Method for producing and cryopreserving lymphocytes, cleaning solution and cryopreserving solution used therefor, and method for producing lymphocytes for transplantation from cryopreserved lymphocytes

Abstract

The present invention provides: a method for cleaning lymphocytes, which comprises cleaning lymphocytes with a physiological aqueous solution containing bicarbonate ions; a method for cryopreserving lymphocytes, which comprises suspending and freezing the lymphocytes obtained by the method in a cryopreservation solution containing a physiological aqueous solution; a cleaning solution for lymphocytes, which contains a physiological aqueous solution containing bicarbonate ions; the cryopreservation solution for lymphocytes comprises a physiological aqueous solution containing bicarbonate ions, glucan, glucose, DMSO and human serum albumin. A composition containing lymphocytes, which contains lymphocytes treated with a physiological aqueous solution containing bicarbonate ions, and the cryopreservation solution; a kit for producing lymphocytes for transplantation, which contains the composition containing lymphocytes and a diluent for transplantation, which contains a physiological aqueous solution containing bicarbonate ions; and the like.

Description

Technical Field

[0001] The present invention relates to a method for washing and cryopreserving lymphocytes such as T cells and natural killer cells (hereinafter sometimes simply referred to as "NK cells") useful in the medical field, a washing solution and a cryopreservation solution therefor, a composition containing lymphocytes washed and cryopreserved using the method or solution, a dilution solution for transplantation for the composition containing lymphocytes, a kit for preparing transplant lymphocytes comprising the composition containing lymphocytes and the dilution solution for transplantation, a method for preparing transplant lymphocytes using the same, and a transplant lymphocyte preparation obtained by the method. The washing solution and the dilution solution for transplantation contain a physiological aqueous solution containing bicarbonate ions, and the cryopreservation solution contains a physiological aqueous solution, preferably a physiological aqueous solution containing bicarbonate ions. Background Art

[0002] As cells involved in cancer immunity, lymphocytes such as T cells, NK cells, and natural killer T (NKT) cells can be mentioned. In recent years, with the development of technologies such as the development of induced pluripotent stem cells (iPS cells) and their differentiation induction methods, and cell gene modification methods, gene modification of pluripotent stem cells such as iPS cells and differentiation induction into target immune cells can be performed, whereby highly functional immune cells can be produced. As a cell drug containing genetically modified immune cells, drugs based on CAR-T cells targeting CD19 (Kymriah and Yescarta) have been approved at present. On the other hand, since NK cells have unique properties different from those of T cells, expectations for immunotherapy using NK cells have also increased, and various clinical trials are actually being conducted on cell drugs using mutant NK cells.

[0003] In order to use immune cells as cell drugs, it is necessary to prepare a large amount of immune cells. There have also been a large number of reports on methods for differentiating and inducing lymphocytes such as T cells and NK cells from pluripotent stem cells and methods for amplifying and culturing lymphocytes. For example, the present applicant has developed a technique for effectively mass-producing NK cells that maintain high activity even after freezing and thawing by forming pluripotent stem cell spheres of appropriate size by three-dimensional suspension culture, replacing the culture medium by perfusion (continuous three-dimensional perfusion culture), differentiating and inducing hematopoietic progenitor cells from the spheres, and three-dimensionally suspending and culturing the obtained hematopoietic progenitor cells to differentiate them into NK cells, and has applied for a patent (Patent Document 10).

[0004] Since the cultivation of cells for transplantation needs to be carried out at the GMP level, when immune cells are used as cell drugs, the cells manufactured and cryopreserved in a cell preparation facility (CPC) are usually transported to a hospital, thawed in the hospital, suspended and diluted in a transplantation medium, and then sent to the operating room for transplantation. As the cryopreservation solution, a solution obtained by adding a cryoprotectant to a culture medium for cell culture is used. However, among dozens of culture medium components, there may be components harmful to the recipient, and it is necessary to wash the cells after thawing and before transplantation, resulting in a loss of time, a decrease in the cell recovery rate and survival rate.

[0005] Regarding these problems, the development of a washing solution and a cryopreservation solution that can remove harmful components by washing the cells before freezing and maintain a high cell recovery rate and survival rate after freezing and thawing is being promoted. As the washing solution and the cryopreservation solution, there are reports of using solutions based on physiological aqueous solutions such as physiological saline and Ringer's solution. For example, it has been reported that in the washing and cryopreservation steps of human mesenchymal stem cells, by using a solution containing human serum albumin (HuSA) and bicarbonate Ringer's solution, the decrease in cell survival rate in "each step" can be inhibited (Patent Document 1). In addition, in order to avoid using cryoprotectants harmful at high concentrations such as dimethyl sulfoxide (DMSO) or using them for cells with low storage stability after cryopreservation such as NK cells, it has been reported that a solution containing glucose and / or HuSA and bicarbonate is used as a preservation solution for long-term storage of cells in a non-frozen state (refrigeration or normal temperature) (Patent Documents 2 and 3). However, there is no reported example of using a bicarbonate solution as a washing solution before cryopreserving lymphocytes, and there is also no example of completely investigating the effect of the washing solution on the recovery rate and survival rate of cells after freezing and thawing (not after washing).

[0006] As a cryopreservation solution for lymphocytes, there are reported examples of using a solution containing, in addition to DMSO, a part of dextran, glucose, HuSA, bicarbonate, and a physiological aqueous solution (for example, physiological saline, Ringer's solution) (Patent Documents 1, 5-9). However, a cryopreservation solution containing all of the aforementioned components or a cryopreservation solution composed of these components has not been disclosed.

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: WO 2009 / 057537

[0010] Patent Document 2: JP 2008-104407 A

[0011] Patent Document 3: WO 2013 / 115322

[0012] Patent Document 4: Japanese Patent Application Laid-Open No. 2018-138565

[0013] Patent Document 5: WO 2011 / 021618

[0014] Patent Document 6: Japanese Patent Application Laid-Open No. 2000-201672

[0015] Patent Document 7: Japanese Patent Application Laid-Open No. 2018-183161

[0016] Patent Document 8: WO 2018 / 084228

[0017] Patent Document 9: Chinese Patent Application Publication No. 112868642

[0018] Patent Document 10: WO 2023 / 145922 Summary of the Invention

[0019] Problems to be Solved by the Invention

[0020] As described above, in order to produce lymphocytes for transplantation, washing solutions and cryopreservation solutions that can maintain a high cell recovery rate and survival rate even after freezing and thawing have been intensively developed, but they are not necessarily satisfactory. In particular, regarding the washing step of lymphocytes for transplantation, maintaining and improving the high cell recovery rate and cell survival rate after freezing and thawing have not been envisaged. Therefore, an object of the present invention is to provide a washing solution and a cryopreservation solution for lymphocytes that can maintain a high cell recovery rate and survival rate even after freezing and thawing, and can produce lymphocytes for transplantation that maintain high physiological activity. Another object of the present invention is to provide a dilution solution for transplantation that can maintain a high cell survival rate even when stored at room temperature until transplantation after thawing a frozen lymphocyte-containing composition obtained using the washing solution and the cryopreservation solution, and to provide a kit for preparing lymphocytes for transplantation comprising the lymphocyte-containing composition and the dilution solution for transplantation, to provide a lymphocyte preparation more suitable for transplantation.

[0021] Means for Solving the Problems

[0022] The present inventors conducted intensive studies to achieve the above object, and as a result, found that if lymphocytes maintained by expansion culture are suspended and allowed to stand (simulating the washing step) in a physiological aqueous solution containing bicarbonate ions such as bicarbonate Ringer's solution and then cryopreserved using a conventionally known cryopreservation solution, surprisingly, the cell recovery rate and cell survival rate after thawing and after resuscitation culture relative to before cryopreservation are significantly increased compared to the case of suspending and allowing to stand in a physiological aqueous solution not containing bicarbonate ions.

[0023] Next, the inventors of the present invention suspended and cryopreserved the lymphocytes maintained in amplified culture in a cryopreservation solution containing dextran, glucose, DMSO, HuSA and a physiological aqueous solution (e.g., physiological saline, bicarbonate Ringer's solution, etc.) at various concentrations, and found that the cryopreservation solution containing the aforementioned components in the respective specified concentration ranges significantly improved the viable cell recovery rate after thawing compared to the general cryopreservation solution. In addition, after being suspended in the cryopreservation solution containing bicarbonate Ringer's solution as a physiological aqueous solution, when the cells were left standing at room temperature for 0-4 hours before cryopreservation, the decrease in the viable cell recovery rate after thawing and resuscitation culture over time due to standing at room temperature was significantly suppressed.

[0024] Furthermore, in order to study the effect of physiological aqueous solutions as a medium (diluent) for transplantation, the inventors thawed the frozen NK cells, diluted the cell suspension with various physiological aqueous solutions and allowed to stand at room temperature for 0-4 hours. The results showed that the recovery rate of live cells was significantly improved when using physiological aqueous solutions containing bicarbonate ions compared with physiological aqueous solutions not containing such ions.

[0025] The present inventors have further conducted studies based on this finding and have completed the present invention.

[0026] That is, the present invention is as follows.

[0027] [Project 1]

[0028] A method for washing lymphocytes, comprising washing lymphocytes with a physiological aqueous solution containing bicarbonate ions.

[0029] [Project 2]

[0030] The method according to item 1, wherein the lymphocytes are T cells or NK cells.

[0031] [Item 3]

[0032] The method according to item 1 or 2, wherein the physiological aqueous solution containing bicarbonate ions is bicarbonate Ringer's solution.

[0033] [Item 4]

[0034] A method for cryopreservation of lymphocytes, comprising suspending the lymphocytes treated by the method according to any one of items 1 to 3 in a cryopreservation solution containing a physiological aqueous solution and freezing the suspension.

[0035] [Item 5]

[0036] The method according to item 4, wherein the physiological aqueous solution is a physiological aqueous solution containing bicarbonate ions.

[0037] [Item 6]

[0038] The method according to item 5, wherein the physiological aqueous solution containing bicarbonate ions is bicarbonate Ringer's solution.

[0039] [Item 7]

[0040] The lymphocytes are washed with a washing solution containing a physiological aqueous solution containing bicarbonate ions.

[0041] [Item 8]

[0042] The lymphocytes are cryopreserved with a cryopreservation solution containing a physiological aqueous solution containing bicarbonate ions, dextran, glucose, DMSO, and human serum albumin.

[0043] [Item 9]

[0044] A composition containing lymphocytes, which comprises lymphocytes treated with a physiological aqueous solution containing bicarbonate ions and the cryopreservation solution described in item 8.

[0045] [Item 10]

[0046] A kit for preparing lymphocytes for transplantation, which comprises: the composition containing lymphocytes described in item 9 and a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions.

[0047] [Item 11]

[0048] A transplantation diluent, which is a transplantation diluent for the composition containing lymphocytes described in item 9 and contains a physiological aqueous solution containing bicarbonate ions.

[0049] [Item 12]

[0050] A method for preparing lymphocytes for transplantation, which comprises diluting the composition containing lymphocytes described in item 9 thawed after cryopreservation with a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions.

[0051] [Item 13]

[0052] A lymphocyte preparation for transplantation, which is obtained by the method described in item 12.

[0053] Advantages of the Invention

[0054] By using the washing solution and cryopreservation solution of the present invention, it is possible to produce lymphocytes for transplantation (e.g., NK cells, T cells, etc.) that maintain a high cell recovery rate and survival rate even after freezing and thawing, and maintain high physiological activity. In addition, by using the composition containing lymphocytes and the transplantation diluent of the present invention, it is possible to provide a lymphocyte preparation that can maintain a high cell survival rate even when stored at room temperature from the time of thawing until transplantation, and is more suitable for transplantation. Detailed implementation mode

[0055] [I]The cleaning method of the present invention and the cleaning solution of the present invention

[0056] The present invention provides a method for washing lymphocytes (hereinafter sometimes referred to as "the washing method of the present invention"), which includes washing lymphocytes with a physiological aqueous solution containing bicarbonate ions.

[0057] In this specification, "washing" means an operation of suspending the cells in a liquid (i.e., the washing solution) in order to remove substances (such as culture medium components) present in the surrounding environment (such as the culture solution) of the cells before washing. The washing of cells can be carried out, for example, by removing the dispersion medium of the cells before washing (such as centrifugation, filtration, etc.) and recovering the suspension of the cells in the washing solution (repeating this operation as needed), or it can also be carried out by replacing the dispersion medium before washing with the washing solution using, for example, an automatic concentration washing device. The manner is not particularly limited.

[0058] (A) Lymphocytes

[0059] The lymphocytes for the washing method of the present invention are not particularly limited, and examples include NK cells, T cells (such as cytotoxic T cells (CTL), helper T cells, regulatory T cells, etc.), B cells, etc. NK cells and T cells are preferred, and NK cells are more preferred. Lymphocytes can be isolated from blood (such as peripheral blood, cord blood, etc.) collected from humans or other mammals (preferably humans) or monocytes isolated therefrom, using conventional methods such as flow cytometry, with the expression of surface antigen markers specifically expressed in the target lymphocytes (such as CD3, CD4, CD8, etc. for T cells, CD56 for NK cells, etc.) as an index.

[0060] In a preferred embodiment, various lymphocytes can be induced to differentiate from lymphocyte progenitors containing pluripotent cells. Examples of such lymphocyte progenitors include iPS cells, embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), embryonic germ stem cells (EG cells), hematopoietic stem cells, multipotent progenitors (MPP) that have lost the ability of self-renewal, myeloid-lymphoid common progenitors (MLP), myeloid progenitors (MP), granulocyte-monocyte progenitors (GMP), macrophage-dendritic cell progenitors (MDP), dendritic cell progenitors (DCP), etc. Preferred examples include pluripotent stem cells such as ES cells and iPS cells.

[0061] As a method for inducing the differentiation of the aforementioned progenitor cells into lymphocytes, various well-known differentiation induction methods can be appropriately selected. For example, as a well-known differentiation induction method for differentiating T cells from human pluripotent stem cells, the method described in Timmermans, F. et al., J. Immunol., 2009, 182, 68879-6888 can be cited. In addition, for example, by the method described in Nishimura, T. et al. Cell Stem Cell, 2013, 12, 114-126, human iPS cells established from human peripheral blood T lymphocytes can be differentiated into T cells. In addition, as a well-known differentiation induction method for differentiating NK cells from human pluripotent stem cells, the methods described in WO 2020 / 086889, Biochem Biophys Res Commun. 515(1):1-8(2019), Methods Mol Biol. 2048:107-119(2019) can be cited.

[0062] T cells can be activated and expanded from samples containing T cells such as PBMCs or T cells induced to differentiate from lymphocyte progenitor cells by conventional methods. For example, in a sample containing T cells, a molecule that interacts with the surface molecules of T cells to promote the activation and / or proliferation of T cells can be added to the culture medium for culturing. As such a molecule, those that specifically bind to CD3 conjugated with TCR and responsible for TCR-mediated signal transduction, surface molecules known as co-stimulatory factors for T cell activation such as CD28, ICOS, CD137, OX40, CD27, GITR, BAFFR, TACI, BMCA, CD40L, etc., and have the function of transducing activation / proliferation signals and co-signals into T cells can be cited. Preferably, an antibody against CD3 and / or an antibody against CD28 can be cited. These molecules can be used alone or in the form of a complex (e.g., TransAct (Milteny Biotech), Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher Scientific)) combined with a carrier (e.g., Dynabeads (registered trademark) (ThermoFisher Scientific)). When performing proliferation stimulation on T cells using an anti-CD3 antibody and / or an anti-CD28 antibody, cytokines such as IL-2, IL-7, IL-15, etc. can be further added to the culture medium.

[0063] NK cells can be activated and expanded by conventional methods from specimens containing T cells such as PBMCs, and NK cells induced to differentiate from lymphocyte progenitors. As such methods, for example, the methods described in WO 2020 / 086889, Biochem Biophys Res Commun. 515(1):1-8(2019), Methods Mol Biol. 2048:107-119(2019) can be cited, but are not limited thereto.

[0064] As described above, the present applicant has developed a method for effectively mass-producing NK cells that maintain high activity even after freezing and thawing by continuously performing three-dimensional perfusion culture to induce hematopoietic progenitor cells to differentiate from pluripotent stem cell spheres and then performing three-dimensional suspension culture on the obtained hematopoietic progenitor cells to differentiate them into NK cells (Japanese Patent Application No. 2022-013646). More specifically, this method is characterized by including:

[0065] (1) a step of forming pluripotent stem cell spheres having an average particle size of 200 μm or more in a first culture medium;

[0066] (2) a step of inducing the pluripotent stem cell spheres formed in step (1) into a cell population containing hematopoietic progenitor cells by three-dimensional culture using a second culture medium; and

[0067] (3) a step of inducing the cell population containing hematopoietic progenitor cells obtained in step (2) into a cell population containing NK cells by three-dimensional culture using a third culture medium:

[0068] And steps (1) to (3) are carried out by the three-dimensional perfusion culture method.

[0069] The three-dimensional suspension culture method is one of the cell culture methods, which is characterized in that while three-dimensionally expanding a carrier for three-dimensional culture of suspended cells, spheres (spheroids), or adherent cells using a stirring paddle or an oscillator, culture is carried out. In addition, the perfusion culture method is one of the continuous culture methods, which can achieve the purpose of continuously replacing the culture medium, that is, supplying new nutrients and removing waste, by supplying a certain amount of new culture medium into the culture solution in the culture vessel while extracting a certain amount of the culture medium. In this method, perfusion culture is preferably carried out continuously. "Carrying out perfusion culture continuously" means that only membrane replacement is carried out between steps, and in the case where a cleaning step usually carried out when changing the culture medium is not involved, the culture medium of the previous step is taken out while supplying the culture medium of the subsequent step.

[0070] The separation membrane used in perfusion culture preferably has a certain pore diameter and properties (e.g., degree of hydrophobicity, etc.) so that the target cell population does not escape outside the culture system. As a suitable pore diameter, for step (1), a pore diameter of 15 - 75 μm can be cited; for step (2), a pore diameter of 45 - 225 μm can be cited; for step (3), a pore diameter of 0.2 - 10 μm can be cited.

[0071] The first medium in step (1) is not particularly limited as long as it is a culture solution used in the maintenance culture of pluripotent stem cells, and for example, a medium capable of feeder-free culture can be used. Preferably, a medium containing a ROCK inhibitor in a medium capable of feeder-free culture can be cited.

[0072] As the second medium in step (2), as long as it is a medium capable of inducing pluripotent stem cells into hematopoietic progenitor cells, it is not particularly limited, and for example, a medium containing vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), and glycogen synthase 3β (GSK3β) inhibitor, a medium containing stem cell factor (SCF) and transforming growth factor β (TGFβ) / Smad inhibitor, a medium containing SCF and Flt3 ligand (Flt3L), etc. can be cited.

[0073] As the third medium in step (3), as long as it is a medium capable of inducing hematopoietic progenitor cells into NK cells, it is not particularly limited, and for example, a medium containing IL-15 and SCF can be cited.

[0074] The average particle diameter of the spheres in step (1) is generally 200 μm or more, and specifically, it is suitably adjusted in the range of 200 - 600 μm. As the culture time required for spheroid formation, for example, it is preferably about 2 - 10 days, more preferably about 4 - 7 days, starting from cell seeding.

[0075] The culture time in step (2) is generally 5 - 20 days, preferably about 10 - 18 days.

[0076] The culture time in step (3) is generally 20 - 60 days, preferably about 25 - 40 days.

[0077] After step (3), an amplification culture step or a maturation step of the obtained NK cells as step (4) can be further implemented. The amplification culture step or the maturation step can be suitably applied using known methods.

[0078] It should be noted that in the above method, by substituting step (3) and implementing a method known per se for inducing the differentiation of T cells from hematopoietic stem cells, T cells can also be induced from pluripotent stem cells.

[0079] (B) Physiological aqueous solution containing bicarbonate ions

[0080] In this specification, "physiological aqueous solution" means an isotonic solution in which the salt and sugar concentrations are adjusted using sodium, potassium, etc. to be approximately the same as the osmotic pressure of body fluids (such as plasma) and cell sap. Examples of such isotonic solutions include physiological saline, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), HEPES-buffered saline, Ringer's solution, Hank's balanced salt solution (HBSS), 5% glucose aqueous solution, and other isotonic solutions. In this specification, "isotonic" means that the osmotic pressure is in the range of 250 to 380 mOsm / L.

[0081] As the physiological aqueous solution containing bicarbonate ions (hereinafter sometimes referred to as "the cleaning solution of the present invention") used in the cleaning method of the present invention, any physiological aqueous solution containing a salt of bicarbonate ions and various cations can be cited. Examples of such bicarbonates include sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, calcium bicarbonate, etc. Preferably, as the physiological aqueous solution containing bicarbonate ions, Ringer's bicarbonate solution, HBSS, etc. can be cited. More preferably, it is Ringer's bicarbonate solution.

[0082] "Ringer's bicarbonate solution" in this specification is a physiological aqueous solution containing bicarbonate ions, sodium ions, potassium ions, calcium ions, and chloride ions as electrolytes, and the bicarbonate ion concentration is 22 to 31 mEq / L, and the sodium ion concentration is 120 to 150 mEq / L. The pH of Ringer's bicarbonate solution can be 6.8 to 7.8. The osmotic pressure ratio relative to physiological saline can be 0.9 to 1.1, but it is not limited thereto. Ringer's bicarbonate solution can further contain magnesium ions and / or citrate ions as electrolytes. When Ringer's bicarbonate solution contains magnesium ions and citrate ions, the concentrations of each electrolyte can be as described below, but it is not limited thereto.

[0083]

[0084] As specific examples of such Ringer's bicarbonate solution, commercially available Bicanate (registered trademark; Otsuka Pharmaceutical Factory, Inc.) and Bicarbon (registered trademark; AY Pharma Co., Ltd.) can be cited. The concentrations of each electrolyte in them are as described below. In addition, the composition (w / v%) of each component (salt) of these Ringer's bicarbonate solutions is shown in Table 8.

[0085] (Bicanate (registered trademark))

[0086]

[0087] (Bicarbon (registered trademark))

[0088]

[0089] As HBSS, HBSS(-) containing bicarbonate ions, sodium ions, potassium ions, chloride ions and phosphate ions as electrolytes and having the electrolyte composition shown in Table 1, and HBSS(+) containing calcium ions and magnesium ions as electrolytes and having the electrolyte composition shown in Table 1 can be mentioned. HBSS(+) is more preferred.

[0090] The cleaning solution of the present invention may contain components other than the above electrolytes. As such components, for example, electrolytes (buffering agents) other than the above such as phosphate ions, lactate ions, acetate ions, sugars such as glucose, galactose, fructose, mannose, maltose, sucrose, and biological source proteins such as serum albumin (for example, HuSA, BSA, etc.), serum globulin, etc. can be mentioned.

[0091] However, in a preferred embodiment, the cleaning solution of the present invention is a physiological aqueous solution containing bicarbonate ions and not containing buffering agents such as phosphate ions, lactate ions, acetate ions, sugars (for example, glucose, etc.), and biological source proteins (for example, HuSA, etc.).

[0092] (C) Cleaning method

[0093] In the cleaning method of the present invention, the method of cleaning lymphocytes with the cleaning solution of the present invention is not particularly limited. For example, it can be carried out by removing the dispersion medium (for example, culture solution) of lymphocytes before cleaning by centrifugation, filtration, etc., and suspending the recovered lymphocytes in the cleaning solution of the present invention (repeating this operation as needed). In a preferred embodiment, for example, an automatic concentration cleaning device that includes a cell filter composed of a hollow fiber membrane module, etc. in a circuit and can transport a cell suspension and a cleaning solution into the circuit is used, and priming (filling the inside of the circuit with the cleaning solution), concentration (transporting the cell suspension into the circuit and removing medium components (for example, low molecules, proteins, etc.) outside the membrane while circulating it inside the hollow fiber membrane), cleaning (transporting the cleaning solution into the circuit and replacing it with the medium components), and recovery (recovering the cell concentrate from which the medium components have been removed) are carried out in a closed system. Thereby, the risk of cell damage and contamination can be reduced, and the cells can be cleaned (removal of medium components) in a dispersed state. Such an automatic concentration cleaning device is commercially available. For example, the Kaneka cell concentration cleaning system (manufactured by Kaneka Corporation) etc. can be used.

[0094] This washing step can be carried out at about 4°C to room temperature. For example, when washing lymphocytes that have been amplified and cultured in a 3L bioreactor using an automatic concentration washing device, although it is expected to take about 2 hours from cell recovery after culture until the end of the washing step, by the washing method of the present invention, the lymphocytes are suspended in the washing solution of the present invention and allowed to stand at room temperature for 4 hours. Compared with the case of suspending in a physiological aqueous solution such as physiological saline that does not contain bicarbonate ions, the decrease in the recovery rate of viable cells and cell viability after freeze-thawing and after resuscitation culture is also significantly inhibited. Therefore, the washing solution and washing method of the present invention are extremely useful in the washing step (removal step of culture medium components) of lymphocytes after amplified culture.

[0095] [II]The cryopreservation method of the present invention and the cryopreservation solution of the present invention

[0096] When the lymphocytes obtained by the washing method of the present invention are suspended and cryopreserved in a cryopreservation solution containing a physiological aqueous solution, the recovery rate of viable cells after thawing is significantly improved compared with the case of cryopreservation using a general commercially available cryopreservation solution such as STEM-CELLBANKER (registered trademark) or CryoStor CS10. Therefore, the present invention also provides a method for cryopreserving lymphocytes (hereinafter, sometimes referred to as "the cryopreservation method of the present invention"), which includes suspending and freezing the lymphocytes treated by the above washing method of the present invention in a cryopreservation solution containing a physiological aqueous solution.

[0097] It is known that washing cultured cells with bicarbonate Ringer's solution (which contains sugars such as glucose and / or HuSA) can inhibit the decrease in cell recovery rate and viability caused by the washing step. However, by washing lymphocytes with a physiological aqueous solution containing bicarbonate ions (preferably not containing sugars and HuSA), and then even when cryopreserving the lymphocytes under the same conditions, the recovery rate and viability of the cells after thawing and after resuscitation culture are improved. This is a surprising insight first discovered by the present inventors. That is, the lymphocytes obtained by the washing method of the present invention have novel and particularly significant inherent characteristics compared with the lymphocytes obtained by the conventional washing method, but it is impossible or almost impractical to distinguish the two by structure or physical properties.

[0098] The physiological aqueous solution used in the cryopreservation method of the present invention is not particularly limited as long as it can improve the recovery rate of viable cells after thawing when lymphocytes obtained by the cleaning method of the present invention are suspended in a cryopreservation solution containing the physiological aqueous solution. Any physiological aqueous solution exemplified in the cleaning method of the present invention can be cited. Preferably, physiological saline or a physiological aqueous solution containing bicarbonate ions can be cited. More preferably, physiological saline or Ringer's bicarbonate solution is used.

[0099] The concentration of the physiological aqueous solution in the cryopreservation solution used in the cryopreservation method of the present invention, for example, in the case of physiological saline, is, for example, 25 to 50% (v / v), preferably 30 to 45% (v / v), more preferably 32 to 41% (v / v), but is not limited thereto. In addition, when the physiological aqueous solution is Ringer's bicarbonate solution, its concentration in the cryopreservation solution is, for example, 35 to 75% (v / v), preferably 40 to 70% (v / v), more preferably 43 to 67% (v / v), but is not limited thereto.

[0100] The cryopreservation solution used in the cryopreservation method of the present invention preferably contains one or more cryoprotectants. Examples of cryoprotectants include, but are not limited to, dextran, DMSO, hydroxyethyl starch (HES), ethylene glycol, glycerol, trehalose, etc. Preferably, dextran and DMSO can be cited.

[0101] Dextran is a polysaccharide composed of D-glucose with an α1→6 bond as the main chain (C6H 10 O5) n . The molecular weight of dextran is not particularly limited as long as it does not penetrate into the cells. For example, as the weight average molecular weight (Mw), 40,000 (dextran 40) to 70,000 (dextran 70) can be cited. Dextran can be produced by known methods such as chemical synthesis, microbial production, and enzyme production. In addition, commercially available products can also be used. Examples of commercially available dextran products include dextran 40 (manufactured by Meito Sangyo Co., Ltd.) and dextran 70 (manufactured by Tokyo Chemical Industry Co., Ltd.).

[0102] Alternatively, derivatives such as dextran sulfate, carboxylated dextran, and diethylaminoethyl (DEAE)-dextran can also be used.

[0103] The dextran or its derivative may also be in the form of a free body or a salt. As their salts, for example, acid addition salts with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, etc., acid addition salts with organic acids such as propionate, tosylate, succinate, oxalate, lactate, tartrate, glycolate, mesylate, butyrate, valerate, citrate, fumarate, maleate, malate, etc., metal salts such as sodium salt, potassium salt, calcium salt, etc., basic salts such as ammonium salt, alkylammonium salt, etc. These salts can form hydrates or solvates. In addition, any one of them can be used alone or two or more of them can be used in a suitable combination.

[0104] As the concentration of dextran contained in the cryopreservation solution, for example, it is 3 to 12% (w / v), preferably 3 to 11% (w / v), more preferably 3.5 to 10.5% (w / v), but it is not limited thereto. In addition, the preferred concentration range may vary depending on the type of physiological aqueous solution used.

[0105] In order to avoid adverse effects on cells, DMSO with as high a purity as possible is preferably used. As the concentration of DMSO contained in the cryopreservation solution, for example, it is 3 to 15% (w / v), preferably 5 to 12% (w / v), more preferably 7.5 to 12% (w / v), but it is not limited thereto. In addition, the preferred concentration range may vary depending on the type of physiological aqueous solution used.

[0106] The cryopreservation solution used in the cryopreservation method of the present invention may contain one or more kinds of saccharides. As the saccharides, for example, hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, tagatose, fucose, fucoidan, rhamnose, etc., pentoses such as ribulose, xylulose, ribose, arabinose, xylose, lyxose, etc. monosaccharides; disaccharides such as sucrose, maltose, etc.; oligosaccharides such as raffinose, stachyose, etc.; polysaccharides such as starch, glycogen, etc., but it is not limited thereto. Glucose is preferred.

[0107] As the concentration of the saccharide contained in the cryopreservation solution, for example, it is 0.03 to 1.2% (w / v), preferably 0.04 to 0.8% (w / v), more preferably 0.05 to 0.7% (w / v), but it is not limited thereto. In addition, the preferred concentration range may vary depending on the type of physiological aqueous solution used.

[0108] The cryopreservation solution used in the cryopreservation method of the present invention may contain one or more proteins. Examples of the protein include plasma-derived proteins such as serum albumin (e.g., HuSA, bovine serum albumin (BSA)), serum globulin, etc., but are not limited thereto. HuSA is preferred. The plasma-derived protein may be purified from animal plasma or produced recombinantly.

[0109] As the concentration of the protein contained in the cryopreservation solution, for example, it is 0.5 to 5% (w / v), preferably 1 to 4% (w / v), more preferably 1.2 to 3.5% (w / v), but is not limited thereto. In addition, the preferred concentration range may vary depending on the type of physiological aqueous solution used.

[0110] In a preferred embodiment, the cryopreservation solution used in the cryopreservation method of the present invention contains a physiological aqueous solution selected from physiological saline or bicarbonate Ringer's solution, dextran, DMSO, glucose, and HuSA.

[0111] When the physiological aqueous solution is physiological saline, the composition of the cryopreservation solution may be:

[0112]

[0113] Preferably:

[0114]

[0115] On the other hand, when the physiological aqueous solution is bicarbonate Ringer's solution, the composition of the cryopreservation solution may be:

[0116]

[0117] Preferably:

[0118]

[0119] As described above, as a cryopreservation solution for lymphocytes, there are reported examples of using a solution containing a part of DMSO, dextran, glucose, HuSA, bicarbonate, and a physiological aqueous solution (e.g., physiological saline, Ringer's solution), but a cryopreservation solution containing all of the foregoing components or a cryopreservation solution composed of these components has not been disclosed.

[0120] Therefore, the present invention also provides a cryopreservation solution for lymphocytes (hereinafter, sometimes referred to as "the cryopreservation solution of the present invention"), which contains a physiological aqueous solution containing bicarbonate ions, dextran, glucose, DMSO, and HuSA.

[0121] As components of the cryopreservation solution of the present invention, namely a physiological aqueous solution containing bicarbonate ions, dextran, glucose, DMSO, and HuSA, those separately described for the cryopreservation method of the present invention can be used. As the physiological aqueous solution containing bicarbonate ions, bicarbonate Ringer's solution is preferably used.

[0122] When the physiological aqueous solution containing bicarbonate ions is bicarbonate Ringer's solution, the composition of the cryopreservation solution of the present invention is preferably as described below.

[0123]

[0124] More preferably:

[0125]

[0126] In the cryopreservation method of the present invention, first, the washing solution is removed from the cell concentrate of lymphocytes obtained by performing the above-described washing method of the present invention, and the cells are resuspended in any of the above cryopreservation solutions. For example, the cell concentrate can be recovered in a centrifuge tube or the like, the cells can be centrifuged to form pellets, and then a cryopreservation solution can be added for suspension. Alternatively, by pumping the cryopreservation solution while withdrawing the washing solution, the washing solution can be continuously replaced with the cryopreservation solution.

[0127] The obtained cell suspension is placed in a cryopreservation tube and frozen in a freezer at -80°C, and then it can be stored in a nitrogen tank in the gas phase or the liquid phase, but it is not limited thereto. For example, the cell suspension is dispensed into cryopreservation vials using an automatic dispensing device, and for example, programmed freezing is performed using a freezer capable of controlling the cooling rate, whereby the freezing process can be optimized.

[0128] For example, lymphocytes amplified and cultured using a 3L-volume bioreactor are subjected to the washing method of the present invention, the washing solution of the obtained cell concentrate is replaced with a cryopreservation solution, the obtained cell suspension is dispensed into a cryopreservation container, and the time required until the start of the freezing process is estimated to be about 2 hours. This operation is carried out at about 4°C to room temperature. Therefore, the cryopreservation solution is required not only to protect the cells from damage during cryopreservation and thawing, but also to be able to stably preserve the cells at about 4°C to room temperature during the time from after washing until the cells are resuspended in the cryopreservation solution and the freezing process is started. As shown in the following examples, when cryopreservation is performed using a cryopreservation solution containing bicarbonate Ringer's solution, compared with the existing cryopreservation solution SCB, the decrease in the time-dependent viable cell recovery rate when the washed lymphocytes are suspended in the cryopreservation solution and left standing at room temperature for 4 hours is significantly suppressed. Therefore, the cryopreservation method and cryopreservation solution of the present invention are extremely useful in the cryopreservation of lymphocytes.

[0129] [III]The composition containing lymphocytes of the present invention, the diluent for transplantation of the present invention, the reagent containing them Cartridge, the method for preparing lymphocytes for transplantation using the same, and the preparation of lymphocytes for transplantation obtained thereby

[0130] Compared with the case of using the existing cryopreservation solution, the recovery rate and cell survival rate of viable cells after thawing and after resuscitation culture of the frozen lymphocyte-containing composition obtained by the cryopreservation method of the present invention described above are significantly improved. That is, the lymphocyte-containing composition achieves advantageous effects as transplant lymphocytes. Therefore, the present invention further provides a lymphocyte-containing composition (hereinafter, sometimes referred to as "the lymphocyte-containing composition of the present invention"), which comprises lymphocytes treated with a physiological aqueous solution containing bicarbonate ions and the above-mentioned cryopreservation solution of the present invention.

[0131] As shown in the examples described later, after thawing the frozen lymphocyte-containing composition obtained by the washing method and cryopreservation method of the present invention, it is diluted with a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions, and after standing at room temperature for 4 hours, resuscitation culture is carried out, and the cell survival rate after culture is evaluated. As a result, a higher cell survival rate was obtained compared with the case of diluting with a physiological aqueous solution not containing bicarbonate ions (for example, physiological saline, acetate Ringer's solution). That is, the physiological aqueous solution containing bicarbonate ions is extremely useful as a transplantation medium. Therefore, the present invention further provides a transplantation diluent, which is a transplantation diluent for the lymphocyte-containing composition of the present invention and contains a physiological aqueous solution containing bicarbonate ions. As the physiological aqueous solution containing bicarbonate ions, the solution used in the above-mentioned washing solution of the present invention can be appropriately selected and used, and preferably Ringer's bicarbonate solution. Furthermore, the present invention further provides a kit for preparing transplant lymphocytes, which comprises: the lymphocyte-containing composition of the present invention and a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions.

[0132] In the above-mentioned kit for preparing transplant lymphocytes, the lymphocyte-containing composition can be provided in a frozen state. For example, it is transported from the CPC to the hospital in a frozen state, thawed, and diluted and suspended with a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions, and can be prepared into a transplant lymphocyte preparation, and can be stably maintained at room temperature until the transplantation surgery. Or, according to another method, the frozen lymphocyte-containing composition can also be thawed at the CPC and immediately diluted and suspended in a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions, and the lymphocytes can be transported to the hospital at room temperature in a non-frozen state, and the transplantation surgery can be quickly performed after arrival.

[0133] Therefore, the present invention further provides a method for preparing lymphocytes for transplantation, which includes diluting the composition containing lymphocytes of the present invention thawed after cryopreservation with a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions. Further, the present invention also provides a lymphocyte preparation for transplantation obtained by this method.

[0134] The lymphocyte preparation for transplantation thus obtained can be administered to a patient, such as a cancer patient, etc., by a known method per se, for example, by the administration methods, dosages and usages used in adoptive immunotherapy, donor lymphocyte infusion therapy, etc.

[0135] Examples are shown below to more specifically illustrate the present invention, but they are only illustrative and the present invention is not limited by them.

[0136] Examples

[0137] [Example 1] Production of NK cells derived from iPS cells

[0138] The iPS cell stock derived from human umbilical cord blood was thawed and inoculated into a flask. After culturing in an environment of 37°C and 5% CO2, the cells were recovered and inoculated into a three-dimensional culture device. After inoculation, perfusion culture was carried out through a membrane filtration filter to form 3D spheres of a certain size. Then, a differentiation induction medium containing addition factors such as BMP4 was added thereto, and 3D culture based on perfusion culture was continued for 12 - 17 days to induce HPC. Furthermore, for the obtained HPC, an NK differentiation induction medium (a medium obtained by adding FBS at a concentration of 5% to AIM V Serum Free Medium (Thermo Fisher scientific) and adding IL-15, IL-7, SCF and Flt3L so that the final concentration reaches 50 ng / mL) was used, inoculated into a three-dimensional culture device, and perfusion culture was carried out for about 40 days to produce a large number of NK cells with high biological activity.

[0139] [Example 2] Comparison of washing solutions in the lymphocyte washing step

[0140] (1) Preparation of NK cells

[0141] The NK cells obtained in Example 1 were centrifuged at 300×g for 5 minutes, and then the culture medium supernatant was removed for recovery. Furthermore, to reach 2.2×10 6Suspended in five physiological aqueous solutions [(1) normal saline (manufactured by Otsuka Pharmaceutical Factory, Inc.), (2) VeenF (Ringer's acetate solution; manufactured by Fuso Pharmaceutical Industries, Ltd.), (3) HESPANDER (Ringer's lactate solution with hydroxyethyl starch and glucose added; manufactured by Otsuka Pharmaceutical Factory, Inc.), (4) HBSS(+) (balanced salt solution), (5) HBSS(-) (balanced salt solution)] at a concentration of cells / mL (the electrolyte compositions of the respective physiological aqueous solutions are shown in Table 1). The NK cell suspension was placed in a 15 mL centrifuge tube and allowed to stand at room temperature. The standing treatment was set as a simulation of the operation time of the concentration and washing steps, with 0 hours set as the comparative example and 2 hours and 4 hours set as the examples.

[0142] [Table 1]

[0143]

[0144] As shown in Table 1, bicarbonate ions in the composition of the balanced salt solution HBSS are not included in "normal saline", "Ringer's acetate solution", and "Ringer's lactate solution with hydroxyethyl starch and glucose added".

[0145] (2) Cryopreservation of NK cell samples after standing treatment

[0146] After standing for 0 hours (immediately after suspension), 2 hours, and 4 hours, the NK cell suspension was centrifuged at 300×g for 5 minutes to remove the supernatant, and then resuspended in STEM-CELLBANKER (registered trademark) GMP grade (SCB, manufactured by Zenogen Pharma) at a concentration of 5.0×10 6 cells / mL. The cell suspension was aliquoted into cryovials and transferred to a BICELL (manufactured by Nippon Freezer Co., Ltd.) in a cryopreservation container and cryopreserved at -80°C.

[0147] (3) Measurement of cell recovery rate and cell viability after thawing and recovery culture

[0148] The NK cells were thawed and used in the medium used in the production of NK cells (a medium prepared by adding FBS at a concentration of 5% to AIM V Serum Free Medium (Thermo Fisher scientific) and adding IL-15, IL-7, SCF, and Flt3L at a final concentration of 50 ng / mL). The cells were re-inoculated into a 6-well plate at a concentration of 1.0×10 6 cells / mL and subjected to recovery culture in an incubator at 37°C and 5% CO2 for 4 days. Then, the density of live cells in the wells was measured, and the live cell recovery rate (refer to formula (I)) and cell viability were calculated.

[0149] Viable cell recovery rate = (Total number of viable cells after thawing and at the end of culture / Total number of viable cells cryopreserved) × 100 (I)

[0150] The results are shown in Table 2.

[0151] [Table 2]

[0152]

[0153] From the results in Table 2, it can be seen that in NK cells suspended in physiological saline, the viable cell recovery rate and cell survival rate of NK cells just after thawing and at the end of culture during the 4-hour static treatment were significantly lower compared with those at 0 hour (without static treatment) and 2-hour static treatment. It should be noted that in actual operation, the operation time for cell recovery and washing steps is set within about 2 hours, but the operation time until cryopreservation treatment after further adding cryopreservation solution is expected to be about 4 hours. When comparing the viable cell recovery rate and cell survival rate after the resuscitation culture of NK cells with 4-hour static treatment, it was confirmed that NK cells suspended in balanced salt solutions such as HBSS had higher viable cell recovery rate and cell survival rate compared with NK cells suspended in physiological saline.

[0154] Based on the above results, it can be considered that in the buffer replacement or washing step of the recovered suspension containing NK cells, a solution with bicarbonate ions is suitable for the recovery and maintenance of the survival rate of NK cells.

[0155] [Example 3] Comparison of washing solutions in the lymphocyte concentration and washing step

[0156] (1) Preparation and static treatment of NK cell suspension

[0157] The NK cells obtained in Example 1 were centrifuged at 300 × g for 5 minutes, and then the culture medium supernatant was removed for recovery. Further, the cells were suspended in 3 kinds of physiological aqueous solutions [(1) physiological saline, (2) Lactec (Ringer's lactate solution; manufactured by Otsuka Pharmaceutical Factory, Inc.), (3) Bicanate (Ringer's bicarbonate solution; manufactured by Otsuka Pharmaceutical Factory, Inc.)] to reach 2.2 × 10 6 cells / mL (the electrolyte compositions of each physiological aqueous solution are shown in Table 3). The NK cell suspension was added to a 15 mL centrifuge tube and left standing at room temperature. The static treatment was set as a simulation of the operation time of the concentration and washing step, with 0 hour set as the comparative example and 2 hours and 4 hours set as the examples.

[0158] As shown in Table 3, Ringer's bicarbonate solution Bicanate contains bicarbonate ions.

[0159] [Table 3]

[0160]

[0161] (2) Cryopreservation of NK cell samples after static treatment

[0162] After standing for 0 hours (just after resuspension), 2 hours, and 4 hours, the NK cell suspension was centrifuged at 300×g for 5 minutes to remove the supernatant, and then resuspended in STEM-CELLBANKER (registered trademark) GMP grade (SCB) at a density of 5.0×10 6 cells / mL. The cell suspension was aliquoted into cryovials, transferred to the BICELL of a cryopreservation container, and cryopreserved at -80°C.

[0163] (3) Determination of viable cell recovery rate and cell viability after thawing and recovery culture

[0164] The NK cells were thawed and cultured in the medium used in the production of NK cells in Example 1 (a medium prepared by adding FBS at a concentration of 5% to AIM V SerumFree Medium (Thermo Fisher scientific) and adding IL-15, IL-7, SCF, and Flt3L to a final concentration of 50 ng / mL) at a density of 1.0×10 6 cells / mL and then re-seeded in a 6-well plate. The cells were cultured in an incubator at 37°C and 5% CO2 for 3 days for recovery culture. Then, the viable cell density in the wells was measured, and the viable cell recovery rate (refer to formula (I)) and cell viability were calculated. The results are shown in Table 4.

[0165] [Table 4]

[0166]

[0167] From the results shown in Table 4, it can be seen that in NK cells suspended in physiological saline, the viable cell recovery rate and cell viability of NK cells just after thawing and after the end of culture during the 4-hour static treatment were significantly lower than those in the 0-hour (no static treatment) and 2-hour static treatment groups.

[0168] When comparing the viable cell recovery rate and cell viability after the end of recovery culture in the case of 4-hour static treatment, it was confirmed that NK cells suspended in bicarbonate Ringer's solution Bicanate had higher viable cell recovery rate and cell viability than NK cells suspended in physiological saline.

[0169] As can be seen from the above results, compared with normal saline without a buffering effect and lactated Ringer's solution without bicarbonate ions, the viable cell recovery rate and survival rate of NK cells were optimized by replacing or washing the buffer of the NK cell suspension with a bicarbonate Ringer's solution having a buffering effect and containing bicarbonate ions.

[0170] [Example 4] Cryopreservation solution for lymphocytes

[0171] (1) Mixing and freezing of NK cell suspension and cryopreservation solution

[0172] The culture solution containing NK cells obtained in Example 1 was centrifuged at 300×g for 5 minutes, and then the culture medium supernatant was removed. The precipitate of NK cells was dispersed by shaking, suspended in normal saline or bicarbonate Ringer's solution, and then the cryopreservation solution with the compositions shown in Table 5 or Table 6 was added and mixed well. After adding the cryopreservation solution, each cell suspension was aliquoted into cryovials, transferred to a cryopreservation container such as Mr.Frosty (manufactured by Nalgene) or BICELL, and frozen at -80°C. In order to compare the performance of the cryopreservation solutions, cells suspended in STEM-CELLBANKER (registered trademark) GMP grade (SCB) were prepared and cryopreserved as control samples.

[0173] [Table 5]

[0174] Composition of cryopreservation solution containing normal saline and viable cell recovery rate (relative value to SCB)

[0175]

[0176] Relative value to SCB: (Viable cell recovery rate of each sample / Viable cell recovery rate of SCB) × 100

[0177] [Table 6]

[0178] Composition of cryopreservation solution containing bicarbonate Ringer's solution and viable cell recovery rate (relative value to SCB)

[0179]

[0180] Relative value to SCB: (Viable cell recovery rate of each sample / Viable cell recovery rate of SCB) × 100

[0181] (2) Thawing and resuscitation culture of cryopreserved NK cells

[0182] After thawing the cryopreserved NK cells obtained in the above step (1), they were suspended in the medium used in the production of NK cells in Example 1 (a medium prepared by adding FBS at a concentration of 5% to AIMV SerumFree Medium (Thermo Fisher scientific) and adding IL-15, IL-7, SCF, and Flt3L to a final concentration of 50 ng / mL) to reach 1.0×10 6 cells / mL and then re-seeded into a 12-well plate. Subsequently, recovery culture was carried out in an incubator under the conditions of 37°C and 5% CO2 for 3 days. Then, the viable cell density and cell viability in the wells were measured, and the viable cell recovery rate of each cryopreservation solution (refer to formula (I)) was calculated as a relative value (SCB relative value) with respect to the viable cell recovery rate of the cells cryopreserved using SCB.

[0183] The results using the cryopreservation solution containing physiological saline are shown in Table 5 (SCB relative value on Day 3 at the end of culture), and the results using the cryopreservation solution containing bicarbonate Ringer's solution are shown in Table 6 (SCB relative value on Day 3 at the end of culture).

[0184] (3) Results

[0185] (3-1) Case of cryopreservation solution containing physiological saline

[0186] When cryopreserving the NK cells prepared in Example 1 using the cryopreservation solution containing physiological saline, compared with the existing cryopreservation solutions SCB and CryoStor CS10 (CS10; manufactured by Charles River Laboratories CellSolutions, Inc.), multiple compositions with significantly higher viable cell recovery rates after thawing can be obtained. The preferred composition is physiological saline: 32.7 - 40.8% (v / v), Dextran 40 (Dex40; manufactured by Meito Sangyo Co., Ltd.): 8.06 - 9.68% (w / v), Glucose (manufactured by Terumo Corporation): 0.045 - 0.675% (w / v), CryoSure DMSO (DMSO; manufactured by WAK-Chemie Medical GmbH): 6.0 - 10.0% (v / v), human serum albumin; (HuSA; manufactured by Japan Blood Products Organization): 1.75 - 2.75% (w / v). (Table 5)

[0187] (3-2) Case of cryopreservation solution containing bicarbonate Ringer's solution

[0188] When cryopreserving the NK cells prepared in Example 1 using a cryopreservation solution containing bicarbonate Ringer's solution, a composition with a significantly higher viable cell recovery rate after thawing can be obtained compared to the existing cryopreservation solution SCB. The preferred composition is bicarbonate Ringer's solution: 44.0 - 66.1% (v / v), Dextran40: 3.60 - 7.44% (w / v), Glucose: 0.09 - 0.25% (w / v), DMSO: 8.5 - 11.0% (v / v), HuSA: 1.875 - 3.375% (w / v).

[0189] (Table 6)

[0190] [Example 5] Study on cryopreservation solution in the time stability of NK cells

[0191] (1) Mixing and freezing operations of NK cell suspension and cryopreservation solution

[0192] The culture solution containing NK cells prepared in Example 1 was centrifuged at 300×g for 5 minutes, and then the culture medium supernatant was removed. The precipitate of NK cells was dissolved by shaking, suspended in physiological saline or bicarbonate Ringer's solution, and then the cryopreservation solution with the compositions shown in Table 7 was added and mixed well. The cell suspension added with the cryopreservation solution was aliquoted into cryogenic containers, allowed to stand at room temperature for the target time (1, 2, 4 hours), and then transferred to cryopreservation containers (Mr.Frosty or BICELL) and frozen at -80°C.

[0193] (2) Thawing and resuscitation culture of cryopreserved NK cells

[0194] After thawing the NK cells, the medium for NK cell expansion culture in Example 1 was used to re-inoculate into a 12-well plate at a density of 1.0×10 6 cells / mL, and resuscitation culture was carried out in an incubator at 37°C and 5% CO2 for 3 days. Then, the viable cell density and cell viability in the wells were measured, and the viable cell recovery rate was calculated in the form of the relative value of SCB (refer to Equation (I)). The results are shown in Table 7 (relative value of SCB at the end of culture Day3).

[0195] [Table 7]

[0196] Table 7 Composition of cryopreservation solution and stability at room temperature standing

[0197]

[0198] Relative value of SCB: (Viable cell recovery rate of each sample / Viable cell recovery rate of SCB) × 100

[0199] Regarding the reduction in the time-dependent viable cell recovery rate during 0 h to 4 h of standing at room temperature after mixing with the cryopreservation solution, when cryopreserving using a cryopreservation solution containing bicarbonate Ringer's solution, a significantly higher viable cell recovery rate can be maintained compared to the existing cryopreservation solution SCB. In particular, the preferred composition is bicarbonate Ringer's solution: 43.2 - 66.10% (v / v), Dextran40: 3.60 - 10.06% (w / v), Glucose: 0.05 - 0.41% (w / v), DMSO: 6.0 - 11.0% (v / v), HuSA: 1.50 - 2.95% (w / v). (Table 7)

[0200] [Example 6] Diluent after Freezing and Thawing of NK Cells

[0201] After thawing the NK cells, 0.1 mL of the cell suspension was diluted and suspended in 10-fold amounts of each physiological aqueous solution, and the stability after standing at room temperature for 1, 2, and 4 hours was evaluated. As a control for the physiological aqueous solution, the medium for resuscitation culture was used. After the standing cell suspension was centrifuged at 300×g for 5 minutes to remove the supernatant, it was resuspended in 2 mL of the medium for NK cell expansion culture in Example 1, 1.9 mL was inoculated into a 12-well plate, and resuscitation culture was carried out in an incubator under the conditions of 37°C and 5% CO2 for 3 days. Then, the viable cell density in the well was measured, and the number of viable cells was counted. As the physiological aqueous solutions, normal saline (manufactured by Otsuka Pharmaceutical Factory, Inc.), Veen D (acetate Ringer's solution; manufactured by Fuso Pharmaceutical Industries, Ltd.), Veen F (acetate Ringer's solution; manufactured by Fuso Pharmaceutical Industries, Ltd.), Bicanate (bicarbonate Ringer's solution; manufactured by Otsuka Pharmaceutical Factory, Inc.), and Bicarbon (bicarbonate Ringer's solution; manufactured by AY Pharma Co., Ltd.) were used for the study (the compositions of each physiological aqueous solution are shown in Table 8).

[0202] [Table 8]

[0203] Composition (%) Normal saline Veen F Veen D Bicanate Bicarbon Glucose - 5 - - Sodium chloride 0.9 0.6 0.6 0.584 0.614 Potassium chloride 0.03 0.03 0.03 0.03 Calcium chloride hydrate - 0.02 0.02 0.022 0.022 Sodium acetate hydrate 0.38 0.38 - - Magnesium chloride - 0.02 0.0102 Sodium bicarbonate - - - 0.235 0.21 Sodium citrate - - - 0.02 0.049 Hydrochloric acid - - - Carbon dioxide - - - -

[0204] The results of the experiments using normal saline, Veen F, and Veen D are shown in Table 9, the results of the experiments using normal saline and Bicanate are shown in Table 10, and the results of the experiments using normal saline, Bicanate, and Bicarbon are shown in Table 11.

[0205] [Table 9]

[0206]

[0207] [Table 10]

[0208]

[0209] [Table 11]

[0210]

[0211] As shown in Tables 9 - 11, after thawing cryopreserved NK cells by the method of Example 4, diluting and suspending them 10 - fold in a physiological aqueous solution and allowing them to stand, the reduction in the recovery of viable cell count over time was evaluated. As a result, when using Bicarbonate Ringer's solution Bicanate and Bicarbon, significantly higher viable cell count recoveries were obtained compared to the cases using physiological saline, Veen D, and Veen F. This indicates that Bicarbonate Ringer's solution, as a diluent for administering NK cell cryopreparations to patients, has the effect of maintaining a high viable cell count.

[0212] Industrial Applicability

[0213] According to the washing method and cryopreservation method of the present invention, transplantable lymphocytes (e.g., NK cells, T cells, etc.) that maintain a high cell recovery rate and survival rate and high physiological activity even after freezing and thawing can be produced. In addition, through the composition containing lymphocytes and the transplant diluent of the present invention, a lymphocyte preparation that can maintain a high cell survival rate even when stored at room temperature from thawing until transplantation and is more suitable for transplantation can be provided. Therefore, the present invention is extremely useful in lymphocyte - based cellular immunotherapy represented by cancer immunotherapy.

[0214] (Cross - reference to related applications)

[0215] This application is based on Japanese Patent Application No. 2022 - 185201 filed in Japan on November 18, 2022, the entire contents of which are incorporated herein by reference.

Claims

1. A method for washing lymphocytes, comprising washing lymphocytes with a physiological aqueous solution containing bicarbonate ions.

2. The method according to claim 1, wherein, The lymphocytes are T cells or NK cells.

3. The method according to claim 1, wherein The physiological aqueous solution containing bicarbonate ions is bicarbonate Ringer's solution.

4. A method for cryopreserving lymphocytes, comprising suspending and freezing the lymphocytes treated by the method according to claim 1 in a cryopreservation solution containing a physiological aqueous solution.

5. The method according to claim 4, wherein, The physiological aqueous solution is a physiological aqueous solution containing bicarbonate ions.

6. The method according to claim 5, wherein, The physiological aqueous solution containing bicarbonate ions is bicarbonate Ringer's solution.

7. A washing solution for lymphocytes, comprising a physiological aqueous solution containing bicarbonate ions.

8. A cryopreservation solution for lymphocytes, comprising a physiological aqueous solution containing bicarbonate ions, dextran, glucose, DMSO, and human serum albumin.

9. A composition containing lymphocytes, comprising lymphocytes treated with a physiological aqueous solution containing bicarbonate ions and the cryopreservation solution according to claim 8.

10. A kit for preparing lymphocytes for transplantation, comprising: the composition containing lymphocytes according to claim 9 and a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions.

11. A transplantation diluent, which is a transplantation diluent for the composition containing lymphocytes according to claim 9 and contains a physiological aqueous solution containing bicarbonate ions.

12. Method for preparing lymphocytes for transplantation, comprising: The composition containing lymphocytes according to claim 9, thawed after cryopreservation, is diluted with a transplantation diluent containing a physiological aqueous solution containing bicarbonate ions.

13. A lymphocyte preparation for transplantation, obtained by the method according to claim 12.

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