An integrated cell culture bag
Patent Information
- Application Number
- CN202522439725.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-18
- Publication Date
- 2026-10-23
- Estimated Expiration
- 2035-11-18
AI Technical Summary
这种多次转换操作不仅繁琐、耗时耗力，更重要的是，每一次开放操作都极大地增加了细胞产品被污染的风险
[0013]1.一体化封闭培养：通过将三个不同容积的培养袋串联集成，实现了从接种、扩增到收获的全过程封闭式培养，无需中途更换培养容器，极大降低了污染风险。
Smart Images
Figure CN224884077U_ABST
Abstract
Description
Technical Field
[0001] This utility model relates to the field of cell culture bag technology, and in particular to an integrated cell culture bag. Background Technology
[0002] During the expansion culture of suspension cells such as natural killer (NK) cells, the cell number and culture volume increase significantly. Currently, this process typically requires sequentially using different sized culture containers, such as switching from T75 culture flasks to T182 culture flasks, and then to 2L culture bags. This multiple conversion operation is not only cumbersome and time-consuming, but more importantly, each open operation greatly increases the risk of cell product contamination.
[0003] Therefore, there is an urgent need in the field for an integrated culture device that can adapt to dynamic changes in cell culture volume and reduce intermediate transfer operations. Utility Model Content
[0004] To solve the above problems, this utility model provides an integrated cell culture bag, the specific technical solution of which is as follows:
[0005] An integrated cell culture bag includes a first culture bag, a second culture bag, and a third culture bag; the first culture bag is provided with a first inlet and a first outlet; the second culture bag is provided with a second inlet, a second outlet, and a second replenishment outlet; the third culture bag is provided with a third inlet, a third outlet, and a third replenishment outlet; the first outlet and the second inlet are connected by a first connecting pipe; the second outlet and the third inlet are connected by a second connecting pipe; the first inlet is connected to a first inlet pipe, the second replenishment outlet is connected to a second replenishment pipe, and the third replenishment outlet is connected to a third replenishment pipe; wherein the volume of the second culture bag is larger than the volume of the first culture bag, and the volume of the third culture bag is larger than the volume of the second culture bag.
[0006] Preferably, the ports of the first injection pipe, the second replenishment pipe, and the third replenishment pipe are all provided with screw caps.
[0007] Preferably, at least one sampling bag is connected to each of the first culture bag, the second culture bag, and the third culture bag.
[0008] Preferably, the connection between the sampling bag and the corresponding culture bag forms a heat-fused connection section.
[0009] Preferably, the first culture bag, the second culture bag, and / or the third culture bag are provided with a plurality of sampling bags.
[0010] Preferably, the first connecting pipe is provided with a first connecting clip for controlling its opening and closing.
[0011] Preferably, the second connecting pipe is provided with a second connecting clip for controlling its opening and closing.
[0012] Compared with the prior art, the present invention has the following beneficial effects:
[0013] 1. Integrated closed culture: By connecting three culture bags of different volumes in series, a closed culture process from inoculation and amplification to harvest is achieved, eliminating the need to change culture containers midway and greatly reducing the risk of contamination.
[0014] 2. Simple and efficient operation: By controlling the clamps on the connecting pipes and the replenishment pipes with screw caps, the cell suspension can be transferred between different culture bags and the culture medium can be replenished, which simplifies the operation steps and saves manpower and time.
[0015] 3. Safe sampling function: The sampling bag is equipped with a heat-fused connection section, which allows for the safe and timely acquisition of samples for counting or quality control testing during the culture process without compromising the aseptic seal of the main culture system. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the overall structure of the integrated cell culture bag in this application;
[0017] Figure 2 yes Figure 1 A magnified structural diagram of point A in the middle.
[0018] Reference numerals: First culture bag 1, first injection port 11, first drain port 12, first injection pipe 13, second culture bag 2, second injection port 21, second drain port 22, second replenishment port 23, second replenishment pipe 24, third culture bag 3, third injection port 31, third drain port 32, third replenishment port 33, third replenishment pipe 34, first connecting pipe 4, first connecting clip 41, second connecting pipe 5, second connecting clip 51, screw cap 6, sampling bag 7, heat-fused connection section 71. Detailed Implementation
[0019] The embodiments of this disclosure will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the embodiments and the accompanying drawings are used to illustrate the principles of this disclosure by way of example, but should not be used to limit the scope of this disclosure. This disclosure can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.
[0020] These embodiments are provided to make the disclosure thorough and complete, and to fully express the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values set forth in these embodiments should be interpreted as exemplary only and not as limiting.
[0021] It should be noted that, in the description of this disclosure, unless otherwise stated, "a plurality of" means two or more; the terms "upper," "lower," "left," "right," "inner," and "outer," etc., indicating orientation or positional relationship, are only for the convenience of describing this disclosure and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this disclosure. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.
[0022] Furthermore, the terms "first," "second," and similar terms used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. "Vertical" is not strictly vertical, but within the permissible margin of error. "Parallel" is not strictly parallel, but within the permissible margin of error. Terms such as "including" or "contains" mean that the element preceding the word encompasses the element listed after the word, and do not exclude the possibility of encompassing other elements as well.
[0023] It should also be noted that, in the description of this disclosure, unless otherwise expressly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this disclosure depending on the specific circumstances. When a particular device is described as being located between a first device and a second device, an intermediary device may or may not be present between the particular device and the first or second device.
[0024] All terms used in this disclosure have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in general dictionaries should be interpreted as having meanings consistent with their meanings in the context of the relevant art, and not as idealized or highly formalized, unless expressly defined herein.
[0025] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.
[0026] like Figures 1 to 2 As shown, an integrated cell culture bag includes a first culture bag 1, a second culture bag 2, and a third culture bag 3. These three culture bags are preferably made of biocompatible polymer materials such as polyethylene or polypropylene composite films.
[0027] The first culture bag 1 is provided with a first injection port 11 and a first drainage port 12. The first injection port 11 is connected to a first injection pipe 13 for injecting the initial cell suspension. The second culture bag 2 is provided with a second injection port 21, a second drainage port 22, and a second replenishment port 23. The second replenishment port 23 is connected to a second replenishment pipe 24 for replenishing culture medium into the second culture bag 2. The third culture bag 3 is provided with a third injection port 31, a third drainage port 32, and a third replenishment port 33. The third drainage port 32 is used for the final harvesting of the cell suspension, and the third replenishment port 33 is connected to a third replenishment pipe 34 for replenishing culture medium into the third culture bag 3.
[0028] The first drain port 12 and the second injection port 21 are connected by a first connecting pipe 4, which is equipped with a first connecting element 41, such as a plastic clamp or a stopcock, to control the opening and closing of the channel. The second drain port 22 and the third injection port 31 are connected by a second connecting pipe 5, which is also equipped with a second connecting element 51.
[0029] Each of the ports of the first injection pipe 13, the second replenishment pipe 24, and the third replenishment pipe 34 is equipped with a screw cap 6. When injection or replenishment is required, the corresponding screw cap 6 is unscrewed to allow operation using a sterile syringe or other device. After the operation is completed, the screw cap 6 is tightened to restore and maintain the system's sealed state.
[0030] At least one sampling bag 7 is connected to each of the first culture bag 1, the second culture bag 2, and the third culture bag 3. The connection between each sampling bag 7 and its corresponding culture bag forms a weak heat-fused joint 71. When sampling is required, a small amount of cell suspension is gently squeezed into the sampling bag 7, and then a dedicated heat-sealing machine is used to heat-seal the joint 71, allowing the sampling bag 7 to be safely separated for testing, while the main culture bag system remains completely sealed and undamaged. To meet the need for multiple samplings at different time points during the culture process, multiple sampling bags 7 can be set on one or more culture bags.
[0031] In one specific application of this invention, the volume of the first culture bag 1 is set to 200 ml, the volume of the second culture bag 2 is set to 500 ml, and the volume of the third culture bag 3 is set to 2000 ml. This specification is particularly suitable for typical culture processes that expand NK cells from an initial volume of tens of milliliters to a harvest volume of nearly two thousand milliliters.
[0032] Initial preparation:
[0033] Device status: The first connecting clip 41 on the first connecting pipe 4 and the second connecting clip 51 on the second connecting pipe 5 are both in the closed state. The screw caps 6 of all injection / replenishment pipes are in the tightened sealed state.
[0034] Phase 1: Initial culture in 1200ml of the first culture bag
[0035] Day 0 vaccination:
[0036] The operator resuscitates and prepares cell suspensions such as umbilical cord blood mononuclear cells (CBMCs).
[0037] Unscrew the screw cap 6 of the first injection tube 13 and use a sterile syringe to inject 30-60 ml of cell suspension into the first culture bag 1 through the first injection port 11.
[0038] After injection, tighten screw cap 6 to restore the seal. Place the entire culture bag in an incubator at 37°C and 5% CO2 to begin incubation.
[0039] Day 4, first sampling and fluid resuscitation:
[0040] Remove the culture bag from the incubator and mix gently.
[0041] A small amount of cell suspension was squeezed from the first culture bag 1 into the connected sampling bag 7, number 14.
[0042] The sampling bag 7 is then separated from the sample bag 7 by heat sealing the heat-fused connection section 71 using a heat sealer. The main culture bag remains completely sealed at this time.
[0043] Based on the counting results, unscrew the screw cap 6 of the first injection tube 13 and use a syringe to add complete culture medium, bringing the total volume to 50-100 ml. After completion, reseal and return to the incubator.
[0044] Day 6:
[0045] Repeat sampling procedure: squeeze the cell suspension into another sampling bag 7 (No. 15) of the first culture bag 1, heat seal and separate, and count.
[0046] Similarly, replenish the fluid through the first injection tube 13 to increase the volume to 100-190ml, seal, and continue culturing. Second stage: Transfer to a second culture bag (2500ml) and continue culturing. First transfer and bag change on day 8:
[0047] Remove the culture bag and mix the cells.
[0048] Open the first connecting piece 41, and using gravity or slight pressure, transfer most of the cell suspension in the first culture bag 1 to the second culture bag 2 through the first connecting tube 4. Only a small amount of suspension is retained in the first culture bag 1 for counting.
[0049] A heat sealer is used to heat seal the first connecting pipe 4 at an appropriate location near the first culture bag 1, thereby separating and discarding the substantially emptied first culture bag 1, including its attached sampling bag 7, from the entire system.
[0050] The small amount of cell suspension retained was counted.
[0051] Unscrew the screw cap 6 of the second replenishment pipe 24 and replenish the culture medium into the second culture bag 2 through the second replenishment port 23 to make the volume reach 200-400ml.
[0052] After sealing, place the culture bag back into the incubator. At this time, the second connecting clip 51 remains closed.
[0053] On day 9, samples were taken and fluid was replenished in culture bag 2.
[0054] After mixing the cells, squeeze a small amount of suspension into the No. 14 sampling bag 7 connected to the second culture bag 2, heat-separate and count.
[0055] Add liquid through the second replenishment tube 24 to increase the volume to 300-490ml, seal, and continue culturing. Third stage: Transfer to the third culture bag (32000ml) for final culture and harvest. Days 11-15: Day 11: Second transfer, bag change, and central control sampling.
[0056] Remove the culture bag and mix the cells.
[0057] Open the second connecting piece 51 to transfer the cell suspension in the second culture bag 2 to the third culture bag 3 through the second connecting pipe 5.
[0058] The second culture bag 2 was sealed and separated using a heat sealer. The small amount of cell suspension remaining in the second culture bag 2 at this point has two uses:
[0059] A portion is squeezed into its No. 15 sampling bag 7. After heat sealing and separation, this sample can be used for more comprehensive intermediate quality control, such as sterility, endotoxin, mycoplasma detection, as well as cell phenotype and killing function analysis.
[0060] The remainder is used for routine cell counting.
[0061] Unscrew the screw cap 6 of the third replenishment pipe 34 and replenish the liquid through the third replenishment port 33, so that the volume inside the third culture bag 3 reaches 500-800ml.
[0062] After sealing, return it to the incubator.
[0063] On day 13, samples were taken and fluid was replenished in culture bag 3:
[0064] After mixing, sampling, heat-sealing separation, and counting are performed using sampling bag 7 (No. 14) of the third culture bag 3.
[0065] Massive replenishment is performed through the third replenishment channel 34 to reach a volume of 1200-1900 ml, and the culture is continued after sealing.
[0066] Final sampling and cell harvesting on day 15:
[0067] Perform a final sampling: use sampling bag 7 (size 15) of the third culture bag 3 to take, heat-seal, and count the cells to calculate the final total cell count.
[0068] Harvesting cells: Through the third drain port 32, all the cell suspension in the third culture bag 3 is discharged into a centrifuge cup or collection container for subsequent centrifugation, washing and preparation steps.
[0069] The technical principles of this utility model have been described above with reference to specific embodiments. These descriptions are merely for explaining the principles of this utility model and should not be construed as limiting the scope of protection of this utility model in any way. Based on this explanation, those skilled in the art can readily conceive of other specific embodiments of this utility model without inventive effort, and these embodiments will all fall within the protection scope of the claims of this utility model.
Claims
1. An integrated cell culture bag, characterized in that, The system includes a first culture bag (1), a second culture bag (2), and a third culture bag (3); the first culture bag (1) is provided with a first injection port (11) and a first drainage port (12); the second culture bag (2) is provided with a second injection port (21), a second drainage port (22), and a second replenishment port (23); the third culture bag (3) is provided with a third injection port (31), a third drainage port (32), and a third replenishment port (33); the first drainage port (12) and the second injection port (21) are connected by a first connecting pipe. The channel (4) is connected; the second drain port (22) and the third injection port (31) are connected through the second connecting pipe (5); the first injection port (11) is connected to the first injection pipe (13), the second replenishment port (23) is connected to the second replenishment pipe (24), and the third replenishment port (33) is connected to the third replenishment pipe (34); wherein, the volume of the second culture bag (2) is greater than the volume of the first culture bag (1), and the volume of the third culture bag (3) is greater than the volume of the second culture bag (2).
2. The integrated cell culture bag according to claim 1, characterized in that, The ports of the first injection pipe (13), the second replenishment pipe (24) and the third replenishment pipe (34) are all provided with screw caps (6).
3. The integrated cell culture bag according to claim 1, characterized in that, At least one sampling bag (7) is connected to each of the first culture bag (1), the second culture bag (2) and the third culture bag (3).
4. The integrated cell culture bag according to claim 3, characterized in that, The connection between the sampling bag (7) and the corresponding culture bag constitutes a heat-fused connection section (71).
5. The integrated cell culture bag according to claim 3, characterized in that, The first culture bag (1), the second culture bag (2) and / or the third culture bag (3) are provided with a plurality of sampling bags (7).
6. The integrated cell culture bag according to claim 1, characterized in that, The first connecting pipe (4) is provided with a first connecting card (41) for controlling its opening and closing.
7. The integrated cell culture bag according to claim 1, characterized in that, The second connecting pipe (5) is provided with a second connecting clip (51) for controlling its opening and closing.