Method for preparing suspension of cells with suppressed mitosis

Abstract

The present invention relates to a method of preparing a suspension of cells with suppressed mitosis, the method comprising the steps of: a) providing cells suspended in a cell culture medium; and b) inhibiting the mitotic activity of the cells in the suspension, thereby obtaining mitotic inhibited cells. The step of inhibiting mitotic activity of cells comprises: subjecting the suspension to a hypotonic treatment followed by exposure of the cells to at least one freeze-thaw cycle, and vice versa; or exposing the cells to two or more freeze-thaw cycles, optionally performing a hypotonic treatment on the suspension prior to freeze-thaw cycles. In the methods described herein, the hypotonic treatment of the suspension comprises the step of adding a liquid, such as water, to the suspension comprising cells to form a hypotonic cell culture medium. The invention also relates to a method for culturing target cells and application of the hypotonic cell culture medium.

Description

Technical Field

[0001] This invention relates to a method for preparing a suspension of mitotically inhibited cells, particularly a suspension of mitotically inhibited feeder cells. The invention also relates to a method for culturing cells, particularly natural killer cells. Furthermore, the invention relates to the use of hypotonic cell culture media. Background Technology

[0002] This background description includes information that can be used to understand the present invention. It is not intended to acknowledge that any information provided herein is prior art or related to the claimed invention, nor is it an admission that any express or implied references to publications are prior art.

[0003] Natural killer (NK) cells are lymphocytes that spontaneously and rapidly respond to a variety of threats, including infected cells, transformed cells, and malignant degenerated cells, also known as tumor cells. One of the defining characteristics of NK cells, their ability to distinguish between healthy and abnormal cells, is achieved through a precise balance between inhibitory and activating receptors on the cell surface. This defining characteristic enables NK cells to perform their primary function, known as "natural killing," by releasing cytotoxic molecules such as perforin and granzymes to destroy target cells.

[0004] Due to their potential applications in cancer immunotherapy, NK cells have attracted significant interest in clinical research. Clinical strategies are being developed, such as adoptive NK cell therapy, which involves reinfusing expanded and activated NK cells from outside the body into the patient to enhance the anti-tumor response. Furthermore, extensive research is being conducted on modulating NK cell activity to optimize the treatment of various diseases.

[0005] Several in vitro NK cell proliferation induction protocols for increasing NK cell numbers have been disclosed in the prior art, and are hereinafter referred to as "NK cell expansion". In vitro expansion of NK cells can be achieved by culturing with a combination of cytokines, supplementing the cell culture medium with small molecules, and stimulating expansion in vitro using cytokines, antibodies, and / or feeder cells.

[0006] NK cell expansion protocols based on feeder cells generate high numbers of NK cells. Feeder cells induce NK cell activation and proliferation with the aid of cytokines through cell surface receptor-ligand interactions (Gurney et al., (2022) Front. In Immunol. Vol. 13). Effective protocols include the use of genetically modified feeder cells transfected with proteins specifically designed to stimulate NK cell proliferation (e.g., K562 cells, an erythroleukemia cell line lacking human leukocyte antigen (HLA) class I). Alternatively, B cell lines that have been naturally immortalized through infection with Epstein-Barr virus can be used in such protocols.

[0007] To use feeder cells for NK cell expansion, the feeder cell lines need to be subjected to high-dose ionizing radiation, typically around 100 Gy. Irradiation of feeder cells inhibits mitosis, preventing them from proliferating (thus avoiding overgrowth of the intended immune cells, such as NK cells, during expansion). A limitation of this method is that companies conducting NK cell expansion must rely on third-party suppliers of irradiated cells, as such irradiation facilities are strictly regulated, costly, and difficult to maintain. Furthermore, the transportation of these irradiated cells places a logistical burden on cell companies.

[0008] In addition, regulatory agencies require stringent safety parameters and risk assessments for the presence of feeder cells in NK cell-based therapies to reduce the risk of contamination of NK cell-based therapies with live feeder cells.

[0009] Therefore, there is an urgent need for an alternative, optionally optimized, method for expanding NK cells to extremely high quantities, preferably sufficient to support clinical trials for patient use, and preferably using feeder cells during the NK cell expansion process. In particular, there is an urgent need for an alternative method for preparing suspensions of mitotically inhibited cells that eliminates the need for high-dose ionizing radiation to inhibit mitosis. Summary of the Invention

[0010] To provide such an alternative method, in a first aspect of the invention, a method for preparing a suspension of mitotically inhibited cells is provided, the method comprising the steps of: a) providing cells suspended in a cell culture medium; and b) inhibiting the mitotic activity of the cells in the suspension provided in step a) to obtain mitotically inhibited cells, wherein the step of inhibiting the mitotic activity of the cells comprises the following steps: The suspension was hypotonic, followed by exposure of the cells to at least one freeze-thaw cycle, or vice versa; or Expose the cells to two or more freeze-thaw cycles, optionally hypotonicizing the suspension prior to the freeze-thaw cycles.

[0011] It should be noted that the hypotonic treatment of the suspension as defined above includes the step of adding a liquid, such as water, to the cell suspension to form a hypotonic cell culture medium. It should also be noted that the process conditions for the step of inhibiting the mitotic activity of the cells are selected such that the resulting mitotically inhibited cells can maintain their desired biological activity. By providing a method for inhibiting cell mitosis while still maintaining their desired biological activity, including the direct use of a suspension of mitotically inhibited cells for, for example, the culture of target cells (such as NK cells).

[0012] In a second aspect, a suspension of mitotically inhibited cells is provided, which can be obtained by the method for preparing a suspension of mitotically inhibited cells as described in this invention.

[0013] As described above, the present invention also relates to, in a third aspect, a method for culturing target cells, the method comprising the following subsequent steps: i) Provide a suspension of mitotically inhibited cells according to the present invention; ii) Provide target cells, then add the target cells to a suspension to form a co-culture of the target cells and mitotically inhibited cells; iii) Expanding target cells in suspension; and iv) Collect the expanded target cells.

[0014] In a fourth aspect of the invention, a hypotonic cell culture medium is provided, wherein the hypotonic cell culture medium comprises: Liquid content (e.g., water) up to 95% by volume, preferably between 10% and 90% by volume, between 40% and 80% by volume, or between 45% and 60% by volume, most preferably about 50% by volume; and Cell culture medium.

[0015] In a fifth aspect, the use of the hypotonic cell culture medium described in the fourth aspect of the invention in a method for inhibiting the mitotic activity of cells is provided. Attached Figure Description

[0016] Figure 1 Growth curves of K562-F012 cells treated with different hypotonic solutions and subjected to 4 freeze-thaw cycles, and untreated K562-F012 cells.

[0017] Figure 2After treatment with different hypotonic solutions, the following steps were performed: Figure 1 The growth curves of K562-F012 cells after four freeze-thaw cycles are shown, but they are not compared with normally expanded K562-F012 cells.

[0018] Figure 3 NK cell expansion is expressed in absolute numbers. NK cells were expanded on K562-F012 cells that had been treated with different hypotonic solutions and / or subjected to different freeze-thaw cycles. Detailed Implementation

[0019] definition This disclosure contains material protected by copyright (such as, but not limited to, diagrams, photographs of devices, or any other aspect of this application that has been or is available for copyright protection in any jurisdiction). The copyright holder does not object to any fax copying of the patent documents or patent disclosures contained in the patent office's patent documents or records, but otherwise reserves all copyright rights.

[0020] Various terms used throughout this specification and claims are relevant to the methods, compositions, uses, and other aspects of the invention. Unless otherwise stated, such terms should have their ordinary meaning in the field to which this invention pertains. Other particularly defined terms should be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar to or equivalent to those described herein may be used for the implementation or testing of the invention, preferred materials and methods are described herein.

[0021] In connection with this invention, the relevant terms are defined below.

[0022] As used herein, the singular forms of the terms “a” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and so on. For example, a method of culturing a cell includes culturing multiple cells (e.g., dozens, hundreds, thousands, tens of thousands, hundreds of thousands, millions or more).

[0023] As used herein, when referring to measurable values ​​such as quantities and time spans, the terms "about" and "approximately" are intended to cover deviations from the specified value of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, because such deviations are applicable to the performance of the disclosed invention. Unless the context clearly indicates otherwise, all numerical values ​​provided herein include those modified by the term "about".

[0024] As used herein, “at least” a specific value refers to that specific value or a larger value. For example, “at least 2” should be understood as having the same meaning as “2 or more,” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term “at most” a specific value refers to that specific value or a smaller value. For example, “at most 5” should be understood as having the same meaning as “5 or fewer,” i.e., 5, 4, 3…-10, -11, etc.

[0025] As used herein, “comprising” should be interpreted as inclusive and open-ended, not exclusive. Specifically, the term and its variations refer to including the specified features, steps, or components, and should not be construed as excluding the presence of other features, steps, or components. The term also covers the narrower scope of “composed of”.

[0026] As used herein, “exemplary,” “for example,” or “such as” means “used as an example, instance, or illustration” and should not be construed as excluding other configurations, including those disclosed herein.

[0027] As used herein, the term "freeze-thaw cycle" refers to a method comprising the following steps: a step of exposing a suspension, cells, or cell population comprising cell culture medium and cells to a temperature equal to or below the freezing point of the suspension or cell population; and a subsequent step of exposing the frozen cells or cell population to a temperature above the freezing point of the suspension or cell population. It is understood herein that the freezing point may vary depending on the cell culture medium used in the suspension comprising the cells. It is understood herein that freeze-thaw cycles affect the proliferation of the cells or cell population, for example, by interfering with the mitotic process of the cells or cell population.

[0028] As used herein, “in vivo” refers to an event occurring inside a subject; “in vitro” and “ex vivo” refer to events occurring outside a subject. For example, an in vitro assay or method covers any assay or method performed outside a subject. An in vitro assay or method covers cell-based assays using live or dead cells. An in vitro assay also covers cell-free assays that do not use intact cells. The terms “in vivo,” “in vitro,” and “ex vivo” are well-known terms in the art.

[0029] As used herein, the term "mitotically inhibited" refers to a phenomenon in which the process of mitosis in a cell is slowed down and / or stopped to the point that the cell's ability to divide, proliferate, and expand is limited. In some cases, as described herein, mitotic inhibition can lead to the inactivation of mitosis in a cell, thereby eliminating any ability of a (healthy) cell to divide through the naturally occurring process. Essentially, the term refers to cells that are intentionally prevented from undergoing cell division or mitosis. Therefore, mitotic inhibition is the process of stopping cell proliferation.

[0030] As used herein, the term "required biological activity" refers to the biological activity of a cell, substance, or compound that is suitable for subsequent use. Required biological activity may include the cellular biological ability to stimulate cell proliferation and expansion. Other required biological activities may include the ability of a cell, substance, or compound to interact with other biological systems, thereby inhibiting the growth of cancer cells, reducing inflammation, targeting specific receptors in the body, promoting certain physiological responses, etc.

[0031] When used in combination, the term "mitotically inhibited cells that maintain desired biological activity" refers to cells that have been treated to stop their division (mitosis inhibition) while still retaining their intended or preferred biological activity. Thus, the growth or behavior of such cells is controlled without impairing their desired function or activity.

[0032] Detailed Explanation This invention is defined herein, and particularly in the appended claims. Subject matter not falling within the scope of the claims does not constitute part of the invention as claimed.

[0033] Any method, use, or composition described herein may be practiced with respect to any other method, use, or composition described herein. The embodiments described in the context of the methods, uses, and / or compositions of this invention may be employed with respect to any other method, use, or composition described herein. Therefore, embodiments relating to a method, use, or composition are equally applicable to other methods, uses, and compositions of this invention.

[0034] Research has found that the method, suspension, and hypotonic cell culture medium according to the present invention provide an optimized method for culturing and expanding NK cells. The inventors were surprised to discover that, by using the method according to the present invention, the step of irradiating feeder cells is redundant during the expansion of NK cells. Therefore, one of the beneficial effects of the present invention is that it eliminates the need to rely on a third party with irradiation facilities to produce irradiated cells.

[0035] The method according to the invention can unexpectedly produce mitotically inactivated cells, thereby ensuring that the cells do not proliferate. Another beneficial effect of the method according to the invention is that it does not rely on irradiation treatment of the cells. This means that the method can also be used for cell lines resistant to ionizing radiation.

[0036] Another unexpected benefit of this invention is that the risk of target cell cultures being contaminated by other living cells is significantly reduced, for example, compared to target cell cultures contaminated with living irradiated cells. Therefore, cultures of cells of interest (e.g., NK cells) are likely to meet the stringent clinical and regulatory requirements set by institutions for contamination with living cells (e.g., living feeder cells).

[0037] This invention relates to a method for preparing a suspension of mitotically inhibited cells, the method comprising the steps of: a) providing cells suspended in a cell culture medium; and b) inhibiting the mitotic activity of the cells in the suspension provided in step a), thereby obtaining mitotically inhibited cells; wherein the step of inhibiting the mitotic activity of the cells includes the following steps: The suspension was hypotonic, followed by exposure of the cells to at least one freeze-thaw cycle, or vice versa; or Expose the cells to two or more freeze-thaw cycles, optionally hypotonicizing the suspension prior to the freeze-thaw cycles.

[0038] The hypotonic treatment of the suspension includes the step of adding a liquid, such as water, to the suspension containing cells to form a hypotonic cell culture medium. Furthermore, the process conditions for the step of inhibiting the mitotic activity of the cells are selected so that the resulting mitotically inhibited cells can maintain their required biological activity.

[0039] In step b) of the method provided herein, mitotic activity of the cells is inhibited by exposing the cells to any of the following processes: After hypotonic treatment of the suspension, expose it to at least one freeze-thaw cycle, or vice versa; or Perform two or more freeze-thaw cycles, optionally subjecting the suspension to hypotonic treatment before the freeze-thaw cycles.

[0040] Studies have found that exposing cells to any of the above processes yields mitotically inhibited cells. Preferably, studies have found that when the process conditions of any process are selected in an optimized manner (e.g., the manner implemented herein), the resulting mitotically inhibited cells are able to maintain the required biological activity.

[0041] In one and / or more embodiments of the present invention, at least one freeze-thaw cycle or two or more freeze-thaw cycles are performed, including two, three or four freeze-thaw cycles, preferably four freeze-thaw cycles.

[0042] In one and / or more embodiments of the present invention, the amount of liquid (such as water) added to the suspension is selected such that the added liquid content of the resulting hypotonic cell culture medium does not exceed 95% by volume, preferably wherein the added liquid content of the hypotonic cell culture medium is between 10% by volume and 90% by volume.

[0043] Preferably, the liquid content (e.g., water content) in the hypotonic culture medium is between 10% and 90% by volume, or between 20% and 80% by volume, or between 25% and 75% by volume, or between 30% and 70% by volume, or between 40% and 60% by volume, more preferably about 50% by volume.

[0044] In one and / or more embodiments of the present invention, the cell culture medium is selected from the group consisting of SCGM, RPMI, MEMα and EMDM, or any one or more combinations thereof, preferably wherein the cell culture medium is SCGM.

[0045] Preferably, the hypotonic culture medium comprises, and preferably mainly consists of, a liquid (e.g., water) and a culture medium. More preferably, the hypotonic culture medium includes a cell culture medium selected from the group consisting of SCGM, RPMI, MEMα, and EMDM, or any one or more combinations thereof.

[0046] In one and / or more embodiments of the present invention, the cells are stored after step b), preferably in a refrigerator, more preferably in a -80°C refrigerator, or in liquid nitrogen.

[0047] In one and / or more embodiments of the present invention, the at least one freeze-thaw cycle includes the following subsequent steps: h) Provide the cells in the suspension provided in step a); i) Cool the suspension to a temperature below its freezing point, preferably to a temperature below -20°C, more preferably below -40°C, and most preferably to a temperature between -40°C and -80°C; j) Heating the suspension to a temperature above its freezing point; and k) Optionally, the cooling and heating in steps i) and j) are repeated once, twice or three times, preferably three times.

[0048] In one and / or more embodiments of the present invention, the proportion of total mitotically inhibited cells generated by freeze-thaw cycles is 90% to 100% of the cells suspended in the cell culture medium.

[0049] In one and / or more embodiments of the present invention, the cells provided in step a) are selected from the group consisting of feeder cells, wherein the feeder cells are capable of stimulating the proliferation of immune cells.

[0050] In one and / or more embodiments of the invention, the feeder cells are provided with transfected ligands for stimulating the proliferation of immune cells.

[0051] The present invention also provides a suspension of mitotically inhibited cells, which can be obtained by the method for preparing a suspension of mitotically inhibited cells according to the present invention.

[0052] The present invention also provides a method for culturing target cells, the method comprising the following steps: i) Provide a suspension of mitotically inhibited cells prepared by the method described in this invention; ii) Provide target cells and then add the target cells to a suspension to form a co-culture of target cells and mitotically inhibited cells; iii) Expanding target cells in suspension; and iv) Collect the expanded target cells.

[0053] In one and / or more embodiments of the present invention, the target cells are selected from the group consisting of natural killer cells (NK cells).

[0054] In one and / or more embodiments of the present invention, during the method of culturing target cells, the ratio of target cells to mitotically inhibited cells is selected to prevent the overgrowth of mitotically inhibited cells.

[0055] In one and / or more embodiments of the present invention, the collected target cells are substantially free of cells with inhibited mitosis.

[0056] In one and / or more embodiments of the present invention, the method further includes the following steps: 1) Providing a suspension of mitotically inhibited cells according to the present invention; and 2) Add the suspension from step 1) to the suspension from step iii).

[0057] The present invention also provides a hypotonic cell culture medium, wherein the hypotonic cell culture medium comprises: Liquid content (e.g., water content) up to 95% by volume, preferably between 10% and 90% by volume, between 40% and 80% by volume, or between 45% and 60% by volume, most preferably about 50% by volume; and Cell culture medium.

[0058] In one and / or more embodiments of the present invention, the hypotonic cell culture medium further includes about 0.5 mg / mL to 5 mg / mL of glucose, preferably about 2 mg / mL.

[0059] In one and / or more embodiments of the present invention, the liquid is water, preferably purified water.

[0060] In one and / or more embodiments of the present invention, the cell culture medium comprises vitamins, amino acids, and / or inorganic salts, preferably: The vitamins selected are inositol, choline chloride, para-aminobenzoic acid, folic acid, nicotinamide, pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, biotin, riboflavin, cyanocobalamin, and combinations thereof. The amino acids are selected from the group consisting of glutamine, arginine, asparagine, cysteine, leucine, isoleucine, lysine hydrochloride, serine, aspartic acid, glutamic acid, hydroxyproline, proline, threonine, tyrosine, valine, histidine, methionine, phenylalanine, glycine, tryptophan, glutathione, and combinations thereof; and Salts are selected from the group consisting of sodium chloride, sodium bicarbonate, disodium phosphate, potassium chloride, magnesium sulfate, calcium nitrate, and combinations thereof.

[0061] The present invention ultimately provides the use of a hypotonic cell culture medium in a method for inhibiting cell mitotic activity, wherein the hypotonic cell culture medium comprises: Liquid content (e.g., water content) up to 95% by volume, preferably between 10% and 90% by volume, between 40% and 80% by volume, or between 45% and 60% by volume, most preferably about 50% by volume; and Cell culture medium.

[0062] In an embodiment of the present invention, the present invention relates to the use of a hypotonic cell culture medium in a method for inhibiting the mitotic activity of cells, wherein the liquid is water, preferably purified water.

[0063] experiment Cells and Culture Media 293FT cells (R70007, Thermo Fisher Scientific) were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (Fetal Calf Serum, FCS, Greiner-Bio-one), GlutaMAX™ stabilized glutamine (Glutamax), sodium pyruvate, and non-essential amino acids (all from Gibco). K562 cells (CCL-243, American Type Culture Collection (ATCC)) were cultured in IMDM medium (Gibco, Glen Island, NE, USA) supplemented with 10% FCS (Greiner-Bio-one) and 1% penicillin-streptomycin (Gibco).

[0064] NK cells were expanded using SCGM medium (CellGenix), which was supplemented with 10% FCS and 200 units / mL of interleukin-2 (IL-2, trade name Proleukin, Clinigen).

[0065] Antibody Anti-IL21-APC antibody or anti-IL21-PE antibody, and anti-4-1BBL-PE-Cy7 antibody (all from Miltenyi) were used to identify transduced K562 cells. Anti-CD3 APC-Vio-770, anti-CD14-PE, anti-CD16-APC, anti-CD19-FITC, and anti-CD56-PerCP-Vio 700 antibodies (all from Miltenyi) were used to identify the expansion of blood lymphocytes and NK cells.

[0066] Construction of F012 feeder cells Genes encoding IL-21 (including the human CD4 transmembrane region) and 4-1BBL (CD137) were sequence-optimized by GeneArt and cloned into the T easy vector pMX (GeneArt). These two genes were then cloned from this vector into the third-generation lentiviral vector pCDH-CMV, which also contains EF1a-green fluorescent protein (GFP, i.e., EF1a-GFP) and the T2A-purinemycin resistance gene. The pCDH vector was mixed with helper packaging plasmids pMDLg / pRRE (Addgene 12251), pRSV-Rev (Addgene 12253), and pMD2.G (Addgene 12259), and transfected into 293FT cells with polyethyleneimine (Polyscience). Within 7 days post-transfection, supernatant from transfected cells was collected every 24 hours. The supernatants were combined, filtered through a 0.45 mm filter, and concentrated using Lenti-X lentiviral concentrator (Clontech). K-562 cells were transduced twice over 2 days using lentivirus encoding membrane-bound IL-21 / 4-1BB ligands (mIL-21 / 4-1BBL). 48 hours later, puromycin (1 mg / mL, Merck Millipore) was added to the cells, and viable cells were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence activated cell sorting (FACS) to confirm the expression of GFP, 4-1BBL, and membrane-bound IL-21 (mbIL-21).

[0067] The ability of a feeder cell bank to promote the expansion of NK cells obtained from CD3-depleted peripheral blood was analyzed. After single-cell cloning via FACS sorting and propagation, 25 K562-mIL-21-4-1BBL-GFP clones were selected to further validate the expression of mIL-21 and 4-1BBL. The 10 clones with the highest expression levels were selected for NK cell expansion experiments. Finally, one clone (F012) exhibited the strongest NK cell expansion-promoting effect within 10 days, and a small cryopreservation bank of this cell line was prepared.

[0068] One cryopreservation tube of cells was thawed and amplified, and tested for mycoplasma, sterility, endotoxins, and exogenous viruses. The results showed negative for pathogens and sterility. The cells were further amplified in a Class B cleanroom conforming to Good Manufacturing Practices (GMP) using 1-liter and 5-liter G-Rex® bioreactors (WilsonWolf) to establish a master cell bank (MCB).

[0069] Preparation of F012 feeder cells for NK cell expansion F012 cells were thawed from the working cell bank and seeded into T175 culture flasks for culture until 300 × 10⁶ cells were obtained. 6 10 cells. Then take 20 × 10 6 Cells were mixed with different hypotonic solutions containing varying ratios of water and SCGM medium / 10% FCS and incubated at 37°C in a 5% CO2 incubator. Cells were collected the following morning and counted using a NucleoCounter NC200 cell counter (Chemometec). Simultaneously, staining was performed using a LIVE / DEAD fixed aqueous dead cell staining kit (Thermo Fisher). FACS analysis was then performed on a MACSQuant to determine the percentage of live and dead cells.

[0070] These K562-F012 cell suspensions were re-inoculated into standard culture medium (IMDM / 10% FCS) and cultured for several days to observe whether the F012 cells proliferated after treatment.

[0071] In other experiments, cells were treated in different hypotonic solutions for 2 hours as described above. The cell suspension was then transferred to cryovials, and the cryovials containing the cell suspension were cooled to -80°C using a ViaFreeze programmed freezing system, then restored to room temperature. This process was repeated 1 to 4 times. Finally, the cell suspension was stored in liquid nitrogen until it was used for NK cell expansion experiments.

[0072] Amplification scheme The blood products were obtained from a blood donation sample (approximately 500 ml) from a blood bank (Sanquin). The blood products were collected into blood bags and loaded into the CliniMACSProdigy fully automated cell processing system (Miltenyi), a closed isolation and culture system.

[0073] Blood was separated using a density gradient (Ficoll), and mononuclear cell fractions were CD3 depleted by magnetic bead sorting. Remaining cells were adjusted to 2 × 10⁶ cells per well. 6NK cells were transferred to 6-well G-Rex deep-well plates (Wilson-Wolf) and cultured in 100 mL of SCGM medium (CellGenix) supplemented with 10% FCS, 1% penicillin-streptomycin, and 500 IU / mL recombinant human IL-2. K562 / mIL21 / 4-1BBL (F012) feeder cells treated with hypotonic solution were added to the G-Rex plates at a 1:1 ratio to stimulate NK cell proliferation. Cells were collected on day 7. NK cells were pooled and washed, and cell yield and viability were determined using a cell counter. Phenotypic analysis was performed using FACS.

[0074] As a control, F012 feeder cells irradiated with 100 to 750 Gy yielded the same results.

[0075] FACS analysis For flow cytometry analysis, cells were collected and washed with phosphate-buffered saline (PBS, Sigma). First, cells were stained in PBS on ice for 30 minutes using the LIVE / DEAD Immobilizable Aqueous Dead Cell Staining Kit (Thermo Fisher Scientific). Cells were then resuspended in 100 μL of PBS, and a pre-determined optimal dilution of a directly labeled monoclonal antibody was added for staining to perform lineage analysis. After incubation for 20 minutes, cells were washed twice with PBS. The cell pellet was resuspended in 200 μL of PBS for flow cytometry analysis, yielding 100,000 cells. Fluorescence signals were read using a Canto II flow cytometer (BD Biosciences), and data were analyzed using FlowJo 10.7 software (TreeStar). The final product was analyzed using 3x10⁻¹⁰ ppm. 6 GFP expression was detected in individual cells to identify residual F012 feeder cells.

[0076] Example 1 The growth curves of K562-F012 cells treated with different hypotonic solutions and subjected to four freeze-thaw cycles, compared to untreated K562-F012 cells, are as follows: Figure 1 As shown, untreated K562-F012 cells proliferated normally, increasing approximately 24-fold within 7 days of culture. In contrast, all cells treated with different hypotonic solutions and subjected to four freeze-thaw cycles ceased proliferation, and their numbers decreased.

[0077] Example 2 Figure 2 It shows something similar to Example 1 and as follows Figure 1The results (growth curves) of K562-F012 cells treated with different hypotonic solutions and subsequently subjected to four freeze-thaw cycles are shown. However, in this example, the treated K562-F012 cells were not compared with normally expanded K562-F012 cells. All cells treated with different hypotonic solutions ceased proliferation, and the cell number decreased. Cells treated with 95% or 100% water as hypotonic solutions showed the most significant decrease in cell number after 7 days of culture (the values ​​are uncertain as they fell below the detection limit of the cell counter). In hypotonic solutions, cells remained present in a detectable number.

[0078] Example 3 Figure 3 The expansion of NK cells is shown in absolute numbers. NK cells were expanded on K562-F012 cells treated with different hypotonic solutions and / or subjected to different numbers of freeze-thaw cycles. Figure 3 The results show that K562-F012 cells treated with hypotonic solutions containing 95% or 100% water cannot achieve effective NK cell expansion.

Claims

1. A method for preparing a suspension of mitotically inhibited cells, the method comprising the following steps: a) Provide cells suspended in cell culture medium; as well as b) Inhibit the mitotic activity of the cells in the suspension provided in step a), thereby obtaining cells with inhibited mitosis. The step of inhibiting the mitotic activity of the cells includes the following steps: The suspension is hypotonic, followed by exposure of the cells to at least one freeze-thaw cycle, or vice versa; or The cells are exposed to two or more freeze-thaw cycles, and optionally the suspension is hypotonic before the freeze-thaw cycles. The hypotonic treatment of the suspension includes the step of adding a liquid, such as water, to the suspension containing the cells to form a hypotonic cell culture medium. The characteristic feature is that the process conditions for the step of inhibiting the mitotic activity of the cells are selected such that the resulting mitotically inhibited cells maintain the required biological activity.

2. The method according to claim 1, wherein, The at least one freeze-thaw cycle or the two or more freeze-thaw cycles, including two, three or four freeze-thaw cycles, preferably four freeze-thaw cycles.

3. The method according to claim 1 or 2, wherein, The amount of liquid added to the suspension is selected such that the content of the added liquid in the resulting hypotonic cell culture medium does not exceed 95% by volume. Preferably, the hypotonic cell culture medium has an added liquid content between 10% and 90% by volume.

4. The method according to any one of the preceding claims, wherein, The cell culture medium is selected from the group consisting of SCGM, RPMI, MEMα and EMDM, or any one or more combinations thereof, preferably, wherein the cell culture medium is SCGM.

5. The method according to any one of the preceding claims, wherein, After step b), the cells are stored, preferably in a refrigerator, more preferably in a -80°C refrigerator, or in liquid nitrogen.

6. The method according to any one of the preceding claims, wherein, The at least one freeze-thaw cycle includes the following subsequent steps: h) Provide the cells in the suspension provided in step a); i) Cool the suspension to a temperature below its freezing point, preferably until the suspension reaches a temperature below -20°C, more preferably below -40°C, and most preferably between -40°C and -80°C; j) Heating the suspension to a temperature above its freezing point; and k) Optionally, the cooling and heating in steps i) and j) are repeated once, twice, or three times, preferably three times.

7. The method according to any one of the preceding claims, wherein, The total number of mitotically inhibited cells produced by the freeze-thaw cycle accounts for 90% to 100% of the cells suspended in the cell culture medium.

8. The method according to any one of the preceding claims, wherein, The cells provided in step a) are selected from the group consisting of feeder cells, wherein the feeder cells are capable of stimulating the proliferation of immune cells.

9. The method according to claim 8, wherein, The feeder cells are provided with transfected ligands to stimulate the proliferation of immune cells.

10. A method for culturing target cells, the method comprising the following subsequent steps: i) Provide a suspension of mitotically inhibited cells prepared by the method of any one of the preceding claims; ii) Provide target cells, and then add the target cells to the suspension to form a co-culture of target cells and mitotically inhibited cells; iii) Expanding the target cells in the suspension; and iv) Collect the expanded target cells.

11. The method according to claim 10, wherein, The target cells were selected from a group composed of natural killer cells.

12. The method according to claim 10 or 11, wherein, During the method of culturing target cells, the ratio of target cells to mitotically inhibited cells is selected to prevent the mitotically inhibited cells from overgrowing.

13. The method according to any one of claims 10 to 12, wherein, The collected target cells contained virtually no cells with inhibited mitosis.

14. The use of hypotonic cell culture media in methods for inhibiting cell mitotic activity, wherein, The hypotonic cell culture medium includes: Liquid content up to 95% by volume, preferably between 10% and 90% by volume, between 40% and 80% by volume, or between 45% and 60% by volume, most preferably about 50% by volume, said liquid content being, for example, water content; and Cell culture medium.

15. The use according to claim 14, wherein, The liquid is water, preferably purified water.

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