A dmsO-free cell cryopreservation solution based on injection grade protection system, methods of use, related products and applications

Abstract

The application discloses a DMSO-free cell cryopreservation solution based on an injection-grade protection system, a use method, related products and application, and belongs to the technical field of immune cell storage. The technical problem to be solved is to provide a cell cryopreservation solution which does not use DMSO and animal-derived components and has higher cell survival rate and higher gamma delta T cell ratio after recovery. The technical solution points are as follows: the cell cryopreservation solution comprises the following components: a colloidal protection component, a cold stabilization component, an antioxidant component, an energy and basic osmotic balance component, an extracellular electrolyte homeostasis regulation component and a transmembrane osmotic pressure regulation component; the osmotic pressure of the cell cryopreservation solution is 280-380 mOsm / kg, the pH of the cell cryopreservation solution is 7.0-7.4; the cell cryopreservation solution does not contain DMSO, animal serum and animal-derived substances. The cell cryopreservation solution is suitable for the storage and transportation of gamma delta T cell treatment products, the storage of an immune cell bank, the cryopreservation of cell treatment before return and the like.

Description

Technical Field

[0001] This invention belongs to the field of immune cell storage technology, specifically relating to a DMSO-free cell cryopreservation solution based on an injection-grade protection system, its usage method, related products and applications. Background Technology

[0002] For understanding the technical content of this invention:

[0003] T lymphocytes (γδ T lymphocytes) are a subset of immune cells with unique biological characteristics, possessing both innate and adaptive immunity. They exhibit core functions such as recognition of non-MHC (Major Histocompatibility Complex) antigens, rapid activation of responses, broad-spectrum anti-tumor killing ability, cytokine release to regulate the immune microenvironment, anti-infection, and immune surveillance. Compared to traditional αβ T cells, γδ T lymphocytes can recognize target cells without antigen presentation. They exert their cytotoxic function directly through receptor-mediated pathways such as NKG2D, TCR-γδ, and CD16, and participate in immune regulation by secreting cytokines such as IFN-γ, TNF-α, and IL-17. Therefore, γδ T lymphocytes show significant clinical application potential in the treatment of solid tumors, hematologic malignancies, viral infection clearance, regulation of autoimmune diseases, and postoperative immune reconstitution. Currently, cell-based immunotherapy technologies are rapidly developing and gradually moving towards standardized preparation, large-scale production, and clinical application. However, cells are highly sensitive to cold damage, osmotic stress, oxidative stress, membrane structure damage, and mitochondrial function impairment during storage and transportation, leading to problems such as decreased viability, functional decline, and weakened amplification capacity after cryopreservation and thawing.

[0004] Currently, most cryopreservation protocols used in clinical practice and research are based on systems containing DMSO and fetal bovine serum (FBS). While DMSO, as a permeabilizing cryoprotectant, has some anti-ice crystal formation effect, it exhibits significant cytotoxicity and must be washed away after thawing, increasing operational complexity and contamination risk, and can trigger adverse clinical infusion reactions (such as hypotension, hepatotoxicity, vascular irritation, and allergic reactions). FBS, as a heterologous animal component, carries risks of exogenous protein immunogenicity, large batch-to-batch variability, and viral or mycoplasma contamination, failing to meet clinical GMP requirements and xeno-free cell preparation requirements. Therefore, constructing a cell cryopreservation system with clearly defined components, clinical re-infusion capability, no animal origin, no washing required, DMSO-free, and compatible with programmed cooling is an urgent need in the field of immune cell preparation storage.

[0005] Relevant patent documents retrieved: This document, published in China (CN107148967A) on September 12, 2017, discloses an antigen-specific T lymphocyte cryopreservation solution, comprising cryopreservation solution A and cryopreservation solution B. Cryopreservation solution A comprises the following components in volume fractions: 30%-40% of Peptide electrolyte injection, 30%-40% of glucose sodium chloride injection, 5%-15% of dextran glucose injection, and 15%-25% of human serum albumin solution. Cryopreservation solution B comprises the following components in volume fractions: 20%-30% of Peptide electrolyte injection, 20%-30% of glucose sodium chloride injection, 5%-15% of dextran glucose injection, 15%-25% of human serum albumin solution, and 10%-20% of dimethyl sulfoxide. Cryopreservation solutions A and B are stored separately, and when used, the volume ratio of cryopreservation solution A to cryopreservation solution B is 1:1. A mixture of these components at a ratio of 0.5–2 is used to form an antigen-specific T lymphocyte cryopreservation solution. This cryopreservation solution can reduce intracellular crystal formation, increase cell viability, and maintain the tumor-killing function of cells. This technology also provides a method for preparing and applying the cryopreservation solution, as well as an antigen-specific T lymphocyte injection solution.

[0006] Relevant non-patent literature retrieved: The journal or book title is "Biotechnology Bulletin," and the article title is "The Influence of Cryopreservation Density on the Cryopreservation Effect of Peripheral Blood Mononuclear Cells," 2019, 35(6): 119-124, published in June 2019. This article discloses a CIK cell cryopreservation solution of autologous plasma:DMSO=9:1. This technique requires the use of a -196℃ gas phase tank to preserve T cells. Summary of the Invention

[0007] The purpose of this invention is to provide: A DMSO-free cell cryopreservation solution based on an injection-grade protection system, and related technologies, to solve the following technical problems or a combination thereof: (1) Reliance on DMSO, animal serum and / or animal serum products leads to risks of reinfusion toxicity, allergic reactions, complex clinical cleaning procedures, and high operational risks; (2) Cells are prone to oxidative damage, membrane rupture, mechanical damage from ice crystals, and osmotic shock during the freezing and thawing process; (3) The ingredients in the formula are from complex sources and the quality of the ingredients is unstable, making it difficult to meet GMP and clinical injection grade standards; (4) The cryopreservation system cannot be well adapted to programmed cooling, resulting in insufficient survival rate and functional retention rate of immune cells after thawing.

[0008] Terminology Explanation Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of standard terms in the relevant field can be found in the following references: General Chemistry (China Agricultural University Press, September 1, 2005, edited by Ding Lijun et al.)

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as aseptic techniques and filtration, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] As used in this article, “cell cryopreservation solution” refers to a solution used for long-term preservation of live cells at low temperatures (typically -196°C liquid nitrogen or -80°C freezer), which typically contains basal culture medium, serum or serum substitutes, and cryoprotectants (such as dimethyl sulfoxide or glycerol) to prevent ice crystal formation from damaging the cells.

[0015] The term "osmotic pressure (of human tissues)" as used in this article refers to the total osmotic concentration of solute particles such as sodium ions, glucose, and proteins contained in the extracellular fluid of human tissues (such as plasma and tissue fluid), usually expressed in milliosmol / L (mOsm / L). Normal human plasma osmotic pressure is approximately 280-310 mOsm / L, primarily maintaining the dynamic balance of water inside and outside cells. When tissue osmotic pressure changes, it can lead to cell dehydration (shrinkage) or water absorption (swelling or even rupture), thereby affecting tissue function.

[0016] The term "weight-volume concentration (of a solution)" as used in this article refers to the percentage of the weight (in grams) of a substance in a solution to the total volume (in milliliters) of the solution. For example, if 1 g of substance A is dissolved in 100 mL of a solution, then the weight-volume concentration of substance A is 1%.

[0017] In a first aspect, the present invention provides a cell cryopreservation solution comprising the following components: Colloidal protective components, cold-stabilizing components, antioxidant components, energy and basic osmotic balance components, extracellular electrolyte homeostasis regulating components, and transmembrane osmotic pressure regulating components; The osmotic pressure of the cell cryopreservation solution is 280-380 mOsm / kg, and the pH of the cell cryopreservation solution is 7.0-7.4. The cell cryopreservation solution does not contain DMSO, animal serum, or substances made from animal serum.

[0018] The colloidal protective components include, but are not limited to, plant-derived recombinant proteins, plant-derived human serum proteins, bacterial-derived recombinant proteins, bacterial-derived human serum proteins, fungal-derived recombinant proteins, and fungal-derived human serum proteins.

[0019] The cold-stable components include, but are not limited to, dextran-39, dextran-40, and dextran-41.

[0020] The antioxidant components include, but are not limited to, N-acetylcysteine, N-acetylcysteine ​​hydrochloride, N-acetylcysteine ​​acetate, and N-acetylcysteine ​​potassium salt. 14 N-acetylcysteine, etc.

[0021] The energy and basic osmotic balance components include, but are not limited to, glucose and hydrated glucose.

[0022] The extracellular electrolyte homeostasis regulating components include, but are not limited to, sodium ions, potassium ions, calcium ions, magnesium ions, lactate ions, acetate ions, bicarbonate ions, chloride ions, hydrogen phosphate ions, dihydrogen phosphate ions, etc., mixed in any proportion to form electrically neutral salts or mixed salts.

[0023] The transmembrane osmotic pressure regulating components include, but are not limited to, mannitol and hydrated mannitol.

[0024] The osmotic pressure is selected from any value within the range of 280-380 mOsm / kg, such as the following values ​​or any sub-range between them: 280 mOsm / kg, 290 mOsm / kg, 300 mOsm / kg, 310 mOsm / kg, 320 mOsm / kg, 330 mOsm / kg, 340 mOsm / kg, 350 mOsm / kg, 360 mOsm / kg, 370 mOsm / kg, and 380 mOsm / kg.

[0025] The pH value is selected from any value in the range of 7.0-7.4, such as the following values ​​or any sub-range between the two: 7.0, 7.1, 7.2, 7.3, 7.4.

[0026] The substances made from animal serum include, but are not limited to: concentrated animal serum, solids formed after drying animal serum, mixtures containing animal serum (including diluted animal serum), and substances obtained by mixing serums from different animals.

[0027] Further, in the cell cryopreservation solution, the colloidal protective component is plant-derived recombinant human serum albumin; the cold-stabilizing component is dextran-40; the antioxidant component is N-acetylcysteine; the energy and basal osmotic balance component is glucose; the transmembrane osmotic pressure regulating component is mannitol; the mass-volume concentration of the colloidal protective component in the cell cryopreservation solution is 5%-20%; the mass-volume concentration of the cold-stabilizing component in the cell cryopreservation solution is 2%-10%; the molar concentration of the antioxidant component in the cell cryopreservation solution is 1-20 mmol / L; the molar concentration of the energy and basal osmotic balance component in the cell cryopreservation solution is 5-20 mmol / L; and the molar concentration of the transmembrane osmotic pressure regulating component in the cell cryopreservation solution is 10-50 mmol / L. The extracellular electrolyte homeostasis regulating component is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, sodium acetate, potassium acetate, calcium acetate, and magnesium acetate.

[0028] The plant-derived recombinant human serum albumin refers to recombinant human serum albumin obtained by expression and purification through a plant cell expression platform. As a specific embodiment of the present invention, the plant-derived recombinant human serum albumin is OsrHSA (Recombinant Human Serum Albumin from Oryza sativa). Replacing all or part of the colloidal protective component with other non-animal-derived recombinant human serum albumin with equivalent functions (such as bacterial-expressed recombinant human serum albumin, fungal-expressed recombinant human serum albumin, etc.) is also considered within the scope of protection of this invention.

[0029] The cold-stabilizing component is dextran-40. Replacing all or part of dextran-40 with other substances having equivalent functions (such as dextran-40 hydrate, dextran-39, etc.) is also considered within the scope of protection of this invention.

[0030] The antioxidant component is N-acetylcysteine. Replacing all or part of N-acetylcysteine ​​with other substances having equivalent functions (such as N-acetylcysteine ​​labeled with a specific isotope, sodium N-acetylcysteine, N-acetylcysteine ​​hydrochloride, etc.) is also considered within the scope of protection of this invention.

[0031] The energy and basic osmotic balance component is glucose. Replacing all or part of the glucose with other substances with equivalent functions (such as glucose labeled with a specific isotope, glucose monohydrate, etc.) is also considered within the scope of protection of this invention.

[0032] The transmembrane osmotic pressure regulating component is mannitol. Replacing all or part of the mannitol with other substances having equivalent functions (such as mannitol labeled with a specific isotope, mannitol hydrate, etc.) is also considered within the scope of protection of this invention.

[0033] The colloidal protective component in the cell cryopreservation solution has a mass-volume concentration of 5%-20%, such as the following concentrations or any range between the two: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%; The cold-stable component has a mass-volume concentration of 2%-10% in the cell cryopreservation solution, for example, the following concentrations or any range between the two: 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; The molar concentration of the antioxidant component in the cell cryopreservation solution is any concentration between 1 and 20 mmol / L, such as the following concentrations or any range between the two: 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L; The molar concentration of energy and basic osmotic balance components in the cell cryopreservation solution is any concentration between 5 and 20 mmol / L, such as the following concentrations or any range between the two: 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L; The transmembrane osmotic pressure regulating component in the cell cryopreservation solution has any concentration within the range of 10-50 mmol / L, such as the following concentrations or any range between two: 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 2 3mmol / L, 24mmol / L, 25mmol / L, 26mmol / L, 27mmol / L, 28mmol / L, 29mmol / L, 30mmol / L, 31mmol / L, 32mmol / L, 33mmol / L, 34mmol / L, 35mmol / L, 36mmol / L, 37mmol / L, 38mmol / L, 39mmol / L, 40mmol / L, 41mmol / L, 42mmol / L, 43mmol / L, 44mmol / L, 45mmol / L, 46mmol / L, 47mmol / L, 48mmol / L, 49mmol / L, 50mmol / L.

[0034] The extracellular electrolyte homeostasis regulating component is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, sodium acetate, potassium acetate, calcium acetate, and magnesium acetate. The sources of the above substances include, but are not limited to, isotonic injectable solutions such as sodium chloride injection, potassium chloride injection, sodium bicarbonate injection, lactated Ringer's solution, acetate Ringer's solution, and phosphate buffer solution.

[0035] In a second aspect, the present invention provides a cell cryopreservation solution composition, the cell cryopreservation solution composition being used to prepare the above-mentioned cell cryopreservation solution; The cell cryopreservation solution composition comprises the following components: Colloidal protective components, cold-stabilizing components, antioxidant components, energy and basic osmotic balance components, extracellular electrolyte homeostasis regulating components, and transmembrane osmotic pressure regulating components; The colloidal protective component is plant-derived recombinant human serum albumin, the cold-stabilizing component is dextran-40, the antioxidant component is N-acetylcysteine, the energy and basal osmotic balance component is glucose, and the transmembrane osmotic pressure regulating component is mannitol. The weight ratio of the colloidal protective component, cold-stabilizing component, antioxidant component, energy and basic osmotic balance component, and transmembrane osmotic pressure regulating component is 50-200:20-100:0.221-4.42:0.9-3.6:1.82-9.1. The extracellular electrolyte homeostasis regulating component enables the cell cryopreservation solution made from the cell cryopreservation solution composition to have an osmotic pressure of 280-380 mOsm / kg and a pH of 7.0-7.4.

[0036] Thirdly, the present invention provides a method for preparing cell cryopreservation solution, comprising the following steps: mixing the above-mentioned cryopreservation solution composition with water, and adjusting the pH and osmotic pressure.

[0037] Fourthly, the present invention provides a cell mixture comprising: (1) Cell; (2) The cell cryopreservation solution mentioned above.

[0038] Preferably, the cell is a T cell.

[0039] The T cells can be classified in different ways as follows: naive T cells, effector T cells and memory T cells; αβ T cells and γδ T cells; regulatory T cells, follicular helper T cells, natural killer T cells, mucosa-associated inertial T cells, killer-like inhibitory receptor positive T cells, etc.

[0040] In one specific embodiment of the present invention, the T cell is a γδT cell.

[0041] Fifthly, the present invention provides a cell cryopreservation kit, comprising the cell cryopreservation solution described above or a combination of the cell cryopreservation solutions described above.

[0042] Sixthly, the present invention provides a method for cell cryopreservation, comprising the following steps: Cells are placed in a cell cryopreservation solution, and the temperature is controlled to obtain cryopreserved cells; The cell cryopreservation solution is the cell cryopreservation solution described above.

[0043] In a seventh aspect, the present invention provides a cell resuscitation method, comprising the following steps: The frozen cells are warmed to 35-40°C; the frozen cells include cells and the cell cryopreservation solution described above.

[0044] Eighthly, the present invention provides the application of the above-described cell cryopreservation solution, cryopreservation solution composition, cell mixture, cell cryopreservation kit, cell cryopreservation method or cell resuscitation method in cell storage.

[0045] In a ninth aspect, the present invention provides the use of the above-described cell mixture in the preparation of medicaments for antitumor, anti-infection, and / or treatment of immune disorders.

[0046] In a tenth aspect, the present invention provides a medicine comprising the above-described cell mixture.

[0047] In one aspect, the present invention provides a cell kit comprising the cell mixture described above.

[0048] Compared with the prior art, the advantages of this invention include: 1. Establish a cryopreservation system for γδT cells based on pure injectable raw materials, achieving DMSO-free, serum-free, animal-derived, and clinically recombinant cell-free reinfusion; 2. Through a five-fold synergistic mechanism of colloidal protection, macromolecular cold stabilization, anti-oxidation, osmotic regulation, and electrolyte homeostasis, the system covers the main damage pathways in cell cryopreservation; 3. Highly compatible with programmed cooling processes, significantly reducing the risk of intracellular ice crystal formation and rewarming lysis; 4. Cell survival rate after resuscitation ≥90%, and retention rate of cytotoxicity, proliferation capacity, and cytokine expression ≥75%; 5. The components are clearly defined and safe, the batches are controllable, and the process can be standardized, making it suitable for GMP-scale production and clinical-grade cell storage; 6. It can be used for clinical reinfusion without washing after resuscitation, significantly improving the accessibility and standardization of cell therapy products. Detailed Implementation

[0049] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0050] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0051] In the following specific embodiments, some of the raw materials used are sourced from the following sources: OsrHSA: Yisheng Biotechnology, catalog number 20901ES10.

[0052] Dextran-40: BN26120.

[0053] N-acetylcysteine: Adamas Life Sciences, catalog number 70601K.

[0054] Lactated Ringer's solution: Source leaf, catalog number R23019, sterile.

[0055] Mannitol (all mannitol used in the following specific embodiments is D-mannitol): Sangon Biotech, product number A600335-0500.

[0056] Fetal bovine serum: Gibco, catalog number A5256701.

[0057] Dextran-70: Bairuiji, catalog number BN26123.

[0058] Glycerin: Adamas Life Sciences, catalog number 66258K.

[0059] Example 1 The formulations of cell cryopreservation solutions, formulations 1-5, are shown in Table 1 below.

[0060] Table 1

[0061] The preparation method of the above-mentioned cell cryopreservation solution is as follows: Under aseptic conditions, weigh out OsrHSA, dextran 40, N-acetylcysteine, glucose, and mannitol. Mix them with half the volume of lactated Ringer's solution and ensure complete dissolution. Measure the pH of the solution. If the pH is below 7.0, add 20% potassium hydroxide solution dropwise until the pH reaches 7.5. Then measure the osmotic pressure. If the osmotic pressure exceeds 380 mOsm / kg, add water until the osmotic pressure is ≥280 mOsm / kg, and measure the pH again. If the pH is still below 7.0, carefully add 20% potassium hydroxide solution dropwise until the pH reaches 7.4, then bring the solution to volume with lactated Ringer's solution. If the osmotic pressure is below 280 mOsm / kg, add water until the osmotic pressure is ≤380 mOsm / kg, and measure the pH again. If the pH value is greater than 7.4 at this point, continue adding lactated Ringer's solution until the pH value is 7.4, and then make up to volume with water for injection.

[0062] The cell cryopreservation solution in Formula 6 is the same as that in Formula 1, except that OsrHSA is replaced with the same amount of fetal bovine serum (the preparation method is to weigh the liquid fetal bovine serum in the formula and mix them, and determine the volume of the entire mixture by adjusting the volume so that the final weight-volume concentration of fetal bovine serum in the entire solution is 11%).

[0063] The cell cryopreservation solution in Formula 7 is the same as that in Formula 1, except that dextran-40 is replaced with the same weight of dextran-70.

[0064] The cell cryopreservation solution in Formula 8 is the same as that in Formula 1, except that mannitol is replaced with glycerol of the same molar concentration.

[0065] The cell cryopreservation solution of Formula 9 differs from that of Formula 1 only in that the weight-volume concentration of OsrHSA is adjusted to 25%, the weight-volume concentration of dextran-40 is adjusted to 1%, the molar concentration of glucose is adjusted to 30 mM, and the molar concentration of mannitol is adjusted to 8 mM. All other aspects are the same.

[0066] The preparation methods for the cell cryopreservation solutions in Formulas 6-9 are similar to those in Formula 1. Their osmotic pressure and pH values ​​are shown in Table 2 below: Table 2

[0067] The cell cryopreservation solution of Formula 10 is the same as that of Formula 1, except that potassium hydroxide solution is added to adjust the pH to 7.7.

[0068] The cell cryopreservation solution of Formula 11 is the cell cryopreservation solution of Example 1 in Chinese Invention Patent Application CN107148967A.

[0069] Example 2 A cell cryopreservation method, comprising the following steps: S1 cells, γδT cells or immune cells containing γδT cells are mixed with the cryopreservation solution at a volume ratio of 1:5 and dispensed into 50mL cryopreservation bags. S2. Cool the dispensed cryopreservation bags to ≤-80℃ at a rate of -1℃ / min; wherein, cooling in the temperature range of -5℃ to -40℃ is linear cooling.

[0070] S3. Transfer to liquid nitrogen for long-term storage.

[0071] To revive the cells, simply remove the cryopreservation bag from the liquid nitrogen and place it directly in sterile water at 37°C to warm it.

[0072] Effect evaluation 1. Evaluation of the cryopreservation and recovery rate of γδT cells under different conditions.

[0073] 1.1 Culture of γδT cells Collect 100 mL of anticoagulated venous blood from a healthy blood donor. Centrifuge at 1500 rpm for 15 min. Add approximately 10 mL of the leukocyte layer to the lymphocyte separation medium and centrifuge at 2000 rpm for 20 min. Collect the PBMC layer, wash with physiological saline, centrifuge at 1500 rpm for 10 min, repeat the washing three times, and count the cells for later use. Add the PBMCs to the culture medium (RPMI 1640 + 10% fetal bovine serum + 5% human AB serum) and adjust the cell count to 1 × 10⁻⁶ cells / mL. 8 / L, place in a 250ml cell culture flask, and culture in a 37℃, 5%CO2 cell culture incubator. Add culture medium as needed based on cell growth.

[0074] PBMCs can be observed to adhere to the culture medium after 24 hours of culture, and large colonies can be seen after 10 days of culture.

[0075] 1.2 Counting of γδT cells Cells cultured for 10 days were collected and centrifuged at 2000 rpm for 20 min to collect cells, and cell counts were performed. γδT cells were stained (FITC anti-human TCR γ / δ, catalog number 331208, BioLegend). The γδT cell ratio was detected using flow cytometry, and this ratio was multiplied by the total cell count to calculate the total number of γδT cells (resulting in A1 cells).

[0076] 1.3. Cell cryopreservation Take the culture medium of formulations 1-11 in Example 1, mix the cells collected after 10 days of culture with the cryopreservation solution according to the method in Example 2, and freeze them in liquid nitrogen.

[0077] 1.4 Cell resuscitation Cells frozen for a specific period of time can be thawed by removing the cryopreservation bag from liquid nitrogen and placing it directly in sterile water at 37°C for warming.

[0078] 1.5 Count of γδT cells after resuscitation 1.5.1 Cell live / dead staining The revived cells were stained using a cell liveness / death staining kit (Calcein-AM / PI, Beyotime, C1371S), and the percentage of surviving cells was detected using flow cytometry.

[0079] Cell viability = Number of cells that only emit green fluorescence and not red fluorescence ÷ Total number of cells detected × 100% 1.5.2 Detection of γδT cell survival rate Take 50% of the total frozen cell volume, centrifuge at 1500 rpm for 10 min, wash 3 times, count the cells, and set aside. Add PBMCs to the culture medium (RPMI 1640 + 10% fetal bovine serum + 5% human AB serum) and adjust the cell count to 1×10⁶ cells / mL. 8 The cells were cultured at 1 / L in a 250ml cell culture flask and incubated at 37℃ in a 5% CO2 cell culture incubator. After 18 hours of culture, the cells were collected by centrifugation at 1500 rpm for 10 min. The γδT cells were counted using the method described in section 1.2 (resulting in A2 cells).

[0080] γδT cell survival rate = 2 × A2 ÷ A1 × 100%.

[0081] 2. Results Evaluation In section 1.3, cell cryopreservation was performed for 10, 30, 100, 150, 250, and 360 days, and cell viability and γδT cell survival rate were measured.

[0082] The results are shown in Tables 3-8 below: In order to control the experimental cost, save experimental materials such as human whole blood, and minimize experimental error, each experimental result below is the average of two parallel experiments (n=2, average).

[0083] Table 3

[0084] Table 4

[0085] Table 5

[0086] Table 6

[0087] Table 7

[0088] Table 8

[0089] Industrial applicability All components of this invention are mature injectable or pharmaceutical grade raw materials with clear pharmacopoeia or clinical application basis. They can be directly adapted to aseptic filling systems and GMP production environments. They are suitable for: storage and transportation of γδT cell therapy products, storage in immune cell banks, cryopreservation before cell therapy reinfusion, and use with programmed cooling equipment. They have significant industrial application value and clinical translation feasibility.

[0090] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A cell cryopreservation solution, characterized in that, Includes the following components: Colloidal protective components, cold-stabilizing components, antioxidant components, energy and basic osmotic balance components, extracellular electrolyte homeostasis regulating components, and transmembrane osmotic pressure regulating components; The osmotic pressure of the cell cryopreservation solution is 280-380 mOsm / kg, and the pH of the cell cryopreservation solution is 7.0-7.

4. The cell cryopreservation solution does not contain DMSO, animal serum, or substances made from animal serum.

2. The cell cryopreservation solution according to claim 1, characterized in that, The colloidal protective component is plant-derived recombinant human serum albumin; The cold-stabilizing component is dextran-40; The antioxidant component is N-acetylcysteine; The energy and basic osmotic balance component is glucose; The transmembrane osmotic pressure regulating component is mannitol; The mass-volume concentration of the colloidal protective component in the cell cryopreservation solution is 5%-20%; The mass-volume concentration of cold-stable components in cell cryopreservation solutions is 2%-10%; The molar concentration of antioxidant components in the cell cryopreservation solution is 1-20 mmol / L; The molar concentration of energy and basic osmotic balance components in the cell cryopreservation solution is 5-20 mmol / L; The molar concentration of the transmembrane osmotic pressure regulating component in the cell cryopreservation solution was 10-50 mmol / L; The extracellular electrolyte homeostasis regulating component is selected from one or more of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, sodium acetate, potassium acetate, calcium acetate, and magnesium acetate.

3. A cell cryopreservation solution composition, characterized in that, The cell cryopreservation solution composition is used to prepare the cell cryopreservation solution according to any one of claims 1-2; The cell cryopreservation solution composition comprises the following components: Colloidal protective components, cold-stabilizing components, antioxidant components, energy and basic osmotic balance components, extracellular electrolyte homeostasis regulating components, and transmembrane osmotic pressure regulating components; The colloidal protective component is plant-derived recombinant human serum albumin, the cold-stabilizing component is dextran-40, the antioxidant component is N-acetylcysteine, the energy and basal osmotic balance component is glucose, and the transmembrane osmotic pressure regulating component is mannitol. The weight ratio of the colloidal protective component, cold-stabilizing component, antioxidant component, energy and basic osmotic balance component, and transmembrane osmotic pressure regulating component is 50-200:20-100:0.221-4.42:0.9-3.6:1.82-9.

1. The extracellular electrolyte homeostasis regulating component enables the cell cryopreservation solution made from the cell cryopreservation solution composition to have an osmotic pressure of 280-380 mOsm / kg and a pH of 7.0-7.

4.

4. A method for preparing a cell cryopreservation solution using the cell cryopreservation solution composition according to claim 3, characterized in that, Includes the following steps: Mix the cryopreservation solution composition with water.

5. A cell mixture, characterized in that, include: (1) Cell; (2) The cell cryopreservation solution according to any one of claims 1-2.

6. A cell cryopreservation kit, characterized in that, Includes the cell cryopreservation solution according to any one of claims 1-2 or the cell cryopreservation solution composition according to claim 3.

7. A method for cell cryopreservation, characterized in that, Includes the following steps: Cells are placed in a cell cryopreservation solution, and the temperature is controlled to obtain cryopreserved cells; The cell cryopreservation solution is the cell cryopreservation solution according to any one of claims 1-2.

8. A method for cell resuscitation, characterized in that, Includes the following steps: The frozen cells are warmed to 35-40°C; the frozen cells include cells and the cell cryopreservation solution according to any one of claims 1-2.

9. The use of the cell cryopreservation solution according to any one of claims 1-2, the cell cryopreservation solution composition according to claim 3, the cell mixture according to claim 5, the cell cryopreservation kit according to claim 6, the cell cryopreservation method according to claim 7, or the cell resuscitation method according to claim 8 in cell storage.

10. Use of the cell mixture of claim 5 in the preparation of medicaments for the treatment of tumors, infections, and / or immune disorders.

11. A drug, characterized in that, Includes the cell mixture described in claim 5.

12. A cell reagent kit, characterized in that, Includes the cell mixture described in claim 5.

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