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

[Technical Field]

[0001] The technical field of the present invention relates to the cryopreservation of cells. This application claims priority based on Japanese Patent Application No. 2023-39124, filed in Japan on March 13, 2023, and the contents of that application are incorporated herein by reference. [Background technology]

[0002] It is known that when cells are cryopreserved, the number of viable cells decreases after thawing. Therefore, efforts are being made to suppress this decrease in the number of viable cells by including cryoprotective agents in cell-containing compositions.

[0003] Dimethyl sulfoxide (DMSO) is commonly used as a cryoprotective agent. On the other hand, it has been reported that hydroxyethyl starch (HES) and albumin are added to the composition to enhance the cryoprotective effect (e.g., Non-Patent Documents 1-3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Stiff et al., Cryobiology. 1983 Feb;20(1):17-24. [Non-Patent Document 2] Stiff et al., Blood. 1987 Oct;70(4):974-8. [Non-Patent Document 3] Luo et al., Leuk Lymphoma. 1995 May;17(5-6):495-9. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The above cryoprotection method had room for improvement in that it included albumin in the cell-containing composition. Albumin is a molecule that can cause biological contamination. [Means for Solving the Problems]

[0006] In the course of repeated research, the inventors of the present invention examined a method for cryopreserving cells. As a result, by adding a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block to a composition, cells could be effectively cryopreserved without adding albumin. Based on this finding, the inventors of the present invention completed the present invention.

[0007] According to one aspect of the present invention, there is provided a composition for use in cryopreserving cells, which contains a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block. By using this composition, cells can be effectively cryopreserved.

[0008] According to one aspect of the present invention, there is provided a composition containing a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block and a cryoprotectant. By using this composition, cells can be effectively cryopreserved.

[0009] According to one aspect of the present invention, there is provided a method for producing cells, which includes a freezing 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 to produce frozen cells. By using this method, effectively cryopreserved cells can be obtained.

[0010] According to one aspect of the present invention, there is provided a method for storing cells, which includes a freezing step of freezing a composition containing 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 stored in an effectively cryopreserved state.

[0011] According to one aspect of the present invention, a method for freezing cells is provided, comprising a freezing step of freezing a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and cells to produce frozen cells. This method allows for effective cryoprotection of cells. [Effects of the Invention]

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

[0013] [Figure 1] This figure shows the results of an investigation into the recovery rate of cells after thawing. [Modes for carrying out the invention]

[0014] The embodiments of the present invention will be described in detail below. To avoid repetition and unnecessary complexity, similar content will be omitted from explanation as appropriate.

[0015] (1) Composition One embodiment of the present invention provides a composition comprising a polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block. Here, the composition may be an additive composition for cell cryoprotection. Using this composition, cells can be effectively cryoprotected. For example, the examples described later show that when polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block was used as a cryoprotective component, the recovery rate of cells after thawing was improved. Another embodiment of the present invention also provides a method for freezing cells using this composition, a method for producing cells, a method for culturing cells, a method for storing cells, and a container containing this composition.

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

[0017] (3) Method According to one embodiment of the present invention, a method is provided which includes 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. This method allows for effective cryoprotection of cells.

[0018] (4) Freezing method According to one embodiment of the present invention, a method for freezing cells is provided, comprising a freezing step of freezing a mixture comprising polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and cells to produce frozen cells. This method allows for effective cryoprotection of cells. The method may also include a production step of contacting a polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and a cell-containing composition to produce a mixture comprising polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and cells.

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

[0020] (6) Storage method According to one embodiment of the present invention, a method for preserving cells is provided, comprising a freezing step of freezing a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and cells to produce frozen cells. This method allows for effective cryopreservation of cells. The method may also include a production step of contacting a polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and a cell-containing composition to produce a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam blocks and polyvinyl acetate blocks and cells.

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

[0022] (8) Storage method According to one embodiment of the present invention, a method for preserving cells is provided, 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. This method allows for the effective preservation of cells in a cryoprotective state. The composition may also contain a cryoprotectant.

[0023] (9) Storage method According to one embodiment of the present invention, a method for preserving cells is provided, 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, into a freezing environment. This method allows for effective cryoprotection of cells during storage. The composition may also contain a cryoprotectant.

[0024] (10) Assistance method According to one embodiment of the present invention, a cryoprotection aid is provided, 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, a cryoprotective composition is provided, comprising a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, for addition to a composition containing a cryoprotectant or cells. This composition can be used to assist or enhance the cryoprotective effect of a cryoprotectant.

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

[0027] (13) How to improve According to one embodiment of the present invention, a method is provided for improving the cryoprotective effect of cells, comprising the steps 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, a method for producing a composition is provided, 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 be used to effectively cryoprotect cells.

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

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

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

[0032] (18) Cell According to one embodiment of the present invention, cells or a population of cells can be obtained by any of the methods described in (3) to (10), (12) to (15) above, or (21) described later. These cells or a population of cells are protected from damage caused by freezing. Therefore, the obtained cells or a population of cells 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 obtained that contains cells or a population of cells obtained by any of the methods described in (3) to (10), (12) to (15) above, or (21) described later. The cells or population of cells contained in this container are protected from damage caused by freezing.

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

[0035] (21) Transplant method According to one embodiment of the present invention, a transplantation method is provided. This transplantation method may include, for example, the step of transplanting cells or a population of such cells obtained by any of the methods described in (3) to (10) or (12) to (15) above, or a pharmaceutical composition containing such cells or a population of such cells, into a target (e.g., a human or a mammal other than a human). With this transplantation method, the disease can be treated by the effective engraftment of the cells. 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, a composition is provided comprising a polyalkylene glycol modified with a copolymer of a polyvinylcaprolactam 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 products According to one embodiment of the present invention, a frozen product of the composition of (1), (2), (11), (16), (20), or (22) is provided. This frozen product exhibits excellent cell recovery rate upon thawing.

[0038] (24) Process In one embodiment of the present invention (including, for example, (3) to (10), (12) to (15) or (21) above), the method may include one or more of the following steps (i) to (iii): (i) a production step of contacting a polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and a cell-containing composition to produce a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and cells; (ii) a freezing step of freezing a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and cells to produce frozen cells; or (iii) a thawing step of thawing frozen cells to produce thawed cells. In another embodiment of the present invention (including, for example, (3) to (10), (12) to (15) or (21) above), the method may include one or more of the following steps (iv) to (vi). (iv) A step of mixing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and a cryoprotectant to produce a mixture; (v) A step of contacting a cell-containing composition with a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block and a cryoprotectant to produce a mixture of polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, a cryoprotectant and cells; (vi) A freezing step of freezing a mixture containing polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, cells and a cryoprotectant to produce frozen cells. In addition, in one embodiment of the present invention (for example, including (3) to (10), (12) to (15) or (21) above), the method may include one or more of the following steps (vii) to (ix).(vii) A step of culturing cells to produce a composition containing cultured cells; (viii) A step of recovering the composition containing cultured cells (for example, a step of separating the composition containing cultured cells into a supernatant and a precipitate fraction by centrifugation to obtain a precipitate fraction); or (ix) A step of contacting a solution containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block with a precipitate fraction containing cells to produce a mixture containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block and cells. In addition, in one embodiment of the present invention (for example, including (3) to (10), (12) to (15) or (21) above), the method may include one or more of the following steps (x) to (xvi). (x) A step of producing a mixture by contacting an aqueous solution containing polyalkylene glycol modified by a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, without cells, with a cell-containing composition and optionally a cryoprotective agent; (xi) A freezing step of producing frozen cells by freezing the mixture containing polyalkylene glycol modified by a copolymer of polyvinyl caprolactam block and polyvinyl acetate block, cells, and optionally a cryoprotective agent; (xii) A step of placing a container containing the frozen cells under non-freezing conditions (thawing conditions); (xiii) A step of transplanting the thawed cells into a living organism; (xiv) A step of culturing the thawed cells; (xv) A step of recovering the cultured cells; or (xvi) A step of 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 embodiments of the present invention (for example, including the methods of (3) to (10), (12) to (15) or (21) above, or the steps of (i) to (xvi)) are adopted, the order is arbitrary and can be determined according to the desired operation. Each method or step of embodiments of the present invention (for example, including the methods of (3) to (10), (12) to (15) or (21) above, or the steps of (i) to (xvi)) may be carried out in vitro or ex vivo.

[0039] In embodiments of the present invention (including, for example, (1) to (24) above), effective cryoprotection includes a good recovery rate of viable cells after freezing and thawing. The recovery rate is 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 demonstrates a survival rate of 70% or higher. The recovery rate may be 70, 75, 80, 85, 90, 95, or 100%, or within the range of any two of these values.Superior or improved recovery rate includes an improved recovery rate compared to conditions without the use of polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block. The recovery rate may be expressed as (number of viable cells in the culture medium after freeze-thawing ÷ number of viable cells in the same culture medium before freezing) × 100 (%). The recovery rate may also be expressed as (number of viable cells in the container after freeze-thawing ÷ number of viable cells in the same container before freezing) × 100 (%).

[0040] In embodiments of the present invention (including, for example, (1) to (24) above), the concentration of polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block in a composition (including, for example, a pre-freezing composition 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, and may be within the 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, (1) to (24) above), the concentration of polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block in a composition (including, for example, a composition before mixing with cells, an additive composition for use after mixing with cells, or an additive composition for use after mixing with a cryoprotectant) 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, and may be within the 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 polyalkylene glycol modified by a copolymer of polyvinyl caprolactam block and polyvinyl acetate block may also contain a cryoprotectant. The composition containing polyalkylene glycol modified by a copolymer of polyvinyl caprolactam block and polyvinyl acetate block may be mixed with a cell-containing composition for cryoprotection of cells. In this case, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or twice the amount of cell-containing composition may be mixed with the composition.

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

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

[0044] In embodiments of the present invention (including, for example, (1) to (24) above), freezing includes placing the composition or container under freezing conditions. Freezing conditions include, for example, placing it below freezing point. Freezing conditions may be, for example, 0, -10, -20, -40, -60, -80, -100, -120, -140, -150, -160, or -180°C or lower, and may be within the range of any two of these values. 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 more, and may be within the range of any two of these values. Freezing may be carried out by placing the composition or container in liquid nitrogen or in a freezer. The composition to be frozen includes a composition containing cells, a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, or a cryoprotectant. Containers subject to freezing include containers containing the composition. Non-freezing conditions include, 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 placed under freezing conditions. Damage includes damage resulting from the freezing of water during freezing. Damage also includes damage to cell membranes due to the pressure caused by the freezing of water.

[0046] In embodiments of the present invention (including, for example, (1) to (24) above), the cryoprotective agent includes, for example, DMSO, HES, glycerol, trehalose, or sorbitol. The cryoprotective agent can be manufactured by known methods. The cryoprotective agent can be commercially available or purchased from manufacturers (e.g., Kyokuto Pharmaceutical Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industries, Ltd., etc.). The concentration of the cryoprotective agent in a composition containing cells and the cryoprotective agent (including, for example, a pre-freezing composition prepared for cryopreservation, a composition immediately before freezing, or a composition at the time of freezing) (if the composition has multiple cryoprotective agents, the concentration per cryoprotective agent (including, for example, DMSO or 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 within the range of two such values. The concentration of the cryoprotectant in a composition (for example, the composition before mixing with cells, or the additive composition for use after mixing with cells) that includes a polyalkylene glycol modified with a copolymer of a polyvinyl caprolactam block and a polyvinyl acetate block, and a cryoprotectant (for example, the composition before mixing with cells, or the additive composition for use after mixing with cells) 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 higher, or within the range of two such values.

[0047] In embodiments of the present invention (including, for example, (1) to (24) above), thawing may be carried out by transferring the cells of the frozen material from freezing conditions to non-freezing conditions. Thawing may also be carried out, for example, by bringing the container containing the frozen material into contact with a water bath at approximately 37 degrees Celsius.

[0048] In embodiments of the present invention (including, for example, (1) to (24) above), the addition involves adding an additional substance to the composition. The addition may be carried out to produce a mixture comprising, for example, a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block and a cell-containing composition. This allows for effective cryoprotection of cells. The addition may also be carried out to produce a mixture comprising, for example, a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block and a composition containing a cryoprotectant. This enhances the cryoprotective effect of the composition containing the 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 culture medium, or an additive. The composition may include, for example, a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, cells, or a cryoprotectant. The composition may be, for example, albumin-free. Free includes a state in which the component of interest is not added to the composition, a state in which the composition does not contain the component of interest at all, or a state in which the composition does not contain the component at a concentration above the detection limit. Albumin-free includes being substantially albumin-free. Additives may be additives to be mixed into a cell-containing composition to enhance the cryoprotective effect of cells. The term additive can be used interchangeably with additive composition. Embodiments of composition can also be applied to mixtures.

[0050] In embodiments of the present invention (including, for example, (1) to (24) above), the frozen product may be produced by placing the composition under freezing conditions. The frozen product includes the composition in a frozen form. 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 (mice, hamsters, etc.), rabbits, dogs, cats, horses, cattle, sheep, pigs, goats, marmosets, etc. The cells include, for example, somatic cells or stem cells. Somatic cells include, for example, cells derived from skin, heart, liver, lungs, stomach, intestines, kidneys, uterus, brain, blood, or mesenchymal tissue. The cells may also include, for example, CD34 + Cells include immune cells (e.g., T cells), regenerative medicine cells, CHO cells, or malignant tumor cells. Stem cells include, for example, pluripotent stem cells, compound pluripotent stem cells, and unipotent stem cells. Pluripotent stem cells include, for example, iPS cells or ES cells. Compound pluripotent stem cells include, for example, mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, and neural stem cells. Unipotent stem cells include, for example, muscle stem cells and pigment stem cells. Hematopoietic stem cells can be collected from the umbilical cord, bone marrow, placenta, and peripheral blood. Human hematopoietic stem cells are CD34 + Includes cells. These cells may be purchased from a manufacturer or distributor (e.g., StemExpress or Lonza). CD34 + Cells or hematopoietic stem cells are obtained from umbilical cord blood, peripheral blood, or bone marrow, and contain CD34 + Cells may be obtained by separating them. Cells may include living cells. Cells may exist in the form of a cell population. A cell population may include, for example, several cells produced by cell division. Any of the cells listed above in the cell population (e.g., CD34) + The percentage of cells may be, for example, 10, 20, 40, 60, 80, or 100%, or within the range of any two of these values. Any of the cells listed above (e.g., CD34) relative to the total cells in the culture medium or container. + The percentage of cells may be, for example, 10, 20, 40, 60, 80, or 100%, or within the range of any two of these values.

[0052] In embodiments of the present invention (including, for example, (1) to (24) above), the culture medium includes a culture medium used for cell culture. The base components of the culture medium may be any common base components, and their composition is not limited. The base components of the culture medium may include, for example, amino acids, peptides, proteins, inorganic salts, vitamins, minerals, or carbon sources (e.g., glucose). The base components of the culture 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 culture medium may include, for example, a basic culture medium for cell culture. Basic culture media include, for example, 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 proliferation medium (Sigma), SYN serum-free medium (SYN H, SYN B) (AbCys SA), SPE IV medium (AbCys SA), and MyeloCult medium (StemCell This includes HPG serum-free medium (Lonza Technologies), UltraCULTURE medium (Lonza), Opti-MEM medium (Gibco Invitrogen, etc.), MEM medium (Gibco Invitrogen, etc.), MEMα (Gibco Invitrogen, etc.), DMEM medium (Gibco Invitrogen, etc.), IMDM medium (Wako Pure Chemical, etc.), PRMI1640 medium (Gibco Invitrogen, etc.), Ham F-12 medium (Gibco, etc.), RD medium, etc.The medium is preferably an albumin (e.g., GenBank accession number AAN17825.1)-free or serum-free medium. In this case, problems such as stable inhibition of undifferentiation and biological contamination can be suppressed. The medium may contain Soluplus (registered trademark) (CAS registration number 402932-23-4). The medium may contain an antibiotic (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 the medium includes culturing the cells in the medium.

[0053] In an embodiment of the present invention (including, for example, the above (1) to (24)), culturing includes incubating the cells under conditions suitable for growth or maintenance. Culturing may include a step of contacting the medium and the cells to produce a cell-containing composition, and a step of incubating the cell-containing composition. The incubating step may be performed with the cell-containing composition being static or stirred. Incubation may be performed under an atmosphere of about 37°C and about 5% CO2. The culturing time of the 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, and may be within the range of any two of these values. This culturing time may be, for example, from 1 to 20 days, from 3 to 17 days, from 4 to 16 days, or from 5 to 14 days. Culturing includes the time for incubating the medium containing the cells.

[0054] In an embodiment of the present invention (including, for example, the above (1) to (24)), the cell density in the composition containing the 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 cells / mL, and may be within the range of any two of these values. This density is, for example, 1 × 10⁻⁶ 3 ~1 × 10 8 , 1 x 10 4 ~1 × 10 7 , 1 x 10 4 ~5×10 6 , or 1 × 10 5 ~5×10 6 Cells / mL may also be acceptable.

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

[0056] In embodiments of the present invention (including, for example, (1) to (24) above), contact may be carried out by mixing a composition containing one substance with another composition containing another substance. Contact between cells and polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block may be carried out by mixing a cell suspension with a solution containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block. Contact between cells and polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block may be carried out by mixing a cell fraction (including, for example, a pellet) with a solution containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block. Contact between cells, polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, and a cryoprotectant may be carried out by mixing a cell suspension or cell fraction with a solution containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, and a 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, one or more of the following steps: separating a composition containing the culture medium and cultured cells into a supernatant and a precipitate fraction by centrifugation to obtain a precipitate fraction; suspending the precipitate fraction containing cells in an aqueous solution to produce a cell suspension; transferring desired cells from a container containing cells to another container; or separating or purifying desired cells. Recovery may be carried out 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 preservation 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, and may be within the range of any two of these values.

[0060] In embodiments of the present invention (including, for example, (1) to (24) above), the container includes tubes, vials, ampoules, or bottles. The container includes a freezing container. The freezing container includes a container that is resistant to damage due to freezing. The container may contain a composition comprising polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, cells, or a cryoprotectant.

[0061] In embodiments of the present invention (including, for example, (1) to (24) above) + This includes the cell 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. In this specification, “or” is used when “at least one” of the items listed in the text can be adopted. The same applies to “or.” In this specification, when “within the range of two values” is specified, that range includes the two values ​​themselves. In this specification, “A to B” includes A and B. In this specification, “(1) to (24) above” includes reference to 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 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 other configurations can be adopted. Furthermore, the present invention can be adopted by combining or independently the configurations or features described in the above embodiments. [Examples]

[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 (hereinafter, CP-1) (Kyokuto Pharmaceutical Co., Ltd.) was used as the 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 instructions for CP-1 (No. 2, HSA plus composition). In the third composition, Soluplus® (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] [Table 1]

[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. Human umbilical cord blood CD34, thawed according to the manufacturer's instructions, was added to the resulting medium. +The cells were suspended and plated in a 96-well plate. The culture medium was changed every 3-4 days. On day 10 of culture, all CD34 cells in the wells were removed. + Cell progeny were collected in 1.5 mL tubes (Eppendorf tubes). The tubes were centrifuged and the supernatant was removed. The precipitate was suspended in physiological saline to obtain a cell suspension. The cell suspension was placed in a cryotube, and an equal volume of each of the compositions in Table 1 (No. 1, 2, or 3) was slowly added. The cryotubes containing the resulting mixtures were stored overnight in a -80°C freezer. The next day, the cryotubes were transferred to liquid nitrogen. After 7 days, the cells were thawed using a water bath, and the number of viable cells was counted.

[0068] The materials used in this experiment are listed below. [Table 2]

[0069] [Table 3]

[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 indicate that the addition of Soluplus improves the cell recovery rate after thawing.

[0071] Traditionally, albumin has been used as a cryoprotective agent, but it is a molecule that can cause biological contamination. Furthermore, albumin has the problem of high raw material costs. However, this experiment demonstrates that Soluplus can be used as a cryoprotective agent instead of albumin. Soluplus can be chemically synthesized and is cheaper than albumin. Moreover, the amount added when used as a cryoprotective agent 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 examples. These examples are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible, and that such modifications also fall within the scope of the present invention. [Industrial applicability]

[0073] According to the present invention, by adding polyalkylene glycol modified with a copolymer of polyvinyl caprolactam block and polyvinyl acetate block to a composition, cells can be effectively cryoprotected without the addition of albumin, making it suitable for industrial use.

Claims

1. A composition for use in cell cryoprotection, comprising a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block, wherein the polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block is a graft copolymer obtained from (i) 50-60 wt% N-vinylcaprolactam, (ii) 25-35 wt% vinyl acetate, and (iii) 10-20 wt% polyethylene glycol (where (i) to (iii) total 100 wt%).

2. The composition according to claim 1, wherein the polyalkylene glycol modified with the copolymer of the polyvinylcaprolactam block and the polyvinyl acetate block is a compound represented by the following structural formula (1). 【Chemistry 1】 (Here, the wt% of l, m, and n are 50-60 wt%, 25-35 wt%, and 10-20 wt%, respectively, and the sum of the wt% of l, m, and n is 100 wt%.)

3. The composition according to claim 1 or 2, wherein the composition is an additive composition, and the addition is carried out to produce a mixture comprising 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 according to claim 1 or 2, further comprising a freeze-protecting agent.

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

6. A method for producing frozen cells, comprising a freezing step of freezing a mixture containing polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block and cells to produce frozen cells, wherein the polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block is a graft copolymer obtained from (i) 50-60 wt% N-vinylcaprolactam, (ii) 25-35 wt% vinyl acetate, and (iii) 10-20 wt% polyethylene glycol (where (i) to (iii) total 100 wt%).

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

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

9. A method for preserving cells, comprising a freezing step of freezing a composition comprising a polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block and cells to produce frozen cells, wherein the polyalkylene glycol modified with a copolymer of polyvinylcaprolactam block and polyvinyl acetate block is a graft copolymer obtained from (i) 50-60 wt% N-vinylcaprolactam, (ii) 25-35 wt% vinyl acetate, and (iii) 10-20 wt% polyethylene glycol (where (i) to (iii) total 100 wt%).

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

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

Images