Cryoprotection of cells

Abstract

In the present invention, a composition for cell cryporotection use is used, the composition containing a poly(alkylene glycol) modified with a copolymer of a poly(vinyl caprolactam) block and a poly(vinyl acetate) block. Alternatively, a cell production method may be used, the method including a freezing step for freezing a mixture containing cells and a poly(alkylene glycol) modified with a copolymer of a poly(vinyl caprolactam) block and a poly(vinyl acetate) block to produce frozen cells.

Description

Cryoprotection of cells

[0001] The technical field of the present invention relates to cryoprotection of cells. This application claims priority to Japanese Patent Application No. 2023-39124, filed on March 13, 2023, the contents of which are incorporated herein by reference.

[0002] It is known that when cells are cryopreserved, the number of viable cells decreases after thawing. Therefore, a cryoprotectant is added to a composition containing cells to suppress the decrease in the number of viable cells.

[0003] Dimethyl sulfoxide (DMSO) is commonly used as a cryoprotectant, but the addition of hydroxyethyl starch (HES) and albumin to the composition has been reported to enhance the cryoprotective effect (e.g., Non-Patent Documents 1 to 3).

[0004] Stiff et al., Cryobiology. 1983 Feb;20(1):17-24.Stiff et al., Blood. 1987 Oct;70(4):974-8.Luo et al., Leuk Lymphoma. 1995 May;17(5-6):495-9.

[0005] The cryoprotection methods described above have room for improvement in that they include albumin in the cell-containing composition, which is a molecule that can be a source of biological contamination.

[0006] The present inventors have conducted extensive research into methods for cryoprotecting cells. As a result, they have found that adding a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to a composition can effectively cryoprotect cells without the addition of albumin. Based on this finding, the present inventors have completed the present invention.

[0007] According to one aspect of the present invention, there is provided a composition for use in cell cryoprotection, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, which can effectively cryoprotect cells.

[0008] According to one aspect of the present invention, there is provided a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cryoprotectant, which can be used to effectively cryoprotect cells.

[0009] According to one aspect of the present invention, there is provided a method for producing cells, which comprises a freezing step of freezing a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells. By using this method, it is possible to obtain effectively cryoprotected cells.

[0010] According to one aspect of the present invention, there is provided a method for preserving cells, comprising the step of freezing a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells to produce frozen cells. By using this method, cells can be effectively preserved in a cryoprotected state.

[0011] According to one aspect of the present invention, there is provided a method for freezing cells, which comprises freezing a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells. By using this method, cells can be effectively cryoprotected.

[0012] According to the present invention, cells can be effectively cryoprotected without the addition of albumin.

[0013] FIG. 1 shows the results of investigating the recovery rate of cells after thawing.

[0014] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0015] (1) Composition According to one embodiment of the present invention, a composition is provided, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The composition may be an additive composition for cell cryoprotection. Use of this composition can effectively cryoprotect cells. For example, the Examples described below demonstrate that the use of a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block as a cryoprotective component resulted in an improved recovery rate of cells after thawing. Furthermore, according to one embodiment of the present invention, there are provided methods for freezing cells, producing cells, culturing cells, and preserving cells using this composition, as well as containers containing this composition.

[0016] (2) Composition According to one embodiment of the present invention, there is provided a composition for use in cell cryoprotection, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. Cells can be effectively cryoprotected using this composition. This composition may also be an additive composition.

[0017] (3) Method According to one embodiment of the present invention, there is provided a method for effectively cryoprotecting cells, the method comprising the step of freezing a mixture containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells.

[0018] (4) Freezing Method According to one embodiment of the present invention, there is provided a method for freezing cells, comprising a freezing step of freezing a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells. This method can effectively cryoprotect cells. This method may also comprise a production step of contacting a cell-containing composition with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0019] (5) Production Method According to one embodiment of the present invention, there is provided a method for producing cells, comprising a freezing step of freezing a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells. This method can effectively obtain cryoprotected cells. This method may also include a production step of contacting a cell-containing composition with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0020] (6) Preservation Method According to one embodiment of the present invention, there is provided a method for preserving cells, comprising a freezing step of freezing a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells. This method allows cells to be effectively preserved in a cryoprotected state. This method may also include a production step of contacting a cell-containing composition with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce a mixture of cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0021] (7) Culturing Method According to one embodiment of the present invention, there is provided a method for culturing cells, comprising: a freezing step of freezing a mixture containing cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce frozen cells; a thawing step of thawing the frozen cells to produce thawed cells; and a culturing step of culturing the thawed cells. This method can effectively obtain cryoprotected cells. This method may also include a producing step of contacting a cell-containing composition with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to produce a mixture containing cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

[0022] (8) Preservation Method According to one embodiment of the present invention, there is provided a method for preserving cells, comprising freezing a composition containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells. Using this method, cells can be effectively cryoprotected and preserved. The composition may also contain a cryoprotectant.

[0023] (9) Preservation Method According to one embodiment of the present invention, there is provided a method for preserving cells, comprising the step of placing a composition containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells in a freezing environment. Using this method, cells can be effectively cryoprotected and preserved. The composition may also contain a cryoprotectant.

[0024] (10) Auxiliary Method According to one embodiment of the present invention, there is provided a method for auxiliary cryoprotection, comprising the step of mixing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, a cryoprotectant, and cells to produce a mixture. This method can be used to assist or enhance the cryoprotective effect of the cryoprotectant on cells.

[0025] (11) Composition According to one embodiment of the present invention, there is provided a composition for supplementing cryoprotection, which comprises a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and is intended to be added to a composition containing a cryoprotectant or cells. The use of this composition can supplement or enhance the cryoprotective effect of a cryoprotectant.

[0026] (12) Enhancement Method According to one embodiment of the present invention, there is provided a method for enhancing cryoprotection, comprising the step of mixing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cryoprotectant to form a mixture. This method can enhance the cryoprotective effect of the cryoprotectant on cells.

[0027] (13) Method for Improving Cryoprotection of Cells According to one embodiment of the present invention, there is provided a method for improving the cryoprotection of cells, the method comprising the step of mixing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, a cryoprotectant, and cells to produce a mixture. By using this method, cells can be effectively cryoprotected.

[0028] (14) Production Method According to one embodiment of the present invention, there is provided a method for producing a composition, comprising the step of mixing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block with a cell-containing composition. The composition obtained by this method can effectively cryoprotect cells.

[0029] (15) Method of Use According to one embodiment of the present invention, there is provided a method of using a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block for use in cell cryoprotection. This method of use can effectively cryoprotect cells. This method of use may be carried out to produce a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cell-containing composition.

[0030] (16) Composition According to one embodiment of the present invention, there is provided a composition comprising 0.001% (w / v) or more of a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The composition can effectively cryoprotect cells.

[0031] (17) Container According to one embodiment of the present invention, a container is provided containing a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. Cells can be effectively cryoprotected using this container. This container may further contain cells or a cryoprotectant. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, cells, and optionally the cryoprotectant may be filled into this container, and the container may be frozen.

[0032] (18) Cells According to one embodiment of the present invention, cells or cell populations thereof are obtained by any of the methods described in (3) to (10), (12) to (15) above, or (21) described below. These cells or cell populations are protected from freezing-induced damage. Therefore, the obtained cells or cell populations can be efficiently used for cell transplantation, cell culture, and the like.

[0033] (19) Container According to one embodiment of the present invention, a container is provided that contains cells or a cell population thereof obtained by the method described in any of (3) to (10), (12) to (15) above, or (21) described below. The cells or cell population thereof contained in this container are protected from damage caused by freezing.

[0034] (20) Pharmaceutical Composition According to one embodiment of the present invention, there is provided a pharmaceutical composition or a method for producing the same, which comprises cells or a cell population thereof obtained by any of the methods described in (3) to (10), (12) to (15) above, or (21) described below.

[0035] (21) Transplantation Method According to one embodiment of the present invention, a transplantation method is provided. This transplantation method may include, for example, a step of transplanting cells or a cell population thereof obtained by the method described in any of (3) to (10) or (12) to (15) above, or a pharmaceutical composition containing the cells or cell population, into a subject (e.g., a human or a mammal other than a human). This transplantation method allows the cells to effectively engraft, thereby treating a disease. This transplantation method may include any of the methods described in (3) to (10) or (12) to (15) above.

[0036] (22) Composition According to one embodiment of the present invention, there is provided a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, a cryoprotectant, or cells, for use in any of the methods described in (3) to (10), (12) to (15), or (21) above.

[0037] (23) Frozen Product According to one embodiment of the present invention, there is provided a frozen product of the composition according to (1), (2), (11), (16), (20), or (22). This frozen product has an excellent cell recovery rate when thawed.

[0038] (24) Steps In one embodiment of the present invention (e.g., including the above (3) to (10), (12) to (15), or (21)), the method may include any one or more of the following steps (i) to (iii): (i) a producing step of contacting a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block with a cell-containing composition to produce a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cells; (ii) a freezing step of freezing the mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cells to produce frozen cells; or (iii) a thawing step of thawing the frozen cells to produce thawed cells. In another embodiment of the present invention (e.g., including the above (3) to (10), (12) to (15), or (21)), the method may include any one or more of the following steps (iv) to (vi). (iv) mixing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cryoprotectant to produce a mixture, (v) contacting a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cryoprotectant with a cell-containing composition to produce a mixture of the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, the cryoprotectant, and cells, and (vi) freezing the mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, the cells, and the cryoprotectant to produce frozen cells. In one embodiment of the present invention (e.g., including the above-mentioned (3) to (10), (12) to (15), or (21)), the method may include any one or more of the following steps (vii) to (ix):(vii) culturing the cells to produce a composition comprising the cultured cells, (viii) recovering the composition comprising the cultured cells (e.g., centrifuging the composition comprising the cultured cells to separate it into a supernatant and a precipitate fraction, and obtaining the precipitate fraction), or (ix) contacting a solution containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block with the precipitate fraction comprising the cells to produce a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cells. In one embodiment of the present invention (e.g., including the above (3) to (10), (12) to (15), or (21)), the method may comprise any one or more of the following steps (x) to (xvi): (x) contacting a cell-free aqueous solution containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block with a cell-containing composition and, optionally, a cryoprotectant to produce a mixture, (xi) freezing a mixture containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, cells, and, optionally, a cryoprotectant to produce frozen cells, (xii) placing a container containing the frozen cells under non-freezing conditions (thawing conditions), (xiii) transplanting the thawed cells into a living organism, (xiv) culturing the thawed cells, (xv) recovering the cultured cells, or (xvi) transferring the cultured cells to a container. Employing one or more of these steps is useful for effectively cryoprotecting cells. When two or more steps from the embodiments of the present invention (e.g., the methods (3) to (10), (12) to (15), or (21) above, or steps (i) to (xvi) included) are employed, they may be performed in any order and the order may be determined depending on the desired procedure. Each method or step from the embodiments of the present invention (e.g., the methods (3) to (10), (12) to (15), or (21) above, or steps (i) to (xvi) included) may be performed in vitro or ex vivo.

[0039] In embodiments of the present invention (including, for example, (1) to (24) above), effective cryoprotection means a high recovery rate of viable cells after freezing and thawing, preferably 70% or higher. This is because FDA guidance (see Guidance for FDA Reviewers and Sponsors. Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs). US Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research. April 2008: 1-39, and Guidance for Industry. Biologics License Applications for Minimally Manipulated, Unrelated Allogeneic Placental / Umbilical Cord Blood Intended for Hematopoietic and Immunologic Reconstitution in Patients with Disorders Affecting the Hematopoietic System. US Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research. March 2014: 1-49) recommends a cryoprotection process that results in a viability of 70% or more. The recovery rate may be 70, 75, 80, 85, 90, 95, or 100%, or may be within a range of any two of these values."Excellent or improved recovery rate" includes an improvement in recovery rate compared to conditions in which a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block is not used. The recovery rate may be expressed as (the number of viable cells in the medium after freezing and thawing divided by the number of viable cells in the same medium before freezing) × 100 (%). The recovery rate may also be expressed as (the number of viable cells in the container after freezing and thawing divided by the number of viable cells in the same container before freezing) × 100 (%).

[0040] In embodiments of the present invention (including, for example, the above (1) to (24)), the concentration of the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block in a composition comprising cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block (including, for example, a composition before freezing prepared for cryopreservation, a composition immediately before freezing, or a composition at the time of freezing) may be, for example, 0.0001, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.015, 0.02, 0.03, 0.05, 0.1, 0.2, 0.5, 1, 2, or 5% (w / v) or more, or may be within a range of any two of these values. This concentration may be, for example, 0.001-0.1% (w / v), 0.002-0.05% (w / v), 0.002-0.01% (w / v), 0.003-0.01% (w / v), 0.003-0.008%, or 0.004-0.006% (w / v).

[0041] In embodiments of the present invention (including, for example, the above (1) to (24)), the concentration of the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block in a composition (including, for example, a composition before mixing with cells, an additive composition for use by mixing with cells, or an additive composition for use by mixing with a cryoprotectant) containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, 0.0002, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.03, 0.04, 0.06, 0.1, 0.2, 0.4, 1, 2, 4, or 10% (w / v) or more, or may be within a range of any two of these values. This concentration may be, for example, 0.002-0.2% (w / v), 0.004-0.1% (w / v), 0.004-0.02% (w / v), 0.006-0.02% (w / v), 0.006-0.016%, or 0.008-0.012% (w / v). The composition containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may also contain a cryoprotectant. The composition containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be mixed with a cell-containing composition for cryoprotection of cells. In this case, the cell-containing composition may be mixed with the composition in an amount of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 times the amount of the composition.

[0042] In the embodiments of the present invention (including, for example, (1) to (24) above), the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a polyethylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a graft copolymer obtained from (i) N-vinyl caprolactam, (ii) vinyl acetate, and (iii) a polyether. The polyether may be polyethylene glycol. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a compound obtained by free radical polymerization of a mixture of the above (i) to (iii). For example, (i) may be 40 to 60 wt%, (ii) may be 15 to 35 wt%, and (iii) may be 10 to 30 wt%. (i) may be, for example, 40, 45, 50, 55, 57, or 60 wt%, or may be within a range of any two of these values. (ii) may be, for example, 15, 20, 25, 30, or 35 wt%, or may be within a range of any two of these values. (iii) may be, for example, 10, 13, 15, 20, 25, or 30 wt%, or may be within a range of any two of these values. The above (i) to (iii) may total 100 wt%. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, a compound represented by the following structural formula (1) or a salt thereof.

[0043] Here, the wt% of l, m, and n may be, for example, 40 to 60 wt%, 15 to 35 wt%, and 10 to 30 wt%, respectively. The wt% of l may be, for example, 40, 45, 50, 55, 57, or 60 wt%, or may be within a range of any two of these values. The wt% of m may be, for example, 15, 20, 25, 30, or 35 wt%, or may be within a range of any two of these values. The wt% of n may be, for example, 10, 13, 15, 20, 25, or 30 wt%, or may be within a range of any two of these values. The wt% of l, m, and n may total 100 wt%. The wt% of l, m, and n may be, for example, 56 to 58 wt%, 29 to 31 wt%, and 12 to 14 wt%, respectively, or may be about 57, about 30, and about 13 wt%, respectively. The degrees of polymerization of l, m, and n may be, for example, 60 to 160, 470 to 1110, and 480 to 1130, respectively. The degree of polymerization of l may be, for example, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160, or may be within a range of any two of these values. The degree of polymerization of m may be, for example, 470, 500, 550, 600, 650, 700, 750, 780, 800, 850, 900, 950, 1000, 1050, or 1110, or may be within a range of any two of these values. The degree of polymerization (n) may be, for example, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1130, or may be within a range of any two of these values. The degree of polymerization may be an average degree of polymerization. The weight average molecular weight (Mw) of the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, 70,000 g / mol or more. This value may be, for example, 70,000, 80,000, 90,000, 100,000, 120,000, 140,000, 150,000, 160,000, or 170,000 g / mol, or may be within a range of any two of these values.This value may be, for example, 70,000 to 170,000 g / mol, 80,000 to 160,000 g / mol, 90,000 to 140,000 g / mol, 100,000 to 130,000 g / mol, 110,000 to 125,000 g / mol, or 117,000 to 119,000 g / mol. This value may be approximately 118,000 g / mol. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be, for example, Soluplus. Soluplus is a compound having the structure of formula (1) above. The l, m, and n of Soluplus may have the wt% or degree of polymerization described above. The polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be produced, for example, by the method described in US 2008 / 0293828 A1, US 2010 / 0204425 A1, or US 2018 / 0305636 A1. Furthermore, the components (i) to (iii) described in these documents may be used.

[0044] In embodiments of the present invention (including, for example, (1) to (24) above), freezing includes placing a composition or a container under freezing conditions. Freezing conditions include, for example, placing the composition or a container at subzero temperatures. The freezing conditions may be, for example, 0, -10, -20, -40, -60, -80, -100, -120, -140, -150, -160, or -180°C or lower, or within a range between any two of these values. The freezing time may be, for example, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 10, 50, or 100 days or longer, or within a range between any two of these values. Freezing may be performed by placing the composition or a container in liquid nitrogen or a freezer. Compositions to be frozen include those containing cells, a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, or a cryoprotectant. The container to be frozen includes the container containing the composition. A non-freezing environment includes, for example, placing the composition or container in an environment above 0°C (e.g., 37°C).

[0045] In embodiments of the present invention (including, for example, (1) to (24) above), cryoprotection includes preventing damage to cells that may occur when exposed to freezing conditions. Damage includes damage caused by freezing of water during freezing. Damage includes damage to cell membranes due to pressure caused by freezing of water.

[0046] In embodiments of the present invention (including, for example, (1) to (24) above), the cryoprotectant includes, for example, DMSO, HES, glycerol, trehalose, or sorbitol. The cryoprotectant can be produced by known methods. Commercially available cryoprotectants are available, and may be purchased from manufacturers (e.g., Kyokuto Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., etc.). The concentration of the cryoprotectant in a composition containing cells and a cryoprotectant (including, for example, a composition prepared for cryopreservation before freezing, a composition immediately before freezing, or a composition at the time of freezing) (when the composition contains multiple cryoprotectants, the concentration per cryoprotectant (e.g., DMSO, HES, etc.)) may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20% (w / v or v / v) or more, or may be within a range between these two values. The concentration of the cryoprotectant in a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cryoprotectant (e.g., a composition before being mixed with cells, or an additive composition to be mixed with cells for use) (if the composition has multiple cryoprotectants, the concentration per cryoprotectant (e.g., including DMSO or HES, etc.)) may be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 40% (w / v or v / v) or more, or may be within a range between these two values.

[0047] In embodiments of the present invention (including, for example, (1) to (24) above), thawing may be performed by transferring the cells from frozen conditions to non-frozen conditions, for example, by contacting a container containing the frozen material with a water bath at approximately 37°C.

[0048] In embodiments of the present invention (including, for example, (1) to (24) above), the term "adding" includes adding an additional substance to a composition. The addition may be carried out, for example, to produce a mixture containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cell-containing composition. This allows for effective cryoprotection of cells. The addition may also be carried out, for example, to produce a mixture containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a composition containing a cryoprotectant. This allows for enhanced cryoprotective effects of the composition containing a cryoprotectant.

[0049] In embodiments of the present invention (including, for example, (1) to (24) above), the composition may be in the form of, for example, a solution, a medium, or an additive. The composition may include, for example, a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, cells, or a cryoprotectant. The composition may be, for example, albumin-free. "Free" includes a state in which the target component is not added to the composition, a state in which the composition is completely free of the target component, or a state in which the composition does not contain the component at a concentration above the detection limit. "Albumin-free" includes being substantially free of albumin. The additive may be an additive to be mixed with a cell-containing composition to enhance the cryoprotective effect of the cells. The term "additive" can be used interchangeably with "composition for addition." Embodiments of the composition can also be applied to mixtures.

[0050] In embodiments of the present invention (including, for example, the above (1) to (24)), the frozen product may be produced by placing the composition under freezing conditions. The frozen product includes a form in which the composition is frozen. The frozen product is preferably albumin-free.

[0051] In embodiments of the present invention (including, for example, (1) to (24) above), the cells may be mammalian cells. Mammals include, for example, humans, monkeys, rodents (such as mice and hamsters), rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, and the like. The cells include, for example, somatic cells or stem cells. Somatic cells include, for example, cells derived from skin, heart, liver, lung, stomach, intestine, kidney, uterus, brain, blood, or mesenchymal tissue. The cells may also be, for example, CD34 + Examples of stem cells include cells, immune cells (e.g., T cells), cells for regenerative medicine, CHO cells, or malignant tumor cells. Stem cells include, for example, pluripotent stem cells, multipotent stem cells, and unipotent stem cells. Pluripotent stem cells include, for example, iPS cells or ES cells. Multipotent stem cells include, for example, mesenchymal stem cells, adipose stem cells, hematopoietic stem cells, and neural stem cells. Unipotent stem cells include, for example, muscle stem cells and melanocyte stem cells. Hematopoietic stem cells can be collected from the umbilical cord, bone marrow, placenta, and peripheral blood. Human hematopoietic stem cells express CD34 + These cells may be purchased from manufacturers or distributors (e.g., StemExpress or Lonza). + Cells or hematopoietic stem cells are isolated from umbilical cord blood, peripheral blood, or bone marrow using CD34 + The cells may be obtained by sorting. The cells include living cells. The cells may exist in the form of a cell population. The cell population includes, for example, a plurality of cells generated by cell division. Any of the cells listed above (e.g., CD34 + The percentage of any of the above-listed cells (e.g., CD34 cells) relative to the total cells in the medium or container may be, for example, 10, 20, 40, 60, 80, or 100%, or may be within a range of any two of these values. + The percentage of cells may be, for example, 10, 20, 40, 60, 80, or 100%, or may be within a range of any two of these values.

[0052] In embodiments of the present invention (including, for example, (1) to (24) above), the medium includes a medium used for culturing cells. The base components of the medium may be any commonly used base components, and the composition is not limited. The base components of the medium may include, for example, amino acids, peptides, proteins, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose). The base components of the medium may include, for example, GlutaMAX, L-alanine L-glutamine dipeptide, L-glutamine, ITS-X, insulin, transferrin (apo), sodium selenite, ethanolamine, or Flt3 ligand. The medium may include, for example, a basal medium for cell culture. Examples of basal media include S-clone SF-3 medium, F12 medium, StemSpan (Stem Cell technologies), STEMα (STEM ALPHA), StemPro-34 serum-free medium (Gibco Invitrogen), StemPro MSC serum-free medium (Invitrogen), HSC-CFU medium (Miltenyl Biotech), S-Clone serum-free medium (SF-02, SF-03, CM-B, SF-B) (Sanko Junyaku), HPGM medium (Sanko Junyaku), AIM V medium (Invitrogen), Marrow MAX bone marrow medium (Invitrogen), KnockOut DMEM / F-12 medium (Invitrogen), Stemline hematopoietic stem cell growth medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), and MyeloCult medium (StemCell Technologies), HPG serum-free medium (Lonza), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen and others), MEM medium (Gibco Invitrogen and others), MEMα (Gibco Invitrogen and others), DMEM medium (Gibco Invitrogen and others), IMDM medium (Wako Pure Chemical and others), PRMI1640 medium (Gibco Invitrogen and others), Ham's F-12 medium (Gibco and others), RD medium, etc.The medium is preferably albumin-free (e.g., GenBank accession number AAN17825.1) or serum-free. In this case, stable inhibition of undifferentiated state and problems of biological contamination can be suppressed. The medium may contain Soluplus® (CAS registration number 402932-23-4). The medium may contain antibiotics (e.g., penicillin or streptomycin). The medium may contain hematopoietic stem cells. The medium may be a liquid medium or a solid medium. Culturing cells using a medium includes culturing cells in the medium.

[0053] In embodiments of the present invention (including, for example, (1) to (24) above), culturing includes incubating cells under conditions suitable for proliferation or maintenance. Culturing may include a step of contacting a medium and cells to produce a cell-containing composition, and a step of incubating the cell-containing composition. The incubating step may be performed while the cell-containing composition is left standing or stirred. Incubation may be performed at about 37°C in an atmosphere of about 5% CO2. The culture time for cells may be, for example, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 17, 18, 20, 30, or 35 days or more, or may be within a range of any two of these values. This culture time may be, for example, 1 to 20 days, 3 to 17 days, 4 to 16 days, or 5 to 14 days. Culturing includes the time for incubating the medium containing the cells.

[0054] In embodiments of the present invention (including, for example, the above (1) to (24)), the cell density in the composition containing cells at the time of freezing is, for example, 1 × 10 3 , 1×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 8×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 1×10 8 , or 1 × 10 9The density may be greater than or equal to 1 x 10 cells / mL, or may be within the range of any two of these values. 3 ~1×10 8 , 1×10 4 ~1×10 7 , 1×10 4 ~5×10 6 , or 1 × 10 5 ~5×10 6 It may be cells / mL.

[0055] In embodiments of the present invention (including, for example, the above (1) to (24)), the cell density in a composition comprising cells and a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, or in a composition comprising cells, a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and a cryoprotectant, is, for example, 1 × 10 3 , 1×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 8×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 1×10 8 , or 1 × 10 9 The density may be greater than or equal to 1 x 10 cells / mL, or may be within the range of any two of these values. 3 ~1×10 8 , 1×10 4 ~1×10 7 , 1×10 4 ~5×10 6 , or 1 × 10 5 ~5×10 6 It may be cells / mL.

[0056] In embodiments of the present invention (including, for example, (1) to (24) above), contacting may be performed by mixing a composition containing a substance with another composition containing another substance. Contacting cells with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be performed by mixing a cell suspension with a solution containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. Contacting cells with a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block may be performed by mixing a cell fraction (including, for example, a pellet) with a solution containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. The contacting of the cells, the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and the cryoprotectant may be carried out by mixing the cell suspension or cell fraction with a solution containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cryoprotectant.

[0057] In embodiments of the present invention (including, for example, (1) to (24) above), the cell-containing composition may be in the form of a cell suspension or a cell fraction. The cell fraction includes, for example, a cell pellet.

[0058] In embodiments of the present invention (including, for example, (1) to (24) above), recovery may include, for example, any one or more of the following steps: centrifuging a composition containing a medium and cultured cells to separate the supernatant and a precipitate fraction to obtain the precipitate fraction; suspending the cell-containing precipitate fraction in an aqueous solution to produce a cell suspension; transferring the desired cells from a container containing the cells to another container; or sorting or purifying the desired cells. Recovery may be performed in a sterile environment.

[0059] In embodiments of the present invention (including, for example, (1) to (24) above), preservation includes cryopreservation. Preservation includes, for example, placing cells, compositions, or containers containing cells or compositions in a freezing environment. The storage period may be, for example, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 10, 50, or 100 days or more, or may be within a range of any two of these values.

[0060] In embodiments of the present invention (including, for example, (1) to (24) above), the container includes a tube, vial, ampoule, bottle, or the like. The container includes a freezing container. The freezing container includes a container that is resistant to breakage due to freezing. The container may contain a composition including a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, cells, or a cryoprotectant.

[0061] In the embodiments of the present invention (including, for example, the above (1) to (24)) + includes the cells being found to be positive for the molecule identified by the term immediately preceding it.

[0062] All publications cited herein are incorporated by reference in their entirety. As used herein, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "or." When "within a range of two values" is stated herein, the range also includes the two values ​​themselves. As used herein, "A to B" includes A and B. As used herein, "the above (1) to (24)" includes reference to any one or more of (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), (22), (23), or (24).

[0063] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may also be adopted. Furthermore, the present invention may be adopted by combining or independently adopting each of the configurations or features described in the above embodiments.

[0064] The present invention will be further explained below with reference to examples, but is not limited to these.

[0065] 1. Cryopreservation Test 1.1 Method Three compositions were prepared as shown in Table 1. CP-1® High Grade (hereafter referred to as CP-1) (Kyokuto Pharmaceutical Industries Co., Ltd.) was used as a solution containing dimethyl sulfoxide (DMSO) and hydroxyethyl starch (HES). In the first composition, human serum albumin (HSA) was not added to CP-1 (No. 1, no HSA composition). In the second composition, HSA was added to CP-1 according to the CP-1 instructions (No. 2, HSA plus composition). In the third composition, Soluplus® (a polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block) was added to CP-1 (No. 3, Soluplus plus composition).

[0066]

[0067] 5 μM 740Y-P, 0.1 μM butizamide, 1 μM UM729, and 0.01% Soluplus® were added to IMDM (Iscove's Modified Dulbecco's Media) containing 1x GlutaMAX, 1x ITS-X, and 10 ng / ml Flt3-ligand. The resulting medium was then inoculated with thawed human umbilical cord blood CD34 cells according to the manufacturer's instructions. + The cells were suspended and plated in a 96-well plate. The medium was changed every 3-4 days. On day 10 of culture, all CD34 +The cell progeny were collected in a 1.5 mL tube (Eppendorf tube). The tube was centrifuged and the supernatant removed. The precipitate was suspended in saline to obtain a cell suspension. The cell suspension was placed in a cryotube, and an equal volume of each of the compositions listed in Table 1 (No. 1, 2, or 3) was slowly added. The cryotube containing the resulting mixture was stored overnight in a -80°C freezer. The next day, the cryotube was transferred to liquid nitrogen. After 7 days, the cells were thawed in a water bath and viable cells were counted.

[0068] The materials used in this experiment are listed below.

[0069]

[0070] 1.2 Results The results are shown in Figure 1. When a mixture of cell suspension and no HSA composition was frozen, the cell recovery rate after thawing was low at approximately 43%. When a mixture of cell suspension and HSA plus composition was frozen, the cell recovery rate after thawing was high at approximately 108%. When a mixture of cell suspension and Soluplus plus composition was frozen, the cell recovery rate after thawing was high at approximately 83%. These results demonstrate that the addition of Soluplus improves the cell recovery rate after thawing.

[0071] Previous attempts have been made to use albumin as a cryoprotectant, but albumin is a molecule that can cause biological contamination. Albumin also has the problem of high raw material costs. However, this experiment demonstrated that Soluplus can be used as a cryoprotectant instead of albumin. Soluplus can be chemically synthesized and is less expensive than albumin. Furthermore, when used as a cryoprotectant, the amount added can be kept to a minimum. Therefore, the use of Soluplus is superior in terms of safety and convenience.

[0072] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

[0073] According to the present invention, by adding a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to a composition, cells can be effectively cryoprotected without adding albumin, and therefore the present invention is industrially applicable.

Claims

1. A composition for use in cell cryoprotection, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

2. 2. The composition according to claim 1, wherein the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block is a compound represented by the following structural formula (1): 【Chemical 1】 (Here, the wt% of l, m, and n are 40 to 60 wt%, 15 to 35 wt%, and 10 to 30 wt%, respectively.)

3. 3. The composition of claim 1 or 2, wherein the composition is an additive composition, and the addition is carried out to produce a mixture containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cell-containing composition.

4. The composition of claim 1 or 2, further comprising a cryoprotectant.

5. The composition according to claim 1 or 2, comprising 0.001% (w / v) or more of a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

6. A method for producing frozen cells, comprising: a freezing step of freezing a mixture of a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells to produce frozen cells.

7. 7. The method for producing the cells described in claim 6, comprising a production step of contacting a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block with a cell-containing composition to produce a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and the cells.

8. The method of claim 7, wherein the producing step comprises contacting a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, a cryoprotectant, and a cell-containing composition to produce a mixture containing the polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, the cryoprotectant, and the cells.

9. A method for preserving cells, comprising a freezing step of freezing a composition comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and cells to produce frozen cells.

10. 10. The method of claim 9, wherein the composition comprises 0.001% (w / v) or more of a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block.

11. The method of claim 9 or 10, wherein the composition comprises a cryoprotectant.

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