A method for recovering and culturing ROBO1 CAR NK cells

Abstract

The present application relates to the fields of bioengineering and technology, and specifically to a method for resuscitating and culturing ROBO1CAR NK cells. The method comprises: resuscitating and culturing the cells using a resuscitation medium, wherein the resuscitation medium is a serum-free HIPP-T009 lymphocyte culture medium containing IL-2 and nicotinamide; performing a primary amplification culture on the resuscitated cells using a first amplification culture medium to obtain primary amplified cells; and performing a secondary amplification culture on the primary amplified cells using a second amplification culture medium to harvest the cells. The method can rapidly resuscitate frozen cells, effectively maintain cell growth and cell killing activity, and expand the culture scale, greatly reducing the culture cycle and production costs.

Description

Technical Field

[0001] The present application relates to the fields of bioengineering and technology, and specifically to a method for resuscitating and culturing ROBO1 CAR NK cells. Background Art

[0002] Cancer immunotherapy has become the fourth leading cancer treatment option after surgery, radiotherapy, and chemotherapy, and is currently the most popular approach. Cancer immunotherapy has completely overturned previous understandings of cancer treatment: from the prior approach of direct tumor cell destruction through external interventional therapy to a strategy of tumor cell elimination through enhancing the patient's own immune system. Cancer immunotherapy primarily works by systemically stimulating and enhancing the normal defenses of the patient's immune system to kill and eliminate tumor cells. Compared to traditional treatments, this emerging approach offers greater targeting and specificity, reduced heterogeneity, minimal toxicity, and remarkable therapeutic efficacy. In particular, the tremendous success of cancer immunotherapy technologies in recent years, such as immune checkpoint inhibitors and chimeric antigen receptor (CAR)-modified immune cells, has propelled cancer treatment onto a faster and more robust trajectory. Clinical results have demonstrated that it is more effective than traditional cancer treatments such as surgery, chemotherapy, radiotherapy, and targeted therapies. It can combine the characteristics of monoclonal antibodies that target and recognize tumor cell surface antigens with the characteristics of immune effector cells that can quickly and effectively kill tumor cells, thereby fully unleashing the maximum potential of immune cells to specifically and efficiently kill tumor cells.

[0003] NK cells (Natural killer cells) are a type of effector cell that plays a vital role in the human immune system. They are named because they can exert their cell-killing effect without prior antigen sensitization. NK cells distinguish between "self" and "non-self" through a "missing-self" recognition pattern, recognizing MHC-I molecules on the surface of target cells through inhibitory receptors on their cell surface, thereby inhibiting NK cell killing. When normal cells express MHC-I molecules, they bind to inhibitory receptors and transmit NK cell inhibitory signals, preventing NK cells from being activated. In tumor cells with malignant lesions, the expression of MHC-I molecules is downregulated or even absent, and NK cells become activated. In addition, NK cells can also initiate cell-killing pathways through an "induced-self" mode. Under the action of activating receptors such as NKG2D and their ligands, NK cells can generate sufficiently strong activation signals that are unrestrained by inhibitory signals and directly activate NK cells. Once activated, NK cells can leave the peripheral blood circulation system under the influence of chemotactic factors, diffuse and infiltrate into sites of infection and tumor lesions, and then exert cytotoxic effects. Simultaneously, NK cells can secrete a large number of cytokines, such as IFN-γ, TNF-α, and GM-CSF, which can recruit and activate DCs, monocytes and macrophages, neutrophils, and other cells, thereby triggering adaptive immune responses. In addition to exerting CAR-dependent cell activity, NK-based chimeric antigen receptor-modified cells can also rely on the activation and activation mode of NK cells themselves. On the one hand, they can directly kill tumor cells by secreting killing-related molecules such as perforin and granzymes, and on the other hand, they can directly or indirectly interact with other immune cells to regulate adaptive immune responses.

[0004] As adoptive CAR NK cell therapy gains increasing attention in cancer treatment, the source of NK cells and the methods for their in vitro expansion and culture are also receiving increasing attention. A suitable NK cell source and in vitro expansion and culture method can, on the one hand, keep CAR NK cells in a highly activated state, ensuring their subsequent clinical therapeutic efficacy. On the other hand, the production scale of each batch of cells needs to meet the clinical use needs of more "allogeneic" patients, while also requiring the quality of each batch of cell products to be controlled. Currently, NK cells are mainly derived from primary NK cells from peripheral blood or umbilical cord blood. The conventional culture method is to extract freshly isolated or frozen peripheral blood mononuclear cells (PBMCs) (or umbilical cord blood mononuclear cells (CB-MNCs), add autologous plasma or human AB serum to the culture medium, and use feeder cells (such as K562 cells) expressing activating cytokines (such as mIL21 and IL15) and ligands (such as 41BBL) for stimulation and co-culture. Although this method has a certain effect on the expansion of NK cells. However, this method still has many limitations and shortcomings: the cell production and preparation process is complicated and involves many steps, the culture scale is limited (primary NK cells have limited in vitro expansion activity, which determines that they can only be expanded to a maximum culture scale of about 1-2L), it is expensive, and there are large batch differences (there are obvious individual differences in the starting cells between autologous or donors). These shortcomings limit the large-scale promotion and clinical application of CAR NK cell therapy in the biomedical field. Summary of the Invention

[0005] In light of this, the present invention provides a method for reviving and culturing ROBO1 CAR NK cells. This method can rapidly revive frozen cells, effectively maintaining cell growth and cytotoxic activity, while also expanding the culture scale, significantly reducing the culture cycle, and lowering production costs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for resuscitation and culture of ROBO1 CAR NK cells, wherein the ROBO1 CAR NK cells are resuscitated and cultured using a resuscitation medium, wherein the resuscitation medium is a HIPP-T009 lymphocyte serum-free medium containing IL2 and nicotinamide.

[0008] Preferably, in the recovery medium, the content of IL2 is 100-1000 IU / mL, and the content of nicotinamide is 1-10 mM.

[0009] In a specific embodiment provided by the present invention, the content of IL2 in the recovery medium is 500 IU / mL, and the content of nicotinamide is 5 mM.

[0010] Preferably, during the recovery culture process, fresh culture medium is replaced every 48 to 72 hours;

[0011] Preferably, during the recovery culture process, fresh culture medium is replaced every 48 to 52 hours. Under such culture conditions, the viability of the recovered cells can quickly reach over 80%.

[0012] More preferably, during the recovery culture process, fresh culture medium is replaced every 48 hours.

[0013] Preferably, the recovery culture conditions are 36-38° C., 4%-6% CO 2 , and the recovery culture time is 9 days or more.

[0014] Preferably, the recovery culture conditions are 37° C., 5% CO 2 , and the recovery culture time is 9 to 16 days.

[0015] The present invention also provides a method for culturing ROBO1 CAR NK cells, comprising the following steps:

[0016] The ROBO1 CAR NK cells were revived and cultured using the above-mentioned resuscitation culture method to obtain revived cells;

[0017] Performing primary expansion culture on the revived cells using the first expansion culture medium to obtain primary expansion cells;

[0018] The primary amplified cells are subjected to secondary amplification culture using a second amplification culture medium, and the cells are harvested.

[0019] Preferably, the first expansion medium is a HIPP-T009 lymphocyte serum-free medium containing IL2.

[0020] Preferably, the second expansion medium is a HIPP-T009 lymphocyte serum-free medium containing IL2 and an anti-clumping agent.

[0021] Preferably, the content of IL2 in the first expansion medium is 50 to 1000 IU / mL;

[0022] Preferably, the content of IL2 in the first expansion medium is 100 to 200 IU / mL. Under this IL2 concentration condition, not only the growth of cells can be maintained, but also their high cell killing activity can be maintained.

[0023] More preferably, the content of IL2 in the first expansion medium is 125 IU / mL;

[0024] As a preference, the seeding density of the primary expansion culture is (3-5)×10 5 cells / mL;

[0025] Preferably, the seeding density of the primary expansion culture is 3×10 5 cells / mL. At this seeding density, the cell killing activity can be maintained at a high level.

[0026] More preferably, the seeding density of the primary expansion culture is 3×10 5 cells / mL.

[0027] Preferably, during the primary expansion culture process, fresh culture medium is replaced every 48 to 76 hours;

[0028] Preferably, during the primary expansion culture process, fresh culture medium is replaced every 72-76 hours. Under these culture conditions, the cells can still maintain a high viable cell density and an optimal total viable cell count, which can reduce production costs and improve production efficiency.

[0029] More preferably, during the primary expansion culture process, fresh culture medium is replaced every 72 hours.

[0030] Preferably, the conditions for the primary expansion culture are 36-38° C., 4%-6% CO 2 , and the duration of the primary expansion culture is 9 days or more.

[0031] Preferably, the conditions for the primary amplification culture are 37° C., 5% CO 2 , and the duration of the primary amplification culture is 9 to 12 days.

[0032] Preferably, the content of IL2 in the second expansion medium is 50 to 1000 IU / mL.

[0033] Preferably, the content of IL2 in the second expansion medium is 100-200 IU / mL.

[0034] More preferably, the content of IL2 in the second expansion medium is 125 IU / mL.

[0035] Preferably, in the second expansion medium, the volume ratio of the anti-clumping agent to the HIPP-T009 lymphocyte serum-free medium is (0.1-10):1000; the addition of the anti-clumping agent can prevent dead cells from agglomerating and maintain cell killing activity.

[0036] Preferably, in the second expansion medium, the volume ratio of the anti-clumping agent to the HIPP-T009 lymphocyte serum-free medium is 1:1000.

[0037] More preferably, in the second expansion medium, the volume ratio of the anti-clumping agent to the HIPP-T009 lymphocyte serum-free medium is 1:1000.

[0038] As a preference, the seeding density of the secondary amplification culture is (3-5)×10 5cells / mL;

[0039] Preferably, the seeding density of the secondary expansion culture is 3×10 5 cells / mL.

[0040] Preferably, during the secondary expansion culture process, fresh culture medium is replaced every 48 to 72 hours;

[0041] Preferably, during the secondary expansion culture process, fresh culture medium is replaced every 48 to 52 hours.

[0042] More preferably, during the secondary expansion culture process, fresh culture medium is replaced every 48 hours.

[0043] Preferably, the secondary expansion culture is a secondary expansion culture of the primary expansion cells using a WAVE wave bioreactor.

[0044] Preferably, the culture conditions of the WAVE bioreactor include: 36-38°C, 4%-6% CO2, swing speed 5-8rpm, swing angle 6-10°, shaking dynamic control 30%-50%, and ventilation flow 0.1-0.2L / min.

[0045] Preferably, the culture conditions of the WAVE bioreactor include: 37° C., 5% CO 2 , a swing speed of 6 rpm, a swing angle of 6 to 8°, a shaking dynamic control of 30% to 50%, and a ventilation flow rate of 0.15 L / min.

[0046] Preferably, the cells are harvested 69 to 76 hours after the culture volume of the cells is expanded to 25 L.

[0047] Preferably, the cells are harvested 72 hours after the culture volume of the cells is expanded to 25 L.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The culture process is relatively simple. The cell culture process provided by the present invention does not require the separation of lymphocytes from autologous or healthy donors, nor does it require viral transfection in each batch of cell product preparation. The CAR NK cell process starts with the recovery of PCB cells. When the cell viability reaches 80%, the cells are expanded and cultured at a fixed live cell inoculation density (using the culture system of T25 → T75 → T175 → T225 → 10L WAVE bag → 50L WAVE bag in sequence);

[0050] (2) Large culture scale and low cost for patients: The culture volume of the CAR NK cells provided by the present invention can reach up to 25L (and a larger culture scale can be obtained by further harvesting in multiple batches), which far exceeds the production scale of primary CAR NK cells, can greatly shorten the culture cycle, significantly reduce production costs, have smaller batch differences, and more controllable quality, which can meet more clinical use needs;

[0051] (3) The specific recovery and culture conditions of the present invention enable the cells to maintain a high proliferation activity and cell viability while maintaining their biological functions, so that the cell harvest volume and density reach a certain scale to meet the clinical use needs of allogeneic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Screening and confirmation experiment results of different serum-free culture media for CAR NK cells; CAR NK cells were cultured in serum-free culture medium for 20 days, and the viable cell density, cell viability and total viable cell count, doubling time, in vitro cell killing activity, and expression of major surface molecules such as CAR, CD56, and NKG2D were recorded; Among them,

[0053] 1-1 shows the viable cell density in different serum-free media;

[0054] 1-2 show the cell viability in different serum-free culture media;

[0055] 1-3 show the cell doubling time in different serum-free media;

[0056] 1-4 show the total number of viable cells in different serum-free media;

[0057] 1-5 show the expression of NKG2A, NKG2D, NKp44, NKp46, CAR, CD56, and CD16 surface molecules of CAR NK cells in three serum-free culture media;

[0058] 1-6 show the killing activity of CAR NK cells against MDA-MB453 target cells in three serum-free culture media;

[0059] Figure 2 : Results of treatment intervals during the CAR NK cell recovery phase. Starting from the second day after cell thawing and recovery, cells were cultured according to the standard recovery and culture procedures. Cells were treated every 2 days and every 3 days, and the treatment interval at which the CAR NK cell viability first reached 80% was compared.

[0060] Figure 3: Live cell seeding density in the CAR NK cell expansion stage; cells were cultured at different live cell seeding densities, and after 72 hours of culture, the in vitro cytotoxicity of cells seeded at different densities was detected by RTCA (Real Time Cellular Analysis) and CCK8 assays; wherein,

[0061] 3-1 shows the killing activity of CARNK cells against MDA-MB453 cells after CAR NK cells obtained at different seeding densities acted on MDA-MB453 target cells for 2.5 hours;

[0062] 3-2 shows the killing activity of CAR NK cells obtained at different seeding densities against MDA-MB453 target cells at 29 hours after 2.5 hours of action;

[0063] Figure 4 : The viable cell density and cell viability of CAR NK cells at different IL2 concentrations during the expansion phase; CAR NK cells were cultured at a rate of 3×10 5 Cells / mL were inoculated with viable cells and the viable cell density and cell viability were recorded every 3 days (72 h) according to the cell treatment interval.

[0064] Figure 5 : The killing activity of CAR NK cells at different IL2 concentrations during the expansion phase; CAR NK cells were cultured at a rate of 3×10 5 Cells were cultured at a viable cell seeding density of cells / mL, and the in vitro killing activity of the cells was detected at the time of harvest at a cell treatment interval of every 3 days (72 h).

[0065] Figure 6 : Treatment interval of CAR NK cell expansion stage; CAR NK cells were cultured at a rate of 3×10 5 cells / mL, and cultured at a cell treatment interval of every 2 or 3 days, and recorded the viable cell density and cell viability of cells cultured in different treatments;

[0066] Figure 7 : Results of secondary expansion culture of CAR NK cells for 48 h (without adding anti-aggregating agent); CAR NK cells were cultured at 3×10 5 cells / mL of viable cells, inoculate 400 mL of cells for culture (containing a final concentration of 125 IU / mL IL2), and observe the status of the cultured cells under a microscope after culturing for 48 h;

[0067] Figure 8 : Results of secondary expansion culture of CAR NK cells for 48 hours (with the addition of anti-aggregant); CAR NK cells were cultured at a rate of 3×10 5cells / mL, inoculate 400 mL of cells for culture (containing a final concentration of 125 IU / mL IL-2 and an anti-agglomerating agent at a dilution of 1:1000), and observe the status of the cultured cells under a microscope after 48 h of culture;

[0068] Figure 9 : Results of secondary expansion culture of CAR NK cells for 72h (with the addition of anti-aggregant); CAR NK cells were cultured at a rate of 3×10 5 cells / mL, inoculate 400 mL of cells for culture (containing a final concentration of 125 IU / mL IL-2 and an anti-agglomerating agent at a dilution of 1:1000), and observe the status of the cultured cells under a microscope after 72 h of culture;

[0069] Figure 10 : CAR NK cell secondary expansion culture system; CAR NK cells were cultured at 3×10 5 Cells were cultured at a viable cell seeding density of 10 cells / mL, expanded and harvested at a 48-h culture treatment interval and a 72-h cell harvesting interval, and the viable cell density and cell viability of the cultured cells were recorded.

[0070] Figure 11 : In vitro killing effect and doubling time of secondary expansion of CAR NK cells; CAR NK cells were cultured at a rate of 3×10 5 Cells were cultured at a viable cell seeding density of 100 cells / mL, expanded and harvested at a 48-h culture treatment interval and a 72-h cell harvesting time. The in vitro killing effect and doubling time of the cells during expansion and harvest were tested, and CAR NK cells cultured statically in T-flasks were used as a control. DETAILED DESCRIPTION

[0071] The present invention discloses a method for the recovery and culture of ROBO1 CAR NK cells. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0072] In the early stage of the present invention, NK92 cells that can be passaged and cultured in vitro were used as the main source of CAR NK cells. Through the construction of CAR genes, CAR gene transfection of NK cells, sorting of CAR NK monoclonal cells, active function verification and preliminary culture, a standardized CAR NK cell library with stable expression of CAR genes was obtained (see the authorized patent CN201811394153.3 of Sichuan Asikeli Biotechnology Co., Ltd.). On this basis, the present invention further explored the screening and confirmation of serum-free culture medium, the live cell seeding density in the expansion and culture stage of ROBO1 CAR NK cells, the optimized combination and concentration optimization of culture medium additives in the expansion and culture stage, the confirmation of the treatment interval time and harvest time in the cell recovery and expansion stages, and established stable CAR NK cell recovery and culture related methods and standard operating procedures.

[0073] Specifically, the present invention includes resuscitation, primary expansion culture (primary expansion culture) and secondary expansion culture (secondary expansion culture) of CAR NK cells, adding different additive components to the culture medium, controlling the cell seeding density, and controlling the cell treatment interval, so that the cells maintain a high expansion activity and cell viability while maintaining their biological functions, and ultimately the cell harvest volume and density reach a certain scale to meet the clinical use needs of allogeneic patients.

[0074] Compared with existing methods for preparing personalized cell therapy products, the cell product preparation method and process route used in the present invention have the advantages of shorter time consumption, more controllable product quality standards, smaller batch differences, and lower preparation costs, and can truly meet the needs of "off-the-shelf" cell therapy drug use.

[0075] Explanation of terms:

[0076] A PCB cell bank (also known as a primary cell bank, PCB) is a stable cell population developed from a single original cell population, or a homogeneous cell population formed through clonal culture, and certified as suitable for the production or testing of biological products. Under special conditions, a certain number of homogeneous cell suspensions are quantitatively and evenly distributed into ampoules or suitable cell cryopreservation tubes and frozen in liquid nitrogen. This primary cell bank is used to establish a master cell bank.

[0077] Cell recovery: refers to the process of thawing cells frozen in liquid nitrogen or a -70°C refrigerator and then re-culturing them to resume cell growth.

[0078] Cell expansion, also known as large-scale cell culture technology, refers to the art of artificially controlling environmental conditions to cultivate useful microbial, animal, and plant cells at high densities on a large scale. It serves as the foundation for the large-scale proliferation of new cells derived from genetic recombination and cell fusion techniques in industrial production, such as food and pharmaceuticals.

[0079] The reagents, instruments, and biological materials used in the present invention can all be obtained through commercial channels.

[0080] The experimental reagents, equipment and suppliers are as follows:

[0081]

[0082]

[0083] The serum-free culture media used in this application (serum-free, no animal-derived components) are all GMP grade.

[0084] The present invention will be further described below in conjunction with the embodiments:

[0085] Example 1 and Comparative Examples 1-7: Serum-free medium for CAR NK cell culture

[0086] The validation process of serum-free culture medium mainly includes the following steps:

[0087] (1) Select CAR NK cells for serum-free acclimation (427A-D5-B3, 427A is the CAR NK project number, D5 and B3 represent the serum-containing cell bank established after cell clone expansion from the monoclonal growth well D5 and the subclonal growth well B3), observe the cell status under a microscope, and perform serum-free acclimation when the cells are in the optimal growth state;

[0088] (2) Collect cells into a centrifuge tube and centrifuge at 175g for 5 minutes;

[0089] (3) Add a small amount of the supernatant from step (2) and gently pipette to resuspend the cells;

[0090] (4) Prepare a sterile 1.5 mL centrifuge tube and label it. Pipette approximately 40 μL of the cell suspension from step (3) and transfer it to the corresponding centrifuge tube. Add 40 μL of AO / PI dye solution and mix well. Pipette 20 μL of the mixture into a cell counting plate, prepare three replicate wells, and use a cell counter to detect the viable cell density and cell viability.

[0091] (5) Take 8 T25 culture flasks respectively, add 5 mL of different serum-free culture media (all containing 1000 IU / mL IL2) into them, and then take a certain number of living cells from the centrifuge tube in step (4) into T25 culture flasks (5 mL / flask, according to the NK92 cell culture instructions recommended by the ATCG official website, the final living cell culture density is (2-3) × 10 5 cells / mL);

[0092] The types of serum-free culture media corresponding to the examples and comparative examples are as follows:

[0093] Example 1: HIPP TM -T009 medium (abbreviated as T009);

[0094] Comparative Example 1: Excell bio NK medium (abbreviated as Excell bio);

[0095] Comparative Example 2: NK002-9 culture medium;

[0096] Comparative Example 3: NK002-10 culture medium;

[0097] Comparative Example 4: NK002-11 culture medium;

[0098] Comparative Example 5: NK002-12 culture medium;

[0099] Comparative Example 6: NK002-13 culture medium;

[0100] Comparative Example 7: NK002-5-2 culture medium.

[0101] (6) After culturing at 37°C in 5% CO2 for approximately 72 h, gently tap the culture flask to mix thoroughly. Pipette approximately 40 μL of cell suspension from each bottle of T25 and transfer it to the corresponding centrifuge tube. Add 40 μL of AO / PI staining solution and mix thoroughly. Pipette 20 μL of the mixture onto a cell counting plate, and count three times. Use a cell counter to detect the viable cell density and cell viability.

[0102] (7) Collect cells from each bottle into a centrifuge tube and centrifuge at 175g for 5 minutes. Add an appropriate amount of centrifugal supernatant and gently pipette to mix and resuspend the cells. At the same time, add 5 mL of the above different serum-free culture media (all containing 1000 IU / mL IL2) to each T25 culture flask. Take about 15×10 5 viable cells to a T25 culture flask (5 mL / flask, the final viable cell culture density was 3×10 5 cells / mL);

[0103] (8) Repeat steps (6)-(7) and culture the CAR NK cells in serum-free culture medium for 20 days. Record the viable cell density, cell viability and total number of viable cells in each bottle of cells at each test. Determine the doubling time of CAR NK cells in different serum-free culture media by culture time and total number of viable cells. At the end of culture, detect the in vitro killing effect of CAR NK cells cultured in different serum-free culture media by RTCA method. Detect the expression of major surface molecules such as CAR, CD56 and NKG2D of CAR NK cells cultured in different serum-free culture media by flow cytometry.

[0104] The experimental results are as follows Figure 1 As shown in the figure, among the 8 serum-free culture media, only T009 and Excel bio culture media can support the large-scale expansion of CAR NK cells while maintaining a high cell viability based on the two main indicators of viable cell number and cell viability. Further investigation of the two indicators of cell killing activity and cell phenotype showed that only the T009 culture medium of Example 1 can maintain the killing activity of CAR NK cells against target cells, and the main cell surface markers did not change significantly. According to the experimental results, T009 serum-free culture medium was used to expand the culture of CAR NK cells, and a PCB cell bank was established.

[0105] The serum-free culture medium used in the following examples was all T009 serum-free culture medium.

[0106] Example 2 and Comparative Example 8: CAR NK cell PCB recovery culture

[0107] 1) Open the thermostatic water bath and set the temperature to 37°C;

[0108] 2) Pipette 4 mL of T009 serum-free medium into a 15 mL centrifuge tube and pre-cool it at 4°C. Also pre-cool an empty 15 mL centrifuge tube.

[0109] 3) Pipette 5 mL of serum-free medium into a T25 cell flask. Add IL-2 to a final concentration of 500 IU / mL and nicotinamide to a final concentration of 5 mM. Tap gently to mix, and place in a CO2 incubator to prewarm.

[0110] 4) Using tweezers, carefully remove the cryovials (named 427A-PCB-T009-0609 and 427A-PCB-T009-0614, where 427A-PCB-T009 is the name of the serum-free cell bank and 0609 and 0614 represent the dates of thawing) from the designated location in the liquid nitrogen tank. Immediately place the vials in a 37°C water bath and gently shake, ensuring the caps are always level.

[0111] 5) When the cells in the cryovial have thawed to the size of soybeans, quickly remove the cryovial and slowly transfer the cell suspension to a new, pre-chilled 15 mL centrifuge tube using a pipette. Then, slowly add the pre-chilled culture medium dropwise to the cell suspension and gently tap to mix.

[0112] 6) Centrifuge at 175g for 5 minutes at 4°C. Discard the supernatant and gently resuspend the cell pellet in pre-warmed culture medium. Mix thoroughly and transfer the pellet to a T25 cell culture flask. Place the flask in a 37°C, 5% CO2 incubator for 24 ± 3 hours.

[0113] 7) On the second day of cell recovery, remove the recovered cells from the CO2 incubator, observe the cell status under an inverted microscope, and count the cells;

[0114] 8) Centrifuge the cell suspension at 175g for 5 minutes, discard the supernatant, add 5 mL of fresh pre-warmed culture medium to resuspend the cell pellet, and then transfer the cells to a T25 culture flask and continue culturing in an incubator.

[0115] 9) In Example 2, the cell treatment interval (replacement of fresh culture medium) was every 2 days, and in Comparative Example 8, the cell treatment interval was every 3 days. The viable cell density and cell viability in the T25 cell culture flask were detected. When the cell viability in the culture flask was less than 80% and the viable cell density was less than 1×10 6 cells / mL, centrifuge the cells and replace with fresh culture medium, maintain the culture volume at 5 mL and continue culturing; when the cell viability in the culture flask is less than 80% and the viable cell density is ≥1×10 6 cells / mL, the cells were centrifuged and replaced with fresh culture medium, and the culture volume was expanded to 10 mL;

[0116] 10) Compare the culture time required for the cell viability to reach above 80%.

[0117] Figure 2 As shown in the figure, it can be seen from the experimental results that when 427A-PCB cells are thawed and revived and treated at intervals of every 2 days, the cell viability can reach more than 80% on the 9th day after cell recovery, while if treated every 3 days, the cell viability still does not reach 80% on the 15th day after recovery.

[0118] Example 3 and Comparative Examples 9-11: Primary Expansion Culture of CAR NK Cells

[0119] The CAR NK cells revived in Example 2 were subjected to primary expansion culture, and the culture medium contained IL2 at a final concentration of 125 IU / mL, and the culture volume was 5 mL for 3 days. The seeding densities corresponding to the example and the comparative example are as follows:

[0120] Example 3: 3×10 5cells / mL;

[0121] Comparative Example 9: 6×10 5 cells / mL;

[0122] Comparative Example 10: 8×10 5 cells / mL;

[0123] Comparative Example 11: 1×10 6 cells / mL.

[0124] The killing activity of CAR NK was detected by RTCA to confirm the effect of different live cell seeding densities on CAR NK cells.

[0125] The experimental results are as follows Figure 3 As shown, according to 3×10 5 cells / mL、6×10 5 cells / mL、8×10 5 cells / mL, 1×10 6 Cells were inoculated at different inoculation densities of cells / mL. The cell killing activity decreased with the increase of inoculation density, showing a negative correlation. Therefore, the live cell inoculation density in the expansion and culture stage of CAR NK cells is preferably 3×10 cells / mL as in Example 3. 5 cells / mL.

[0126] Example 4 and Comparative Examples 12-16: Primary Expansion Culture of CAR NK Cells

[0127] The CAR NK cells recovered from Example 2 were cultured for primary expansion, and the seeding density of live cells was 3×10 5 cells / mL, the CAR NK cells were cultured in an incubator for 9 days, treated once every 3 days, and the culture volume was 5 mL. The final concentrations of IL2 corresponding to the examples and comparative examples were as follows:

[0128] Example 4: 125 IU / mL;

[0129] Comparative Example 12: 0 IU / mL;

[0130] Comparative Example 13: 62.5 IU / mL;

[0131] Comparative Example 14: 250 IU / mL;

[0132] Comparative Example 15: 500 IU / mL;

[0133] Comparative Example 16: 1000 IU / mL.

[0134] The cell status was observed under an inverted microscope during each treatment, and the live cell density and cell viability were detected using a cell counter, while the cell killing activity was detected using RTCA.

[0135] The experimental results are as follows Figure 4 and Figure 5 As shown, different concentrations of IL2 were added to the cell expansion medium, and the three indicators of viable cell density, cell viability and cytotoxicity were simultaneously judged. In Example 4, the IL2 concentration of 125 IU / mL not only maintained the growth viability of CAR NK cells, but also maintained the cytotoxicity of CAR NK cells against target cells, and at the same time, enabled the CAR NK cells to have a higher expansion multiple during the culture process.

[0136] Example 5 and Comparative Example 17: Primary Expansion Culture of CAR NK Cells

[0137] The seeding density of live cells was 3×10 5 cells / mL, and the CAR NK cells were cultured in an incubator at treatment intervals of every 2 days (Comparative Example 17) and every 3 days (Example 5), and the culture volume was always 5 mL (containing a final concentration of 125 IU / mL IL2), for a total of 12 days. During each treatment, the cells were removed from the incubator, the cell state was observed under an inverted microscope, and then a small amount of the suspension was gently pipetted and mixed, and the viable cell density and cell viability were detected using a cell counter.

[0138] The experimental results are as follows Figure 6 As shown, in the primary expansion stage of cells, the cell viability is not much different when the cells are treated every 3 days compared with when they are treated every 2 days. The total number of viable cells treated every 3 days is slightly higher than that treated every 2 days. Considering the cost and efficiency, the treatment interval in the primary expansion culture stage of CAR NK cells in Example 5 is more favorable.

[0139] Example 6: Secondary Expansion Culture of CAR NK Cells

[0140] The CAR NK cells obtained in Example 5 were subjected to secondary expansion culture, which mainly included the following contents:

[0141] 1) The secondary expansion of CAR NK cells is mainly carried out using WAVE bioreactors, and 10L disposable cell culture bags and 50L disposable cell culture bags are gradually used for expansion. The maximum culture volume can be expanded to 25L.

[0142] 2) According to the live cell seeding density of 3×10 5cells / mL, inoculated 400mL of cells (cell viability ≥80%) for culture (containing a final concentration of 125IU / mL IL2), set the WAVE culture parameters to 37°C, 5% CO2, Rocking: 6RPM, 6-8° and 30-50% motion, ventilation at 0.15L / min, and after 48h of cell inoculation and culture, observe the status of the cultured cells under a sampling microscope (the results are shown in Figure 2). Figure 7 The results showed that under the sampling microscope, there were flocculent dead cell clumps (visible to the naked eye as foreign matter), and the dead cell clumps in the centrifuge tube settled quickly.

[0143] At the same time, an anti-agglomerant with a volume ratio of 1:1000 was further added to the serum-free culture medium, and the same volume and density of CAR NK cells were inoculated. After 48h and 72h of cell inoculation, the state of the cultured cells was observed under a sampling microscope (the results were shown in Table 2). Figure 8 and Figure 9 shown). Figure 8 It can be seen that at the 48-h cell treatment interval, the cells are in better condition, with rounder and more translucent cells, indicating that the cells are in the logarithmic growth phase; Figure 9 The results showed that at a 72-hour treatment interval, although the cell count was high, the cells were irregular in morphology, poorly rounded, and opaque under microscopy, indicating that the cells were in the plateau growth phase. Based on these experimental results, the treatment interval for secondary expansion of CAR NK cells was determined to be 48 hours.

[0144] According to the above-determined live cell inoculation density, culture medium additive types and contents, and treatment intervals, the cells were gradually expanded from 400 mL to 25 L. The live cell density and cell viability of the cells during the expansion culture were recorded. At the same time, RTCA was used to detect the cell killing activity and doubling time during the expansion culture process (compared with the cells cultured statically in T flasks). The results are as follows Figure 10 and Figure 11 As shown, the seeding density of live cells was 3×10 5 cells / mL, 400 mL of cells were initially inoculated in the WAVE culture system for culture, and the cell treatment interval was 50 ± 2 hours, with a fixed cell seeding density of 3 × 10 5 cells / mL, cells were expanded from 400mL to 25L, and the cells were harvested at the time of expansion to 25L (according to 3×10 5The viable cell density and cell growth rate were recorded 72±3 hours after culture. The results showed that CAR NK cells were greatly expanded in the WAVE bioreactor system and the cells remained in the optimal growth state. At the same time, the killing activity was compared with the cells cultured statically in T bottles. Both had the same cell killing activity against target cells, indicating that the cells cultured in the WAVE system are fully functional and can be used for subsequent preparation freezing and patient transfusion.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for culturing ROBO1 CAR NK cells, characterized in that: The steps include: S1, ROBO1 CAR NK cells are revived and cultured using a recovery medium, wherein the recovery medium is a HIPP-T009 lymphocyte serum-free medium containing IL2 and nicotinamide; In the recovery medium, the content of IL2 is 500 IU / mL, and the content of nicotinamide is 5 mM; During the recovery culture process, fresh culture medium is replaced every 48 hours to obtain recovered cells; S2, performing primary expansion culture on the revived cells using the first expansion medium to obtain primary expanded cells; Performing secondary expansion culture on the primary expanded cells using a second expansion medium, and harvesting the cells; The first expansion medium is a HIPP-T009 lymphocyte serum-free medium containing IL2; the content of IL2 in the first expansion medium is 125 IU / mL; The seeding density of the primary expansion culture was 3×10 5 cells / mL; During the primary expansion culture process, fresh culture medium was replaced every 72 hours; The second expansion medium is a HIPP-T009 lymphocyte serum-free medium containing IL2 and an anti-clumping agent; the content of IL2 in the second expansion medium is 125 IU / mL; The seeding density of the secondary expansion culture was 3×10 5 cells / mL; During the secondary amplification culture process, fresh culture medium was replaced every 48 hours; The time for harvesting cells is 72 hours after the culture volume of cells is expanded to 25L.

2. The culture method according to claim 1, wherein In the second expansion medium, the volume ratio of the anti-clumping agent to the HIPP-T009 lymphocyte serum-free medium is (0.1-10):1000.

3. The culture method according to claim 2, wherein In the second expansion medium, the volume ratio of the anti-clumping agent to the HIPP-T009 lymphocyte serum-free medium is 1:1000.

4. The culture method according to claim 1, wherein The secondary expansion culture is to use a WAVE wave bioreactor to perform secondary expansion culture on the primary expansion cells; The culture conditions of the WAVE bioreactor include: 36-38°C, 4%-6% CO2, swing speed 5-8rpm, swing angle 6-10°, shaking dynamic control 30%-50%, and ventilation flow 0.1-0.2L / min.

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