reagent port, and the other end is connected to the reagent storage tank. The reagent storage tank stores the transfection reagent diluent; the reagent infusion pump pumps the transfection reagent diluent from the reagent storage tank into the reagent bag through the reagent infusion tubing after both the nucleic acid bag and the reagent bag are placed on the workbench.

[0011] In some implementations, the transfection mixture preparation apparatus further includes an air compressor and an air supply pipe. The connecting bag also has an air vent communicating with its interior. The air compressor is located adjacent to the workbench, and one end of the air supply pipe is connected to the connecting bag through the air vent, while the other end is connected to the air compressor. The air vent is located between the nucleic acid wells and the reagent wells. Specifically, the air compressor is used to draw in and compress outside air before pumping the exogenous nucleic acid diluent and transfection reagent diluent into the bag. The air supply pipe is used to deliver the compressed air into the bag and to release the air from the bag after pumping the exogenous nucleic acid diluent and transfection reagent diluent into the bag. Furthermore, the transfection mixture preparation apparatus also includes a disc filter. The air supply pipe is connected to the air compressor through the disc filter, which is used to sterilize the air entering the bag.

[0012] In some implementations, the transfection mixture preparation device further includes a first sampling tube, a first needle-free sampler, and a first sample tube. The nucleic acid bag also has a first sampling hole that communicates with the interior. One end of the first sampling tube is connected to the nucleic acid bag through the first sampling hole, and the other end of the first sampling tube is connected to the first needle-free sampler. The first needle-free sampler is used to connect to a first Luer syringe to extract the transfection mixture or exogenous nucleic acid diluent from the nucleic acid bag and inject it into the first sample tube.

[0013] In some implementations, the transfection mixture preparation device further includes a second sampling tube, a second needleless sampler, and a second sample tube. The reagent bag also has a second sampling port connected to the interior. One end of the second sampling tube is connected to the reagent bag through the second sampling port, and the other end of the second sampling tube is connected to the second needleless sampler. The second needleless sampler is used to connect to a second Luer syringe to draw transfection reagent diluent from the reagent bag and inject it into the second sample tube.

[0014] A second aspect of this application provides another apparatus for preparing a transfection mixture, comprising a workbench, a nucleic acid bag, a reagent bag, and at least one connecting tube, the two ends of which are respectively connected to the nucleic acid bag and the reagent bag, both of which are placed on the workbench. The nucleic acid bag is used to contain exogenous nucleic acid diluent; the reagent bag is used to contain transfection reagent diluent; the reagent bag is also used to detach from the workbench under external force and move away from the workbench to pour the transfection reagent diluent into the nucleic acid bag, thereby mixing the transfection reagent diluent with the exogenous nucleic acid diluent to obtain a transfection mixture.

[0015] A third aspect of this application provides yet another transfection mixture preparation apparatus, comprising a worktable and a bag body. The bag body is functionally divided into three parts: a nucleic acid bag, a reagent bag, and a connecting bag. The two ends of the connecting bag are respectively connected to the nucleic acid bag and the reagent bag. Both the nucleic acid bag and the reagent bag are placed on the worktable. The nucleic acid bag is used to contain exogenous nucleic acid diluent; the reagent bag is a 2D bag used to contain transfection reagents; the reagent bag is also designed to detach from the worktable under external force and move away from the worktable to pour the transfection reagents into the nucleic acid bag, thereby mixing the transfection reagents with the exogenous nucleic acid diluents to obtain the transfection mixture.

[0016] In some implementations, the transfection mixture preparation device also includes a clip that is attached to the reagent bag along the direction from the reagent bag to the nucleic acid bag to isolate the internal space of the reagent bag.

[0017] The fourth aspect of this application provides a method for preparing a transfection mixture, which is applied to the transfection mixture preparation apparatus mentioned in any one of the first, second, and third aspects of this application. The method for preparing the transfection mixture includes: placing an exogenous nucleic acid diluent in a nucleic acid bag; placing a transfection reagent or a transfection reagent diluent in a reagent bag connected to the nucleic acid bag, wherein, when the reagent bag is a 2D bag, the reagent bag contains the transfection reagent; moving the reagent bag to a position higher than the nucleic acid bag, so that the transfection reagent or transfection reagent diluent in the reagent bag flows into the nucleic acid bag and mixes with the exogenous nucleic acid diluent in the nucleic acid bag to obtain a transfection mixture.

[0018] This application provides a gene transfection system comprising a transfection infusion pump, a transfection infusion tubing, a reactor, and a transfection mixture preparation apparatus as described in any one of the first, second, and third aspects of this application. The reactor and the transfection infusion pump are both located adjacent to the workbench in the transfection mixture preparation apparatus. The nucleic acid bag in the transfection mixture preparation apparatus also has a transfection well communicating with its interior. One end of the transfection infusion tubing is connected to the nucleic acid bag through the transfection well, and the other end of the transfection infusion tubing is connected to the reactor through the transfection infusion pump. Specifically, the reactor is used to contain cells to be transfected; the transfection infusion pump is used to pump the transfection mixture from the nucleic acid bag into the reactor through the transfection infusion tubing when the nucleic acid bag contains the transfection mixture, so that the transfection mixture binds to the cells to be transfected; the reactor is also used to culture the cells to be transfected that have bound the transfection mixture.

[0019] The sixth aspect of this application provides a gene transfection method applied to the gene transfection system mentioned in the fifth aspect of this application, and the gene transfection method includes: preparing a transfection mixture using a transfection mixture preparation device; delivering the transfection mixture to a reactor and combining it with cells to be transfected; and culturing the cells to be transfected that have combined with the transfection mixture in the reactor.

[0020] The transfection mixture preparation apparatus provided in the first aspect of this application includes a workbench and a bag body. The bag body is divided into a nucleic acid bag, a reagent bag, and a connecting bag. The two ends of the connecting bag are respectively connected to the nucleic acid bag and the reagent bag. The nucleic acid bag is used to contain exogenous nucleic acid diluent, and the reagent bag is used to contain transfection reagent diluent. The reagent bag can be moved to a position higher than the nucleic acid bag under the action of external force, so that the transfection reagent diluent in the reagent bag flows into the nucleic acid bag through the connecting bag and mixes with the exogenous nucleic acid diluent in the nucleic acid bag, thus obtaining the transfection mixture. Therefore, in preparing the transfection mixture, this application first moves the reagent bag containing the transfection reagent diluent to a higher position than the nucleic acid bag containing the exogenous nucleic acid diluent. Then, the transfection reagent diluent in the reagent bag is poured into the nucleic acid bag by tilting. This tilting method can disregard the volume of the reactor used to contain and culture the cells to be transfected. In this case, regardless of the size of the reactor, that is, regardless of the volume of the required transfection mixture, or regardless of the volume of the required transfection reagent diluent and exogenous nucleic acid diluent, the transfection reagent diluent can be added to the exogenous nucleic acid diluent in a very short time. That is, the transfection reagent diluent and the exogenous nucleic acid diluent can be mixed in a very short time, thereby eliminating the influence of the reactor volume on the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent, thus ensuring the transfection efficiency of the transfection mixture on the cells to be transfected.

[0021] The transfection mixture preparation apparatus provided in the second and third aspects of this application is structurally similar to the transfection mixture preparation apparatus provided in the first aspect of this application and is the same in principle for preparing transfection mixtures. That is, these three transfection mixture preparation apparatuses are based on the same inventive concept, which gives them all the advantages of the transfection mixture preparation apparatus provided in the first aspect of this application.

[0022] The transfection mixture preparation method provided in the fourth aspect of this application is applied in any one of the transfection mixture preparation apparatuses provided in the first, second and third aspects of this application. That is, it is implemented based on any one of the three transfection mixture preparation apparatuses, and therefore has all the advantages of the three transfection mixture preparation apparatuses.

[0023] The gene transfection system provided in the fifth aspect of this application includes the transfection mixture preparation apparatus provided in any one of the first, second and third aspects of this application, and therefore possesses all the advantages of the three transfection mixture preparation apparatuses.

[0024] The gene transfection method provided in the sixth aspect of this application is applied to the gene transfection system provided in the fifth aspect of this application. Since the gene transfection system includes the transfection mixture preparation device provided in any one of the first, second and third aspects of this application, the gene transfection method has all the advantages of the three transfection mixture preparation devices. Attached Figure Description

[0025] To more clearly illustrate the related technologies or the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the related technologies or the embodiments of this application will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application, and not all embodiments. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the transfection mixture preparation apparatus provided in the embodiments of this application;

[0027] Figure 2 A schematic diagram of the structure of the bag provided in an embodiment of this application;

[0028] Figure 3 This is another structural schematic diagram of the bag body provided in an embodiment of this application;

[0029] Figure 4 A schematic flowchart illustrating the method for preparing the transfection mixture provided in the embodiments of this application;

[0030] Figure 5 This is a schematic diagram of the structure of the gene transfection system provided in the embodiments of this application;

[0031] Figure 6 This is a schematic flowchart of the gene transfection method provided in the embodiments of this application.

[0032] The markings in the above figures represent: 110-workbench, 120-bag body, 121-nucleic acid bag, 122-reagent bag, 123-connecting bag, 1211-first mounting hole, 1212-first sampling hole, 1213-transfection hole, 1221-second mounting hole, 1222-second sampling hole, 1231-nucleic acid well, 1232-reagent well, 1233-vent, 130-first isolation sleeve, 140-first stirrer, 150-second isolation sleeve, 160-second stirrer, 170-transfection infusion pump, 180-transfection infusion tubing, 190-reactor. Detailed Implementation

[0033] Typically, the mixing time between the transfection reagent diluent and the exogenous nucleic acid diluent during the preparation of the transfection mixture significantly affects the transfection efficiency of the transfection mixture on the cells to be transfected; the longer the mixing time, the lower the transfection efficiency. In related technologies, for small-scale experiments, a pipette can be used to add the transfection reagent diluent to the exogenous nucleic acid diluent. This process is instantaneous, thus avoiding the low transfection efficiency problem caused by long mixing time. However, in actual gene transfection, the cells to be transfected are usually cultured in a large-volume reactor, which increases the volume of the required transfection mixture. This means that both the transfection reagent diluent and the exogenous nucleic acid diluent are required. In this case, the transfection reagent diluent cannot be added to the exogenous nucleic acid diluent instantaneously as in small-scale experiments, resulting in a longer mixing time and lower transfection efficiency of the transfection mixture. For example, when the reactor is 200L, the required volume of the transfection mixture is 12-20L, which means 6-10L of transfection reagent diluent needs to be added to 6-10L of exogenous nucleic acid diluent. This process cannot be instantaneous and requires at least 2.5 minutes. Therefore, the mixing time between the transfection reagent diluent and the exogenous nucleic acid diluent is affected by the reactor volume. The larger the reactor volume, the longer the mixing time, and the lower the transfection efficiency of the transfection mixture on the cells to be transfected.

[0034] In view of this, the present application proposes a transfection mixture preparation apparatus and a gene transfection system using the transfection mixture preparation apparatus in the embodiments below. When preparing the transfection mixture using the transfection mixture preparation apparatus, the mixing of the transfection reagent diluent and the exogenous nucleic acid diluent can be achieved regardless of the reactor volume. That is, no matter how large the reactor volume is, the transfection reagent diluent and the exogenous nucleic acid diluent can be mixed in a very short time, thereby eliminating the influence of the reactor volume on the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent, thereby improving the transfection efficiency of the transfection mixture on the cells to be transfected, thus avoiding the above-mentioned drawbacks existing in related technologies.

[0035] To make the objectives, technical solutions, and advantages of this application more apparent and understandable, this application will be clearly and completely described below in conjunction with its embodiments and corresponding drawings. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. It should be understood that the embodiments of this application described below are only for explaining this application and are not intended to limit this application. That is, all other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application. Furthermore, the technical features involved in the various embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0036] Please see Figure 1 , Figure 1 This is a schematic diagram of the transfection mixture preparation device, in which... Figure 1 (a) is a schematic diagram of the transfection mixture preparation device before the transfection reagent diluent is added to the exogenous nucleic acid diluent. Figure 1 (b) is a schematic diagram of the transfection mixture preparation device when the transfection reagent diluent is added to the exogenous nucleic acid diluent. This embodiment provides a transfection mixture preparation device, which includes a workbench 110 and a bag body 120. The bag body 120 includes a nucleic acid bag 121, a reagent bag 122, and a connecting bag 123. The nucleic acid bag 121 has a nucleic acid port (not shown) communicating with the interior, and the reagent bag 122 has a reagent port (not shown) communicating with the interior. The two opposite ends of the connecting bag 123 have connecting ports (not shown) communicating with the interior. The two opposite ends of the connecting bag 123 are respectively connected to the nucleic acid bag 121 and the reagent bag 122, and the two connecting ports are respectively connected to the nucleic acid port and the reagent port, that is, the nucleic acid bag 121 and the reagent bag 122 are connected through the connecting bag 123. In addition, it should be noted that the bag body 120 in this embodiment is a one-piece molded cuboid, except that the internal space of the bag body 120 is divided into the nucleic acid bag 121, the reagent bag 122, and the connecting bag 123 according to function. It should also be noted that both the nucleic acid bag 121 and the reagent bag 122 are 3D bags. For example, the nucleic acid bag 121 has a volume of 20L and the reagent bag 122 has a volume of 10L.

[0037] Specifically, the nucleic acid bag 121 is used to contain exogenous nucleic acid diluent and is placed on the workbench 110 with the nucleic acid port facing away from the workbench 110; the reagent bag 122 is used to contain transfection reagent diluent and is placed on the workbench 110 with the reagent port facing away from the workbench 110; the reagent bag 122 is also used to detach from the workbench 110 under external force and move away from the workbench 110, thereby making itself higher than the nucleic acid bag 121, so that the transfection reagent diluent inside it flows out from the reagent port and flows into the nucleic acid bag 121 through the connecting bag 123 and the nucleic acid port, so as to mix with the exogenous nucleic acid diluent in the nucleic acid bag 121 and obtain a transfection mixture accordingly. For easier understanding, please refer to Figure 1 , Figure 1 The gray arrow in (a) indicates the direction of movement of reagent bag 122. Figure 1 The gray arrow in (b) indicates the direction of flow of the transfection reagent diluent in reagent bag 122 to nucleic acid bag 121.

[0038] In other words, the process of preparing the transfection mixture in this embodiment includes: placing the nucleic acid bag 121 on the workbench 110 with the nucleic acid port facing away from the workbench 110; placing the reagent bag 122 on the workbench 110 with the reagent port facing away from the workbench 110; placing the exogenous nucleic acid diluent in the nucleic acid bag 121, for example, by pumping the exogenous nucleic acid diluent into the nucleic acid bag 121; placing the transfection reagent diluent in the reagent bag 122, for example, by pumping the transfection reagent diluent into the reagent bag 122; moving the reagent bag 122 a certain distance away from the workbench 110 and keeping it suspended in the air, so that the reagent bag 122 is higher than the nucleic acid bag 121. At this time, the transfection reagent diluent will flow out from the reagent bag 122 at the higher position and flow along the connecting bag 123 into the nucleic acid bag 121 at the lower position. The transfection reagent diluent flowing into the nucleic acid bag 121 will mix with the exogenous nucleic acid diluent in the nucleic acid bag 121, and finally form a transfection mixture. In addition, after the transfection mixture is formed in the nucleic acid bag 121, the transfection mixture can be introduced into a reactor containing cells to be transfected, so that the cells to be transfected in the reactor combine with the transfection mixture. Then, the cells to be transfected with the transfection mixture are cultured in the reactor to achieve gene transfection of the cells to be transfected.

[0039] In this embodiment, when the nucleic acid bag 121 is placed on the workbench 110, the nucleic acid port of the nucleic acid bag 121 faces away from the workbench 110. In this way, after the exogenous nucleic acid diluent is pumped into the nucleic acid bag 121, it can be prevented from entering the connecting bag 123 through the nucleic acid port, or even from entering the reagent bag 122 through the connecting bag 123. Similarly, when the reagent bag 122 is placed on the workbench 110, the reagent port of the reagent bag 122 also faces away from the workbench 110. In this way, after the transfection reagent diluent is pumped into the reagent bag 122, it can be prevented from entering the connecting bag 123 through the reagent port, or even from entering the nucleic acid bag 121 through the connecting bag 123. Furthermore, after placing both the nucleic acid bag 121 and the reagent bag 122 flat on the workbench 110, since the nucleic acid port of the nucleic acid bag 121 and the reagent port of the reagent bag 122 both face away from the workbench 110, the orientation of the connecting bag 123 is such that the middle is higher than both ends, that is, the connecting bag 123 is in the shape of a "parabola" or an "arch". This orientation of the connecting bag 123 can further prevent the exogenous nucleic acid diluent in the nucleic acid bag 121 from entering the connecting bag 123 through the nucleic acid port, or even from entering the reagent bag 122 through the connecting bag 123; and the transfection reagent diluent in the reagent bag 122 from entering the connecting bag 123 through the reagent port, or even from entering the nucleic acid bag 121 through the connecting bag 123.

[0040] In this embodiment, the exogenous nucleic acid used in the exogenous nucleic acid diluent can be any type of nucleic acid commonly used in the art, including but not limited to RNA and DNA (such as plasmid DNA, linear double-stranded DNA, etc.). The specific choice can be made according to actual needs, and this application does not impose a unique limitation. The transfection reagent used in the transfection reagent diluent can be any type of transfection reagent commonly used in the art, including but not limited to liposome-based transfection reagents (such as Oligofectamine and Lipofectamine 2000 / 3000 from Invitrogen), non-liposome-based transfection reagents (such as PEI, FuGENE HD, and FuGENE 4K), and nanomaterial-based transfection reagents (such as Rfect V2). The specific choice can be made according to actual needs, and this application does not impose a unique limitation. Furthermore, it should be noted that the exogenous nucleic acid diluent is actually a mixture of exogenous nucleic acid with DMEM (Dupuyck modified Eagle medium), other serum-free suspension 293 CD medium, PBS (phosphate buffered saline), or a sodium chloride solution of a certain concentration. The process of mixing the two is the dilution process of the exogenous nucleic acid. Similarly, the transfection reagent diluent is actually a mixture of the transfection reagent with DMEM, other serum-free suspension 293 CD medium, PBS, or a sodium chloride solution of a certain concentration. The process of mixing the two is the dilution process of the transfection reagent. It should also be noted that the diluents used in the dilution process of the exogenous nucleic acid and transfection reagent mentioned in this paragraph are selected according to actual needs, and this application does not impose a unique limitation on them; however, the diluents used in the dilution process of the exogenous nucleic acid and transfection reagent should be the same.

[0041] As can be seen from the above, in this embodiment, when preparing the transfection mixture, the reagent bag 122 containing the transfection reagent diluent is first moved to a position higher than the nucleic acid bag 121 containing the exogenous nucleic acid diluent. Then, the transfection reagent diluent in the reagent bag 122 is poured into the nucleic acid bag 121 by tilting. This tilting method can disregard the volume of the reactor used to contain and culture the cells to be transfected. In this case, no matter how large the reactor is, that is, no matter how large the volume of the required transfection mixture is, or no matter how large the volumes of the required transfection reagent diluent and exogenous nucleic acid diluent are, the transfection reagent diluent can be added to the exogenous nucleic acid diluent in a very short time. That is, the transfection reagent diluent and the exogenous nucleic acid diluent can be mixed in a very short time, thereby eliminating the influence of the reactor volume on the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent, thus ensuring the transfection efficiency of the transfection mixture on the cells to be transfected.

[0042] In some embodiments, please refer to Figure 2 and Figure 3 , Figure 2 This is a structural diagram of the bag. Figure 3 This is another structural diagram of the bag body. The bottom center of the nucleic acid bag 121 has a first mounting hole 1211 communicating with the interior and opposite to the nucleic acid port. The bottom center of the reagent bag 122 has a second mounting hole 1221 communicating with the interior and opposite to the reagent port. Based on this, the transfection mixture preparation device, in addition to the structure given above, also includes a first motor (not shown), a second motor (not shown), a first isolation sleeve 130, a second isolation sleeve 150, a first stirring paddle 140, and a second stirring paddle 160. The first motor and the second motor are arranged opposite each other and spaced apart on the worktable 110. The first isolation sleeve 130 and the second isolation sleeve 150 are respectively disposed within the first mounting hole 1211 and the second mounting hole 1221. The first stirring paddle 140 and the second stirring paddle 160 are respectively located within the nucleic acid bag 121 and the reagent bag 122. The first stirring paddle 140 is disposed on the first isolation sleeve 130, and the second stirring paddle 160 is disposed on the second isolation sleeve 150.

[0043] In these embodiments, a first hollow protrusion (not shown) extending outward from the nucleic acid bag 121 is formed in the middle of the first isolation sleeve 130, and a first boss (not shown) is formed in the middle of the first stirring paddle 140. The first boss is inserted into the first protrusion so that the first stirring paddle 140 and the first isolation sleeve 130 are rotatably engaged; a second hollow protrusion (not shown) extending outward from the reagent bag 122 is formed in the middle of the second isolation sleeve 150, and a second boss (not shown) is formed in the middle of the second stirring paddle 160. The second boss is inserted into the second protrusion so that the second stirring paddle 160 and the second isolation sleeve 150 are rotatably engaged; a first motor has a first recess (not shown) adapted to the first protrusion, and a second motor has a second recess (not shown) adapted to the second protrusion.

[0044] Specifically, the first motor is used to align its first concave hole with the first protrusion of the first isolation sleeve 130 when the nucleic acid bag 121 is placed on the worktable 110, and to drive the first stirring paddle 140 to rotate using electromagnetic levitation. When the nucleic acid bag 121 contains exogenous nucleic acid diluent, the rotating first stirring paddle 140 stirs the exogenous nucleic acid diluent within the nucleic acid bag 121. When the nucleic acid bag 121 contains a mixture of exogenous nucleic acid diluent and transfection reagent diluent, the rotating first stirring paddle 140 stirs the mixed exogenous nucleic acid diluent and transfection reagent diluent within the nucleic acid bag 121, thus obtaining a transfection mixture. The second motor is used to align its second concave hole with the second protrusion of the second isolation sleeve 150 when the reagent bag 122 is placed on the worktable 110, and to drive the second stirring paddle 160 to rotate using electromagnetic levitation. When the reagent bag 122 contains transfection reagent diluent, the rotating second stirring paddle 160 stirs the transfection reagent diluent within the reagent bag 122.

[0045] In other words, during the preparation of the transfection mixture, when the nucleic acid bag 121 is placed on the workbench 110, the first concave hole of the first motor will fit with the first protrusion of the first isolation sleeve 130. Then, the exogenous nucleic acid diluent can be pumped into the nucleic acid bag 121. The first motor will use electromagnetic levitation to drive the first stirring paddle 140, which is rotated in conjunction with the first isolation sleeve 130, to stir the exogenous nucleic acid diluent in the nucleic acid bag 121, so that the exogenous nucleic acid in the exogenous nucleic acid diluent is fully mixed with the corresponding diluent (i.e., DMEM, other serum-free suspension 293 CD medium, PBS, or a certain concentration of sodium chloride solution as described above), so that the exogenous nucleic acid is fully diluted. This is because only fully diluted exogenous nucleic acid can be used to prepare the transfection mixture.

[0046] When the reagent bag 122 is placed on the workbench 110, the second concave hole of the second motor will fit with the second protrusion of the second isolation sleeve 150. Then, the transfection reagent diluent can be pumped into the reagent bag 122. The second motor will use electromagnetic levitation to drive the second stirring paddle 160, which is rotated in conjunction with the second isolation sleeve 150, to stir the transfection reagent diluent in the reagent bag 122, so that the transfection reagent in the transfection reagent diluent is fully mixed with the corresponding diluent, that is, the transfection reagent is fully diluted. This is because only fully diluted transfection reagent can be used to prepare transfection mixture.

[0047] When the reagent bag 122 is moved a certain distance away from the workbench 110 and stops in the air, the second motor will disconnect from the drive connection of the second stirring paddle 160 and separate from the second isolation sleeve 150. That is, the second protrusion of the second isolation sleeve 150 will detach from the second concave hole of the second motor. Then, the transfection reagent diluent in the reagent bag 122 can be poured into the nucleic acid bag 121, and the first stirring paddle 140 inside the nucleic acid bag 121 can be rotated by the first motor to stir the mixed exogenous nucleic acid diluent and transfection reagent diluent in the nucleic acid bag 121, so that the exogenous nucleic acid diluent and transfection reagent diluent in the nucleic acid bag 121 are fully mixed, so as to obtain the transfection mixture for gene transfection.

[0048] In some embodiments, still refer to Figure 2 as well as Figure 3The connecting bag 123 also has a nucleic acid well 1231 connected to the interior and close to the nucleic acid bag 121. Based on this, the transfection mixture preparation apparatus, in addition to the structure described above, also includes a nucleic acid storage tank (not shown), a nucleic acid infusion tube (not shown), and a nucleic acid infusion pump (not shown). Both the nucleic acid storage tank and the nucleic acid infusion pump are mounted on the workbench 110. One end of the nucleic acid infusion tube is connected to the connecting bag 123 through the nucleic acid well 1231, and the other end is connected to the nucleic acid storage tank through the nucleic acid infusion pump. Specifically, the nucleic acid storage tank is used to store exogenous nucleic acid diluent; the nucleic acid infusion pump is used to introduce the exogenous nucleic acid diluent from the nucleic acid storage tank into the nucleic acid infusion tube after the nucleic acid bag 121 and reagent bag 122 are both flatly mounted on the workbench 110, and then deliver it to the connecting bag 123 through the nucleic acid infusion tube, so that the exogenous nucleic acid diluent delivered to the connecting bag 123 flows into the nucleic acid bag 121 through the nucleic acid well. It should be noted that the nucleic acid infusion pump can be any device with liquid delivery function commonly used in the field, such as centrifugal pumps, plunger pumps, air compressors, vacuum pumps, peristaltic pumps, and metering pumps. The specific choice can be made according to actual needs, and this application does not impose a unique limitation. In addition, exemplarily, the nucleic acid infusion tubing consists of a bag buckle, a silicone tube, a tube clamp, and a male and female plug; wherein, the bag buckle is an SBB8 bag buckle, and the silicone tube is a platinum-cured silicone tube with an outer diameter of 19.1 mm, an inner diameter of 12.7 mm, and a length of 100 cm.

[0049] In other words, during the preparation of the transfection mixture, after the nucleic acid bag 121 and reagent bag 122 are placed flat on the workbench 110, the exogenous nucleic acid diluent in the nucleic acid storage tank is introduced into the nucleic acid infusion tube using a nucleic acid infusion pump and then transported to the connecting bag 123 through the nucleic acid infusion tube. Since the connecting bag 123 is positioned with the middle higher than both ends when the nucleic acid bag 121 and reagent bag 122 are placed flat on the workbench 110, the end of the connecting bag 123 near the nucleic acid bag 121 is tilted towards the nucleic acid bag 121. At the same time, since the nucleic acid hole 1231 on the connecting bag 123 is close to the nucleic acid bag 121, the exogenous nucleic acid diluent transported to the connecting bag 123 will flow into the nucleic acid bag 121 due to the tendency of the end of the connecting bag 123 near the nucleic acid bag 121 to tilt towards the nucleic acid bag 121. Of course, in order to shorten the time for pumping exogenous nucleic acid diluent into the bag 120, in other embodiments, two or more sets of nucleic acid wells 1231 and nucleic acid infusion tubes can be set, and exogenous nucleic acid diluent can be pumped into the bag 120 simultaneously using multiple sets of nucleic acid wells 1231 and nucleic acid infusion tubes. As for the number of sets of nucleic acid wells 1231 and nucleic acid infusion tubes, it is set according to actual needs, and this application does not make a unique limitation on it.

[0050] Furthermore, the connecting bag 123 also has a reagent port 1232 communicating with the interior and close to the reagent bag 122. In addition to the structure described above, the transfection mixture preparation apparatus also includes a reagent storage tank (not shown), a reagent infusion tube (not shown), and a reagent infusion pump (not shown). Both the reagent storage tank and the reagent infusion pump are mounted on the workbench 110. One end of the reagent infusion tube is connected to the connecting bag 123 through the reagent port 1232, and the other end is connected to the reagent storage tank through the reagent infusion pump. Specifically, the reagent storage tank is used to store the transfection reagent diluent; the reagent infusion pump is used to introduce the transfection reagent diluent from the reagent storage tank into the reagent infusion tube after the nucleic acid bag 121 and the reagent bag 122 are both flatly mounted on the workbench 110, and then deliver it to the connecting bag 123 through the reagent infusion tube, so that the transfection reagent diluent delivered to the connecting bag 123 flows into the reagent bag 122 through the reagent port. It should be noted that the reagent infusion pump can be any device commonly used in the art that has liquid delivery capabilities, such as centrifugal pumps, plunger pumps, air compressors, vacuum pumps, peristaltic pumps, and metering pumps. The specific choice depends on actual needs, and this application does not impose a single limitation. Furthermore, exemplarily, the reagent infusion tubing consists of a bag buckle, silicone tubing, tubing clamps, and male and female plugs; wherein the bag buckle is an SBB8 bag buckle, and the silicone tubing is a platinum-cured silicone tubing with an outer diameter of 19.1 mm, an inner diameter of 12.7 mm, and a length of 100 cm.

[0051] In other words, during the preparation of the transfection mixture, after the nucleic acid bag 121 and reagent bag 122 are placed flat on the workbench 110, the transfection reagent diluent in the reagent storage tank is introduced into the reagent infusion tube using a reagent infusion pump and then delivered to the connecting bag 123 through the reagent infusion tube. Since the connecting bag 123 is positioned with the middle higher than both ends when the nucleic acid bag 121 and reagent bag 122 are placed flat on the workbench 110, the end of the connecting bag 123 near the reagent bag 122 is inclined towards the reagent bag 122. At the same time, since the reagent hole 1232 on the connecting bag 123 is close to the reagent bag 122, the transfection reagent diluent delivered to the connecting bag 123 will flow into the reagent bag 122 due to the tendency of the end of the connecting bag 123 near the reagent bag 122 to flow into the reagent bag 122. Of course, in order to shorten the time for pumping the transfection reagent diluent into the bag 120, in other embodiments, two or more sets of reagent holes 1232 and reagent infusion tubes can be provided, and multiple sets of reagent holes 1232 and reagent infusion tubes can be used to pump the transfection reagent diluent into the bag 120 simultaneously. As for the number of sets of reagent holes 1232 and reagent infusion tubes, it is set according to actual needs, and this application does not make a unique limitation on it.

[0052] In some embodiments, still refer to Figure 2 as well as Figure 3The connecting bag 123 also has an air vent 1233 that communicates with the interior and is located between the nucleic acid well 1231 and the reagent well 1232. In addition to the structure described above, the transfection mixture preparation apparatus also includes an air compressor (not shown), an air supply tube (not shown), and a disc filter (not shown). The air compressor is located adjacent to the workbench 110. One end of the air supply tube is connected to the connecting bag 123 through the air vent 1233, and the other end of the air supply tube is connected to the disc filter. The disc filter is connected to the air compressor. Specifically, the air compressor can draw in and compress outside air before pumping the exogenous nucleic acid diluent and transfection reagent diluent into the connecting bag 123. The compressed air enters the connecting bag 123 through a disc filter and an air supply tube, causing the bag 120 to expand. After the exogenous nucleic acid diluent and transfection reagent diluent are pumped into the connecting bag 123, the air inside the bag 120 can be released through the air supply tube and disc filter, isolating the inside of the bag 120 from the external environment and causing the bag 120 to contract, preventing the exogenous nucleic acid diluent and transfection reagent diluent pumped into the bag 120 from bursting. The disc filter can sterilize the air entering the connecting bag 123, thereby ensuring that the inside of the bag 120 is sterile as a whole.

[0053] In other words, during the preparation of the transfection mixture, after the nucleic acid bag 121 and reagent bag 122 are placed flat on the workbench 110, an air compressor is used to fill the connecting bag 123 with air through a disc filter and an air supply tube, causing the bag body 120 (i.e., the connected nucleic acid bag 121, connecting bag 123, and reagent bag 122) to expand, thus preparing for the subsequent pumping of exogenous nucleic acid dilution and transfection reagent dilution. After the exogenous nucleic acid dilution is pumped into the connecting bag 123 using a nucleic acid infusion pump and the pumped exogenous nucleic acid dilution is contained in the nucleic acid bag 121, and the transfection reagent dilution is pumped into the connecting bag 123 using a reagent infusion pump and the pumped transfection reagent dilution is contained in the reagent bag 122, the air in the bag body 120 needs to be released through the air supply tube and disc filter, causing the bag body 120 to contract, thereby preventing the exogenous nucleic acid dilution and transfection reagent dilution pumped into the bag body 120 from bursting the bag body 120. In addition, it should be noted that the air supply pipe is usually equipped with a pipe clamp. When it is necessary to release the air in the bag 120, simply open the pipe clamp on the air supply pipe without operating the air compressor.

[0054] It is understandable that installing a disc filter between the gas supply pipe and the air compressor can effectively filter the air supplied to the connecting bag 123 through the gas supply pipe, thereby preventing the air entering the bag 120 from contaminating the exogenous nucleic acid diluent and transfection reagent diluent inside the bag 120, and ensuring the quality of the final transfection mixture formed in the nucleic acid bag 121, which is equivalent to ensuring the transfection effect of the cells to be transfected. In addition, it should be noted that the installation between the gas supply pipe and the air compressor is not limited to a disc filter. In other embodiments, other filters with filtration functions commonly used in the art can also be installed between the gas supply pipe and the air compressor, such as air filters, activated carbon filters, disc filters, HEPA filters, electrostatic filters, photocatalytic filters, etc. The specific choice can be made according to actual needs, and this application does not make a unique limitation. For example, if the filter is used as part of the gas supply pipe, then the gas supply pipe can be composed of a bag buckle, a silicone tube, a tube clamp, and a filter; wherein the bag buckle is an SBB4 bag buckle, the tube clamp is a Robert clamp, the filter is a disc filter, and the silicone tube is a platinum vulcanized silicone tube with a specification of 9.6mm outer diameter, 6.4mm inner diameter, and 15cm length.

[0055] In some embodiments, still refer to Figure 2 and Figure 3 The nucleic acid bag 121 also has a first sampling port 1212 communicating with the interior. Based on this, the transfection mixture preparation apparatus, in addition to the structure given above, also includes a first needle-free sampler (not shown), a first sampling tube (not shown), and a first sample tube (not shown). One end of the first sampling tube is connected to the nucleic acid bag 121 through the first sampling port 1212, and the other end of the first sampling tube is connected to the first needle-free sampler. Specifically, the first needle-free sampler is used to connect to a first Luer syringe to extract the transfection mixture or exogenous nucleic acid diluent from the nucleic acid bag 121 through the first Luer syringe, and to inject the extracted transfection mixture or exogenous nucleic acid diluent into the first sample tube through the first Luer syringe. For example, if the first needleless sampler is used as part of the first sampling tube, then the first sampling tube may be composed of a bag buckle, a silicone tube, a tube clamp, a connector, the first needleless sampler, and a first needleless protective cap; wherein, the bag buckle is an SBB4 bag buckle, the tube clamp is a Robert clamp, the connector is a Luer connector, and the silicone tube is a platinum vulcanized silicone tube with a specification of 9.6 mm outer diameter, 6.4 mm inner diameter, and 10 cm length.

[0056] In other words, during the preparation of the transfection mixture, after the exogenous nucleic acid diluent is pumped into the nucleic acid bag 121 using a nucleic acid infusion pump and the exogenous nucleic acid diluent in the nucleic acid bag 121 is stirred by the first stirring paddle 140 driven by the first motor, the exogenous nucleic acid diluent can be extracted from the nucleic acid bag 121 by connecting the first needleless sampler to the first Luer syringe. The extracted exogenous nucleic acid diluent is then injected into the first sample tube as a sample. Subsequently, the staff can test the sample in the first sample tube to determine the quality of the exogenous nucleic acid diluent, such as whether it is sterile. In addition, after pouring the transfection reagent diluent from reagent bag 122 into nucleic acid bag 121 and using the first motor to drive the first stirring paddle 140 to stir the exogenous nucleic acid diluent and transfection reagent diluent mixed in nucleic acid bag 121, it is necessary to let it stand. After standing, the first needleless sampler can be connected to the first Luer syringe to extract the transfection mixture from nucleic acid bag 121, and the extracted transfection mixture is injected from the first Luer syringe into the first sample tube as a sample. Then, the staff can test the sample in the first sample tube to determine the quality of the transfection mixture.

[0057] Furthermore, the reagent bag 122 also has a second sampling port 1222 communicating with the interior. In addition to the structure described above, the transfection mixture preparation apparatus also includes a second needle-free sampler (not shown), a second sampling tube (not shown), and a second sample tube (not shown). One end of the second sampling tube is connected to the reagent bag 122 through the second sampling port 1222, and the other end of the second sampling tube is connected to the second needle-free sampler. Specifically, the second needle-free sampler is used to connect to a second Luer syringe to extract the transfection reagent diluent from the reagent bag 122 through the second Luer syringe, and to inject the extracted transfection reagent diluent into the second sample tube through the second Luer syringe. In other words, during the preparation of the transfection mixture, after the transfection reagent diluent is pumped into the reagent bag 122 by the reagent infusion pump and the second stirring paddle 160 driven by the second motor is used to stir the transfection reagent diluent in the reagent bag 122, the second needleless sampler can be connected to the second Luer syringe to extract the transfection reagent diluent from the reagent bag 122. The extracted transfection reagent diluent is then injected into the second sample tube through the second Luer syringe as a sample. Subsequently, the staff can test the sample in the second sample tube to determine the quality of the transfection reagent diluent. For example, if the second needleless sampler is used as part of the second sampling tube, then the second sampling tube can be composed of a bag buckle, a silicone tube, a tube clamp, a connector, the second needleless sampler, and a second needleless protective cap; wherein, the bag buckle is an SBB4 bag buckle, the tube clamp is a Robert clamp, the connector is a Luer connector, and the silicone tube is a platinum vulcanized silicone tube with a specification of 9.6 mm outer diameter, 6.4 mm inner diameter, and 10 cm length.

[0058] Compared to the transfection mixture preparation apparatus provided in the previous embodiment, this embodiment provides another transfection mixture preparation apparatus. This apparatus is structurally similar to the one provided in the previous embodiment, and the principle of preparing the transfection mixture is the same. The difference lies in that the connecting bag 123 is replaced with at least one connecting tube, and it includes two independent nucleic acid bags 121 and reagent bags 122. Therefore, the transfection mixture preparation apparatus provided in this embodiment includes a workbench 110, nucleic acid bags 121, reagent bags 122, and at least one connecting tube. The nucleic acid bag 121 has at least one nucleic acid port communicating with its interior, and the reagent bag 122 has at least one reagent port communicating with its interior. The number of connecting tubes, nucleic acid ports, and reagent ports is the same. The two ends of the connecting tube are respectively connected to the nucleic acid bag 121 and the reagent bag 122, and the two opposite ports are respectively connected to the nucleic acid port and the reagent port. In other words, there is a one-to-one correspondence between the connecting tube, the nucleic acid port, and the reagent port.

[0059] Specifically, the nucleic acid bag 121 is used to contain exogenous nucleic acid diluent and is placed on the workbench 110 with the nucleic acid port facing away from the workbench 110; the reagent bag 122 is used to contain transfection reagent diluent and is placed on the workbench 110 with the reagent port facing away from the workbench 110; the connecting tube is used to connect the nucleic acid bag 121 and the reagent bag 122; the reagent bag 122 is also used to detach from the workbench 110 under the action of external force and move in the direction away from the workbench 110, so that it is higher than the nucleic acid bag 121, and the transfection reagent diluent inside it flows out from the reagent port and flows into the nucleic acid bag 121 through the connecting tube and the nucleic acid port in turn, so as to mix with the exogenous nucleic acid diluent in the nucleic acid bag 121 to obtain a transfection mixture. It is understandable that the more connecting tubes connecting the nucleic acid bag 121 and the reagent bag 122, the faster the transfection reagent diluent in the reagent bag 122 is poured into the nucleic acid bag 121, and the shorter the time consumed. This means the mixing time between the exogenous nucleic acid diluent and the transfection reagent diluent is also shorter, resulting in higher transfection efficiency of the transfection mixture for the cells to be transfected. The number of connecting tubes between the nucleic acid bag 121 and the reagent bag 122 can be set according to actual needs, and this application does not impose a unique limitation on this. Of course, increasing the diameter of the connecting tubes can also speed up the pouring of the transfection reagent diluent from the reagent bag 122 into the nucleic acid bag 121. Therefore, in other embodiments, the diameter of the connecting tubes can be appropriately increased to shorten the mixing time between the exogenous nucleic acid diluent and the transfection reagent diluent, thereby improving transfection efficiency.

[0060] Compared to the transfection mixture preparation apparatus provided in the previous two embodiments, this embodiment provides another transfection mixture preparation apparatus. This apparatus is structurally similar to the one provided in the first embodiment, and the principle of preparing the transfection mixture is the same. The difference lies in that the reagent bag 122 is set as a 2D bag and used to contain undiluted transfection reagent. Therefore, the transfection mixture preparation apparatus provided in this embodiment includes a workbench 110 and a bag body 120. The bag body 120 is functionally divided into three parts: a nucleic acid bag 121, a reagent bag 122, and a connecting bag 123. The nucleic acid bag 121 has a nucleic acid port communicating with the interior, the reagent bag 122 has a reagent port communicating with the interior, and the two ends of the connecting bag 123 have connecting ports communicating with the interior. The two ends of the connecting bag 123 are connected to the nucleic acid bag 121 and the reagent bag 122, respectively, and the two connecting ports of the connecting bag 123 are connected to the nucleic acid port and the reagent port, respectively.

[0061] Specifically, the nucleic acid bag 121 is used to contain exogenous nucleic acid diluent and is placed on the workbench 110 with the nucleic acid port facing away from the workbench 110; the reagent bag 122 is a 2D bag used to contain transfection reagent and is placed on the workbench 110 with the reagent port facing away from the workbench 110 or facing the nucleic acid bag 121; the connecting bag 123 is used to connect the nucleic acid bag 121 and the reagent bag 122; the reagent bag 122 is also used to detach from the workbench 110 under the action of external force and move away from the workbench 110, so that it is higher than the nucleic acid bag 121, and the transfection reagent inside it slides out from the reagent port and slides into the nucleic acid bag 121 through the connecting bag 123 and the nucleic acid port in turn, so as to mix with the exogenous nucleic acid diluent in the nucleic acid bag 121 to obtain a transfection mixture. In this embodiment, the reagent port can be located on the upper surface of the reagent bag 122 or on the edge of the reagent bag 122, depending on actual needs; this application does not impose a unique limitation on this. When the reagent port is located on the upper surface of the reagent bag 122, after placing the reagent bag 122 on the workbench 110, the reagent port of the reagent bag 122 faces away from the workbench 110. When the reagent port is located on the edge of the reagent bag 122, after placing the reagent bag 122 on the workbench 110, the reagent port of the reagent bag 122 faces the nucleic acid bag 121. Preferably, to ensure smooth pouring of the transfection reagent from the reagent bag 122 into the nucleic acid bag 121, the reagent port is located on the edge of the reagent bag 122.

[0062] Understandably, the nucleic acid bag 121 is a 3D bag used to contain the exogenous nucleic acid diluent, while the reagent bag 122 is a 2D bag used to contain the undiluted transfection reagent. In this way, all the diluents (i.e., DMEM, other serum-free suspension 293 CD medium, PBS, or a certain concentration of sodium chloride solution described above) can be used to dilute the exogenous nucleic acid, while also saving materials and simplifying the bag manufacturing process. Since the 2D reagent bag 122 contains the undiluted transfection reagent, and the undiluted transfection reagent does not require stirring, this embodiment does not need to include the second motor, the second isolation sleeve 150, and the second stirring paddle 160 of the first embodiment, thereby simplifying the structure of the transfection mixture preparation device and reducing the production cost of the transfection mixture preparation device.

[0063] In some embodiments, the reagent port is located at the edge of the reagent bag 122, such that when the reagent bag 122 is placed on the worktable 110, the reagent port faces the nucleic acid bag 121. Based on this, the transfection mixture preparation apparatus, in addition to the structure described above, also includes a clip (not shown), which is used to clamp the reagent bag 122 along the direction from the reagent bag 122 to the nucleic acid bag 121, thereby isolating the internal space of the reagent bag 122. It is understood that using a clip to isolate the internal space of the 2D reagent bag 122 can effectively prevent partial mixing of the exogenous nucleic acid diluent and the transfection reagent diluent during the separate pumping of the exogenous nucleic acid diluent and the transfection reagent diluent, thus preventing any impact on the final transfection mixture preparation effect.

[0064] Please see Figure 4 , Figure 4 This is a flowchart illustrating a method for preparing a transfection mixture. This embodiment provides a method for preparing a transfection mixture, applicable to any of the transfection mixture preparation devices described above, and includes the following steps 401 to 403 (abbreviated as S401 to S403): S401, placing exogenous nucleic acid diluent in a nucleic acid bag; S402, placing transfection reagent or transfection reagent diluent in a reagent bag connected to the nucleic acid bag; S403, moving the reagent bag to a position higher than the nucleic acid bag, allowing the transfection reagent or transfection reagent diluent in the reagent bag to flow into the nucleic acid bag and mix with the exogenous nucleic acid diluent in the nucleic acid bag to obtain a transfection mixture; wherein, when the reagent bag is a 3D bag, the reagent bag contains the transfection reagent diluent, and when the reagent bag is a 2D bag, the reagent bag contains the transfection reagent. It should be noted that for any omissions in the description of the transfection mixture preparation method, please refer to the relevant descriptions of each transfection mixture preparation device above; these will not be repeated here.

[0065] Please see Figure 5 , Figure 5This is a schematic diagram of a gene transfection system. This embodiment provides a gene transfection system including a reactor 190, a transfection infusion pump 170, a transfection infusion tube 180, and any of the transfection mixture preparation devices described above. The reactor 190 and the transfection infusion pump 170 are both located adjacent to the workbench 110 in the transfection mixture preparation device. The nucleic acid bag 121 in the transfection mixture preparation device also has a transfection well 1213 communicating with its interior (see [link to documentation]). Figure 2 or Figure 3 One end of the transfection infusion tube 180 is connected to the nucleic acid bag 121 through the transfection port 1213, and the other end of the transfection infusion tube 180 is connected to the reactor 190 through the transfection infusion pump 170. Exemplarily, the transfection infusion tube 180 consists of a bag buckle, a tube clamp, a silicone tube, and a female / male plug; wherein the bag buckle is an SBB4 bag buckle, the tube clamp is a Robert clamp, and the silicone tube is a C-Flex thermoplastic tube with an outer diameter of 11.1 mm, an inner diameter of 6.4 mm, and a length of 100 cm. Furthermore, it should be noted that the transfection infusion pump 170 can be any device commonly used in the art that has liquid delivery capabilities, such as a centrifugal pump, plunger pump, air compressor, vacuum pump, peristaltic pump, and metering pump, etc., and can be selected according to actual needs; this application does not impose a unique limitation on this.

[0066] Specifically, reactor 190 is used to contain cells to be transfected; transfection infusion pump 170 is used to introduce the transfection mixture in nucleic acid bag 121 into transfection infusion tube 180 when the nucleic acid bag 121 contains the transfection mixture, and then deliver it to reactor 190 through transfection infusion tube 180, so that the transfection mixture combines with the cells to be transfected in reactor 190; reactor 190 is also used to culture the cells to be transfected that have combined with the transfection mixture, thereby achieving transfection of the cells to be transfected. That is to say, after the transfection mixture is formed in nucleic acid bag 121, the transfection infusion pump 170 is used to extract the transfection mixture from nucleic acid bag 121, and the extracted transfection mixture is pumped into reactor 190 through transfection infusion tube 180, so that the cells to be transfected in reactor 190 combine with the transfection mixture, and then the cells to be transfected that have combined with the transfection mixture are cultured in reactor 190, thereby achieving gene transfection of the cells to be transfected.

[0067] Please see Figure 6 , Figure 6This is a flowchart illustrating a gene transfection method. This embodiment provides a gene transfection method applied to the gene transfection system described above, and includes the following steps 601 to 603 (abbreviated as S601 to S603): S601, preparing a transfection mixture using a transfection mixture preparation device; S602, conveying the transfection mixture to a reactor and combining it with the cells to be transfected; S603, culturing the cells to be transfected containing the transfection mixture in the reactor. It should be noted that for any aspects not covered in the description of the gene transfection method, please refer to the relevant descriptions of the gene transfection system and each transfection mixture preparation device above; these will not be repeated here.

[0068] The above embodiments are merely preferred implementations of this application and are not the only limitations on the transfection mixture preparation device, gene transfection system, and other related contents. Those skilled in the art can flexibly configure these embodiments based on actual application scenarios. It is understood that through the implementation of the above embodiments of this application, a transfection mixture preparation device is constructed using the workbench 110 and the bag 120. The bag 120 is generally divided into a nucleic acid bag 121, a reagent bag 122, and a connecting bag 123. The two ends of the connecting bag 123 are respectively connected to the nucleic acid bag 121 and the reagent bag 122. The connecting bag 123 is used to connect the nucleic acid bag 121 and the reagent bag 122. The nucleic acid bag 121 is used to contain exogenous nucleic acid diluent, and the reagent bag 122 is used to contain transfection reagent diluent. The reagent bag 122 can be moved to a position higher than the nucleic acid bag 121 under external force, allowing the transfection reagent diluent in the reagent bag 122 to flow into the nucleic acid bag 121 through the connecting bag 123 and mix with the exogenous nucleic acid diluent in the nucleic acid bag 121, ultimately obtaining the transfection mixture. Therefore, in preparing the transfection mixture, this application first moves the reagent bag 122 containing the transfection reagent diluent to a position higher than the nucleic acid bag 121 containing the exogenous nucleic acid diluent. Then, the transfection reagent diluent in the reagent bag 122 is poured into the nucleic acid bag 121 by tilting. This tilting method can disregard the volume of the reactor 190 used to contain and culture the cells to be transfected. In this case, no matter how large the volume of the reactor 190 is, that is, no matter how large the volume of the required transfection mixture is, or no matter how large the volumes of the required transfection reagent diluent and the exogenous nucleic acid diluent are, the transfection reagent diluent can be added to the exogenous nucleic acid diluent in a very short time. That is, the transfection reagent diluent and the exogenous nucleic acid diluent can be mixed in a very short time, thereby eliminating the influence of the volume of the reactor 190 on the mixing time of the transfection reagent diluent and the exogenous nucleic acid diluent, thus ensuring the transfection efficiency of the transfection mixture on the cells to be transfected.

[0069] It should be noted that the several embodiments shown above in this application are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. It should also be noted that in the textual description of this application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply such an actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements may include not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus; and, without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0070] Furthermore, those skilled in the art can implement or use this application by practicing the several embodiments shown above. Various modifications to the embodiments shown above will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments not shown without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the several embodiments shown above, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An apparatus for preparing a transfection mixture, characterized in that, The system includes a workbench and a bag body. The bag body is functionally divided into three parts: a nucleic acid bag, a reagent bag, and a connecting bag. The two ends of the connecting bag are connected to the nucleic acid bag and the reagent bag, respectively. Both the nucleic acid bag and the reagent bag are placed on the workbench. The nucleic acid bag is used to contain exogenous nucleic acid diluent; The reagent bag is used to contain the diluent for the transfection reagent; The reagent bag is also used to detach from the workbench under external force and move away from the workbench to pour the transfection reagent diluent into the nucleic acid bag, so that the transfection reagent diluent mixes with the exogenous nucleic acid diluent and a transfection mixture is obtained accordingly.

2. The transfection mixture preparation apparatus according to claim 1, characterized in that, The nucleic acid bag has a nucleic acid port communicating with the interior, the reagent bag has a reagent port communicating with the interior, and the two ends of the connecting bag each have a connecting port communicating with the interior, and the two connecting ports of the connecting bag are respectively connected to the nucleic acid port and the reagent port.

3. The transfection mixture preparation apparatus according to claim 2, characterized in that, When both the nucleic acid bag and the reagent bag are placed on the workbench, both the nucleic acid port and the reagent port face away from the workbench.

4. The transfection mixture preparation apparatus according to claim 2, characterized in that, It also includes a first motor, a first isolation sleeve, and a first stirring paddle. The nucleic acid bag has a first mounting hole communicating with the interior. The first motor is disposed on the worktable. The first isolation sleeve is disposed in the first mounting hole. The first stirring paddle is located inside the nucleic acid bag. The first stirring paddle is disposed on the first isolation sleeve and rotates with the first isolation sleeve. The first motor is used to connect with the first isolation sleeve when the nucleic acid bag is placed on the worktable, and to drive the first stirring paddle to rotate using magnetic force.

5. The transfection mixture preparation apparatus according to claim 4, characterized in that, The first mounting hole is located at the center of the bottom of the nucleic acid bag and is opposite to the nucleic acid port.

6. The transfection mixture preparation apparatus according to claim 4, characterized in that, The first isolation sleeve has a hollow first protrusion extending outward from the nucleic acid bag in the middle, and the first stirring paddle has a first boss in the middle. The first boss is inserted into the first protrusion so that the first stirring paddle and the first isolation sleeve can rotate together.

7. The transfection mixture preparation apparatus according to claim 6, characterized in that, The first motor has a first recess that is adapted to the first protrusion; The first motor is specifically used to make the first concave hole fit into the first protrusion when the nucleic acid bag is placed on the worktable.

8. The apparatus for preparing transfection mixture according to claim 2, characterized in that, It also includes a second motor, a second isolation sleeve, and a second stirring paddle. The reagent bag has a second mounting hole that communicates with the interior. The second motor is mounted on the worktable. The second isolation sleeve is mounted in the second mounting hole. The second stirring paddle is located inside the reagent bag. The second stirring paddle is mounted on the second isolation sleeve and rotates with the second isolation sleeve. The second motor is used to connect with the second isolation sleeve when the reagent bag is placed on the worktable, and to drive the second stirring paddle to rotate using magnetic force.

9. The apparatus for preparing transfection mixture according to claim 8, characterized in that, The second mounting hole is located at the center of the bottom of the reagent bag and is opposite to the reagent port.

10. The apparatus for preparing transfection mixture according to claim 8, characterized in that, The second isolation sleeve has a hollow second protrusion extending outward from the reagent bag in the middle, and the second stirring paddle has a second boss in the middle. The second boss is inserted into the second protrusion so that the second stirring paddle and the second isolation sleeve can rotate together.

11. The apparatus for preparing transfection mixture according to claim 10, characterized in that, The second motor has a second recess that is adapted to the second protrusion; The second motor is specifically used to make the second concave hole fit into the second protrusion when the reagent bag is placed on the worktable.

12. The apparatus for preparing transfection mixture according to claim 2, characterized in that, When both the nucleic acid bag and the reagent bag are placed on the worktable, the middle of the connecting bag is higher than both ends.

13. The transfection mixture preparation apparatus according to claim 12, characterized in that, It also includes a nucleic acid storage tank, a nucleic acid infusion tube, and a nucleic acid infusion pump. The connecting bag also has a nucleic acid port that communicates with the interior and is close to the nucleic acid port. The nucleic acid storage tank and the nucleic acid infusion pump are both mounted on the workbench. One end of the nucleic acid infusion tube is connected to the connecting bag through the nucleic acid port, and the other end of the nucleic acid infusion tube is connected to the nucleic acid storage tank. The nucleic acid storage tank is used to store the exogenous nucleic acid diluent; The nucleic acid infusion pump is used to pump the exogenous nucleic acid diluent from the nucleic acid storage tank into the nucleic acid bag through the nucleic acid infusion tube after the nucleic acid bag and the reagent bag are placed on the workbench.

14. The apparatus for preparing transfection mixture according to claim 13, characterized in that, It also includes a reagent storage tank, a reagent infusion tube, and a reagent infusion pump. The connecting bag also has a reagent port communicating with the interior and close to the reagent inlet. The reagent storage tank and the reagent infusion pump are both mounted on the workbench. One end of the reagent infusion tube is connected to the connecting bag through the reagent port, and the other end of the reagent infusion tube is connected to the reagent storage tank. The reagent storage tank is used to store the diluent of the transfection reagent; The reagent infusion pump is used to pump the transfection reagent diluent from the reagent storage tank into the reagent bag through the reagent infusion tube after both the nucleic acid bag and the reagent bag are placed on the workbench.

15. The apparatus for preparing transfection mixture according to claim 14, characterized in that, It also includes an air compressor and an air supply pipe. The connecting bag also has an air hole communicating with its interior. The air compressor is located adjacent to the workbench. One end of the air supply pipe is connected to the connecting bag through the air hole, and the other end of the air supply pipe is connected to the air compressor. Wherein: The air compressor is used to draw in and compress outside air before pumping the exogenous nucleic acid diluent and the transfection reagent diluent into the bag. The air supply pipe is used to deliver compressed air from the air compressor into the bag body, and to release the air inside the bag body after the exogenous nucleic acid diluent and the transfection reagent diluent are pumped into the bag body.

16. The apparatus for preparing transfection mixture according to claim 15, characterized in that, It also includes a disc filter, through which the air supply pipe is connected to the air compressor, and the disc filter is used to sterilize and filter the air entering the bag.

17. The apparatus for preparing transfection mixture according to claim 15, characterized in that, The pores are located between the nucleic acid wells and the reagent wells.

18. The apparatus for preparing transfection mixture according to claim 1, characterized in that, It also includes a first sampling tube, a first needle-free sampler, and a first sample tube. The nucleic acid bag also has a first sampling hole that communicates with the interior. One end of the first sampling tube is connected to the nucleic acid bag through the first sampling hole, and the other end of the first sampling tube is connected to the first needle-free sampler. The first needleless sampler is used to connect to a first Luer syringe to extract the transfection mixture or the exogenous nucleic acid diluent from the nucleic acid bag and inject it into the first sample tube.

19. The apparatus for preparing transfection mixture according to claim 1, characterized in that, It also includes a second sampling tube, a second needleless sampler, and a second sample tube. The reagent bag also has a second sampling hole that communicates with the interior. One end of the second sampling tube is connected to the reagent bag through the second sampling hole, and the other end of the second sampling tube is connected to the second needleless sampler. The second needleless sampler is used to connect to a second Luer syringe to draw the transfection reagent diluent from the reagent bag and inject it into the second sample tube.

20. An apparatus for preparing a transfection mixture, characterized in that, The system includes a workbench, a nucleic acid bag, a reagent bag, and at least one connecting tube. The two ends of the connecting tube are respectively connected to the nucleic acid bag and the reagent bag. Both the nucleic acid bag and the reagent bag are placed on the workbench. The nucleic acid bag is used to contain exogenous nucleic acid diluent; The reagent bag is used to contain the diluent for the transfection reagent; The reagent bag is also used to detach from the workbench under external force and move away from the workbench to pour the transfection reagent diluent into the nucleic acid bag, so that the transfection reagent diluent mixes with the exogenous nucleic acid diluent and a transfection mixture is obtained accordingly.

21. An apparatus for preparing a transfection mixture, characterized in that, The system includes a workbench and a bag body. The bag body is functionally divided into three parts: a nucleic acid bag, a reagent bag, and a connecting bag. The two ends of the connecting bag are connected to both the nucleic acid bag and the reagent bag. Both the nucleic acid bag and the reagent bag are placed on the workbench. The nucleic acid bag is used to contain exogenous nucleic acid diluent; The reagent bag is a 2D bag used to contain transfection reagents; The reagent bag is also used to detach from the workbench under external force and move away from the workbench to pour the transfection reagent into the nucleic acid bag, so that the transfection reagent is mixed with the exogenous nucleic acid diluent and a transfection mixture is obtained accordingly.

22. The apparatus for preparing transfection mixture according to claim 21, characterized in that, It also includes a clip, which is used to clamp onto the reagent bag along the direction from the reagent bag to the nucleic acid bag, so as to separate the internal space of the reagent bag.

23. A method for preparing a transfection mixture, characterized in that, An apparatus for preparing a transfection mixture according to any one of claims 1 to 19, or the apparatus for preparing a transfection mixture according to claim 20, or the apparatus for preparing a transfection mixture according to claim 21 or 22, comprising: Place the exogenous nucleic acid diluent in a nucleic acid bag; The transfection reagent or transfection reagent diluent is placed in a reagent bag connected to the nucleic acid bag; wherein, when the reagent bag is a 2D bag, the reagent bag contains the transfection reagent; The reagent bag is moved to a position higher than the nucleic acid bag, so that the transfection reagent or the diluent of the transfection reagent in the reagent bag flows into the nucleic acid bag and mixes with the exogenous nucleic acid diluent in the nucleic acid bag to obtain a transfection mixture.

24. A gene transfection system, characterized in that, The apparatus includes a reactor, a transfection infusion pump, a transfection infusion tubing, and a transfection mixture preparation device according to any one of claims 1 to 19, or the transfection mixture preparation device according to claim 20, or the transfection mixture preparation device according to claim 21 or 22. The reactor and the transfection infusion pump are both located adjacent to the workbench in the transfection mixture preparation device. The nucleic acid bag in the transfection mixture preparation device also has a transfection port communicating internally. One end of the transfection infusion tubing is connected to the nucleic acid bag through the transfection port, and the other end of the transfection infusion tubing is connected to the reactor. Wherein: The reactor is used to contain cells to be transfected; The transfection infusion pump is used to pump the transfection mixture in the nucleic acid bag into the reactor through the transfection infusion tube when the nucleic acid bag contains the transfection mixture, so that the transfection mixture combines with the cells to be transfected. The reactor is also used to culture the cells to be transfected, which are incorporating the transfection mixture.

25. A gene transfection method, characterized in that, The gene transfection method, applied in the gene transfection system of claim 24, comprises: Transfection mixtures were prepared using a transfection mixture preparation apparatus. The transfection mixture is delivered to the reactor and combined with the cells to be transfected; The cells to be transfected, incorporating the transfection mixture, are cultured using the reactor.

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