Method of preparing a suspension of mitotically suppressed cells

Abstract

The invention relates to a method of preparing a suspension of mitotically suppressed cells, comprising the steps of a) providing cells suspended in a cell culture medium, and b) suppressing the mitotic activity of the cells in the suspension, thereby obtaining mitotically suppressed cells. The step of suppressing the mitotic activity of the cells comprises the step of hypotonic treating the suspension, followed by exposing the cells to at least one freeze-thaw cycle, or vice versa or exposing the cells to two or more freeze-thaw cycles, optionally preceded by a hypotonic treatment of the suspension. In the method of the present invention the hypotonic treatment of the suspension comprises the step of adding liquid, such as water, to the suspension comprising the cells to form a hypotonic cell culture medium. The invention further relates to a method for culturing cells of interest and to the use of a hypotonic cell culture medium.

Description

Method of preparing a suspension of mitotically suppressed cellsField of the invention

[0001] The invention relates to a method of preparing a suspension of mitotically suppressed cells, in particular mitotically suppressed feeder cells. The invention further relates to a method for culturing cells, in particular natural killer cells. Further the invention relates to the use of a hypotonic cell culture medium.Background

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Natural killer cells (NK cells) are a type of lymphocyte which exhibit a spontaneous and rapid response to various threats, including infected, transformed or malignantly degenerated cells, also known as tumour cells. One of the defining features of NK cells, namely their ability to discriminate between healthy and abnormal cells, is achieved through a delicate balance of inhibitory and activating receptors on the cellular surface. Through this defining feature NK cells are able to perform their primary function, called “natural killing”, which involves the destruction of target cells through the release of cytotoxic molecules, such as perforin and granzymes.

[0004] NK cells have garnered significant interest in clinical research for their potential use in cancer immunotherapy. Clinical strategies are being developed such as adoptive NK cell therapy, where ex vivo expanded and activated NK cells are infused back into patients to enhance anti-tumor responses. Additionally, the modulation of NK cell activity to improve treatments for various diseases is widely investigated.

[0005] Several protocols have been described to induce NK cell proliferation in vitro to increase the number of NK cells, hereinafter also described as "NK cell expansion". NK cells can be expanded in vitro by cultivating with combinations of cytokines, bysupplementing cell culture media with small molecules and by using cytokines, antibodies and / or feeder cells to stimulate expansion in vitro.

[0006] Feeder cell based NK cell expansion protocols generate high NK cell numbers. Feeder cells induce activation and proliferation of NK cells via cell surface receptorligand interactions, supported by cytokines (Gurney et al., (2022) Front. In Immunol. Vol. 13). Effective protocols comprise the use of genetically modified feeder cells that have been transfected with proteins that stimulate proliferation of NK cells specifically (e.g. (K562, an erythroleukemia cell line deficient in HLA class I)). Alternatively, B cell lines naturally immortalized by infection with Epstein-Barr Virus may be used in such protocols.

[0007] To use feeder cells in NK cell expansion the feeder cell line is exposed to high doses of ionizing radiation, often about 100 Gy. By irradiating feeder cells the mitosis of the cells is suppressed so that they do not proliferate (and thereby overgrow the intended immune cells, such as NK cells during expansion). A limitation of such methods is that companies expanding NK cells have to rely on third parties to provide irradiated cells since such radiation facilities are strictly regulated, expensive, and difficult to maintain. Also, the transport of such irradiated cells is a logistic burden for cell companies.

[0008] Additionally, regulatory authorities ask for strict safety parameters and risk assessment on the presence of the feeder cells in NK cell based therapies in order to reduce the risk that NK cell based therapies are contaminated with viable feeder cells.

[0009] Therefore there is a need for alternative, optionally improved, methods for expanding NK cells which allows to expand NK cells to very high numbers, preferably to numbers which are sufficient to serve clinical trials for clinical application of the expanded NK cells in patients and preferentially by utilizing feeder cells in the expansion of the NK cells. In particular, there is a need for an alternative method for preparing a suspension of mitotically suppressed cells, wherein the mitotically suppression of cells using high doses of ionizing radiation is no longer required.Summary of the invention

[0010] In order to provide such alternative method, in a first aspect of the invention a method of preparing a suspension of mitotically suppressed cells is provided, wherein the method comprising the steps of a) providing cells suspended in a cell culture medium, and b) suppressing the mitotic activity of the cells in the suspension provided in step a), thereby obtaining mitotically suppressed cells, wherein the step of suppressing the mitotic activity of the cells comprises the step of: hypotonic treating the suspension, followed by exposing the cells to at least one freeze-thaw cycle, or vice versa; or exposing the cells to two or more freeze-thaw cycles, optionally preceded by a hypotonic treatment of the suspension.

[0011] It is noted that the above defined hypotonic treatment of the suspension comprises the step of adding liquid, e.g. water, to the suspension comprising the cells to form a hypotonic cell culture medium. It is further noted that the process conditions of the step of suppressing the mitotic activity of the cells are selected such that the resulting mitotically suppressed cells maintain the desired biological activity. By providing a method wherein the cells are mitotically suppressed but do maintain their desired biological activity, the suspension comprising the mitotically suppressed cells can be directly used in, for example, culturing cells of interest, such as NK cells.

[0012] In a second aspect there is provided for a suspension of mitotically suppressed cells obtainable by the method of preparing a suspension of mitotically suppressed cells in accordance with the invention.

[0013] As already noted above, the present invention further relates, in a third aspect there is provided for a method for culturing cells of interest, wherein the method comprising the subsequent steps of: i) providing the suspension of mitotically suppressed cells in accordance with the invention; ii) providing cells of interest and subsequently adding the cells of interest to the suspension to form a co-culture of cells of interest and mitotically suppressed cells;iii) expanding the cells of interest in the suspension; and iv) harvesting the expanded cells of interest.

[0014] In a fourth aspect of the present invention a hypotonic cell culture medium is provided, wherein the hypotonic cell culture medium comprises: a liquid content, such as water, of up to 95 vol-%, preferably between 10 vol.% and 90 vol.%, between 40 vol. % and 80 vol.%, or between 45 vol.% and 60 vol.% liquid, most preferably about 50 vol.% liquid; and a cell culture medium.

[0015] In a fifth aspect there is provided for the use of the hypotonic cell culture medium of the fourth aspect of the present invention in a method for suppressing the mitotic activity of cells.Figure description

[0016] Figure 1 : growth curve of K562-F012 cells treated with different hypotonic solutions and 4 freeze / thaw cycles vs non treated K562-F012 cells.

[0017] Figure 2: growth curve of K562-F012 cells treated with different hypotonic solutions followed by 4 freeze / thaw cycles as shown in Figure 1 but not compared to normally expanding K562-F012 cells.

[0018] Figure 3: NK cell expansion in absolute numbers. NK cells were expanded on different hypotonic solution treated K562-F012 cells and / or treated with different numbers of freeze thaw cycles.Definitions

[0019] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0020] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0021] For purposes of the present invention, the following terms are defined below.

[0022] As used herein, the singular form terms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. For example, a method for culturing a cell includes the culturing of a plurality of cells (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more).

[0023] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.”

[0024] As used herein, “at least” a particular value means that particular value or more. For example, "at least 2" is understood to be the same 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 particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11 , etc.

[0025] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps, or components. It also encompasses the more limiting “to consist of.”

[0026] As used herein, "exemplary", “for example” or “such as” means "serving 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 that consists of one step wherein a suspension comprising a cell culture medium and cells, or a cell, or cell population is exposed to a temperature that is at or below the freezing point of said suspension or cell (population), and one subsequent step of exposing the frozen cell or cell population to a temperature that is above the freezing point of said suspension or cell (population). It is contemplated herein that the freezing point may differ per cell culture medium used in the suspension comprising cells. It is contemplated herein that a freeze-thaw cycle affects the proliferation of said cell or cell population, for example by interfering with mitotic processes of the cell or cell population.

[0028] As used herein, "in vivo" refers to an event that takes place in a subject's body; "in vitro" and “ex vivo" refer to an event that takes places outside of a subject's body”. For example, an in vitro assay or method encompasses any assay or method conducted outside of a subject. In vitro assays or methods encompass cell-based assays in which cells, alive or dead, are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed. The terms "in vivo", “in vitro" and “ex vivo" are well known in the art.

[0029] As used herein, the term “mitotically suppressed” refers to a phenomenon wherein the mitotic processes of a cell are slowed down and / or halted to the extent that the cell is limited in its ability to divide, proliferate and expand. In some cases, as also described herein, mitotic suppression of a cell may lead to the inactivation of mitosis in a cell, thereby annulling any ability of the (healthy) cell to divide by natural occurring processes. In essence, the term relates to cells that have been intentionallyprevented from undergoing cell division or mitosis. As such mitotic suppression is a process to halt cell proliferation.

[0030] As used herein, the term “desired biological activity” refers to a biological activity of a cell, substance or compound which biological activity is suitable for further use. A desired biological activity may include the biological capability of a cell to stimulate cell proliferation and expansion. Another desired biological activity may include the ability of cells, substance or compound to interact with other biological systems in order to inhibiting the growth of cancer cells, reducing inflammation, targeting specific receptors in the body, promoting certain physiological responses or the like.

[0031] When combined, the term “mitotically suppressed cells that maintain the desired biological activity” refers to cells that have been treated to stop their division (mitotically suppressed) while still retaining their intended or preferred biological activity. As such, for those cells the cell growth or behaviour of those cells is controlled without compromising the desired function or activity of those cells.Detailed description

[0032] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0033] It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0034] It was found that the methods, suspension and hypotonic cell culture medium according to the invention provide for improved methods for culturing and expanding NK cells. It was surprisingly found by the inventors that, by using the methodsaccording to the invention, the step of irradiation of feeder cells is redundant in the processes of expanding NK cells. One of the benefits of the invention thus is that there is no need to rely on third parties having radiation facilities to allow for the producing of irradiated cells.

[0035] Methods in accordance to the invention surprisingly produce mitotically inactivated cells, thereby ensuring that the cells will not proliferate. One of the benefits provided by the method in accordance to the invention is that the method does not rely on the use of irradiation of cells. This means that the method can also be used on cell lines that are resistant to ionizing radiation.

[0036] A further surprising benefit of the present invention is that the contamination of a culture of cells of interest by other viable cells is significantly reduced, for example in comparison to a culture of cells of interest by viable irradiated cells. As such, it is contemplated that a culture of cells of interest, for example NK cells, will likely meet strict clinical and regulatory requirements set by authorities with respect to contamination by viable cells, for example viable feeder cells.

[0037] The invention is related to a method of preparing a suspension of mitotically suppressed cells, the method comprising the steps of a) providing cells suspended in a cell culture medium, and b) suppressing the mitotic activity of the cells in the suspension provided in step a), thereby obtaining mitotically suppressed cells, wherein the step of suppressing the mitotic activity of the cells comprises the step of: hypotonic treating the suspension, followed by exposing the cells to at least one freeze-thaw cycle, or vice versa; or exposing the cells to two or more freeze-thaw cycles, optionally preceded by a hypotonic treatment of the suspension.

[0038] The hypotonic treatment of the suspension comprises the step of adding liquid, such as water, to the suspension comprising the cells to form a hypotonic cell culture medium. Further, the process conditions of the step of suppressing the mitotic activity of the cells are selected such that the resulting mitotically suppressed cells maintain their desired biological activity.

[0039] In step b) of the method provided herein the cells are suppressed in their mitotic activity by exposing the cells to either process of: at least one freeze-thaw cycle, preceded by a hypotonic treatment of the suspension, or vice versa; or two or more freeze-thaw cycles, optionally preceded by a hypotonic treatment of the suspension.

[0040] By exposing the cells to either one of these processes it was found that mitotically suppressed cells were obtained. Preferably, it was found that, when process conditions of either one of the processes are selected in an optimal manner, for example as embodied herein, the resulting mitotically suppressed cells maintain the desired biological activity.

[0041] In an embodiment of the invention and / or embodiments thereof the at least one freeze-thaw cycle or the two or more freeze-thaw cycles comprise two, three or four freeze-thaw cycles, preferably four freeze-thaw cycles.

[0042] In an embodiment of the invention and / or embodiments thereof 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 vol-%, preferably wherein the hypotonic cell culture medium has an added liquid content of between 10 vol-% and 90 vol-%.

[0043] It is preferred that the liquid content, e.g. water content, of the hypotonic culture is between 10 vol-% and 90-vol% or between 20 vol-% and 80-vol%, or between 25 vol-% and 75 vol-%, or between 30 vol-% and 70 vol-%, or between 40 vol-% and 60 vol-%, more preferably about 50 vol-%.

[0044] In an embodiment of the invention and / or embodiments thereof the cell culture medium is selected from the group consisting of SCGM, RPMI, MEM a and EMDM, or a combination of any one or more, preferably wherein the cell culture medium is SCGM.

[0045] It is preferred that the hypotonic culture medium comprises, preferably consist essentially of, liquid, such as water, and a culture medium. It is further preferred that the hypotonic culture medium comprises a cell culture medium is selected from the group consisting of SCGM, RPMI, MEM a and EMDM, or a combination of any one or more.

[0046] In an embodiment of the invention and / or embodiments thereof after step b) the cells are stored, preferably in a freezer, preferably a -80°C freezer, or in liquid nitrogen.

[0047] In an embodiment of the invention and / or embodiments thereof the at least one freeze-thaw cycle comprises the subsequent steps of: h) providing the cells in the suspension provided in step a); i) cooling the suspension to a temperature below its freezing point, preferably until the suspension reaches a temperature of below -20°C, more preferably below -40°C, most preferably reaches a temperature between -40°C and -80°C; j) heating the suspension to a temperature above its freezing point; and k) optionally, repeating the cooling and heating of steps i) and j) one, two or three times, preferably three times.

[0048] In an embodiment of the invention and / or embodiments thereof the percentage of total mitotically suppressed cells by the freeze-thaw cycle is of between 90% and 100% of the cells suspended in the cell culture medium.

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

[0050] In an embodiment of the invention and / or embodiments thereof the feeder cells are provided with transfected ligands for stimulating immune cell proliferation.

[0051] The invention further provides for a suspension of mitotically suppressed cells obtainable by the method of preparing a suspension mitotically suppressed cells in accordance with the invention.

[0052] The invention further provides for a method for culturing cells of interest, the method comprising the subsequent steps of: i) providing a suspension of mitotically suppressed cells prepared by the method according to the invention; ii) providing cells of interest and subsequently adding the cells of interest to the suspension to form a co-culture of cells of interest and mitotically suppressed cells; iii) expanding the cells of interest in the suspension; and iv) harvesting the expanded cells of interest.

[0053] In an embodiment of the invention and / or embodiments thereof the cells of interest are selected from the group consisting of natural killer cells (NK cells).

[0054] In an embodiment of the invention and / or embodiments thereof, during the method of culturing cells of interest, the ratio of cells of interest and the mitotically suppressed cells is selected such that overgrowth of mitotically suppressed cells is prevented.

[0055] In an embodiment of the invention and / or embodiments thereof the harvested cells of interest are substantially free of mitotically suppressed cells.

[0056] In an embodiment of the invention and / or embodiments thereof the method further comprises the steps of:1) providing another suspension of mitotically suppressed cells of mitotically suppressed cells according to the invention; and2) adding the suspension of step 1) to the suspension of step iii).

[0057] The invention further provides for a hypotonic cell culture medium, wherein the hypotonic cell culture medium comprises: a liquid content, such as a water content, of up to 95 vol-%, preferably between 10 vol.% and 90 vol.%, between 40 vol. % and 80 vol.%, or between 45 vol.% and 60 vol.% liquid, most preferably about 50 vol.% liquid; and a cell culture medium.

[0058] In an embodiment of the invention and / or embodiments thereof the hypotonic cell culture medium further comprises about 0.5 mg / mL to 5 mg / mL glucose, preferably about 2 mg / mL.

[0059] In an embodiment of the invention and / or embodiments thereof the liquid is water, preferably purified water.

[0060] In an embodiment of the invention and / or embodiments thereof the cell culture medium comprises vitamins, amino acids and / or salts, preferably wherein: the vitamins are selected from the group consisting of i-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, cystine, leucine, isoleucine, lysine hydrochloride, serine, aspartic acid, glutamic acid, hydroxyproline, proline, threonine, tyrosine, valine, histidine, methionine, phenylalanine, glycine, tryptophan, glutathione and combinations thereof; and the salts are selected from the group consisting of sodium chloride, sodium bicarbonate, disodium phosphate, potassium chloride, magnesium sulphate, calcium nitrate and combinations thereof.

[0061] The invention finally provides for a use of the hypotonic cell culture medium in a method for suppressing the mitotic activity of cells, wherein the hypotonic cell culture medium comprises: a liquid content, such as a water content, of up to 95 vol-%, preferably between 10 vol.% and 90 vol.%, between 40 vol. % and 80 vol.%, or between 45 vol.% and 60 vol.% liquid, most preferably about 50 vol.% liquid; and a cell culture medium.

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

[0063] 293FT cells (R70007; Thermo Fisher) in DMEM high glucose, supplemented with 10% FCS (Greiner-Bio-one), Glutamax, Sodium pyruvate and Non-Essential Amino Acids (all Gibco). K562 cells (CCL-243, ATCC) were cultured in IMDM (Gibco, Grand Island, NE, USA) supplemented with 10% FCS (Greiner-Bio-one) and 1% Pen- strep (Gibco).

[0064] NK cells were expanded in SCGM medium (CellGenix) in the presence of 10% FCS, and 200U / ml IL-2 (Proleukin, Clinigen).Antibodies

[0065] Anti-IL21-APC or PE, Anti 4-1 BBL-PE-Cy7 (both Miltenyi) were used to identify transduced K562 cells. Anti-CD3 APC-Vio-770, Anti-CD14-PE, Anti-CD16-APC, Anti- CD19-FITC, Anti-CD56-PerCP-Vio 700 antibodies (all Miltenyi) were used to identify blood lymphocytes and NK cell expansion.Establishment of F012 Feeder cells

[0066] The genes encoding IL-21 (including the human CD4 transmembrane region) and 4-1 BBL (CD137) were sequence optimized by GeneArt and cloned into the T easy vector pMX (GeneArt). From this vector, the 2 genes were cloned into a 3rd generation lentiviral vector, pCDH-CMV, which further contained EF1a-GFP and a T2A-puromycin resistance gene. Together with the helper packaging plasmids pMDLg / pRRE (Addgene 12251), pRSV-Rev (Addgene 12253) and pMD2.G (Addgene 12259) the pCDH vector was transfected in 293FT with the addition of poly ethylenimine (Polyscience). Supernatant from the transfected cells was harvested every 24 hours for 7 days, pooled, passaged through a 0.45 .m filter and concentrated with Lenti-X Concentrator (Clontech). K-562 cells were transduced twice with the mlL-21 / 4-1 BBL encoding lentivirus in the presence of protamine sulphate (10 .g / ml) for 2 days. After 48 hours, puromycin (1 .g / .1 I Merck Millipore) was added to the cells and living cells were analysed by RT-PCR and FACS analysis to confirm the expression of GFP, 4- 1 BBL and mblL-21.

[0067] The feeder cell pool was analysed to promote NK cell expansion obtained from CD3 depleted peripheral blood. After single cell cloning by FACS sorting and propagation, 25 clones of K562-mlL-21-4-1 BBL-GFP were chosen for further verification of mlL-21 and 4-1 BBL expression and of the best expressors 10 were chosen for NK cell expansion assays. Finally, one clone (F012) was selected that most potently promoted NK cell expansion in 10 days and a small frozen stock was made of this cell line.

[0068] One vial was thawed and expanded and tested for mycoplasma, sterility, endotoxin, and adventitious virus and found to be negative for pathogens and sterile. Cells were further expanded in 1 L and 5L G-Rex® bioreactors (Wilson Wolf) in a B- grade clean-room under Good Manufacturing Practice to create a Master Cell Bank.F012 Feeder cell preparation for NK cell expansion

[0069] F012 cells were thawed from a working cell stock in T 175 flasks until 300x106cells were obtained. Then 20x106cells were mixed with different hypotonic solutions containing different mixes of water and SCGM medium / 10% FCS and incubated in a 5% CO2 incubator at 37°C. Next morning, cells were harvested, counted on a NucleoCounter NC200 (Chemometec) as well as using the LIVE / DEAD fixable aqua dead cell stain dye (Thermo Fisher) followed by FACS analysis on a MACSQuant to determine percentages of living and dead cells.

[0070] These K562-F012 cell suspensions were reseeded in regular culture medium (IMDM / 10% FCS) and cultured for several days to observe possible growth of F012 cells after treatments.

[0071] In other experiments, cells were treated as above for 2 hours in different hypotonic solutions followed by transferring cell solutions to cryovials and subjecting the vials to 1 to 4 freezing / thawing cycles in a ViaFreeze by bringing the vials with cell solution to -80°C and return to room temperature. Finally, cell solutions were stored in the liquid nitrogen until use in NK cell expansion assays.

[0072] Blood products were obtained through blood donations (± 500 ml) from a blood bank (Sanquin). The blood product was collected in a bag and loaded on CliniMACS Prodigy (Miltenyi), which is a closed isolation and culture system.

[0073] Blood is subjected to density gradient (Ficoll) separation and the mononuclear fraction is CD3 depleted by magnetic bead sorting. The remaining cells (normalized to 2x106NK cells per well) are transferred to 6-well G-Rex Deep Well Plate (Wilson Wolf) and cultured in 100ml SCGM medium (CellGenix) supplemented with 10% FCS, 1 % penicillin-streptomycin and 500 lll / mL recombinant human IL-2. To this G-Rex plate hypotonical solution treated K562 / mlL21 / 4-1 BBL (F012) feeder cells in a 1 :1 ratio was added to stimulate NK cell proliferation. Cells were harvested on day 7. The NK cells are combined and washed, followed by determining yield and viability on the NucleoCounter followed by FACS analysis for phenotyping.

[0074] As controls we used 100 Gy to 750 Gy irradiated F012 feeder cells that gave the same results.FACS analysis

[0075] For flow cytometric analysis, cells were harvested, washed with PBS (Sigma) and first stained with Live / Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) in PBS on ice for 30 min. Then cells were resuspended in 100 pL PBS and stained with directly labelled mAb for lineage analysis at predetermined optimal dilution. Cells were washed twice with PBS after a 20 min incubation. Cell pellets were resuspended in 200 pL PBS for flow cytometric analysis acquiring 100.000 cells. Fluorescence was read on a Cantoll flow cytometer (BD Biosciences). Data was analysed with FlowJo 10.7 (TreeStar) software. 3x106cells of the final products were tested for GFP expression to detect absence of F012 feeder cells.

[0076] Growth curve of K562-F012 cells treated with different hypotonic solutions and followed by 4 freeze / thaw cycles versus non treated K562-F012 cells are shown in Figure 1. Non treated K562-F012 cells expanded regularly and expanded about 24-fold in 7 days of culture. In contrast all cells treated with different hypotonic solutions and 4 freeze / thaw cycles stopped in proliferation and reduced in number.

[0077] Figure 2 shows the results (growth curve) of K562-F012 cells treated with different hypotonic solutions followed by 4 freeze / thaw cycles similar to example 1 and shown in Figure 1. However, in this example the treated K562-F012 cells are not compared to normally expanding K562-F012 cells. All cells treated with different hypotonic solutions stopped in proliferation and reduced in number. Cells treated with 95% or 100% water as hypotonic solution, decreased most in cell number after 7 days (values are less certain as they fall below the detection limit of the nucleocounter). In the less hypotonic solutions cells remained present in detectable numbers.

[0078] Figure 3 shows the NK cell expansion in absolute numbers. NK cells were expanded on different hypotonic solution treated K562-F012 cells and / or treated with different numbers of freeze thaw cycles. As it can be seen from the results as depicted in Figure 3, treated K562-F012 with a hypotonic solution comprising 95% or 100% water did not result in an efficient NK cell expansion.

Claims

CLAIMS1. A method of preparing a suspension of mitotically suppressed cells, the method comprising the steps of: a) providing cells suspended in a cell culture medium; and b) suppressing the mitotic activity of the cells in the suspension provided in step a), thereby obtaining mitotically suppressed cells, wherein the step of suppressing the mitotic activity of the cells comprises the step of: hypotonic treating the suspension, followed by exposing the cells to at least one freeze-thaw cycle, or vice versa; or exposing the cells to two or more freeze-thaw cycles, optionally preceded by a hypotonic treatment of the suspension, wherein the hypotonic treatment of the suspension comprises the step of adding a liquid, such as water, to the suspension comprising the cells to form a hypotonic cell culture medium, characterised in that the process conditions of the step of suppressing the mitotic activity of the cells are selected such that the resulting mitotically suppressed cells maintain the desired 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 comprise 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 the liquid added to the suspension is selected such that the added liquid content of the resulting hypotonic cell culture medium does not exceed 95 vol-%, preferably wherein the hypotonic cell culture medium has an added liquid content of between 10 vol-% and 90 vol-%.

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

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

6. The method according to any of the preceding claims, wherein the at least one freeze-thaw cycle comprises the subsequent steps of: h) providing the cells in the suspension provided in step a); i) cooling the suspension to a temperature below its freezing point, preferably until the suspension reaches a temperature of below -20°C, more preferably below -40°C, most preferably reaches a temperature between -40°C and -80°C; j) heating the suspension to a temperature above its freezing point; and k) optionally, repeating the cooling and heating of steps i) and j) one, two or three times, preferably three times.

7. The method according to any of the preceding claims, wherein the percentage of total mitotically suppressed cells by the freeze-thaw cycle is of between 90% and 100% of the cells suspended in the cell culture medium.

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

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

10. A method for culturing cells of interest, the method comprising the subsequent steps of: i) providing a suspension of mitotically suppressed cells prepared by the method according to any of the preceding claims; ii) providing cells of interest and subsequently adding the cells of interest to the suspension to form a co-culture of cells of interest and mitotically suppressed cells; iii) expanding the cells of interest in the suspension; and iv) harvesting the expanded cells of interest.

11. The method according to claim 10, wherein the cells of interest are selected from the group consisting of natural killer cells.

12. The method according to claim 10 or 11 , wherein, during the method of culturing cells of interest, the ratio of cells of interest and the mitotically suppressed cells is selected such that overgrowth of mitotically suppressed cells is prevented.

13. The method according to any of claims 10-12, wherein the harvested cells of interest are substantially free of mitotically suppressed cells.

14. Use of a hypotonic cell culture medium in a method for suppressing the mitotic activity of cells, wherein the hypotonic cell culture medium comprises: a liquid content, such as a water content, of up to 95 vol-%, preferably between 10 vol.% and 90 vol.%, between 40 vol. % and 80 vol.%, or between 45 vol.% and 60 vol.% liquid, most preferably about 50 vol.% liquid; and a cell culture medium.

15. Use according to claim 14, wherein the liquid is water, preferably purified water.

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