Fully automated suspension cell culture bag kit

Abstract

The present invention provides a fully automated suspension cell culture bag kit, which includes a primary cell storage bag, an initial cell activation bag, a waste liquid bag, and a cell culture bag. Each bag is connected via a three-way connector and multiple tubing. With the cell culture bag kit of this invention, in the suspension cell culture process, there is no need to manually transfer cells from the flask into the bag, significantly reducing the risk of contamination in the cell culture process.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims priority to Chinese Patent Applications No. 2023218446223, No. 2023108631538, No. 202310860536X, No. 202310864234X, No. 2023218491534, No. 2023218446257, No. 2023218491708, filed on July 14, 2023, and Chinese Patent Application No. 2024211814534, filed on May 28, 2024. The disclosure content thereof is incorporated herein by reference and is equivalent to that fully described herein.

[0002] The present invention relates to the field of cell culture, particularly to a culture bag kit for culturing suspension cells, and more specifically to a bag body structure with different uses and related connection methods.

Background Art

[0003] Conventional culture of suspension cells (such as NK cells, 293, CHO, K562) is mainly carried out by using a cell culture bag in addition to a cell culture flask. By adding initial cells / inoculum and the corresponding medium into the cell culture flask or the cell culture bag, a cell culture environment is formed, and growth and propagation are maintained under an appropriate environment. Currently, when the initial amount of cells is small, the cells are cultured using a cell culture flask. After the number of cells is sufficient, they are transferred into a new culture bag and continue to be cultured until the required number of cells is amplified. This culture method is mainly carried out manually and requires multiple liquid and medium transfer operations. The operation is cumbersome, and the risk of bacterial infection is likely to increase.

[0004] Simultaneously, in the cell culture process, it is necessary to add culture medium from a flask or bag to a culture bag. Currently, male and female Luer screw connectors are used to connect different bags, but this can sometimes cause entanglement of the tubing. Therefore, a safer and more reliable connection method needs to be designed to improve operational efficiency and safety.

[0005] Existing tubes connecting different bags rely on screw connections. After prolonged connection, a certain rotational force is present in the tube, which can lead to detachment or leakage at the screw connection point, potentially causing contamination and affecting actual cell culture results. [Overview of the project] [Problems that the invention aims to solve]

[0006] In view of the technical problems raised in the background art, the present invention provides a fully automated suspension cell culture bag kit to solve at least one of the technical problems mentioned in the background art. [Means for solving the problem]

[0007] To achieve the above objective, the present invention provides the following technical solutions. The present invention relates to a fully automated suspension cell activation culture kit, and the cell culture bag kit includes: Used for preserving primary cells, the primary cell preservation bag includes a primary cell preservation bag body, an injection port, and an outlet. Used to activate early suspension cells, the early cell activation bag includes an early cell activation bag body, a coating solution inlet, a culture medium and cell inlet, and a liquid outlet. Used to collect waste liquid from an initial cell activation bag, the waste liquid bag includes the waste liquid bag body and an inlet, Used for large-volume cell culture, the cell culture bag includes a cell culture bag body, an inlet, and a sampling port. Here, the outlet of the primary cell storage bag is connected via a tube to the culture medium and cell inlet of the initial cell activation bag, and the liquid outlet of the initial cell activation bag is connected via a tube to the waste liquid bag inlet and the cell culture bag inlet, respectively.

[0008] A more preferable technical option is to use a heparin sodium cap as at least one of the injection port of the primary cell preservation bag or the injection port of the initial cell activation bag coating solution.

[0009] A more preferred technical option is for the cell culture bag to further include a cell density measurement port, the cell density measurement port being made of a light-transmitting material, and the cell density being measured by the absorbance of the liquid inside the culture bag.

[0010] A further preferred technical proposal includes a four-way connector, in which three connectors are each connected to three culture medium inflow tubes, and the fourth connector of the four-way connector is connected via a tube to the culture medium and cell inlet of an initial cell activation bag.

[0011] A further preferred technical proposal includes a three-way connector A, which is used to connect a tube connected to the outlet of the primary cell storage bag, a tube connected to the fourth connector of the four-way connector, and a culture medium and cell inlet tube of the initial cell activation bag.

[0012] A further preferred technical proposal includes a three-way connector B, which is used to connect to the liquid outlet tube of the initial cell activation bag, the waste liquid bag inlet tube, and the cell culture bag inlet tube.

[0013] A more preferred technical option is to connect at least one of the following to a tube using a Luer connector: the outlet of the primary cell storage bag, the culture medium and cell inlet and liquid outlet of the initial cell activation bag, and the cell culture bag inlet.

[0014] A more preferable technical option is for the tube to be made of silicone.

[0015] A more preferable technical proposal is that at least one of the aforementioned primary cell storage bags, initial cell activation bags, waste liquid bags, and cell culture bags is made of a waterproof and breathable FEP film.

[0016] A more preferable technical option is for the cell culture bag to have a volume of 2L or more.

[0017] A further preferred technical proposal is that the cell density measurement port is made of FEP material and includes an upper light-transmitting hole and a lower light-transmitting hole that are opposite each other, and the upper light-transmitting hole and the lower light-transmitting hole are blind holes.

[0018] A more preferable technical option is that at least one of the primary cell storage bag, initial cell activation bag, and cell culture bag has rounded corners.

[0019] In a more preferred technical configuration, the initial cell activation bag is convex in shape, with a narrow upper end and a wide lower end, and all corners protruding outwards from the lumen of the convex initial cell activation bag are rounded, with the coating solution inlet, culture medium and cell inlet, and liquid outlet located at the upper end of the convex shape.

[0020] A further preferred technical proposal includes a quick connector, the quick connector comprising a male Luer connector and a female Luer connector that mates with the male Luer connector, where, The male Luer connector includes a first male mating portion, a first engaging plate, a first gripping portion, a first tube connector, and a first flow path passing through the first male mating portion, the first engaging plate, the first gripping portion, and the first tube connector, and the first tube connector is used to connect to a tube. The female Luer connector includes a first female mating portion, a first engaging portion, and a second tube connector, the first female mating portion being provided with a first cavity into which a first male mating portion is inserted, the second tube connector being provided with a second flow path, and further including a quick connector, the quick connector including a male Luer connector and a female Luer connector that mates and connects with the male Luer connector, where, The male Luer connector includes a first male mating portion, a first engaging plate, a first gripping portion, a first tube connector, and a first flow path passing through the first male mating portion, the first engaging plate, the first gripping portion, and the first tube connector, and the first tube connector is used to connect to a tube. The female Luer connector includes a first female mating portion, a first engaging portion, and a second tube connector, wherein the first female mating portion is provided with a first cavity into which a first male mating portion is inserted, the second tube connector is provided with a second flow path, the second flow path communicates with the first cavity, and the second tube connector is used to connect to a tube. The first engaging plate and the first engaging portion can be engaged and connected to fix the male Luer connector and the female Luer connector, and to connect the first flow path and the second flow path. The quick connector is installed at the end of the tube between the four-way connector and the three-way connector A, and / or the tube between the three-way connector B and the cell culture bag inlet, and / or the culture medium inlet tube.

[0021] In a further preferred technical proposal, the first engaging portion is a first engaging block having at least two L-shaped cross-sections provided on the end face of the first female mating portion, the first engaging plate is provided with at least two first notches corresponding to the positions of the first engaging block, the first engaging block can pass through the first notches and, after rotating by a predetermined angle, engage with the first engaging plate, thereby engaging and connecting the male Luer connector and the female Luer connector.

[0022] As a more preferred technical solution, at least one first annular groove is provided on the outer periphery of the first male fitting portion, and a first sealing ring is provided in the first annular groove. The first sealing ring is in pressure contact and fitting with the inner side wall of the first cavity of the first female fitting portion. And / or, a first sealing ring is provided on the end surface of the first male fitting portion facing the female Luer connector. The first sealing ring can be in pressure contact between the bottom wall of the first cavity of the female Luer connector and the end surface of the first male fitting portion.

[0023] As a more preferred technical solution, it further includes a male Luer plug that is fitted and connected to the female Luer connector. The male Luer plug includes a second male fitting portion, a second engaging plate, and a second gripping portion. At least two second cutouts corresponding to the position of the first engaging block are provided on the second engaging plate. The first engaging block passes through beyond the second cutout and can be engaged with the second engaging plate after rotating a predetermined angle, thereby engaging and connecting the male Luer plug and the female Luer connector.

[0024] As a more preferred technical solution, at least one second annular groove is provided on the outer periphery of the second male fitting portion, and a second sealing ring is provided in the second annular groove. The second sealing ring is in pressure contact and fitting with the inner side wall of the first cavity of the first female fitting portion. And / or, a second sealing ring is provided on the end surface of the second male fitting portion facing the female Luer connector. The second sealing ring can be in pressure contact between the bottom wall of the first cavity of the female Luer connector and the end surface of the second male fitting portion.

[0025] As a more preferred technical solution, it further includes a female Luer plug that is fitted and connected to the male Luer connector. The male Luer plug includes a second female fitting portion and a second engaging portion. The second engaging portion is at least two second engaging blocks with an L-shaped cross-section provided on the end surface of the second female fitting portion. The second engaging block passes through beyond the first cutout and can be engaged with the first engaging plate after rotating a predetermined angle, thereby engaging and connecting the male Luer plug and the male Luer connector.

[0026] A further preferred technical example is a first engagement plate between adjacent first notches, the surface facing the first gripping portion having a thickened wedge-shaped or trapezoidal inclined surface. And / or, the surface of the second engagement plate between adjacent second notches that faces the second gripping portion has a thickened wedge-shaped or trapezoidal bevel. [Effects of the Invention]

[0027] The present invention solves the drawback of existing suspension cell cultures, which require multiple manual transfers of liquid, and provides a fully automated suspension cell activation culture kit system. It guarantees a sterile environment for cell culture, and, when combined with other equipment, allows for observation of cell density without sampling, thus overcoming the risk of cross-contamination of cells during artificial manipulation processes.

[0028] Furthermore, by using the quick connector of the present invention, when connecting two tubes, the male Luer connector and the female Luer connector are quickly engaged using the first engaging plate and first engaging part, thereby achieving communication between the tubes. This allows for connection of the tubes with only a slight rotation angle, preventing entanglement of the tubes. The rotational engagement structure makes the connection faster and more robust, improving the efficiency of liquid transfer. Connecting two different types of bag bodies using this quick engagement method ensures a stable and tight connection of the tubes. Also, when liquid transfer is not required, after separating the male Luer connector and the female Luer connector, the male Luer plug is quickly plugged into the female Luer connector to seal the tube on the female Luer connector side, preventing liquid leakage from the tube on the female Luer connector side. The male Luer plug is then quickly plugged into the male Luer connector to seal the tube on the male Luer connector side, preventing liquid leakage from the tube on the male Luer connector side. The structure is simpler and easier to operate, significantly improving the effectiveness of tube connection while simultaneously reducing the risk of contamination. [Brief explanation of the drawing]

[0029] [Figure 1]This is a schematic diagram of the structure of a fully automated suspension cell culture bag kit according to one embodiment of the present invention. [Figure 2] This is a schematic diagram of the structure of one side of the cell density measurement port in the present invention. [Figure 3] This is a schematic diagram of the structure of the other side of the cell density measurement port in the present invention. [Figure 4] This is a top view of the cell density measurement port in the present invention. [Figure 5] This is a cross-sectional view along line AA in Figure 4. [Figure 6] This is a schematic diagram of the structure of a fully automated suspension cell culture bag kit according to another embodiment of the present invention. [Figure 7] This is a schematic diagram of the structure of the male Luer connector of the quick connector used in the present invention. [Figure 8] This is a schematic diagram of the structure of the female Luer connector of the quick connector used in the present invention. [Figure 9] This is a schematic diagram of the structure after connecting the male Luer connector and female Luer connector of the quick connector used in the present invention. [Figure 10] This is a schematic diagram of the structure of the male luer plug of the quick connector used in the present invention. [Figure 11] This is a schematic diagram of the structure after connecting the female Luer connector and male Luer plug of the quick connector used in the present invention. [Figure 12] This is a schematic diagram of the structure of the male luer plug of the quick connector used in the present invention. [Figure 13] This is a schematic diagram of the structure after connecting the male Luer connector and male Luer plug of the quick connector used in the present invention. [Modes for carrying out the invention]

[0030] The present invention will be described in detail below, with reference to the drawings and examples, to further clarify the purpose, technical proposal and advantages of the present invention, and to provide a detailed description of the features and exemplary embodiments of various aspects of the present invention. It should be understood that the specific embodiments described herein are configured solely to interpret the patent of the present invention and not to limit it. For those skilled in the art, the present invention can be implemented even if some of these specific details are omitted. The following description of embodiments is intended to provide a better understanding of the present invention by illustrating examples of the present invention.

[0031] In this specification, the terms "1," "2," etc., are used merely to distinguish one entity or operation from another, and do not necessarily require or imply that there is an actual relationship or order between these entities or operations. Furthermore, the terms "includes," "contains," or any variation thereof are intended to be non-exclusive, so that a process, method, article, or apparatus containing a set of elements includes not only those elements but also other elements not explicitly listed, or further elements specific to such process, method, article, or apparatus. Unless further limited, the elements limited by the statement "includes" do not preclude the existence of other identical elements within a process, method, article, or apparatus containing such elements.

[0032] Example 1 Referring to Figure 1, the fully automated suspension cell culture bag kit of the present invention includes a primary cell storage bag 1, an initial cell activation bag 2, a waste liquid bag 3, and a cell culture bag 4. Preferably, the bag bodies of the primary cell storage bag 1, initial cell activation bag 2, waste liquid bag 3, and cell culture bag 4 are made of a waterproof and breathable FEP film. The waterproof and breathable FEP film has stable chemical properties, is non-cytotoxic and non-tissue toxic, is beneficial for cell proliferation, and is a transparent material, making microscopic observation easy.

[0033] Here, the primary cell storage bag 1 is used to store primary cells, and its volume is preferably 20 ml. The primary cell storage bag 1 includes a primary cell storage bag body 101, an injection port 102, and an outlet port 103. The initial cell activation bag 2 is used to activate initial suspension cells, and the initial cell activation bag 2 includes an initial cell activation bag body 201, a coating solution inlet 202, a culture medium and cell inlet 203, and a liquid outlet port 204. The waste liquid bag 3 is used to collect waste liquid from the initial cell activation bag, and the waste liquid bag 3 includes a waste liquid bag body 301 and an inlet 302. The cell culture bag 4 is used to perform large-volume cell culture, and the volume of the cell culture bag 4 is preferably 2 L or more. The cell culture bag 4 includes a cell culture bag body 401, an inlet 402, and a sampling port 403.

[0034] The primary cell storage bag 1, initial cell activation bag 2, waste liquid bag 3, and cell culture bag 4 are connected via a three-way connector A 601, a three-way connector B 602, and a plurality of tubes 501 to 507, the tubes 501 to 507 preferably being made of silicone tubing.

[0035] Specifically, the outlet 103 of the primary cell storage bag 1 is connected to the culture medium and cell inlet 203 of the initial cell activation bag 2 via tubes 501 and 502, and the liquid outlet 204 of the initial cell activation bag 2 is connected to the inlet 302 of the waste liquid bag 3 and the cell culture bag inlet 402 via tubes 503, 504 and 505, respectively.

[0036] Here, tubes 501 and 502 are connected via a three-way connector A601, and tubes 503, 504, and 505 are connected via a three-way connector B602.

[0037] Furthermore, heparin sodium caps are used as the injection port 102 of the primary cell preservation bag 1 and the coating solution injection port 202 of the initial cell activation bag 2. The injection port is a channel for manually adding primary cells into the bag, reducing contact between the inside of the bag and the external environment and mitigating the risk of contamination. The injection port is a channel for manually adding the coating solution, allowing the liquid to be added directly into the bag, preventing loss of the coating solution within the tube, which is advantageous for more effective coating of the bag material and improves the initial cell activation effect.

[0038] Furthermore, the cell culture bag 4 further includes a cell density measurement port 404, which is made of a light-transmitting material, and the cell density can be measured by the absorbance of the liquid inside the culture bag.

[0039] Specifically, as shown in Figure 2, the cell density measurement port 404 includes a connector 4041 and a measurement port 4042, where the connector 4041 is used to seal and connect to the cell culture bag 4, and more preferably, the cell density measurement port 404 is made of FEP material.

[0040] Preferably, the connecting portion 4041 is rhomboid in shape, has a through hole 40411 in the center, and communicates with the cavity 40421 of the measuring portion 4042.

[0041] More preferably, the measuring section 4042 includes a bottom wall, an upper wall, a lower wall, and an arc-shaped side wall connecting the upper wall and the lower wall, and preferably the upper wall and the lower wall are flat. Preferably, the upper wall and the lower wall are provided with an upper light-transmitting hole 40431 and a lower light-transmitting hole 40432 that are opposite to each other in the vertical direction, and both the upper light-transmitting hole 40431 and the lower light-transmitting hole 40432 are blind holes, that is, recessed grooves provided on the surfaces of the upper wall and the lower wall, and do not penetrate to the cavity 40421. More preferably, the diameter of the upper light-transmitting hole 40431 and the lower light-transmitting hole 40432 is 2 to 4 mm, more preferably 3 mm, and the thickness of the light-transmitting hole is 0.1 to 0.3 mm, more preferably 0.2 mm.

[0042] The cell density measurement port 404 of the FEP material of the present invention has the advantage of having better light transmission and reproducibility compared to general plastic materials. The light transmission intensity of the cell density measurement port was measured multiple times for the same culture sample, and the measurement results of cell density measurement ports of different materials are shown in the following comparison table.

[0043] [Table 1]

[0044] Measuring cell density using a non-contact method that utilizes the absorbance of the culture medium at the cell density measurement port for the same culture sample at different culture durations yielded virtually no difference in results compared to the conventional method of first removing the culture medium and then measuring cell density using a flow cytometer. Details are shown in the comparison table below.

[0045] [Table 2]

[0046] Furthermore, the cell culture bag kit further includes a four-way connector 701, three of which are connected to three culture medium inflow tubes 506, and the fourth connector of the four-way connector is connected to the culture medium and cell inlet 203 of the initial cell activation bag 2 via tubes 507, 502 and a three-way connector A 601.

[0047] Furthermore, the three-way connector A 601 is connected to tube 501 connected to the outlet 103 of the primary cell storage bag 1, tube 507 connected to the fourth connector of the four-way connector 701, and tube 502 connected to the culture medium and cell inlet of the initial cell activation bag 2.

[0048] The three-way connector B 602 described above is connected to tube 503, which is connected to the liquid outlet of the initial cell activation bag 2, tube 504, which is connected to the waste liquid bag inlet, and tube 505, which is connected to the cell culture bag inlet.

[0049] Furthermore, the outlet 103 of the primary cell storage bag, the culture medium and cell inlet 203 of the initial cell activation bag, the liquid outlet 204, and the cell culture bag inlet 402 are connected to tubes using Luer connectors.

[0050] Example 2 Referring to Figure 6, this is a fully automated suspension cell culture bag kit according to another embodiment of the present invention. Components identical to those in Example 1 are given the same names and reference numerals, and the description of duplicate components is omitted. The following will focus on the differences from Example 1 and will be explained in detail.

[0051] Specifically, compared to Example 1, the initial cell activation bag 2 uses a special shape, with rounded corners at the edges of the lumen of the initial cell activation bag 2 to prevent the occurrence of dead space. More preferably, the initial cell activation bag 2 is convex (or gourd-shaped), with a narrow upper end and a wide lower end, as shown in Figure 6. Preferably, the convex initial cell activation bag body 201 is formed by heat welding on a single rectangular or square bag body, with the center of the bag body 201 being a cavity for containing initial cells; more preferably, all corners protruding outward from the lumen of the convex initial cell activation bag are rounded, and the two corners protruding inward may be right angles or rounded. The upper end of the convex shape of the initial cell activation bag 2 is provided with a coating solution inlet 202, a culture medium and cell inlet 203, and a liquid outlet 204.

[0052] In this embodiment, the convex-shaped initial cell activation bag 2 has the following advantages compared to the rectangular bag body of Example 1: 1. When the connection ports for the coating solution inlet 202, culture medium and cell inlet 203, and liquid outlet 204 are welded to the rectangular bag body, severe wrinkles occur throughout the bag body, and this worsens after sterilization. On the other hand, an essential condition for coating activation is to keep the cell inoculation interface flat. Otherwise, the wrinkles in the bag body cause cell aggregation, inhibiting the inoculation of cells and cytokines at the bottom, and affecting the activation level. Therefore, in order to ensure the flatness of the bottom of the bag body, it is designed in a convex shape with a narrow upper end and a wide lower end, and even after welding each connection port to the upper end, no wrinkles occur at the lower end. 2. When transferring the cell mixture in the rectangular bag body, the liquid is not completely transferred to the cell culture bag, and there is a tendency for excess liquid to remain. By changing the rectangle to the convex shape of Example 2, this problem can be effectively solved.

[0053] Using the same fresh peripheral blood sample, the rectangular bag from Example 1 and the convex-shaped bag from Example 2 were cultured under the same conditions for the same number of days. Comparison of various parameters confirmed that the convex-shaped bag was superior to the rectangular bag. For detailed results, please refer to the comparison table below.

[0054] [Table 3]

[0055] More preferably, based on the same principle as described above, all corners of the primary cell storage bag 1 and cell culture bag 4 are also designed to be rounded in order to prevent wrinkle formation in the bag body and cell aggregation or retention due to the appearance of dead space.

[0056] In a more preferred embodiment, a quick connector 8 is added in the middle of tubes 505 and 507 (as shown in Figures 7-13), and this quick connector 8 includes a male Luer connector 81 and a female Luer connector 82. Here, Figure 7 is a schematic diagram of the structure of the male Luer connector 81, Figure 8 is a schematic diagram of the structure of the female Luer connector 82, and Figure 9 shows the male Luer connector 81 and the female Luer connector 82 in a mated connection state.

[0057] Specifically, referring to Figure 7, the male Luer connector 81 includes a first male mating portion 811, a first engaging plate 812, a first gripping portion 813, and a first tube connector 814, where the first tube connector 814 is used to connect tubes 505 together or tubes 507 together, and the first male mating portion 811 is used to insert into the female mating portion of the female Luer connector 82 and female Luer plug 84, which will be described later, and to connect them. A first flow path 815 is provided that passes through the central axis of the first male mating portion 811, the first engaging plate 812, the first gripping portion 813, and the first tube connector 814 in order to communicate with the connected tubes 505 or 507.

[0058] The first engaging plate 812 is disc-shaped, and at least two first notches 8121 are provided on the edge of the disc. Preferably, the two first notches 8121 are spaced apart, and more preferably, the two first notches 8121 are arranged on the edge of the disc in a mirror-image symmetrical manner with respect to the center of the disc. Even more preferably, if there are three or more first notches 8121, they are arranged at equal intervals on the circumferential edge of the disc.

[0059] Preferably, the first tube connector 814 is pagoda-shaped to facilitate a sealed connection with tube 505 or 507.

[0060] Referring to Figure 8, the female Luer connector 82 includes a first female mating portion 821, a first engaging portion 822, and a second tube connector 823. The first female mating portion 821 is provided with a first cavity 824 into which the first male mating portion 811 is inserted, and the second tube connector 823 is provided with a second flow path 825, which communicates with the first cavity 824. The second tube connector 823 is used to connect to the other tube 505 or 507.

[0061] Preferably, the first cavity 824 of the first female mating portion 821 is cylindrical, with one end of the first cavity 824 connected to the second tube connector 823 and the other end open.

[0062] The first engaging portion 822 of the female Luer connector 82 is provided on the end face 8211 of the first female mating portion 821, which is away from the second tube connector 823, and this end face 8211 is the end face facing outward from the cavity wall at one end of the opening of the cylindrical first cavity 824.

[0063] Furthermore, the first engaging portion 822 is a first engaging block 8221 having at least two L-shaped cross-sections, provided on the end face of the first female fitting portion 821. Preferably, the two first engaging blocks 8221 are spaced apart, and more preferably, the two first engaging blocks 8221 are arranged on the end face 8211 of the first female fitting portion 8211 in a mirror-image symmetrical manner with respect to the circular center of the end face. Even more preferably, if there are three or more first engaging blocks 8221, they are arranged at equal intervals on the end face 8211.

[0064] Preferably, the second tube connector 823 is pagoda-shaped to facilitate a sealed connection with tube 505 or 507.

[0065] As shown in Figure 9, the first engagement plate 812 of the male Luer connector 81 and the first engagement portion 822 of the female Luer connector 82 are connected by engaging the first notch 8121 and the first engagement block 8221 with each other, thereby fixing the male Luer connector 81 and the female Luer connector 82 together, and also connecting the first flow path 815 and the second flow path 825.

[0066] Specifically, taking two first notches 8121 and two first engagement blocks 8221 as an example, when connecting a male Luer connector 81 and a female Luer connector 82, first the first male mating portion 811 of the male Luer connector 81 is inserted into the first female mating portion 821 of the female Luer connector 82, and the first engagement block 8221 of the female Luer connector 82 is aligned with the first notch 8121 of the male Luer connector 81. Once the first engagement block 8221 passes beyond the first notch 8121 along the axial direction of the connector, the male Luer connector and / or female Luer connector are rotated by a predetermined angle, after which the first engagement block 8221 engages with the position on the first engagement plate 812 where the first notch 8121 is not provided, thereby engaging and connecting the male Luer connector 81 and the female Luer connector 82 (see Figure 9). This connection structure allows for quick connection, enabling rapid connection and locking between the male Luer connector 81 and the female Luer connector 82 without the need for large rotation angles.

[0067] More preferably, the outer circumference of the first male mating portion 811 is provided with at least one first annular groove 8111, and a first seal ring (not shown) is provided in the first annular groove 8111, preferably, when installed in the first annular groove 8111, the first seal ring protrudes from the outer surface of the first male mating portion 811 and fits tightly into the inner wall of the first cavity 824 of the first female mating portion 821, thereby achieving a sealed connection between the male Luer connector 81 and the female Luer connector 82. Preferably, the first seal ring is a silicone rubber ring.

[0068] As an optional embodiment, the first seal ring may be positioned on the end face of the first male mating portion 811 facing the female Luer connector 82. This allows the first seal ring to be pressed against the bottom wall of the first cavity 824 of the female Luer connector 82 and the end face of the first male mating portion 811 when connecting the male Luer connector 81 and the female Luer connector 82, thereby achieving a sealed connection between the male Luer connector 81 and the female Luer connector 82. More preferably, the first seal ring is installed in an annular groove provided on the end face of the first male mating portion and protrudes from the end face of the first male mating portion.

[0069] Furthermore, as shown in Figure 10, the quick connector 8 further includes a male luer plug 83 that mates with a female luer connector 82, the male luer plug 83 including a second male mating portion 831, a second engagement plate 832 and a second gripping portion 833, the second engagement plate 832 having at least two second notches 8321 corresponding to the positions of the first engagement block 8221, the first engagement block 8221 passing over the second notches 8321 and, after rotating by a predetermined angle, becoming engaged with the second engagement plate 832, thereby engaging and connecting the male luer plug 83 and the female luer connector 82. This allows the female luer connector 82 to be sealed using the male luer plug 83 after separating the male luer connector 81 and the female luer connector 82 (see Figure 11).

[0070] Preferably, the male luer plug 83 either does not have a flow path inside, or it has a flow path inside that is closed at one end.

[0071] Preferably, the outer circumference of the second male mating portion 831 is provided with at least one second annular groove 8311, and a second seal ring (not shown) is provided in the second annular groove 8311. Preferably, when installed in the second annular groove 8311, the second seal ring protrudes from the outer surface of the second male mating portion 831, and the second seal ring press-fits against the inner wall of the first cavity 824 of the first female mating portion 821.

[0072] As an optional embodiment, the second seal ring may be installed on the end face of the second male mating portion 831 facing the female Luer connector 82. This ensures that when the male Luer plug 83 and the female Luer connector 82 are brought into contact, the second seal ring is pressed against the bottom wall of the first cavity 824 of the female Luer connector 82 and the end face of the second male mating portion 831, thereby achieving a sealed connection between the male Luer plug 83 and the female Luer connector 82 and achieving a sealed enclosure of the female Luer connector 82. More preferably, the second seal ring is positioned in an annular groove provided on the end face of the second male mating portion and protrudes from the end face of the second male mating portion.

[0073] Furthermore, as shown in Figure 12, the quick connectors 8, 8' further include a female luer plug 84 that mates with a male luer connector 81, the female luer plug 84 including a second female mating portion 841 and a second engaging portion 842, the second engaging portion 842 is a second engaging block 8421 provided on the end face 8411 of the second female mating portion 841, having at least two L-shaped cross-sections, the second engaging block 8421 passes over the first notch 8121 and, after rotating by a predetermined angle, is engageable with the first engaging plate 812, thereby engaging the female luer plug 84 and the male luer connector 81 with each other so that the male luer connector 81 can be sealed using the female luer plug 84 (see Figure 13).

[0074] Preferably, the second female fitting portion 841 is cylindrical and has a second cavity 843 inside, with one end of the second cavity 843 closed and the other end open, and the L-shaped second engagement block 8421 is installed on the end face 8411 of the opening end of the second cavity 843 of the second female fitting portion 842.

[0075] More preferably, the surfaces of the first engaging plate 812 and / or second engaging plate 832 of the male Luer connector 81 that engage and contact with the L-shaped first engaging block 8221 and / or L-shaped second engaging block 8421 have a wedge-shaped or trapezoidal bevel, that is, on the surface of the engaging plate facing the gripping portions 813 and 833, the thickness of the engaging plate gradually increases from the first notch 8121 and the second notch 8321 toward the intermediate portion between adjacent notches. More preferably, the thickness is kept constant in the intermediate portion between two adjacent notches, thereby enabling a locking connection by the wedge-shaped or trapezoidal bevel of the engaging plate when the engaging block passes over the notch and rotates by a predetermined angle, and enabling fastening of the male Luer connector 81 and the female Luer connector 82. Furthermore, when combined with an embodiment in which the first and second seal rings are installed on the end face of the male mating portion, the seal rings are pressed against each other simultaneously with fastening, further improving the sealing effect.

[0076] In the above embodiment, when connecting two tubes 505 together or two tubes 507 together, the tube quick connector 8 connects the first tube connector of the male Luer connector 81 to the tube at one end, and the female Luer connector 82 to the tube at the other end. Then, the male Luer connector 81 and the female Luer connector 82 are quickly connected and locked together by the first engaging plate 812 and the first engaging part 822, thereby achieving communication between the tubes 505 and 507. The rotational engagement structure ensures a quick and robust connection, improving the efficiency of liquid transfer. By connecting different bag bodies using this quick engagement method, a stable and tight connection of tubes can be achieved. If liquid transfer is not required, the male Luer connector 81 and the female Luer connector 82 are separated, the male Luer plug 83 is quickly connected to the female Luer connector 82 to seal the tube on the female Luer connector 82 side and prevent liquid leakage from the tube at one end on the female Luer connector 82 side, and the female Luer plug 84 is quickly connected to the male Luer connector 81 to seal the tube on the male Luer connector 81 side and prevent liquid leakage from the tube at one end on the male Luer connector 81 side.

[0077] Furthermore, another quick connector 8' is installed at the end of the tube 506, and the quick connector 8' includes the female Luer connector 82 and a male Luer plug 83 (see Figure 11) that mates with the female Luer connector 82, or includes the male Luer connector 81 and a female Luer plug 84 (see Figure 13) that mates with the male Luer connector 81, thereby enabling rapid sealing of the tube 506.

[0078] In the process of using the cell culture bag kit product according to the present invention, the initial preparation work is completed simply by connecting the culture medium to be used to the inlet, injecting primary cells into the cell culture bag, and injecting the antibody coating solution into the initial cell activation bag. The entire kit of bags is placed in an automated culture workstation, and cell transfer between bags and culture medium replenishment are performed via the interconnection of tubes and bags. This significantly reduces manual steps and improves the overall efficiency of cell culture.

[0079] It will be apparent to those skilled in the art that various modifications and changes can be made to embodiments of the present invention without departing from the spirit and scope of the invention. Accordingly, if such modifications and changes fall within the scope of the claims and equivalents of the present invention, the present invention shall also include such modifications and changes. [Explanation of symbols]

[0080] 1: Primary cell preservation bag 101: Original cell preservation bag body 102: Injection port 103: Exit 2: Initial cell activation bag 201: Initial cell activation bag body 202: Coating liquid inlet 203: Culture medium and cell inlet 204: Liquid outlet 3: Waste liquid bag 301: Waste liquid bag body 302:Inlet 4: Cell culture bag 401: Cell culture bag body 402:Inlet 403: Sampling port 404:Cell density measurement port 4041: Connection part 40411: Through hole 4042:Measurement part 40421: Cavity 40431: Upper light transmission hole 40432: Bottom light transmission hole 501~507: Tube 601: 3-way connector A 602: 3-way connector B 701: 4-way connector 8, 8': Quick connector 81: Male Luer Connector 811: First male mating part 8111: First annular groove 812: First engaging plate 8121: First notch 813: 1st grip part 814: First tube connector 815: First channel 82: Female Luer Connector 821: First female mating section 8211: End face 822: First engaging part 8221: First engagement block 823: Second tube connector 824: First Cavity 825: Second channel 83: Male Luer Plug 831: Second male mating part 8311: Second annular groove 832: Second engaging plate 8321: Second notch 833:Second grip part 84: Female Luer Plug 841: Second female mating section 8411: End face 842: Second engaging part 8421: Second engagement block 843: Second cavity.

Claims

1. This is a fully automated suspension cell culture bag kit, and the cell culture bag kit is Used for preserving primary cells, the primary cell preservation bag includes a primary cell preservation bag body, an injection port, and an outlet. Used to activate early suspension cells, the early cell activation bag includes an early cell activation bag body, a coating solution inlet, a culture medium and cell inlet, and a liquid outlet. Used to collect waste liquid from an initial cell activation bag, the waste liquid bag includes the waste liquid bag body and an inlet, Used for large-volume cell culture, the cell culture bag includes a cell culture bag body, an inlet, and a sampling port. A fully automated suspension cell culture bag kit characterized in that the outlet of the primary cell storage bag is connected via a tube to the culture medium and cell inlet of the initial cell activation bag, and the liquid outlet of the initial cell activation bag is connected via a tube to the waste liquid bag inlet and the cell culture bag inlet, respectively.

2. The fully automated suspension cell culture bag kit according to claim 1, characterized in that a heparin sodium cap is used as at least one of the injection port of the primary cell preservation bag and the initial cell activation bag coating solution injection port.

3. The fully automated suspension cell culture bag kit according to claim 1, wherein the cell culture bag further includes a cell density measurement port, the cell density measurement port is made of a light-transmitting material, and the cell density can be measured by the absorbance of the liquid inside the culture bag.

4. The fully automated suspension cell culture bag kit according to claim 1, further comprising a four-way connector, wherein three connectors in the four-way connector are each connected to three culture medium inflow tubes, and the fourth connector of the four-way connector is connected via a tube to the culture medium and cell inlet of the initial cell activation bag.

5. The fully automated suspension cell culture bag kit according to claim 4, further comprising a three-way connector A, wherein the three-way connector A is used to connect a tube connected to the outlet of the primary cell storage bag, a tube connected to the fourth connector of the four-way connector, and a culture medium and cell inlet tube of the initial cell activation bag.

6. The fully automated suspension cell culture bag kit according to claim 5, further comprising a three-way connector B, the three-way connector B being used to connect to the liquid outlet tube of the initial cell activation bag, the waste liquid bag inlet tube, and the cell culture bag inlet tube.

7. The fully automated suspension cell culture bag kit according to claim 6, characterized in that at least one of the following is connected to a tube using a Luer connector: the outlet of the primary cell storage bag, the culture medium and cell inlet and liquid outlet of the initial cell activation bag, and the cell culture bag inlet.

8. The fully automated suspension cell culture bag kit according to any one of claims 1 to 7, characterized in that the tube is made of a silicone tube.

9. The fully automated suspension cell culture bag kit according to any one of claims 1 to 7, characterized in that at least one of the aforementioned primary cell storage bag, initial cell activation bag, waste liquid bag, and cell culture bag is made of a waterproof and breathable FEP film.

10. The fully automated suspension cell culture bag kit according to any one of claims 1 to 7, characterized in that the volume of the cell culture bag is 2 L or more.

11. The fully automated suspension cell culture bag kit according to claim 3, characterized in that the cell density measurement port is made of FEP material and includes an upper light-transmitting pore and a lower light-transmitting pore that are opposite each other, and the upper light-transmitting pore and the lower light-transmitting pore are blind holes.

12. The fully automated suspension cell culture bag kit according to any one of claims 1 to 7, characterized in that at least one of the primary cell storage bag, initial cell activation bag, and cell culture bag has rounded corners.

13. The fully automated suspension cell culture bag kit according to claim 12, characterized in that the initial cell activation bag has a convex shape with a narrow upper end and a wide lower end, all corners protruding to the outside of the lumen of the convex initial cell activation bag are rounded, and the coating solution inlet, culture medium and cell inlet, and liquid outlet are provided at the upper end of the convex shape.

14. Furthermore, it includes a quick connector, the quick connector including a male Luer connector and a female Luer connector that mates with the male Luer connector, where, The male Luer connector includes a first male mating portion, a first engagement plate, a first gripping portion, a first tube connector, and a first flow path passing through the first male mating portion, the first engagement plate, the first gripping portion, and the first tube connector, and the first tube connector is used to connect to a tube. The female Luer connector includes a first female mating portion, a first engaging portion, and a second tube connector. The first female mating portion is provided with a first cavity into which a first male mating portion is inserted. The second tube connector is provided with a second flow path, the second flow path communicating with the first cavity, and the second tube connector is used to connect to a tube. The first engaging plate and the first engaging portion can be engaged and connected to fix the male Luer connector and the female Luer connector, and to communicate the first flow path and the second flow path. The fully automated suspension cell culture bag kit according to claim 6, characterized in that the quick connector is installed at the end of the tube between the four-way connector and the three-way connector A, and / or the tube between the three-way connector B and the cell culture bag inlet, and / or the end of the culture medium inlet tube.

15. The fully automated suspension cell culture bag kit according to claim 14, characterized in that the first engaging portion is a first engaging block having at least two L-shaped cross-sections provided on the end face of the first female mating portion, the first engaging plate is provided with at least two first notches corresponding to the positions of the first engaging block, the first engaging block can pass over the first notches and, after rotating by a predetermined angle, engage with the first engaging plate, thereby engaging and connecting the male Luer connector and the female Luer connector.

16. At least one first annular groove is provided on the outer circumference of the first male fitting portion, and a first seal ring is provided in the first annular groove, and the first seal ring is pressure-fitted with the inner wall of the first cavity of the first female fitting portion. The fully automated suspension cell culture bag kit according to claim 14, characterized in that and / or, a first seal ring is provided on the end face of the first male mating portion facing the female Luer connector, and the first seal ring is press-fittable between the bottom wall of the first cavity of the female Luer connector and the end face of the first male mating portion.

17. The fully automated suspension cell culture bag kit according to claim 15, further comprising a male luer plug that mates with a female luer connector, wherein the male luer plug comprises a second male mating portion, a second engagement plate, and a second gripping portion, the second engagement plate being provided with at least two second notches corresponding to the positions of the first engagement block, the first engagement block passing over the second notches and, after rotating by a predetermined angle, becoming engaged with the second engagement plate, thereby engaging and connecting the male luer plug and the female luer connector.

18. At least one second annular groove is provided on the outer circumference of the second male fitting portion, and a second seal ring is provided in the second annular groove, and the second seal ring is press-fitted with the inner wall of the first cavity of the first female fitting portion. The fully automated suspension cell culture bag kit according to claim 17, characterized in that and / or, a second seal ring is provided on the end face of the second male mating portion facing the female Luer connector, and the second seal ring is press-fittable between the bottom wall of the first cavity of the female Luer connector and the end face of the second male mating portion.

19. The fully automated suspension cell culture bag kit according to claim 15, further comprising a female luer plug that mates with a male luer connector, wherein the male luer plug comprises a second female mating portion and a second engaging portion, the second engaging portion being a second engaging block provided on the end face of the second female mating portion and having at least two L-shaped cross-sections, the second engaging block passing over a first notch and, after rotating by a predetermined angle, becoming engageable with a first engaging plate, thereby engaging and connecting the male luer plug and the male luer connector.

20. The surface of the first engagement plate between adjacent first notches that faces the first gripping portion has a thickened wedge-shaped or trapezoidal inclined surface. The fully automated suspension cell culture bag kit according to claim 15 or 19, characterized in that the surface of the second engagement plate between adjacent second notches that faces the second gripping portion has a thickened wedge-shaped or trapezoidal slope.

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