Culture device for large-scale immune cells

Abstract

The invention discloses a culture device for large-scale immune cells, comprising: a first container for culturing an inoculated cell suspension; the first container is connected with a second container through a pipeline, a culture medium is contained in the second container, and the cell suspension and the culture medium are circularly conveyed between the first container and the second container; a swing mechanism is arranged below the first container, and an air supply mechanism is arranged on the second container; by arranging the first container and the second container, a cell suspension and a culture medium are mixed and circularly conveyed between the first container and the second container, meanwhile, the first container is driven by the swing mechanism to move to prevent cells from clustering, and gas is conveyed into the second container through the gas supply mechanism, so that the cells obtain sufficient gas and nutrition; the cell proliferation quantity is increased.

Description

Technical Field

[0001] This invention relates to the field of immune cell culture technology, and more particularly to a device for large-scale immune cell culture. Background Technology

[0002] NK cells, short for natural killer cells, are a subset of lymphocytes with unique functions in the human immune system. They are a core component of the innate immune system. NK cells are sensitive to changes in the external environment and require strict control of culture conditions, such as temperature, CO2 concentration, humidity, and nutrient supply.

[0003] Although NK cells possess a certain proliferative capacity, large-scale in vitro expansion still faces challenges such as difficulty and limited efficiency. Compared to T cells, NK cells have a relatively low expansion rate, typically 10-30 times, which is insufficient to meet the clinical demand for large quantities of NK cells. The main reason for these problems is that existing culture devices generally require multiple treatments with various consumables such as culture media, cytokines, and reagent kits before static culture. During this process, cells tend to clump together, preventing the inner cells from accessing gas and nutrients. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a device for large-scale immune cell culture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a device for large-scale immune cell culture, comprising:

[0006] First container,

[0007] The first container is used for culturing the inoculated cell suspension;

[0008] The first container is connected to the second container via a pipeline. The second container contains culture medium, allowing the cell suspension and the culture medium to circulate between the first container and the second container.

[0009] A swing mechanism is provided below the first container, and a gas supply mechanism is provided on the second container.

[0010] As a further description of the above technical solution: the first container includes an upper cover and a lower cover, the upper cover and the lower cover cooperate to form a receiving cavity, and the material of the first container is polystyrene.

[0011] As a further description of the above technical solution: a microporous membrane is provided on the inner side of the receiving cavity, which divides the receiving cavity into an upper cavity and a lower cavity; the microporous membrane is made of polyester material with a pore size of 0.2-12μm.

[0012] As a further description of the above technical solution: the upper cover and the lower cover are provided with connection ports, which are respectively connected to the upper cavity and the lower cavity, so as to inoculate the first container with different cell suspensions.

[0013] As a further description of the above technical solution: the connection port is provided with a sealing cap that is adapted to the outer diameter, so as to seal the first container.

[0014] As a further description of the above technical solution: the input end of the second container is connected to a cell filter, and the cell filter is connected to the output end of the first container.

[0015] As a further description of the above technical solution: a first peristaltic pump is also provided between the cell filter and the output end of the first container.

[0016] As a further description of the above technical solution: the output end of the second container is provided with a second peristaltic pump, and the second peristaltic pump is connected to the input end above the first container through a pipeline.

[0017] As a further description of the above technical solution: the swing mechanism includes a rocking bed, which is connected to the first container and drives the first container to rotate or swing. The rocking bed is provided with support legs underneath.

[0018] As a further description of the above technical solution: the gas supply mechanism includes a gas supply pump, which is disposed below the second container to supply gas to the cell suspension entering the second container.

[0019] The above technical solution has the following advantages or beneficial effects:

[0020] By setting up a first container and a second container, the cell suspension and culture medium are mixed and circulated between the first and second containers. At the same time, the first container is moved by a swing mechanism to prevent the cells from clumping together. Gas is delivered to the second container by a gas supply mechanism so that the cells can obtain sufficient gas and nutrients, thereby increasing the number of cells proliferating. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a perspective view of the cultivation device proposed in this invention;

[0023] Figure 2 This is a side view of the culture device proposed in this invention;

[0024] Figure 3 This is a cross-sectional view of the first container in this invention.

[0025] Legend:

[0026] 1. First container; 101. Top cover; 102. Bottom cover; 103. Microporous membrane; 104. Connection port; 105. Sealing cap; 2. Piping; 3. Second container; 4. Cell filter; 5. First peristaltic pump; 6. Second peristaltic pump; 7. Shaker; 8. Support legs; 9. Air supply pump. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Reference Figures 1-3 An embodiment of the present invention provides a device for large-scale immune cell culture, comprising: a first container 1, the first container 1 being used to culture a cell suspension; the first container 1 being connected to a second container 3 via a pipe 2, the second container 3 containing a culture medium, so that the cell suspension and the culture medium are circulated between the first container 1 and the second container 3; a swinging mechanism is provided below the first container 1, and a gas supply mechanism is provided on the second container 3.

[0029] In this embodiment, by setting up a first container 1 and a second container 3, the cell suspension and culture medium are mixed and circulated between the first container 1 and the second container 3. At the same time, the first container 1 is moved by a swing mechanism to prevent the cells from clumping together. Gas is delivered into the second container 3 by a gas supply mechanism so that the cells can obtain sufficient gas and nutrients and increase the number of cell proliferations.

[0030] The first container 1 includes an upper cover 101 and a lower cover 102, which together form a receiving cavity. The material of the first container 1 is polystyrene.

[0031] In this embodiment, the first container 1 forms a receiving cavity through the upper cover 101 and the lower cover 102. The first container 1 is made of optically transparent polystyrene (PS) for easy observation. The first container 1 is a disposable consumable, sterilized by gamma rays, and is a non-pyrogenic source. It has multiple capacity specifications from 2L to 10L, which can be selected according to actual needs.

[0032] A microporous membrane 103 is provided on the inner side of the receiving cavity, which divides the receiving cavity into an upper cavity and a lower cavity; the microporous membrane 103 is made of polyester material with a pore size of 0.2-12μm.

[0033] In this embodiment, a microporous membrane 103 is disposed on the four side walls of the upper cover 101. The microporous membrane 103 divides the first container 1 into two layers: an upper cavity and a lower cavity, allowing for the exchange of substances between cells and preventing cell contamination that could lead to impurities in the target cells, thus achieving the purpose of co-culture. The microporous membrane 103 allows cells to absorb / permeate molecules from their basal and top surfaces, culturing them in a manner closer to the in vivo environment. This maximizes the simulation of the in vivo environment and enables the co-culture of different cells, allowing for better observation of interactions between cells and between cells and the culture environment. By detecting the relationships between different cytokines, the effects of substances secreted by cells on the growth and metabolism of other cells can be studied. The material of the microporous membrane 103 can be polycarbonate (PC) or polyethylene terephthalate (PET). The pore size of the microporous membrane 103 is 0.2-12μm, which can be selected according to the cell size and whether the cells need to pass through the membrane pores. For example, a 0.4μm pore size membrane can be used for cell co-culture, while a 3, 5 or 8μm pore size membrane is required for chemotaxis assays, cell migration and invasion assays.

[0034] Furthermore, the culture surfaces of the microporous membrane 103 and the first container 1 are treated with tissue culture or matrix gel coating, so that the microporous membrane 103 is suitable for both adherent cell culture and suspension cell culture.

[0035] The upper cover 101 and the lower cover 102 are provided with connection ports 104, which are connected to the upper cavity and the lower cavity respectively, so as to inoculate the first container 1 with different cell suspensions.

[0036] In this embodiment, the interior of the first container 1 is divided into an upper cavity and a lower cavity by a microporous membrane 103. Different cell suspensions can be inoculated into the upper cavity and the lower cavity through the two connecting ports 104 respectively, so as to achieve co-culture of different types of immune cells. The microporous membrane 103 allows small molecules to pass through, prevents cell mixing, and achieves selective permeation of cytokines and nutrients.

[0037] A sealing cap 105 adapted to the outer diameter is provided on the connection port 104 to seal the first container 1.

[0038] In this embodiment, the sealing cap 105 may be made of rubber and be press-fitted with the outer diameter of the connection port 104, or the connection port 104 may have an external thread and the sealing cap 105 may have an internal thread, and the sealing may be achieved through threaded connection.

[0039] The input end of the second container 3 is connected to a cell filter 4, which is connected to the output end of the first container 1. A first peristaltic pump 5 is also provided between the cell filter 4 and the output end of the first container 1. A second peristaltic pump 6 is provided at the output end of the second container 3, and the second peristaltic pump 6 is connected to the input end above the first container 1 through a pipeline.

[0040] In this embodiment, the second container 3, with a capacity of 500-1000 ml, is used to store the culture medium. The first peristaltic pump 5 pumps the cell suspension from the first container 1 into the cell filter 4, where it is filtered and then enters the second container 3. The second peristaltic pump 6 pumps the mixture containing the culture medium and cell suspension back from the second container 3 to the first container 1, thus achieving nutrient renewal. The two peristaltic pumps can control the input and output of the first container 1, thereby controlling the circulation rate to meet the needs of different culture stages. The cell filter 4 is used to filter cell debris and metabolic waste generated during the culture process, preventing excessive cell aggregation and clumping, maintaining unobstructed circulation in the tubing 2, and avoiding blockage.

[0041] Furthermore, the rotational speeds of the first peristaltic pump 5 and the second peristaltic pump 6 are 50-150 rpm / min, referring to...

[0042] Table 1 shows the effect of the flow rates of the first peristaltic pump 5 and the second peristaltic pump 6 on cell viability:

[0043]

[0044] Table 1

[0045] When the peristaltic pump inlet and outlet flow rates are 200 rpm / min, cell viability decreases by 5%, and cell damage is significant and unacceptable. When the peristaltic pump flow rate is less than 150 rpm / min, cell viability decreases by 2%, which is a minor and acceptable impact. When the peristaltic pump flow rate is 10 rpm / min, the cumulative culture medium volume in 24 hours is 96 ml, which is too small and easily causes instability in the circulating culture system; this parameter is not advisable. Based on the analysis, the peristaltic pump flow rate can be selected between 50 rpm / min and 150 rpm / min according to the NK cell culture capacity of 2L-10L. For a 2L culture system, 50 rpm / min is preferred, and for a 10L culture system, 150 rpm / min is preferred.

[0046] The swing mechanism includes a rocking bed 7, which is connected to the first container 1 and drives the first container 1 to rotate or swing. The rocking bed 7 is provided with support legs 8 below it.

[0047] In this embodiment, the first container 1 is rotated or oscillated by the shaker 7, and parameters such as rotation frequency and speed can be adjusted to increase liquid flow, improve the contact efficiency between cells and nutrients, prevent cells from clumping, maintain a uniform culture environment, and the support legs 8 provide stable support and reduce vibration interference.

[0048] Refer to Table 2 for the effect of shaker speed on cell viability.

[0049]

[0050] Table 2

[0051] When the shaker speed is 90 rpm / min, a lot of bubbles will be generated in the first container 1, which are not easy to dissolve and have a significant impact on cell viability, which is unacceptable. When the shaker speed is 10 rpm / min, the liquid in the first container 1 will not mix evenly due to the low speed. Based on the above data analysis, the optimal speed of the shaker 7 is 30-60 rpm / min.

[0052] The gas supply mechanism includes a gas pump 9, which is located below the second container 3 to supply gas to the cell suspension entering the second container 3.

[0053] In this embodiment, the gas supply pump 9 provides suitable gas to the culture medium in the second container 3 to maintain the pH stability and dissolved oxygen level of the culture device. The gas diffuses through the second container 3 to the first container 1 to ensure a consistent gas environment throughout the entire culture device.

[0054] Referring to Table 3, the effect of air supply pump flow rate on cell viability is shown.

[0055]

[0056] When the gas supply pump flow rate is greater than 24 rpm / min, bubbles are generated in the liquid in the first container 1, which are difficult to dissolve and cause damage to the cells; when the gas supply flow rate is 1 rpm / min, the pH value in the first container 1 is low at 7.0, and the gas supply is insufficient; based on the above data analysis, the gas supply pump flow rate can be selected within 6-12 rpm / min.

[0057] During the culture process, cell proliferation fold, cell viability, and cell phenotype (such as the expression of markers like CD3-CD56+ and CD16+CD56+) were monitored. The test results of the culture apparatus (A) in this embodiment and the existing commercial container culture apparatus (B) are shown in Tables 8-9.

[0058]

[0059]

[0060] Table 8

[0061]

[0062]

[0063] Table 9

[0064] As can be seen from the data in Tables 8 and 9, NK cells obtained from the same batch of cells with the same seeding concentration are superior to those from commercial container culture devices in terms of proliferation capacity, cell viability, and cell phenotypic expression.

[0065] In one specific embodiment, UC-MSC umbilical cord mesenchymal stem cells P5-P8 passage utilization culture medium were prepared into a cell suspension with a cell concentration of 1*102. 5 ~5*10 7 The cells were irradiated with cobalt-60 at a dose of 50 Gy per ml, and then cryopreserved in liquid nitrogen.

[0066] Resuscitated MSC cells were then fed at a rate of 2*10-1 4 ~1*10 5 cells / cm 2 For inoculation, cells were seeded into the lower cavity of container 1 and cultured for 16–24 hours. The MSC culture medium was then removed, and the bottom layer of cells was washed with PBS buffer. NK cells cultured for days 7–9 were then transferred to the upper cavity of container 1 at a concentration of 1–3 x 10⁻⁶ cells / mL. 6Cells / ml were cultured for 5-9 days, with the first peristaltic pump 5 and the second peristaltic pump 6 operating at 50-150 rpm / min, the shaker 7 at 30-60 rpm / min, and the air supply pump 9 at 6-12 rpm / min. Then, upper NK cells were harvested. The harvested NK cells were co-cultured with target cells (K562) for cytotoxicity testing.

[0067] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0068] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A device for large-scale culture of immune cells, characterized in that, include: First container (1), The first container (1) is used to culture the inoculated cell suspension; The first container (1) is connected to the second container (3) through a pipe (2). The second container (3) contains a culture medium, so that the cell suspension and the culture medium are circulated between the first container (1) and the second container (3). A swing mechanism is provided below the first container (1), and a gas supply mechanism is provided on the second container (3).

2. The culture apparatus according to claim 1, characterized in that: The first container (1) includes an upper cover (101) and a lower cover (102), which together form a receiving cavity. The material of the first container (1) is polystyrene.

3. The culture apparatus according to claim 2, characterized in that: A microporous membrane (103) is provided on the inner side of the receiving cavity, which divides the receiving cavity into an upper cavity and a lower cavity; the microporous membrane (103) is made of polyester material with a pore size of 0.2-12μm.

4. The culture apparatus according to claim 3, characterized in that: The upper cover (101) and the lower cover (102) are provided with connection ports (104), which are respectively connected to the upper cavity and the lower cavity, so as to inoculate the first container (1) with different cell suspensions.

5. The culture apparatus according to claim 4, characterized in that: The connection port (104) is provided with a sealing cap (105) that is adapted to the outer diameter, for sealing the first container (1).

6. The culture apparatus according to claim 1, characterized in that: The input end of the second container (3) is connected to a cell filter (4), which is connected to the output end of the first container (1).

7. The culture apparatus according to claim 6, characterized in that: A first peristaltic pump (5) is also provided between the cell filter (4) and the output end of the first container (1).

8. The culture apparatus according to claim 1, characterized in that: The output end of the second container (3) is provided with a second peristaltic pump (6), which is connected to the input end above the first container (1) through a pipeline.

9. The culture apparatus according to claim 1, characterized in that: The swing mechanism includes a rocking bed (7), which is connected to the first container (1) and drives the first container (1) to rotate or swing. The rocking bed (7) is provided with support legs (8) below it.

10. The culture apparatus according to claim 1, characterized in that: The gas supply mechanism includes a gas pump (9), which is located below the second container (3) to supply gas to the cell suspension entering the second container (3).

Images