Cell factory device from virus packaging to harvesting

Abstract

The utility model relates to the technical field of biology, in particular to a cell factory device from virus packaging to virus harvesting. The utility model relates to a cell factory device from virus packaging to harvesting, which realizes closed transfer of liquid, can directly transfer a culture medium or cells into a cell factory or a bioreactor through a connecting pipeline in a sterile environment, and is quicker, simpler, more convenient and more reliable in operation. The risk of exogenous pollution in the drug development and production process is greatly reduced, and meanwhile, accurate configuration and incubation of a transfection reagent and plasmid compound are solved, so that the compounding effect of the transfection reagent and plasmids is better and more uniform; and through the clarification filter, the turbidity of the feed liquid is reduced, the purity of the sample is improved, the production efficiency and the product quality are improved, the continuous production of the stable sample is facilitated, and the next purification production is facilitated.

Description

Technical Field

[0001] The utility model relates to the field of biotechnology, in particular to a cell factory device with functions from virus packaging to harvesting. Background Art

[0002] Small-scale production for R&D purposes involves producing adherent cells using culture dishes, flasks, or cell factories. When transitioning to clinical trials, large quantities of lentiviral vectors are required, and cell factories are often necessary.

[0003] A cell factory is a sophisticated cell culture device with a multi-layer structure. It maximizes the culture surface area within a limited space, saving significant plant space and enabling expanded production capacity without requiring any plant modifications. Common cell factory models include 2-layer, 4-layer, 5-layer, 10-layer, and 40-layer models. The 10-layer cell factory, abbreviated as CF10, is the predominant culture device used in the industry.

[0004] The current general method for using CF10 is to first pour the prepared culture medium into the cell factory, tighten the sealing cap, and then place the cell factory on its side with the sealing cap tube facing down. After standing, the culture medium is evenly distributed to each layer to balance the liquid level. The CF10 is then slowly lowered to a horizontal position and placed in a CO2 incubator for incubation. When using CF10 for large-scale batch production, although multiple CF10s can be operated simultaneously to achieve the desired results, greatly improving production efficiency, the open operation increases the risk of exogenous contamination during liquid transfer. In addition, because the binding of transfection reagents and plasmids is significantly affected by temperature, it can lead to poor sample uniformity. Utility Model Content

[0005] The purpose of the utility model is to provide a cell factory device with functions from virus packaging to harvesting. The cell factory parallel closed system of the present application has high sealing performance, is not easily contaminated by external sources and has high sample addition uniformity.

[0006] This application provides a cell factory device with functions from virus packaging to harvesting:

[0007] A cell factory device capable of processing from virus packaging to harvesting comprises a multi-way valve, at least one sample loading container, a thermal incubation device for transferring heat to the at least one sample loading container, a first drive line connected to the at least one sample loading container, a first container for containing a transfection mixture, a second drive line connected to the first container, and at least two cell factory components connected in parallel to the multi-way valve.

[0008] The first drive pipeline and the second drive pipeline are independently or jointly connected to the multi-way valve; the first drive pipeline and the second drive pipeline are respectively provided with a first clamping member and a second clamping member;

[0009] The at least one loading container includes a first loading part for storing transfection reagents and plasmids and having its temperature stabilized by a thermal incubation device, a second loading part for storing culture medium, and a third loading part for storing seed cells; wherein the first loading part is provided with a third clamping stop, the second loading part is provided with a fourth clamping stop, and the third loading part is provided with a fifth clamping stop.

[0010] By providing a cell factory device with functions from virus packaging to harvesting, closed liquid transfer is achieved. Culture medium or cells can also be directly transferred to the cell factory through connecting pipes under a sterile environment. The system is faster, simpler and more reliable to operate, greatly reducing the risk of exogenous contamination during drug development and production. At the same time, it solves the precise configuration and incubation of transfection complexes. Under the constant temperature conditions of the thermal incubation device, the transfection reagent and plasmid complex better and more uniformly, thereby improving the stability and uniformity of the product.

[0011] Furthermore, the first driving pipeline includes a first peristaltic pump and a first pipeline, and the first peristaltic pump is connected to the multi-way valve through the first pipeline; the second driving pipeline includes a second peristaltic pump and a second pipeline, and the second peristaltic pump is connected to the multi-way valve through the second pipeline.

[0012] Furthermore, the thermal incubation device is a magnetic heating stirrer.

[0013] Furthermore, the cell factory component includes a cell factory and a third pipeline; the cell factory is connected to the multi-way valve through the third pipeline.

[0014] Furthermore, the cell factory is provided with a filter element; the filter element includes a filter adapter cover, a silicone tube and an air filter element; one end of the filter adapter cover is connected to the cell factory, the other end of the filter adapter cover is connected to the silicone tube, and the end of the silicone tube away from the cell factory is connected to the air filter element; the connection part between the air filter element and the silicone tube is provided with an anti-detachment part.

[0015] Through this technical solution, an air filter with a 0.2µm pore size is attached to the right outlet of the cell factory to ensure air exchange between the cell factory and the outside world. The filter is disc-shaped and has a barbed connection in the center to facilitate connection with the silicone tubing.

[0016] Furthermore, the number of the cell factories is two to four; and the third pipeline is a TPE thermoplastic pipe.

[0017] Furthermore, the length and inner diameter of the third pipeline are equal; and the inner diameters of the interfaces connecting the multi-way valve and the cell factory are consistent.

[0018] Through the above technical solution, the problem of cell plating in a closed piping system with uniform density distribution is solved, which can achieve good uniformity of cell inoculation density and volume consistency of added transfection materials in each cell factory, reduce process errors and shorten operation time. Too many cell factories will lead to a decrease in the consistency of the number of cells input into the cell factories; production according to this system can ensure the efficiency of virus packaging and the stability of virus production.

[0019] Furthermore, the cell factory component also includes a sixth clamping member; the sixth clamping member is arranged on the third pipeline.

[0020] Furthermore, the cell factory device with functions from virus packaging to harvesting also includes a clarification filter, which is arranged on the second pipeline and connected to the first container; the clarification filter is a velvet-based pre-filter.

[0021] Furthermore, the pore size of the fleece-based pre-filter is less than 1 micron.

[0022] The above technical solution can effectively intercept cells and their fragments, reduce the turbidity of the collected liquid, and control it below 20NTU, which is helpful for the next purification experiment, such as ultrafiltration or chromatography experiments; secondly, its excellent filtration capacity leads to a significant reduction in the required filtration area, fundamentally reducing the consumption of the filter.

[0023] Furthermore, the cell factory device with functions from virus packaging to harvesting also includes a storage pumping assembly, which includes several storage containers, a fourth pipeline and a third peristaltic pump. The storage container is connected to the first sample adding component through the fourth pipeline; the third peristaltic pump is arranged on the fourth pipeline and connected to the storage container.

[0024] Beneficial effects: The utility model provides a closed system for a cell factory, which realizes the closed transfer of liquids and can also transfer culture medium or cells directly to the cell factory through connecting pipes in a sterile environment. The system is faster, simpler and more reliable to operate, greatly reducing the risk of exogenous contamination in the drug development and production process, and at the same time solves the precise configuration and incubation of the transfection reagent and plasmid complex, so that the complex effect of the transfection reagent and plasmid is better and more uniform; and through the clarification filter, the turbidity of the feed liquid is reduced, the purity of the sample is improved, the production efficiency and product quality are improved, which helps to connect the production stability samples and facilitates the next step of purification production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is a schematic diagram of the connection of a cell factory device from virus packaging to harvesting in the present invention;

[0026] Figure numerals: 1. multi-way valve; 2. sample adding container; 3. hot incubation device; 4. first drive pipeline; 5. second drive pipeline; 6. first container; 7. cell factory assembly; 8. clarification filter; 9. storage material pumping assembly; 21. first sample adding part; 22. second sample adding part; 23. third sample adding part; 41. first clamping part; 42. first peristaltic pump; 43. first pipeline; 51. second clamping part; 52. second peristaltic pump; 53. second pipeline; 71. cell factory; 72. third pipeline; 73. sixth clamping part; 74. filter element; 91. storage container; 92. fourth pipeline; 93. third peristaltic pump; 211. third clamping part; 221. fourth clamping part; 231. fifth clamping part; 741. filter adapter cover; 742. silicone tube; 743. air filter element. DETAILED DESCRIPTION

[0027] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] Example

[0029] A cell factory 71 parallel closed system, as shown in the schematic diagram Figure 1 , including a multi-way valve 1 (using a five-way valve), three sample loading containers 2 (using liquid storage bottles), a thermal incubation device 3 (using a magnetic heating stirrer) for transferring heat to at least one sample loading container 2, a first driving pipeline 4 connected to at least one sample loading container 2, a first container 6 (using a liquid storage bag) for receiving a transfection mixture, a second driving pipeline 5 connected to the first container 6, and at least two cell factory components 7 (four in this embodiment) connected in parallel to the multi-way valve 1, a clarification filter 8 arranged on the second driving pipeline 5 and a storage pumping component 9 connected to at least one sample loading container 2; the clarification filter 8 (using a velvet-based pre-filter with a pore size of 0.45 μm) is used to filter the received transfection mixture flowing into the first container 6, and the storage pumping component 9 is used to store transfection reagents or plasmids.

[0030] The first drive pipeline 4 and the second drive pipeline 5 are independently or jointly communicated with the multi-way valve 1 ; the first drive pipeline 4 and the second drive pipeline 5 are respectively provided with a first clamping member 41 and a second clamping member 51 .

[0031] The sample loading container 2 includes a first sample loading part 21 for storing transfection reagents and plasmids and having its temperature stabilized by a thermal incubation device 3, a second sample loading part 22 for storing culture medium, and a third sample loading part 23 for storing seed cells; wherein, the first sample loading part 21 is provided with a third clamping part 211, the second sample loading part 22 is provided with a fourth clamping part 221, and the third sample loading part 23 is provided with a fifth clamping part 231.

[0032] The first driving pipeline 4 includes a first peristaltic pump 42 and a first pipeline 43, and the first peristaltic pump 42 is connected to the multi-way valve 1 through the first pipeline 43; the second driving pipeline 5 includes a second peristaltic pump 52 and a second pipeline 53, and the second peristaltic pump 52 is connected to the multi-way valve 1 through the second pipeline 53.

[0033] The first pipeline 43 of the second driving pipeline 5 is connected to the first sample adding component 21 , the second sample adding component 22 and the third sample adding component 23 through a four-way valve.

[0034] The thermal incubation device 3 is a magnetic heating stirrer. The thermal incubation device 3 adopts a magnetic heating stirrer. The magnetic heating stirrer is a temperature-controllable magnetic stirring device including a magnetic rotor. The temperature-controllable magnetic stirring device realizes constant temperature control of the transfection reagent and plasmid mixture.

[0035] The cell factory assembly 7 includes a cell factory 71 (using CF10), a third pipeline 72 and a sixth clamping member 73 ; the cell factory 71 is connected to the multi-way valve 1 (using a five-way valve) through the third pipeline 72 .

[0036] The cell factory 71 is provided with a filter element 74; the filter element 74 includes a filter adapter cover 741, a silicone tube 742 and an air filter element 743 (with a pore size of 0.2um); one end of the filter adapter cover 741 is connected to the cell factory 71, and the other end of the filter adapter cover 741 is connected to the silicone tube 742, and the end of the silicone tube 742 away from the cell factory 71 is connected to the air filter element 743; the connection part between the air filter element 743 and the silicone tube 742 is provided with an anti-detachment part (an anti-detachment protrusion is used in this embodiment).

[0037] There are four cell factories 71 ; the third pipeline 72 is a TPE thermoplastic pipe.

[0038] The length and inner diameter of the third pipeline 72 are equal; the inner diameter of the interface of the multi-way valve 1 connected to the cell factory 71 is the same.

[0039] The sixth clamping member 73 is disposed on the third pipeline 72 to control the opening and closing of the third pipeline 72 , thereby controlling the flow of the contents in the third pipeline 72 .

[0040] The clarification filter 8 is disposed on the second pipeline 53 and is connected to the first container 6 .

[0041] The storage pumping assembly 9 includes two storage containers 91 (shaking bottles, respectively containing transfection reagent and plasmid), a fourth pipeline 92 and a third peristaltic pump 93. The storage containers 91 are connected to the first sample adding part 21 through the fourth pipeline 92; the third peristaltic pump 93 is arranged on the fourth pipeline 92 and connected to the storage container 91.

[0042] The first clamping member 41 , the second clamping member 51 , the third clamping member 211 , the fourth clamping member 221 , the fifth clamping member 231 and the sixth clamping member 73 are all plastic clamps used to control the closing and opening of the pipeline.

[0043] The pipelines and connectors involved in the utility model are all disposable quick connector pipelines.

[0044] In the present invention, both the liquid storage bottle and the shaking bottle are provided with air holes, and the air holes are provided with air filter elements 743.

[0045] In other embodiments, the storage container 91 may not be provided, and the measured transfection reagent and plasmid may be directly added into the storage container 91 .

[0046] The specific implementation operations are as follows:

[0047] Seed cell inoculation:

[0048] Open the valve of the four-way valve and the fifth clamping piece 231, ensure that the third sample adding piece 23 (using a liquid storage bottle) storing the seed cells is in the open state, the second sample adding piece 22 (using a liquid storage bottle) storing the culture medium and the first sample adding piece 21 (using a liquid storage bottle) storing the transfection reagent and plasmid are in the closed state, the first clamping piece 41 is opened, the second clamping piece 51 is clamped, the valve of the multi-way valve 1 (using a five-way valve) and the sixth clamping piece 73 are fully opened, start the first peristaltic pump 42, and add the seed cell suspension in the third sample adding piece 23 to the cell factory 71.

[0049] After the sample addition is completed, the first peristaltic pump 42 is paused to clamp the fifth clamping member 231 , the fourth clamping member 221 is opened, and the first peristaltic pump 42 is started to add the cell culture medium in the second sample addition member 22 to the cell factory 71 .

[0050] After sample addition is complete, the first peristaltic pump 42 is paused and the fourth clamp 221 is closed. A tube sealer is used to cut the third tube 72 connected to the cell factory 71 and heat-seal the end. The cell suspension in the cell factory 71 is then mixed and placed in a CO2 incubator for continued culture until the cells reach a certain confluence or density.

[0051] The closed system and cultivation process of multiple cell factories 71 in parallel solves the problem of cell plating in a closed piping system, achieves uniform density distribution, ensures that the number of cells input into each cell factory 71 is consistent, reduces process errors and shortens operation time.

[0052] Add transfection reagent and plasmid:

[0053] The transfection reagent and plasmid are placed in the storage container 91 (using a shaking bottle) respectively, and the third peristaltic pump 93 is started to transport the transfection reagent and plasmid to the first sample adding part 21 (liquid storage bottle) respectively. The magnetic rotor stirring and constant temperature heating incubation of the thermal incubation device 3 ensure sufficient and efficient polymerization of the nucleic acid and transfection reagent complex, thereby improving the uniformity, stability and quality of the product.

[0054] Transfection experiments:

[0055] Replace the new third pipeline 72 and reconnect the cell factory 71 after cultivation in the carbon dioxide incubator with the multi-way valve 1 (using a five-way valve); open the third clamp 211, ensure that the first sample adding part 21 (liquid storage bottle) storing the transfection reagent and plasmid is in the open state, the second sample adding part 22 storing the culture medium and the third sample adding part 23 storing the seed cells are in the closed state, the valve of the multi-way valve 1 (using a five-way valve) and the sixth clamp 73 are fully opened, open the first clamp 41 and start the first peristaltic pump 42, and add the transfection reagent and plasmid mixed suspension in the first sample adding part 21 to the cell factory 71. After the addition is completed, pause the first peristaltic pump 42 and close the third clamp 211. Use a tube sealing machine to cut off the third pipeline 72 connected to the cell factory 71 and heat seal the end, then mix the cell suspension in the cell factory 71, place it in a carbon dioxide incubator, and continue to culture until toxin production.

[0056] Most current cell factory 71 systems can only achieve closed packaging and culture processes, and are unable to perform fully closed, semi-automatic production starting from the mixed incubation of plasmids and transfection reagents.

[0057] The system of the present application realizes fully enclosed aseptic operation from the incubation of nucleic acids and transfection reagents to the transfer to various cell engineering processes, controllable incubation temperature, and precise control of the configuration of the transfection complex.

[0058] Medium exchange or harvest:

[0059] Close the first clamp 41, the third clamp 211, the fourth clamp 221, and the fifth clamp 231, replace the third pipeline 72, open the second clamp 51 and the multi-way valve 1 (using a five-way valve) to ensure that the pipeline between the cell factory 71 and the first container 6 is connected. Start the second peristaltic pump 52 to transfer the culture medium in each cell factory 71 to the first container 6 (liquid storage bag). When the harvest is complete, close the second peristaltic pump 52 and the second clamp 51 to achieve a sterile, sealed, and single-use harvest. If you need to culture and harvest again, simply repeat the above process to continue to produce toxins.

[0060] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A cell factory device with functions from virus packaging to harvesting, characterized in that: include A multi-way valve (1), at least one sample loading container (2), a heat incubation device (3) for transferring heat to the at least one sample loading container (2), a first drive pipeline (4) in communication with the at least one sample loading container (2), a first container (6) for receiving a transfection mixture, a second drive pipeline (5) in communication with the first container (6), and at least two cell factory components (7) connected in parallel to the multi-way valve (1); The first drive pipeline (4) and the second drive pipeline (5) are independently or jointly connected to the multi-way valve (1); the first drive pipeline (4) and the second drive pipeline (5) are respectively provided with a first clamping member (41) and a second clamping member (51); The at least one sample loading container (2) comprises a first sample loading part (21) for storing transfection reagents and plasmids and having its temperature stabilized by a thermal incubation device (3), a second sample loading part (22) for storing culture medium, and a third sample loading part (23) for storing seed cells; wherein the first sample loading part (21) is provided with a third clamping part (211), the second sample loading part (22) is provided with a fourth clamping part (221), and the third sample loading part (23) is provided with a fifth clamping part (231).

2. A cell factory device with functions from virus packaging to harvesting according to claim 1, characterized in that: The first drive pipeline (4) includes a first peristaltic pump (42) and a first pipeline (43), and the first peristaltic pump (42) is connected to the multi-way valve (1) through the first pipeline (43); the second drive pipeline (5) includes a second peristaltic pump (52) and a second pipeline (53), and the second peristaltic pump (52) is connected to the multi-way valve (1) through the second pipeline (53).

3. The cell factory device according to claim 1, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The thermal incubation device (3) is a magnetic heating stirrer.

4. The cell factory device according to claim 1, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The cell factory component (7) comprises a cell factory (71) and a third pipeline (72); the cell factory (71) is connected to the multi-way valve (1) via the third pipeline (72).

5. The cell factory device according to claim 4, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The cell factory (71) is provided with a filter element (74); the filter element (74) comprises a filter adapter cover (741), a silicone tube (742) and an air filter element (743); one end of the filter adapter cover (741) is connected to the cell factory (71), the other end of the filter adapter cover (741) is connected to the silicone tube (742), and the end of the silicone tube (742) away from the cell factory (71) is connected to the air filter element (743); the connection portion between the air filter element (743) and the silicone tube (742) is provided with an anti-detachment portion.

6. The cell factory device according to claim 4, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The number of the cell factories (71) is two to four; the third pipeline (72) is a TPE thermoplastic pipe.

7. The cell factory device according to claim 4, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The length and inner diameter of the third pipeline (72) are equal; the inner diameters of the interfaces connecting the multi-way valve (1) and the cell factory (71) are consistent.

8. The cell factory device according to claim 4, which is capable of performing processes from virus packaging to harvesting, is characterized in that: The cell factory component (7) further comprises a sixth clamping member (73); the sixth clamping member (73) is arranged on the third pipeline (72).

9. The cell factory device according to claim 2, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The cell factory device with functions from virus packaging to harvesting further comprises a clarification filter (8), wherein the clarification filter (8) is arranged on the second pipeline (53) and is connected to the first container (6); the clarification filter (8) is a velvet-based pre-filter.

10. The cell factory device according to claim 1, which is capable of performing operations from virus packaging to harvesting, is characterized in that: The cell factory device with functions from virus packaging to harvesting further comprises a storage pumping assembly (9), wherein the storage pumping assembly (9) comprises a plurality of storage containers (91), a fourth pipeline (92) and a third peristaltic pump (93), wherein the storage container (91) is connected to the first sample adding component (21) via the fourth pipeline (92); and the third peristaltic pump (93) is arranged on the fourth pipeline (92) and is connected to the storage container (91).

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