CN-122003496-A - Method for preparing a suspension of mitotically inhibited cells

CN122003496ACN 122003496 ACN122003496 ACN 122003496ACN-122003496-A

Abstract

The present invention relates to a method for preparing a suspension of mitotically inhibited cells 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 mitotically inhibited cells. The step of inhibiting the mitotic activity of the cells comprises subjecting the suspension to a hypotonic treatment followed by exposing the cells to at least one freeze-thaw cycle and vice versa, or exposing the cells to two or more freeze-thaw cycles, optionally subjecting the suspension to a hypotonic treatment prior to the freeze-thaw cycles. In the method of the invention, 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. The invention also relates to a method for culturing target cells and the use of the hypotonic cell culture medium.

Inventors

Wilfred Thomas. Vincent. Helmholtz
Gladys Marinus Joseph Bosch
Eric Young Velvers

Assignees

奇玛斯有限公司

Dates

Publication Date: 20260508
Application Date: 20240816
Priority Date: 20230823

Claims (15)

1. A method of 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 said cells in said suspension provided in step a), thereby obtaining mitotically inhibited cells, Wherein the step of inhibiting the mitotic activity of the cell comprises the steps of: subjecting said suspension to hypotonic treatment followed by exposing said cells to at least one freeze-thawing cycle or vice versa, or Exposing the cells to two or more freeze-thaw cycles, optionally hypotonic treating the suspension prior to the freeze-thaw cycles, Wherein said hypotonic treatment of said suspension comprises the step of adding a liquid to said suspension comprising said cells to form a hypotonic cell culture medium, the liquid is for example water which is used for the treatment of the skin, Characterized in that the process conditions of the step of inhibiting the mitotic activity of the cells are selected such that the resulting mitotically inhibited 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 comprises 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 added liquid in the resulting hypotonic cell culture medium does not exceed 95 vol.%, preferably wherein the hypotonic cell culture medium has a content of added liquid between 10 and 90 vol.%.
4. The method of any one 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 thereof, preferably wherein the cell culture medium is SCGM.
5. The method according to any of the preceding claims, wherein the cells are stored after step b), preferably in a refrigerator, more preferably in a-80 ℃ refrigerator, or in liquid nitrogen.
6. The method according to any of the preceding claims, wherein the at least one freeze-thaw cycle comprises the following subsequent steps: 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 below-20 ℃, more preferably below-40 ℃, most preferably between-40 ℃ and-80 ℃; j) Heating said suspension to a temperature above its freezing point, and K) Optionally, the cooling and heating of steps i) and j) is repeated 1, 2 or 3 times, preferably 3 times.
7. The method of any one of the preceding claims, wherein the total mitotically inhibited cells produced by the freeze-thaw cycle account 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 layer cells, wherein the feeder layer cells are capable of stimulating immune cell proliferation.
9. The method of claim 8, wherein the feeder cells are provided with a transfected ligand for stimulating immune cell proliferation.
10. A method of culturing a target cell, the method comprising the following subsequent steps: i) Providing a suspension of mitotically inhibited cells prepared by the method of any one of the preceding claims; ii) providing a target cell, followed by adding the target cell to the suspension to form a co-culture of the target cell and the mitotically inhibited cell; iii) Amplifying the target cells in the suspension, and Iv) collecting the expanded target cells.
11. The method of claim 10, wherein the target cell is selected from the group consisting of natural killer cells.
12. The method of claim 10 or 11, wherein the ratio of target cells to the mitotically inhibited cells is selected to prevent overgrowth of the mitotically inhibited cells during the method of culturing target cells.
13. The method of any one of claims 10 to 12, wherein the target cells collected are substantially free of mitotically inhibited cells.
14. Use of a hypotonic cell culture medium in a method for inhibiting mitotic activity of a cell, wherein the hypotonic cell culture medium comprises: A liquid content of up to 95% by volume, preferably between 10% and 90% by volume, 40% and 80% by volume, or 45% and 60% by volume, most preferably about 50% by volume, of a liquid, for example a water content, and Cell culture medium.
15. Use according to claim 14, wherein the liquid is water, preferably purified water.

Description

Method for preparing a suspension of mitotically inhibited cells Technical Field The present invention relates to a method of preparing a suspension of mitotically inhibited cells, in particular a suspension of mitotically inhibited feeder cells. The invention also relates to a method of culturing a cell, in particular a natural killer cell. The invention also relates to the use of hypotonic cell culture media. Background This background description contains information that may be used to understand the present application. It is not an admission that any of the information provided by the present application is prior art, or relevant to the presently claimed application, nor that any publication specifically or implicitly referenced is prior art. Natural Killer (NK) cells are lymphocytes that respond spontaneously and rapidly to a variety of threats, including infected cells, transformed cells, malignant degenerated cells, also known as tumor cells. One of the defining characteristics of NK cells, namely its ability to distinguish healthy cells from abnormal cells, is achieved by a delicate balance between cell surface inhibitory and activating receptors. By virtue of this defined feature, NK cells are able to perform their main function known as "natural killing", namely to achieve destruction of target cells by releasing cytotoxic molecules such as perforin, granzyme, etc. NK cells have attracted great interest in clinical research due to their potential use in cancer immunotherapy. Clinical strategies are under development, such as adoptive NK cell therapies that reinfusion ex vivo expanded and activated NK cells into patients to enhance anti-tumor responses. In addition, studies on the regulation of NK cell activity to optimize the treatment of various diseases have also been widely conducted. Several protocols for inducing NK cell proliferation in vitro to increase the number of NK cells, also described below as "NK cell proliferation", have been disclosed in the prior art. In vitro expansion of NK cells can be achieved by culturing using a combination of cytokines, supplementation of small molecules in cell culture media, and in vitro stimulation of expansion by using cytokines, antibodies and/or feeder cells. Feeder cell-based NK cell expansion protocols produce 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, 2029, immunology front end, volume 13) (Gurney et al, (2022) front In immunol. Vol. 13). An effective approach involves the use of genetically modified feeder cells (e.g., K562 cells, an erythroleukemia cell line lacking Human Leukocyte Antigen (HLA) class I) that have been transfected with proteins that specifically stimulate NK cell proliferation. Alternatively, B cell lines naturally immortalized by infection with Epstein-Barr Virus (Epstein-Barr Virus) may be used in such a regimen. To use feeder cells for NK cell expansion, a high dose of ionizing radiation is applied to the feeder cell line, typically at a dose of about 100 Gray (Gy). By irradiating feeder cells, the mitosis of the cells is inhibited, rendering them unable to proliferate (thereby avoiding overgrowth of the intended immune cells, such as NK cells, during expansion). The limitation of such methods is that enterprises that develop NK cell expansion must rely on third parties to provide irradiated cells, as such irradiation facilities are tightly regulated, costly and difficult to maintain. In addition, the transport of such irradiated cells can also place a logistic burden on the cell enterprise. In addition, regulatory authorities require strict safety parameters and risk assessment for the presence of feeder cells in NK cell-based therapies to reduce the risk of NK cell-based therapies being contaminated with live feeder cells. Thus, there is a need for an alternative method of NK cell expansion, optionally optimized, that is capable of expanding NK cells to a very high number, preferably to a number sufficient to support clinical trials of expanded NK cells for clinical use in patients, and preferably using feeder cells during NK cell expansion. In particular, there is a need for an alternative method of preparing a suspension of mitotically inhibited cells that eliminates the need for mitotically inhibiting the cells using high doses of ionizing radiation. Disclosure of Invention To provide such an alternative method, in a first aspect of the invention there is provided a method of 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) to thereby obtain mitotically inhibited cells, wherein the step of inhibiting the mitotic activity of the cells comprises the steps of: subjecting the suspension to hypotonic treatment fo