HEK-293T cell suspension domestication method

Abstract

The invention provides an HEK-293T cell suspension domestication method. The method comprises the following steps: carrying out gradient dilution to obtain an HEK-293T monoclonal cell strain; the method comprises the following steps: screening at least one cell strain with high growth rate and / or high virus packaging efficiency from HEK-293T monoclonal cell strains as a cell strain to be suspended and domesticated; and transferring the to-be-suspended domesticated cell strain into a suspension culture medium for subculture, and screening out the HEK-293T cell for suspension culture based on the cell caking rate and / or cell viability after subculture. According to the suspension domestication method, the serum-free culture medium is adopted, the HEK-293T cells which are good in growth state and dispersity and high in stability can be obtained, and lentiviruses obtained through suspension culture of the HEK-293T cells are high in packaging amount and high in T cell infection capacity. In addition, the suspension domestication method is easy and convenient to operate, low in cost and suitable for large-scale application.

Description

Technical Field

[0001] The present application relates to the field of biology. Specifically, the present application relates to a method for acclimating HEK-293T cells by suspension. Background Art

[0002] HEK-293T, known in Chinese as human embryonic kidney 293T cell line, is a highly transfectable and highly expressive cell line widely used in scientific research and industry. HEK-293T cells are commonly used in lentiviral vector production. The currently used adherent culture process has the following issues: 1) The operation is cumbersome, with the trypsin digestion process significantly impacting cell growth, significantly affecting stability and consistency between batches; 2) It relies on fetal bovine serum (FBS), an animal-derived material that is expensive and increases the safety risks and production costs of drug production. FBS batch-to-batch variability further challenges the consistency of viral vector production processes; and 3) The adherent cell production process takes up a large amount of space and is complex to operate, making it difficult to scale up.

[0003] With advances in biotechnology and bioprocessing, suspension cell production techniques are constantly being developed and optimized. These techniques offer advantages such as ease of scale-up, low cost, and user-friendly production operations. However, the adaptation of HEK-293T cells to suspension culture remains a topic of research. Summary of the Invention

[0004] This application aims to solve, at least to some extent, the technical problems existing in the prior art. To this end, this application proposes a method for acclimating HEK-293T cells in suspension, HEK-293T cells for suspension culture, and a method for producing viruses. The suspension acclimation method of this application uses serum-free culture medium to obtain HEK-293T cells with good growth state, dispersion, and strong stability. The lentivirus obtained by suspension culture using these HEK-293T cells has a high packaging yield and strong ability to infect T cells. In addition, the suspension acclimation method and virus production method of this application are simple to operate, low-cost, and suitable for large-scale applications.

[0005] In one aspect, the present application provides a method for acclimating HEK-293T cells to suspension culture. According to an embodiment of the present application, the method comprises: step S1: obtaining a HEK-293T monoclonal cell line by gradient dilution; step S2: screening at least one cell line with a fast growth rate and / or high viral packaging efficiency from the HEK-293T monoclonal cell line as the cell line to be suspended and acclimated; step S3: transferring the cell line to be suspended and acclimated to suspension culture medium for subculture, and screening HEK-293T cells for suspension culture based on the cell clumping rate and / or cell viability after subculture; wherein the suspension culture medium comprises at least one of Transpro CD 01plus medium, Aplo CD293 medium, and Aplo293 medium.

[0006] According to the method of the embodiment of the present application, first, a HEK-293T cell monoclonal cell strain with high homogeneity and stability is obtained by gradient dilution. Then, the monoclonal cell strain is further screened, and the screening indicators include growth rate and / or virus packaging efficiency. A cell strain with a fast growth rate can reach the required cell density in a shorter time, thereby significantly shortening the production cycle and improving production efficiency. A cell strain with a high virus packaging efficiency can more effectively produce a viral vector with high infectivity. Then, the cell strain to be suspended and acclimated obtained by screening is subjected to suspension subculture so as to convert the HEK-293T cells from the adherent growth mode to the suspended growth mode, and a cell strain with a low agglomeration rate and / or a high cell viability is selected as the target cell strain to ensure that the cells have good dispersibility and high survival rate during the suspension culture process, thereby improving the growth efficiency and stability of the cells. Transpro CD 01plus medium, Aplo CD293 medium, and Aplo 293 medium are commonly used for adherent culture. The inventors of the present application have found through extensive experiments that these three media can be used for suspension culture. 293T cells have better cell viability, proliferation rate, and cell stability in these media, and their clumping degree is relatively low.

[0007] According to the embodiments of the present application, the above-mentioned HEK-293T cell suspension acclimation method may also have the following additional technical features:

[0008] According to an embodiment of the present application, the suspension culture medium includes Transpro CD 01plus culture medium.

[0009] According to an embodiment of the present application, the suspension culture medium includes an anti-agglomerating agent, and the anti-agglomerating agent includes at least one of dextran sulfate, ferric citrate and sodium ferric EDTA.

[0010] According to an embodiment of the present application, the concentration of the anti-agglomerating agent in the suspension culture medium is 0.05 g / L to 0.4 g / L.

[0011] According to an embodiment of the present application, step S2 includes: step S2-1: subculturing the HEK-293T cell monoclonal cell line and selecting at least one cell line with a fast growth rate; step S2-2: packaging each HEK-293T cell line screened out in step S2-1 with a lentivirus, and then infecting the same batch of HEK-293T cells with each group of packaged lentiviruses. After culturing, signal detection is performed to screen out at least one cell line with a high signal intensity as the cell line to be suspended and acclimated.

[0012] According to the examples of the present application, each of the cell lines is co-transfected with a basic packaging plasmid and a plasmid for generating a signal marker, so as to package the lentivirus.

[0013] According to an embodiment of the present application, the plasmid for generating a signal marker is selected from a plasmid encoding a fluorescent protein gene.

[0014] According to an embodiment of the present application, the basic packaging plasmid includes a VSVG envelope protein plasmid, a REV auxiliary protein plasmid and an RRE sequence plasmid.

[0015] According to an embodiment of the present application, the mass ratio of the plasmid for generating the signal marker, the VSVG envelope protein plasmid, the REV auxiliary protein plasmid and the RRE sequence plasmid is (0.5-2): (0.5-2): (0.5-2): (0.5-2).

[0016] According to an embodiment of the present application, in step S3, the viable cell density of the cell line to be suspended and acclimated is first transferred to the suspension culture medium is 5×10 5 cells / mL~5×10 6 cells / mL, subcultured every 2 to 4 days, with 4×10 5 ~5×10 5 The suspension medium was replaced with fresh one at a viable cell density of cells / mL.

[0017] According to an embodiment of the present application, in step S3, the volume of the suspension culture medium for the first transfer is 10-25 mL / 125 mL, and the volume of the fresh suspension culture medium for each subculture is 25-35 mL / 125 mL.

[0018] According to the embodiments of the present application, the subculture adopts an oscillating culture method with a rotation speed of 100 rpm to 150 rpm, a culture temperature of 35° C. to 39° C., and a CO2 concentration of 5% to 10%.

[0019] According to the embodiment of the present application, each generation is cultured to a viable cell density of 3×10 6 cells / mL or more, preferably 3×10 6 ~5×10 6 cells / mL, and then passage was performed.

[0020] According to an embodiment of the present application, when the subculture is performed until the cell agglomeration rate is not higher than a first preset value and / or the cell viability is not lower than a second preset value, HEK-293T cells suitable for suspension culture are obtained; the first preset value is 18% to 25%; the second preset value is 85% to 95%.

[0021] According to an embodiment of the present application, when the subculture is performed until the cell agglomeration rate is not higher than a first preset value and the cell viability is not lower than a second preset value, cells with a high proliferation rate are further screened to obtain HEK-293T cells for suspension culture.

[0022] According to an embodiment of the present application, when the cell clumping rate after the subculture is higher than the first preset value, the cells after the subculture are further subcultured for at least one generation until the cell clumping rate is no higher than the first preset value, thereby obtaining HEK-293T cells for suspension culture.

[0023] According to an embodiment of the present application, step S3 includes: placing the cell line to be suspended and acclimated, DMEM culture medium and fetal bovine serum in a well plate, 35°C to 39°C, CO2 concentration of 4% to 6%, static culture, subculture once every 2 to 4 days, and subculture until the cell viability reaches more than 95%, according to 8×10 5 cells / mL~2×10 6 cells / mL were transferred to the suspension culture medium, the liquid volume was 15-25mL / 125mL, the rotation speed was 100rpm-120rpm, the culture temperature was 36℃-38℃, the CO2 concentration was 7%-9%, and the culture was carried out for 2-4 days. 6 cells / mL, and the passage was carried out with the following conditions for each passage: 4×10 5 ~5×10 5 cells / mL, replace the fresh suspension culture medium with a liquid volume of 28-32 mL / 125 mL, a rotation speed of 100 rpm-120 rpm, a culture temperature of 36° C.-38° C., a CO2 concentration of 7%-9%, and culture once after 2-4 days; when the cell clumping rate is 18%-25% and the cell viability is not less than 90%, HEK-293T cells for suspension culture are obtained.

[0024] In another aspect of the present application, the present application provides HEK-293T cells for suspension culture. According to an embodiment of the present application, the HEK-293T cells for suspension culture are obtained by the HEK-293T cell suspension acclimation method described above.

[0025] In another aspect, the present application provides a method for culturing HEK-293T cells in suspension. According to an embodiment of the present application, the method comprises: culturing the HEK-293T cells obtained by the HEK-293T cell suspension acclimation method described above in suspension culture medium; the suspension culture medium is selected from the suspension culture medium used in the HEK-293T cell suspension acclimation method described above.

[0026] In another aspect, the present application provides a method for producing a virus. According to an embodiment of the present application, the method comprises: transfecting a lentiviral packaging plasmid into HEK-293T cells obtained by the HEK-293T cell suspension acclimation method described above, and performing suspension culture; wherein the suspension culture medium is selected from the suspension culture medium used in the HEK-293T cell suspension acclimation method described above.

[0027] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0029] Figure 1 A flow chart of a method for acclimating HEK-293T cells in suspension according to one embodiment of the present application is shown;

[0030] Figure 2 The fluorescence value analysis graph of different clones of Example 1 is shown;

[0031] Figure 3 Electron micrographs of cell agglomeration of the 020# monoclonal cell culture at passages 1 and 3 of acclimation in Example 2 are shown: (A) acclimation passage 1 (0.1 g / L anti-agglomerant), (B) acclimation passage 3 (0.1 g / L anti-agglomerant), and (C) acclimation passage 3 (0.2 g / L anti-agglomerant).

[0032] Figure 4Electron micrographs showing the difference between the addition and absence of an anti-agglomerating agent in subculture of the 020# monoclonal clone of Example 2: (A) 293T-P14-XF-020#-240613-1, (B) 293T-P12-XF-020#-240607-1-DS, (C) 293T-P14-XF-020#-240613-2, (D) 293T-P12-XF-020#-240607-2-DS;

[0033] Figure 5 The graph showing the trend of the change in viable cell density of 020# and 015# over growth time in Example 4 is shown;

[0034] Figure 6 The figure shows the trend analysis of the cell viability of 020# and 015# cells over the growth time in Example 4;

[0035] Figure 7 The flow cytometric analysis of the virus concentrates of the 020# and 015# monoclonal cell lines in Example 6 for T cell infection is shown. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0038] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0039] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0040] The present application proposes a method for acclimating HEK-293T cells to suspension, HEK-293T cells for suspension culture, a method for culturing HEK-293T cells in suspension, and a method for producing viruses, which will be described in detail below.

[0041] HEK-293T cell suspension acclimation method

[0042] In one aspect of the present application, the present application proposes a method for acclimating HEK-293T cells by suspension. Figure 1 , the method includes: S1 gradient dilution to obtain HEK-293T monoclonal cell lines, S2 screening cell lines with fast growth rate and / or high virus packaging efficiency, and S3 subculture in suspension culture medium. Therefore, the suspension acclimation method of the present application adopts serum-free culture medium to obtain HEK-293T cells with good growth state, dispersion and strong stability. After acclimation, the cells are passaged 15 times, and the difference in doubling value between generations is <10%; the lentivirus packaging volume obtained by suspension culture of the HEK-293T cells is high and the ability to infect T cells is strong. In addition, the suspension acclimation method and virus production method of the present application are simple to operate and low in cost. Compared with traditional adherent culture, the cell culture cost can be reduced by 50% (material cost), which is suitable for large-scale production.

[0043] HEK-293T cell monoclonal cell line was obtained by S1 serial dilution

[0044] Monoclonal refers to a homogenous cell population induced or isolated from a single parent cell or cell line. HEK-293T cell monoclonal cell lines are isolated and screened to obtain highly homogeneous and stable cell lines.

[0045] According to an embodiment of the present application, in step S1, a monoclonal HEK-293T cell line is screened by the limiting dilution method. The limiting dilution method mainly involves performing a gradient dilution of a cell suspension, culturing the diluted cells separately, selecting a well in which only one cell is growing, and collecting the cell to obtain a monoclonal cell line.

[0046] In some embodiments, the method of screening HEK-293T monoclonal cell lines comprises:

[0047] First, the cell suspension is diluted to different gradients to obtain a series of cell dilutions with different dilutions, and then these cell dilutions are distributed into multi-well plates (such as 96-well plates); then, the well plates are placed in a cell culture incubator for culture. The cells in each well will gradually grow and divide. Under microscopic observation, the cell wells in which a single cell has grown into a single cell population are selected and the cells are collected as monoclonal cell lines.

[0048] S2 Screening of cell lines with fast growth rate and / or high virus packaging efficiency

[0049] In this step, at least one cell line with a fast growth rate and / or high viral packaging efficiency is selected from the HEK-293T cell monoclonal cell line as the cell line to be suspended and acclimated. The monoclonal cell line is further screened, and the screening criteria include growth rate and / or viral packaging efficiency. Cell lines with a fast growth rate can reach the required cell density in a shorter time, thereby significantly shortening the production cycle and improving production efficiency. Cell lines with high viral packaging efficiency can more effectively produce infectious viral vectors. Exemplarily, when HEK-293T cell monoclonal cell lines are screened for growth rate / viral packaging efficiency, the growth rate / viral packaging efficiency of each cell line is ranked, and the 1st to Nth cell lines with the highest growth rate / viral packaging efficiency are selected as the cell lines to be suspended and acclimated, where N is an integer from 1 to 100, for example, an integer from 1 to 10, an integer from 1 to 20, an integer from 1 to 40, an integer from 1 to 50, an integer from 1 to 60, an integer from 1 to 80, etc., and can be flexibly selected according to actual needs.

[0050] According to an embodiment of the present application, step S2 includes:

[0051] Step S2-1: Subculture the HEK-293T cell monoclonal cell line and select at least one cell line with a fast growth rate. In some embodiments, the method for screening at least one cell line with a fast growth rate comprises: transferring the screened HEK-293T monoclonal cell line to a new multi-well plate, culturing for 2 to 4 days, screening out cell wells with a confluence greater than 60%, and transferring them to new cell wells (the size of the cell wells of each passage is larger than that of the previous generation to provide a larger culture space, which is conducive to cell proliferation, and the cell density is 1×10 4 ~5×10 4 cells / cm 2 ) Every 2-4 days, cells in good growth condition are passaged. When the cell confluence reaches 90% or above, the cells are digested and transferred to new wells according to the cell culture method. The cells are cultured for 2-4 days. Slow-growing cell lines are discarded, and the remaining cell lines are transferred to plates for expansion culture for 2-4 days and stored. This method can obtain cell lines with high cell viability and fast growth rate, which facilitates the subsequent suspension acclimation.

[0052] Step S2-2: Each HEK-293T cell line screened out in step S2-1 is packaged with a lentivirus, and then each group of packaged lentivirus is used to infect the same batch of HEK-293T cells. After culture, signal detection is performed to screen out at least one cell line with high signal intensity as the cell line to be suspended and acclimated. Thus, the lentivirus is packaged and infected with the virus to screen out cell lines with high packaging efficiency and strong infection ability. Specifically, after the HEK-293T cell line is packaged with the lentivirus, the virus stock solution is collected. Take the same volume of various virus stocks and infect the HEK-293T cell lines that produce the virus, collect the detection signals, and the signal intensity represents the titer of the virus stock solution, that is, the ability of the cell to package the virus.

[0053] According to the examples of the present application, each of the cell lines is co-transfected with a basic packaging plasmid and a plasmid for generating a signal marker, so as to package the lentivirus.

[0054] According to an embodiment of the present application, the plasmid for generating the signal marker is selected from a plasmid encoding a fluorescent protein gene. This facilitates labeling of cells. The fluorescent protein can be mCherry, GFP, EGFP, BFP, RFP, YFP, etc.

[0055] According to an embodiment of the present application, the basic packaging plasmid includes a VSVG envelope protein plasmid, a REV auxiliary protein plasmid and an RRE sequence plasmid, thereby facilitating intracellular packaging to form a virus.

[0056] According to an embodiment of the present application, the mass ratio of the plasmid for generating the signal marker, the VSVG envelope protein plasmid, the REV auxiliary protein plasmid, and the RRE sequence plasmid is (0.5-2):(0.5-2):(0.5-2):(0.5-2). As a result, the expression levels of various plasmids in cells are better, thereby improving the packaging efficiency and yield of the virus.

[0057] Subculture in S3 suspension medium

[0058] In this step, the cell lines to be suspended and adapted are transferred to a suspension culture medium for subculture. Based on the cell clumping rate and / or cell viability after subculture, HEK-293T cells for suspension culture are screened. The screened cell lines to be suspended and adapted are subcultured in suspension to transition the HEK-293T cells from an adherent growth mode to a suspension growth mode. Using the clumping rate and / or cell viability of each cell line as evaluation indicators, cells with good dispersibility and high survival rate during suspension culture are screened as target cells.

[0059] According to embodiments of the present application, the suspension culture medium includes at least one of Transpro CD 01plus medium, Aplo CD293 medium, and Aplo 293 medium. These three culture media are typically used for adherent culture. The inventors of this application have discovered through extensive experiments that these three media can be used for suspension culture. These media exhibit superior cell viability, proliferation rate, and stability, with relatively low clumping, in 293T cells. In some preferred embodiments, the suspension culture medium includes Transpro CD01plus medium.

[0060] According to an embodiment of the present application, the suspension culture medium includes an anti-agglomerating agent. During the suspension culture process, by adding the anti-agglomerating agent, the aggregation and clumping of cells can be reduced, thereby improving the dispersion of cells, helping cells to be more evenly distributed in the culture medium, increasing the contact area between cells and the culture medium, promoting the absorption of nutrients and the discharge of metabolic waste, thereby improving the growth efficiency and viability of cells, and reducing the growth inhibition and death of cells caused by clumping, thereby improving the survival rate and stability of cells.

[0061] According to an embodiment of the present application, the anti-agglomerating agent includes at least one of dextran sulfate, ferric citrate, sodium ferric EDTA, and a commercial anti-agglomerating agent (such as an anti-clumping agent). Among them, dextran sulfate is preferred because it has a better anti-agglomerating effect and is low in cost.

[0062] According to an embodiment of the present application, the concentration of the anti-agglomerating agent in the suspension culture medium is 0.05 g / L to 0.4 g / L. Exemplarily, the concentration of the anti-agglomerating agent is 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, and 0.4 g / L. In this way, HEK-293T cell agglomeration can be effectively inhibited, and its growth rate and viability can be increased. It can also avoid the toxicity to cells caused by excessive addition of anti-agglomerating agents, which may affect cell metabolism and function and even cause culture failure.

[0063] According to an embodiment of the present application, the suspension culture medium further comprises glutamine, thereby providing energy for cell growth.

[0064] According to the examples of the present application, the concentration of glutamine in the suspension culture medium is 1mM to 5mM. Exemplarily, the concentration of glutamine is 1mM, 2mM, 3mM, 4mM, or 5mM. This ensures that the energy required for normal cell growth and metabolism is sufficient, while preventing excessive addition of glutamine from increasing the metabolic burden and even affecting the stability of the cell growth environment, leading to slow cell growth or metabolic abnormalities.

[0065] According to an embodiment of the present application, in step S3, the viable cell density of the cell line to be suspended and acclimated is first transferred to the suspension culture medium is 5×10 5 cells / mL~5×10 6 cells / mL, subcultured every 2 to 4 days, with 4×10 5 ~5×10 5 Cells / mL viable cell density should be replaced with fresh suspension culture medium. The initial transfer can cause a significant number of cells to die due to the sudden change in culture conditions, so it is important to ensure that sufficient cells are transferred to ensure that more cells adapted to suspension culture survive. The transfer density can be reduced during subsequent passages as the cells gradually adapt to the suspension culture environment.

[0066] According to an embodiment of the present application, in step S3, the liquid volume of the suspension culture medium for the first transfer is 10 to 25 mL / 125 mL, for example, 10 mL / 125 mL, 12 mL / 125 mL, 15 mL / 125 mL, 16 mL / 125 mL, 18 mL / 125 mL, 20 mL / 125 mL, 22 mL / 125 mL, and 25 mL / 125 mL; the liquid volume of the fresh suspension culture medium replaced each time is 25 to 35 mL / 125 mL, for example, 25 mL / 125 mL, 26 mL / 125 mL, 28 mL / 125 mL, 30 mL / 125 mL, 32 mL / 125 mL, 34 mL / 125 mL, and 35 mL / 125 mL. Thus, the cells can obtain sufficient nutrients and oxygen during the suspension culture process, while reducing mechanical shear force and aggregation between cells, and the cells are evenly distributed, thereby improving the survival rate, growth rate, and stability of the cells.

[0067] According to an embodiment of the present application, the subculture adopts a shaking culture method with a rotation speed of 100 rpm to 150 rpm, a culture temperature of 35°C to 39°C, and a CO2 concentration of 5% to 10%. For example, the rotation speed is 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, and 150 rpm, the culture temperature is 35°C, 36°C, 37°C, 38°C, and 39°C, and the CO2 concentration is 5%, 6%, 7%, 8%, 9%, and 10%. This is conducive to the growth of HEK-293T cells.

[0068] According to the embodiment of the present application, each generation is cultured to a viable cell density of 3×10 6 cells / mL or more, preferably 3×10 6 ~5×10 6 cells / mL (e.g. 3×10 6 cells / mL、3.5×10 6 cells / mL、4×10 6 cells / mL、4.5×10 6 cells / mL、5×10 6 cells / mL), passage is performed. Thus, the cells are more active and have a better proliferation rate. When passage is made to a new culture system, they can better adapt to the environment and maintain a better growth state. If the cell density is too high, the probability of intercellular contact will increase, resulting in cells easily agglomerating. In addition, when using a cell counter for detection, if the cell density is too high, two or more cells that are in contact with each other will be mistakenly judged as a cell cluster, resulting in a higher cell agglomeration rate detection value.

[0069] According to an embodiment of the present application, when the subculture is performed until the cell clumping rate is no higher than a first preset value and / or the cell viability is no lower than a second preset value, HEK-293T cells suitable for suspension culture are obtained; the first preset value is 18% to 25%, for example, 18%, 20%, 22%, 24%, or 25%; the second preset value is 85% to 95%, for example, 85%, 86%, 88%, 90%, 92%, 94%, or 95%. Thus, the cells obtained by screening have a low clumping rate and / or a high cell viability and can be used in suspension culture.

[0070] According to an embodiment of the present application, when the subculture is performed until the cell clumping rate is no higher than the first preset value and the cell viability is no lower than the second preset value, cells with a high proliferation rate are further screened to obtain HEK-293T cells for suspension culture. In some embodiments, HEK-293T cells for suspension culture are screened using cell viability, cell clumping rate, and cell proliferation rate as evaluation indicators. Specifically, cells with a cell viability not lower than the second preset value are screened first. On the basis of satisfying the cell viability, cells with a cell clumping rate that meets the standard (no higher than the first preset value) are considered. The proliferation rates corresponding to the re-screened cells are then sorted, and cells with a high cell proliferation rate are selected as target cells.

[0071] In some embodiments, the "proliferation rate" is related to the "viable cell density". It is generally believed that under the same culture conditions, a higher viable cell density indicates a faster proliferation rate.

[0072] According to an embodiment of the present application, when the cell clumping rate after the subculture is higher than the first preset value, the cells after the subculture are further subcultured for at least one generation until the cell clumping rate is no higher than the first preset value, thereby obtaining HEK-293T cells for suspension culture.

[0073] In some embodiments, when the cell agglomeration rate after the subculture is higher than the first preset value, the cells after the subculture are further subcultured for one generation, and the following two situations occur:

[0074] 1) when the cell clumping rate decreases and the cell viability does not decrease, if the cell clumping rate is not higher than the first preset value, obtaining HEK-293T cells for suspension culture; if the cell clumping rate is higher than the first preset value, continuing the subculture;

[0075] 2) If the cell clumping rate does not decrease and / or the cell viability decreases, it indicates that the cells are in poor condition in the suspension culture system and should be discarded without further subculturing.

[0076] According to an embodiment of the present application, step S3 includes: placing the cell line to be suspended, DMEM medium and fetal bovine serum (FBS, added in an amount of 8% to 12% of the mass of the DMEM medium) and then placing the packaged lentivirus in a well plate or square bottle, 35°C to 39°C, CO2 concentration of 4% to 6%, static culture, subculture once every 2 to 4 days, and subculture until the cell viability reaches more than 95%, according to 8×10 5 cells / mL~2×10 6 cells / mL of viable cells were transferred to the suspension culture medium, the liquid volume was 15-25mL / 125mL, the rotation speed was 100rpm-120rpm, the culture temperature was 36℃-38℃, the CO2 concentration was 7%-9%, and the culture was carried out for 2-4 days. 6 cells / mL, and the passage was carried out with the following conditions for each passage: 4×10 5 ~5×10 5 cells / mL, the viable cell density is replaced with fresh suspension culture medium, the liquid volume is 28-32 mL / 125 mL, the rotation speed is 100 rpm-120 rpm, the culture temperature is 36°C-38°C, the CO2 concentration is 7%-9%, and the cells are subcultured once after 2-3 days of culture; when the cell clumping rate is 18%-25% and the cell viability is not less than 90%, HEK-293T cells for suspension culture are obtained. Thus, using this method, cells with low clumping rate and / or high cell viability can be obtained, which can be applied to suspension culture.

[0077] HEK-293T cells for suspension culture

[0078] In another aspect, this application provides HEK-293T cells for suspension culture. According to an embodiment of this application, the HEK-293T cells for suspension culture are obtained using the aforementioned HEK-293T cell suspension acclimation method. Thus, the HEK-293T cells for suspension culture described herein can be cultured in suspension, are less prone to clumping in the suspension culture environment, have high cell viability, and are highly stable. After acclimation, the cells can be passaged 15 times with a doubling difference of less than 10% between passages.

[0079] It should be noted that the features and advantages described above for the HEK-293T cell suspension acclimation method are also applicable to the HEK-293T cells used for suspension culture and will not be repeated here.

[0080] Method for culturing HEK-293T cells in suspension

[0081] In another aspect of the present application, the present application proposes a method for culturing HEK-293T cells in suspension. According to an embodiment of the present application, the method comprises: suspending and culturing the HEK-293T cells obtained by the HEK-293T cell suspension acclimation method described above using a suspension culture medium; the suspension culture medium is selected from the suspension culture medium in the HEK-293T cell suspension acclimation method described above. Thus, the method can be used to achieve HEK-293T cell suspension culture, which is conducive to achieving high-density growth of HEK-293T cells and increasing cell yield, thereby meeting the needs of large-scale production of biological products, such as viral vectors, recombinant proteins, etc.; it can also reduce dependence on the surface area of the culture vessel and reduce the culture cost. Compared with traditional adherent culture, the cell culture cost can be reduced by 50% (material cost), the culture efficiency is improved, and it is suitable for large-scale production.

[0082] It should be noted that the features and advantages described above for the HEK-293T cell suspension acclimation method are also applicable to the present method for culturing HEK-293T cells in suspension and will not be described in detail here.

[0083] Methods for producing viruses

[0084] In another aspect of the present application, the present application proposes a method for producing viruses. According to an embodiment of the present application, the method comprises: transfecting a lentiviral packaging plasmid into HEK-293T cells obtained by the HEK-293T cell suspension acclimation method described above, and performing suspension culture; wherein the suspension culture medium is selected from the suspension culture medium in the HEK-293T cell suspension acclimation method described above. Thus, the method of the present application can achieve large-scale virus production with high production efficiency and low cost. Compared with traditional adherent culture, the material cost required to produce the same amount of virus is reduced by more than 70%. In addition, the produced virus has a strong infectivity, and the positive rate of infected T cells can reach about 60%, which reaches and exceeds the positive rate of adherent packaged viruses, and has high application value.

[0085] According to an embodiment of the present application, the method comprises: preparing 1×10 6 ~5×10 6 cells / ml cell fluid, add lentiviral packaging plasmid and PEI (Polyethylenimine), 100-150rpm, 35℃-39℃, CO2 concentration of 5%-10%; culture for 18-22h, feed Transpro feed1 medium at a ratio of 1%-5%, and continue to culture for 2-3 days, and collect the virus.

[0086] It should be noted that the characteristics and advantages described above for the HEK-293T cell suspension acclimation method are also applicable to this virus production method and will not be repeated here.

[0087] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0088] The definition of cell generations embodied in the embodiments of this application is: transferring new culture medium at a certain density is one generation; the number of passages is the number of times the culture has been subcultured. During the cell culture process, passage (diluting the cell suspension into fresh culture medium, and each replacement is considered a generation [passage]) is used to distinguish cells of different generations, and can be interchanged with "P".

[0089] Example 1 - Dilution and separation screening of monoclonal cell lines

[0090] HEK-293T cells were revived and cultured in DMEM medium + 10% FBS until the cell confluence reached about 85% and the cell density was 2.5E+04 cells / cm 2 Passage and continue culturing until the cells are in good condition.

[0091] Use DMEM medium + 10% FBS to perform limiting dilution of the above-mentioned cells in good condition to a cell density of 3.75E+02 cells / mL, and plate them on 96-well cell culture plates at 180μl / well cell solution (i.e. 0.2 cells per well), with a total of 10 plates, named: 293T-P00-240331-01~10, and culture them statically at 37°C and 5% CO2. Change the medium once after 5 days. After 7 days of culture, the cell wells that have grown into a single cell population are screened under a microscope and marked. Discard non-proliferated cells, and the remaining cells are passaged to 48-well plates; after culturing the 48-well plates for 3 days, the cell wells with a confluence of more than 60% are screened under a microscope for counting and passaged to 24-well plates; after culturing the 24-well plates for 4 days, observe under a microscope to eliminate non-proliferated single clones, and the remaining cells are digested and counted at 2E+04 cells / cm 2 Subculture to 12-well plates; after culturing for 4 days, the confluence of the 12-well plates was observed under a microscope to be above 90%, and the cells were digested and counted according to 2E+04 cells / cm 2 Subculture to 6-well plates; remove the 6-well plates after three days of culture, discard the two slow-growing cell lines, and calculate the cell density at 2.00E+04 cells / cm 2 The cells were expanded to 10 cm dishes and incubated at 37°C, 5% CO2 for 3 days. Observation and evaluation were performed under an inverted microscope. A total of 33 monoclonal cell lines were obtained and stored as individual clones, designated 293T-P05-240430-001 to 033-YD, in liquid nitrogen. The clones were then plated in 6-well plates for lentiviral packaging.

[0092] The mCherry fluorescent protein expression plasmid pmCherry was constructed and co-packaged with the three basic packaging plasmids pMD2.G, pRSV-REV and pRRE at a ratio of 1:1:1:1 for lentivirus. PEI (mother solution concentration 1 mg / mL) was used as the auxiliary transfection reagent, and the total plasmid mass: PEI mass = 1:2. The culture medium used was 3% FBS+DMEM. The transfection culture conditions were 37°C and 5% CO2; the medium was changed after 6 hours and the co-culture was carried out for 48 hours. Each monoclonal cell line was individually packaged with lentivirus and the virus stock solution was harvested. The same volume of virus stock solution was used to infect the 293T cells producing the virus in a 96-well plate (the same 293T cells were used at a rate of 1.58E+05 cells / cm 2 The cells were plated in a 96-well plate and incubated at 37°C in 5% CO2 for approximately 24 hours. After 24 hours of incubation, the fluorescence value was detected using a microplate reader with an excitation wavelength of 588 nm and an emission wavelength of 613 nm. Figure 2The five monoclonal strains with the highest fluorescence values were screened out: 007#, 008#, 015#, 020# and 027#.

[0093] Example 2—Suspension acclimation of 020# monoclonal cell line

[0094] 1. Cell recovery and adherent culture

[0095] 1) Take 293T-P05-240430-020-YD (020#), quickly thaw at 37°C, transfer to DMEM medium + 10% FBS, centrifuge, discard the supernatant, resuspend in DMEM medium + 10% FBS, transfer to a T75 cell flask, spread evenly, name it 293T-P06-240517-020, and culture at 37°C with 5% CO2.

[0096] 2) After 3 days of culture, discard the old cell culture medium, rinse with DPBS, and digest with TrypLE Select (1×) at 37°C for 2-3 minutes. Once the cells are detached from the bottom of the flask, add DMEM medium + 10% FBS to terminate the digestion and gently pipette to disperse the cells. Take a small amount of cell suspension and stain with solution for counting and viability testing. Then, subculture the cells into new T75 flasks. Name the cells 293T-P07-240520-020 and incubate at 37°C with 5% CO2.

[0097] 3) After 3 days of culture, the cells were digested and collected using the same method as in step 2). The cell viability was 95.41%, indicating that the cells could be switched to suspension culture.

[0098] 2. Suspension acclimation culture

[0099] 1) First transfer to suspension culture screening medium

[0100] Four culture media were used: Transpro CD 01plus (Dongning Biotechnology), VP-SFM (gibico), Aplo CD293 (Yishengke), and Aplo 293 (Yishengke). The cells collected in step 1 (3) were transferred to suspension culture at a concentration of 1E+06 cells / mL in 125mL shake flasks with a volume of 20mL / flask. Culture conditions were 37°C, 110 rpm, and 8% CO2. Cell status was monitored daily. After 3 days of culture, the sampled cell fluid was added to an equal volume of 0.2% trypan blue staining solution, mixed thoroughly, placed in a cell counting plate, and inserted into a cell viability analyzer to obtain monitoring results.

[0101] Table 1. Cultivation of 020# monoclonal clone in different suspension media

[0102] culture medium Cell viability% Viable cell density cells / mL Agglomeration rate% Transpro CD 01plus 95.74 4.13E+06 78.09 VP-SFM 18.06 2.45E+04 9.33 Aplo CD293 72.95 1.21E+06 91.32 Aplo 293 83.86 1.75E+06 79.4

[0103] result:

[0104] A. The viability of 293T cells in VP-SFM medium was significantly low, and the cells did not proliferate, so they were abandoned;

[0105] B. The cell viability in Aplo CD293 and Aplo 293 culture media was relatively low, and the cell proliferation was approximately 1-fold after 3 days of culture, which was relatively slow.

[0106] C. Cells grew normally using Transpro CD 01plus medium, with viability maintained above 90%. Cell density tripled after three days of culture. Continue subculturing and monitoring.

[0107] 2) Suspension culture subculture

[0108] After comparing the four culture media, Transpro CD 01plus showed a clear advantage. Subculture studies will be conducted on it in the future.

[0109] In the passage investigation, the cells in step 2 (1) were cultured in Transpro CD 01plus medium for 3 days before being passaged. The culture medium used for passage culture was Transpro CD 01plus medium, to which the anti-agglomerating agent dextran sulfate was added at two ratios of 0.1 g / L and 0.2 g / L. The first passage was at a density of 5E+05 cells / mL, and the subsequent passages were at a density of 4E+05 cells / mL. 125 mL shaking flasks were used, and the liquid volume was 30 mL / bottle. The culture conditions were 37°C, 110 rpm, and 8% CO2. The monitoring results showed that the acclimated 293T cells grew stably (the doubling time was about 2.9±0.15 in 3 days), the agglomeration rate gradually decreased, and the cells were well dispersed after acclimation for 3 generations, and the agglomeration rate dropped to below 20%. See the comparative diagram for details. Figure 3 .

[0110] 3. After suspension acclimation is completed, examine whether the anti-agglomeration agent can be removed

[0111] To determine whether the anti-clumping agent could be removed during subsequent passages, cells were passaged in 30 mL / 125 mL shake flasks at a density of 4E+0.5 cells / mL. Culture conditions remained unchanged, and growth differences were compared between continued passages and those without the anti-clumping agent. Data from the acclimation process and monitoring after the removal of the anti-clumping agent are shown in Table 2.

[0112] Table 2.020# monoclonal cell suspension culture results in Transpro CD 01plus

[0113]

[0114] Note: Parallel 1 was added with 0.1 g / L dextran sulfate, and replicate 2 was added with 0.2 g / L dextran sulfate. The cell doubling value* refers to the cell doubling value from the time of inoculation at 4E+05 cells / mL to the end of culture day 3.

[0115] Table 3. Results of investigation on the effect of removing anti-agglomeration agent after suspension acclimation of 020# monoclonal cell line

[0116]

[0117]

[0118] Note: "-DS" indicates no anti-agglomerating agent was added. Cell doublings* refer to the cell doublings from the time of seeding at 4E5 cells / mL to the end of 3 days of culture.

[0119] According to the observation results after 4 passages, there was no obvious difference between the first two generations of cells after the anti-agglomeration agent was withdrawn, a small amount of aggregation occurred in the third generation without the addition of anti-agglomeration agent, and the fourth generation had obvious aggregation ( Figure 4 In contrast, cells that were subcultured with the addition of an anti-agglomerating agent showed stable growth and good dispersion.

[0120] 4 Summary

[0121] Monoclonal 293T cell line 020# was acclimated to suspension culture using Transpro CD 01plus medium supplemented with 0.1g / L or 0.2g / L anti-clumping agent. Culture conditions were 37°C, 110 rpm, and 8% CO2. When adherent culture reached a viability of over 95%, cells were directly switched to suspension culture. During the initial transition to suspension culture, the cell density was controlled at 1E+06 cells / mL. Cells were passaged every three days. Subsequently, cells were passaged at a density of 4E+05 to 5E+05 cells / mL for three generations until the clumping rate dropped below 20%, good dispersibility, and stable growth were achieved. The anti-clumping agent was not removed during continued culture, as long as the above culture conditions were met.

[0122] Example 3—Suspension acclimation of 008#, 015#, and 027# monoclonal strains

[0123] Cells 293T-P05-240430-015 / 008 / 027-YD (008#, 015#, 027#) were revived and resuspended in DMEM medium + 10% FBS and cultured in T75 flasks. Cell viability was monitored and maintained above 95% for 3 days. Cells were then transferred to suspension culture at a viable cell density of 1E+06 cells / mL using a Transpro CD 01plus + 0.1 g / L dextran sulfate (DSS) + 4 mM glutamine formulation at 37°C, 110 rpm, and 8% CO2. Cell viability and density were monitored after 3 days of culture and passaged at a viable cell density of 4E+05 cells / mL. A 30 mL / 125 mL volume was used for the acclimation process. After three generations of acclimation (P08-P10), the clumping rate of 027# did not reach the expected level below 25%. Therefore, all three clones were acclimated for an additional generation (P11). The clumping rate, cell viability, and viable cell density all met the standards, making them suitable for use as the target cells. The monitoring data for the suspension acclimation of the three clones are shown in Table 4.

[0124] Table 4 Suspension culture of 008#, 015# and 027# monoclonal clones

[0125]

[0126]

[0127] Furthermore, the acclimated P11 cells were passaged at a viable cell density of 4E+05 cells / mL to examine the differences between removing the anti-clumping agent and continuing to add the agent (maintaining a final concentration of 0.1 g / L). The results showed that, for the three monoclonal clones, cell viability was relatively low when the anti-clumping agent was removed at the same passage after acclimation, and the viable cell density was slightly lower for the same culture time. By the third passage after removing the anti-clumping agent, the viability of 008# cells dropped below 80%, while the clumping rate reached over 40%, significantly lower than the control group in which the anti-clumping agent was continued.

[0128] Example 4—Growth Curve Assay

[0129] Successfully acclimated monoclonal cell lines 020# and 015# were revived and cultured in Transpro CD01plus + 0.1g / L DSS + 4mM glutamine. Cells were seeded into fresh culture medium at a density of 4E+0.5 cells / mL, and shake flasks were filled with 30mL / 250mL of culture medium. Culture conditions were: 37°C, 110 rpm, 8% CO2. Cells were sampled daily from inoculation until day 9 of culture to measure viable cell density and viability. Monitoring data are shown in Table 5 below. Figure 5 and Figure 6 .

[0130] Table 5. Growth curve measurement data

[0131]

[0132] Results showed that 015# grew faster, reaching its highest viable cell density on day 5, while 020# reached its peak on day 6. The peak viable cell density of 020# was significantly higher, approaching 1E+07 cells / mL. Cell viability data showed that the viable cell density remained above 94% until the peak, after which it gradually declined. Cell diameters were approximately 17μm, with 020# being relatively larger. Regarding the clumping rate, 015# was relatively stable, generally maintaining below 30%. The clumping rate also increased at higher cell densities, with 020# having a higher clumping rate at the same cell density.

[0133] Example 5020# and 015# suspension culture passage stability study

[0134] The cells 293T-P13-XF-020#-240610-1-YD and 293T-P14-XF-015#-240709-YD stored after 020# monoclonal acclimation were revived and inoculated into 20mL / 125mL shake flasks respectively. The suspension culture medium was the same as in Example 3. The culture conditions were 110rpm, 37°C, and 8% CO2. After three days of culture, samples were taken to monitor parameters such as viability and cell density, and the cells were subcultured and inoculated into 30mL / 125mL shake flasks at a viable cell density of 4E+05cells / mL for a total of 16 passages. The seed-keeping passages for 020# were P15 / 20 / 27 / 30, and the seed-keeping passages for 015# were P16 / 21 / 28 / 31. The cells were compared and investigated as follows:

[0135] 1. Comparison of growth conditions

[0136] Cell growth doublings were relatively stable, with 015# doubling slightly faster overall than 020#. During passage studies, the cell viability of both clones remained above 94.5%, with 015# showing more stable cell viability. Aggregation rates were generally stable, with 015# exhibiting lower agglomeration than 020#, indicating better dispersibility.

[0137] 2. Comparison of packaged lentivirus

[0138] Monoclonal 015# and 020# cells were selected for the packaging of the lentivirus LVS2-D72-CTMI4. Five plasmids were required: pMD2.G-VSVG, pRSV-REV, pRRE, pLV-CAR1, and p2G-S2. The plasmid ratio was 1:1:1:1:0.5.

[0139] Two shake flasks (20 mL / 125 mL) were packaged, each containing cells cultured to a viability of over 95%. Cells were harvested by centrifugation and resuspended in Transpro CD 01 plus (4 mM glutamine) to a viable cell density of 2E+06 cells / mL. Plasmid solution A and the PEI cotransfection agent were prepared in Transpro CD 01 plus (4 mM glutamine) at a plasmid dosage of 3 μg / mL and a PEI to plasmid mass ratio of 2.25:1. 20 hours after the packaged virus was transfected, Transprofeed 1 (Dongning Bio) was added at 2% (v / v). Two and three days after packaging, one vial of virus was collected, the virus solution was concentrated with PEG, and finally resuspended in CTS-complete medium at 1 / 10 the original volume (i.e., a 10-fold concentration). Finally, the infectious titer of the packaged virus concentrates before and after passage was measured. The results are shown in Table 7.

[0140] Table 7. Comparison of packaging efficiency of LVP1-CAR1 virus before and after passage in suspension culture 293T

[0141]

[0142] The 2-day titer of the virus packaging cycle of the two monoclonal cells at different generations was higher than the 3-day titer; the efficiency of lentivirus packaging did not decrease after passaging compared with before passaging, indicating that the two monoclonal cell lines had good genetic stability.

[0143] Example 6—Suspension packaging of lentivirus and infection of T cells

[0144] The 2d virus concentrate harvested in Example 5 was used to infect T cells. Taking the synchronously adherent packaged virus as a reference, the infection steps are as follows: 5E+05 cells / well of T cells activated for 3 days were added to a 24-well cell culture plate, resuspended with CTS complete medium to a cell density of 1E+06 cells / ml, and the corresponding amount of virus was added according to MOI=20. The infection aid lenti Boost was added at a ratio of 4μl / ml. After gently mixing, the cells were cultured at 37°C and 5% CO2. The cells were observed every day, and samples were taken after 1 day to measure the cell viability and density. The cells had proliferated by about 2 times, and the viability was about 85%. The cells were transferred to 6-well plates supplemented with culture medium for expansion for 2 days. The positive rate was detected by flow cytometry on the 4th day after T cell infection. The flow cytometry results are as follows. Figure 7 and shown in Table 8.

[0145] Table 8 Virus infection ability of T cells

[0146]

[0147] The results showed that the T cell infection rate of the suspension-packaged virus was significantly higher than that of the adherent-packaged virus. Comparing the two monoclonal clones, 020# had a slightly higher infection efficiency than 015# (showing the same trend before and after passage), and the virus titer packaged with 015# was significantly higher than that with 020#. This indicates that using the same virus packaging process, 015# can harvest more virus, which in turn infects more T cells. This example demonstrates that while individual HEK-293T monoclonal cells cultured using the same suspension-cultured process may vary, they all exhibit significant advantages in high yield and positive rates when applied to lentiviral packaging.

[0148] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for acclimating HEK-293T cells by suspension, characterized in that: include: Step S1: obtain HEK-293T monoclonal cell line by gradient dilution; Step S2: selecting at least one cell line with a fast growth rate and / or high virus packaging efficiency from the HEK-293T monoclonal cell line as the cell line to be suspended and acclimated; Step S3: transferring the cell line to be suspended and acclimated to a suspension culture medium for subculture, and selecting HEK-293T cells for suspension culture based on the cell aggregation rate and / or cell viability after subculture; Wherein, the suspension culture medium comprises at least one of Transpro CD 01plus culture medium, Aplo CD293 culture medium and Aplo293 culture medium.

2. The method according to claim 1, characterized in that The suspension culture medium includes Transpro CD 01plus culture medium.

3. The method according to claim 1 or 2, characterized in that The suspension culture medium includes an anti-agglomerating agent, wherein the anti-agglomerating agent includes at least one of dextran sulfate, ferric citrate and sodium ferric EDTA; Optionally, the concentration of the anti-agglomerating agent in the suspension culture medium is 0.05 g / L to 0.4 g / L.

4. The method according to claim 1, wherein Step S2 includes: Step S2-1: subculturing the HEK-293T monoclonal cell line and selecting at least one cell line with a fast growth rate; Step S2-2: Each HEK-293T cell line screened in step S2-1 is packaged with a lentivirus, and each group of packaged lentivirus is used to infect the same batch of HEK-293T cells. After culture, signal detection is performed to screen at least one cell line with a high signal intensity as the cell line to be suspended and acclimated.

5. The method according to claim 1, wherein In step S3, the viable cell density of the cell line to be suspended and acclimated is first transferred to the suspension culture medium at 5×10 5 cells / mL~5×10 6 cells / mL, subcultured every 2 to 4 days, with 4×10 5 ~5×10 5 The suspension medium was replaced with fresh one at a viable cell density of cells / mL.

6. The method according to claim 1, characterized in that In step S3, the volume of the suspension culture medium for the first transfer is 10-25 mL / 125 mL, and the volume of the fresh suspension culture medium for each subculture is 25-35 mL / 125 mL; Optionally, the subculture adopts a shaking culture method with a rotation speed of 100 rpm to 150 rpm, a culture temperature of 35° C. to 39° C., and a CO2 concentration of 5% to 10%; Optionally, culture each passage to a viable cell density of 3 × 10 6 cells / mL or above, preferably 3×10 6 ~5×10 6 cells / mL, passage was performed; Optionally, when the subculture is performed until the cell agglomeration rate is no higher than a first preset value and / or the cell viability is no lower than a second preset value, HEK-293T cells for suspension culture are obtained; The first preset value is 18% to 25%; the second preset value is 85% to 95%; Optionally, when the subculture is performed until the cell agglomeration rate is no higher than a first preset value and the cell viability is no lower than a second preset value, further screening cells with a high proliferation rate to obtain HEK-293T cells for suspension culture; Optionally, when the cell clumping rate after the subculture is higher than the first preset value, the cells after the subculture are further subcultured for at least one generation until the cell clumping rate is no higher than the first preset value, thereby obtaining HEK-293T cells for suspension culture.

7. The method according to claim 1 or 6, characterized in that Step S3 includes: The cell line to be suspended and acclimated, DMEM medium and fetal bovine serum were placed in a well plate or square bottle, and cultured at 35°C to 39°C, with a CO2 concentration of 4% to 6%. The cells were passaged every 2 to 4 days. After the cell viability reached more than 95%, the cells were cultured at 8×10 5 cells / mL~2×10 6 cells / mL were transferred to the suspension culture medium, the liquid volume was 15-25mL / 125mL, the rotation speed was 100rpm-120rpm, the culture temperature was 36℃-38℃, the CO2 concentration was 7%-9%, and the culture was carried out for 2-4 days. 6 cells / mL, and the passage was carried out with the following conditions for each passage: 4×10 5 ~5×10 5 cells / mL, replace the fresh suspension culture medium with a liquid volume of 28-32 mL / 125 mL, a rotation speed of 100 rpm-120 rpm, a culture temperature of 36° C.-38° C., a CO2 concentration of 7%-9%, and culture once after 2-3 days; when the cell clumping rate is 18%-25% and the cell viability is not less than 90%, HEK-293T cells for suspension culture are obtained.

8. A HEK-293T cell for suspension culture, characterized in that: The HEK-293T cells for suspension culture are obtained by the HEK-293T cell suspension acclimation method according to any one of claims 1 to 7.

9. A method for culturing HEK-293T cells in suspension, characterized in that: include: The HEK-293T cells obtained by the HEK-293T cell suspension acclimation method according to any one of claims 1 to 7 are suspended and cultured in a suspension medium; The suspension culture medium is selected from the suspension culture medium in the HEK-293T cell suspension acclimation method according to any one of claims 1 to 7.

10. A method for producing a virus, characterized in that include: transfecting the lentiviral packaging plasmid into the HEK-293T cells obtained by the HEK-293T cell suspension acclimation method according to any one of claims 1 to 7, and performing suspension culture; Wherein, the suspension culture medium is selected from the suspension culture medium in the HEK-293T cell suspension acclimation method according to any one of claims 1 to 7.

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