Ultralow-temperature preservative special for blood T cells and application of ultralow-temperature preservative

Abstract

The invention discloses a special ultralow-temperature preservative for blood T cells and application of the special ultralow-temperature preservative, and the special ultralow-temperature preservative comprises the following components in percentage by mass and volume: an osmotic pressure regulating system (5-10% of glycerol and 10-20% of glycolalcohol which are compounded); an antioxidant protection system (10%-20% of human serum albumin and 0.1%-1% of glutathione); a nutrition supply agent (a cell culture medium containing glucose, amino acid, vitamin and electrolyte); a pH stable buffer pair (0.5%-3% of HEPES and 0.1%-1% of sodium bicarbonate); and a cell membrane enhancer (1%-1% of cholesterol and 0.05%-0.5% of phosphatidylcholine). The pH value of the preservative is 7.2-7.4, the preservative is suitable for long-time cryopreservation of blood T cells at the ultralow temperature of-80 DEG C to-196 DEG C, and the biological activity and functional integrity of the T cells can be effectively maintained. The invention also relates to an application of the preservative in preparation of T cell preparations preserved at ultra-low temperature, and the survival rate and functional integrity of cells after cryopreservation can be obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of blood T cell preservation, and in particular to a special ultra-low temperature preservation agent for blood T cells and its application. Background Art

[0002] In biomedical research and clinical applications, ultra-low temperature cryopreservation of T cells is crucial for immunotherapy, cell transplantation, and various related research. According to 2023 statistics from Nature Biotechnology, over 2,000 T cell therapies have entered clinical trials worldwide. Commercial products, for example, place extremely high demands on the long-term storage stability of cell preparations. Currently, commonly used cell cryopreservation solutions, mostly based on simple glycerol or DMSO (dimethyl sulfoxide), can achieve a certain degree of cell preservation, but they have numerous limitations. For example, the widely used dimethyl sulfoxide (DMSO) concentration often reaches 10%, which can easily induce cell osmotic shock and adverse reactions in patients after infusion (such as chills and nausea); conventional slow freezing methods (-1°C / min) can easily form intracellular ice crystals, destroying the cell membrane and mitochondrial structure, leading to an increased apoptosis rate after recovery; the freezing process induces oxidative stress, resulting in down-regulation of T cell surface markers (such as CD3 / CD28) and reduced killing activity (IFN-γ secretion); cells are easily damaged by swelling or shrinkage due to sudden changes in osmotic pressure during freezing and thawing, resulting in impaired cell activity and function; cell membranes are easily damaged in ultra-low temperature environments, resulting in reduced activity of T cells after freezing and low survival rates, and many other problems, which limit its effectiveness and reliability in subsequent applications. There is an urgent need to develop a T cell ultra-low temperature cryopreservation agent with better performance to overcome the shortcomings of existing technologies and meet the growing demand for the preservation of highly active T cells. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a special ultra-low temperature preservation agent suitable for blood T cell preparations to maintain cell biological activity, in view of the problems that exist in the cell freezing solution in the prior art when cells are stored for a long time under ultra-low temperature conditions, such as low cell survival rate, large changes in T cell subpopulation ratios, and obvious decline in immune function.

[0004] In order to solve the problems of the prior art, the present invention provides the following technical solutions: In a first aspect, the present invention provides a cryopreservative for blood T cells, comprising the following components by mass volume percentage: Osmotic pressure regulation system: 5%-10% glycerol and 10%-20% mannitol; b. Antioxidant protection system: 10%-20% human serum albumin and 0.1%-1% glutathione form an antioxidant pair; c. Cell membrane strengthener: 0.1% - 1% cholesterol and 0.05% - 0.5% phosphatidylcholine; d. pH stabilizing buffer pair: 0.5% - 3% HEPES and 0.1% - 1% sodium bicarbonate to construct the buffer system; e. Nutrient supply: cell culture medium, balance.

[0005] The solution of the present invention has a synergistic effect through the osmotic pressure synergy of high-concentration mannitol and glycerol, the dual-pathway protection of antioxidant and membrane stabilization of the human serum albumin / glutathione group, and the system design of nutrition-buffer-membrane strengthening, which can maintain the activity of blood T cells for a long time under ultra-low temperature conditions and reduce cell damage.

[0006] In the osmotic pressure regulation system of the present invention, glycerol and mannitol act as cell protectants. Glycerol, as an osmotic protectant, can partially replace the osmotic pressure regulation function of DMSO, but its freezing point inhibition ability is relatively weak. Mannitol, as a non-permeable macromolecule, complements glycerol by reducing ice crystal formation through extracellular dehydration while preventing excessive intracellular dehydration. Within the present ratio of glycerol to mannitol, it is possible to achieve a transmembrane osmotic pressure balance similar to that of DMSO.

[0007] Human albumin and glutathione are two important antioxidants in the antioxidant defense system. Human albumin not only provides colloidal osmotic pressure but also absorbs free radicals, reducing oxidative damage during freeze-thaw cycles. Glutathione, a key endogenous antioxidant, scavenges reactive oxygen species within cells, protecting them from oxidative stress. These two antioxidants work synergistically to form a comprehensive antioxidant defense network.

[0008] The cholesterol in the cell membrane strengthener enhances membrane rigidity, preventing membrane brittleness at low temperatures, while phosphatidylcholine maintains membrane fluidity, compensating for the decrease in membrane fluidity caused by the absence of DMSO. Furthermore, the synergistic protection of the antioxidant system reduces membrane lipid peroxidation damage, indirectly enhancing membrane stability.

[0009] The cell culture medium used in the nutrient supply agent is rich in glucose, amino acids, vitamins, and electrolytes, providing cells with essential nutrients and energy sources. Although cell metabolism slows during cryopreservation, cells still require basic nutritional support. The composition of the cell culture medium is designed to meet the nutritional needs of T cells during cryopreservation and recovery, maintaining pre-cryopreservation cell viability and reducing activation of apoptotic signaling. Furthermore, electrolytes (such as Na⁺ / K⁺) synergistically regulate osmotic pressure with mannitol, avoiding the limitations of relying solely on small-molecule osmotic agents.

[0010] The pH-stabilizing buffer pair consists of HEPES and sodium bicarbonate. HEPES is an excellent biological buffer with strong buffering capacity within the physiological pH range, capable of maintaining a stable pH environment during cryopreservation. Sodium bicarbonate, as a secondary buffer, forms a complementary buffering system with HEPES, jointly maintaining the pH of the preservative at 7.2-7.4, a pH close to the human physiological environment and conducive to cell survival.

[0011] Preferably, the mass volume percentage of glycerol in the osmotic pressure regulating system is 6%-8%, for example: 6%, 7%, 8%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here; the mass volume percentage of mannitol is 15%-18%, for example: 15%, 16%, 17%, 18%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0012] Preferably, the mass volume percentage of human serum albumin in the antioxidant protection system is 15%-18%, for example: 15%, 16%, 17%, 18%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here; the mass volume percentage of glutathione is 0.5%-0.8%, for example: 0.5%, 0.6%, 0.7%, 0.8%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0013] Preferably, the mass volume percentage of cholesterol in the cell membrane strengthening agent is 0.5%-0.8%, for example: 0.5%, 0.6%, 0.7%, 0.8%, etc., and other specific values ​​within this numerical range can be selected, which will not be repeated here; the mass volume percentage of phosphatidylcholine is 0.1-0.4%, for example: 0.1%, 0.2%, 0.3%, 0.4%, etc., and other specific values ​​within this numerical range can be selected, which will not be repeated here; Preferably, the mass volume percentage of HEPES in the pH stabilizing buffer is 1%-2%, for example: 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here; the mass volume percentage of sodium bicarbonate is 0.3%-0.6%, for example: 0.3%, 0.4%, 0.5%, 0.6%, etc., and other specific point values ​​within this numerical range can be selected, which will not be repeated here.

[0014] Preferably, the cell culture medium is selected from any one of RPMI 1640, DMEM / F12, and IMDM.

[0015] In a second aspect, the present invention provides use of the above-mentioned cell preservative in preserving blood T cells.

[0016] Preferably, the method for applying the cell preservative in preserving cells is as follows: taking a blood T cell suspension, 7 / ml into cryopreservation tubes, placed in a programmed cooling box, placed in a -80℃ low-temperature refrigerator overnight, and then transferred to an ultra-low temperature container for storage; The program cooling box is a T storage universal program cooling box.

[0017] In a third aspect, the present invention provides the use of the above-mentioned blood T cell-specific ultra-low temperature preservation agent in the preparation of ultra-low temperature preserved blood T cell preparations; the low temperature is -80°C to -196°C.

[0018] In a fourth aspect, the present invention provides a blood T cell preparation preserved at an ultra-low temperature, characterized in that it is composed of blood T cells and the above-mentioned blood T cell-specific ultra-low temperature preservation agent, and the ultra-low temperature is -80°C to -196°C.

[0019] The beneficial effects of the present invention are: 1. The present invention utilizes the osmotic pressure synergy of high-concentration mannitol and glycerol, the dual-pathway protection of antioxidant and membrane stabilization provided by the human albumin / glutathione combination, and the system design of nutrition-buffering-membrane strengthening to achieve a synergistic effect, maintaining blood T cell activity for a long time under ultra-low temperature conditions and reducing cell damage.

[0020] 2. Ultra-low temperature long-term storage: The preservative of this invention can achieve stable storage of T cells for 12 months at ultra-low temperatures, providing technical support for cell preparation and clinical treatment; 3. High activity maintenance ability: Experimental verification shows that after 12 months, the T cell survival rate of cells using this cell cryopreservation agent reached 96.7%, while that of the control group was only 78.1%; the proportions of CD3+, CD4+, and CD8+ T cell subsets changed little, while the control group showed significant changes; 4. Significant cost-effectiveness: The raw material cost of the preservative is low, the preparation process is simple, and it can be produced on a large scale, reducing the cost of cell preparation storage and application; 5. High safety: All ingredients have good biocompatibility and have no obvious toxic side effects on blood T cells. Cells can be stored for a long time in an ultra-low temperature environment without affecting cell phenotype and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The cell morphology under a 4x microscope on the 1st, 3rd, and 7th day after cell recovery after T cells were frozen using the cell preservative in Example 2 for 12 months. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings and embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0023] The processes, conditions, reagents, experimental methods, etc. for carrying out the present invention, except for the contents specifically mentioned below, are all common knowledge and common general knowledge in the field and are not particularly limited by the present invention. The experimental methods without specific conditions in the examples are generally based on conventional conditions or conditions recommended by the manufacturer.

[0024] Unless otherwise specified, all technical and scientific terms used in this specification have the same meanings as those generally understood by those skilled in the art to which this invention belongs. However, in the event of any conflict, the present specification, including the definitions, shall prevail.

[0025] The sources of the main reagents and consumables in the following examples are shown in Table 1 Table 1 Information on the sources of main reagents

[0026] Example 1 A blood T cell cryopreservative, calculated by weight volume percentage (w / v), comprises the following components: a. Osmotic pressure adjustment system: 5% glycerol and 10% mannitol; b. Antioxidant protection system: 10% human serum albumin and 0.1% glutathione; c. Cell membrane enhancer: 1% cholesterol and 0.05% phosphatidylcholine; d. pH stabilizing buffer pair: 0.5% HEPES and 0.1% sodium bicarbonate; e. Nutrient supply agent: DMEM / F12 culture medium The preparation method of the preservative is as follows: First, under sterile conditions, the culture medium is pre-cooled in a low-temperature environment (4°C); glycerol, mannitol, human serum albumin, glutathione, cholesterol and phosphatidylcholine are slowly added in sequence, stirred to dissolve and mix, then HEPES and sodium bicarbonate are added, mixed thoroughly, the pH value is adjusted to 7.3, and the culture medium is sterilized by filtration through a 0.22 μm filter membrane to prepare the cryopreservative.

[0027] Example 2 A blood cell cryopreservative, calculated by weight volume percentage (w / v), comprising the following components: a. Osmotic pressure adjustment system: 8% glycerol and 15% mannitol; b. Antioxidant protection system: 15% human serum albumin and 0.5% glutathione; c. Cell membrane strengthener: 0.5% cholesterol and 0.25% phosphatidylcholine; d. pH stabilizing buffer pair: 1.5% HEPES and 0.5% sodium bicarbonate; e. Nutrient supply agent: RPMI 1640 medium The preparation method is the same as that of Example 1 The pH value of the preservative is 7.2.

[0028] Example 3 A blood cell cryopreservative, calculated by weight volume percentage (w / v), comprising the following components: a. Osmotic pressure adjustment system: 10% glycerol and 20% mannitol; b. Antioxidant protection system: 20% human serum albumin and 1% glutathione; c. Cell membrane strengthener: 0.1% cholesterol and 0.5% phosphatidylcholine; d. pH stabilizing buffer pair: 3% HEPES and 1% sodium bicarbonate; e. Nutrient supply agent: IMDM medium The preparation method is the same as that of Example 1 The pH value of the preservative is 7.2.

[0029] Example 4 Application of cryopreservatives in T cell storage Peripheral blood was collected from donors, and T cells were isolated using magnetic bead sorting. The isolated T cells were randomly divided into an experimental group and a control group. The experimental group cells were mixed with the cryopreservative prepared in Examples 1-3 at a 1:1 volume ratio, suspending the cells at a concentration of 1×10^7 cells / mL. The control group cells were mixed with a conventional cell cryopreservative (DMSO + serum) at the same ratio. The cells were stored at ultra-low temperatures (-196°C) for 1, 3, 6, and 12 months, followed by cell recovery.

[0030] After cell recovery, cell viability was assessed using AOPI, and the proportions of CD3+, CD4+, and CD8+ T cell subsets were analyzed by flow cytometry to assess cell bioactivity and immune function. Cell morphology was also observed using an inverted microscope to assess cell morphological integrity. The results are shown in Tables 2-3.

[0031] Table 2 T cell viability after cryopreservation

[0032] Table 3 Proportions of T cell subsets after cryopreservation

[0033] The cell morphology was observed under an inverted microscope. After 1, 3, 6, and 12 months of storage, the cell membranes of the cells in the Example 1-3 groups were intact and the cell morphology was normal. The cell membranes of the cells in the control group were damaged and the cells were shrunken. Figure 1 The cell morphology under a 4x microscope on the first, third, and seventh days after cell recovery after T cells were cryopreserved with the cell preservative of Example 2 for 12 months.

[0034] The above experimental results show that the cell cryopreservative of the present invention can achieve stable preservation of T cells at ultra-low temperatures. After 1, 3, 6, and 12 months, the T cell survival rate using this cell cryopreservative reached 96%, and the proportions of CD3+, CD4+, and CD8+ T cell subsets changed little. In contrast, the cell survival rate of the control group using conventional preservation fluid was only 78%, and key immune indicators decreased significantly.

[0035] Comparative Example 1 The difference between this comparative example and Example 2 is that the osmotic pressure regulating system is 8% glycerol and 5% mannitol, and the rest is the same as Example 2.

[0036] Comparative Example 2 The difference between this comparative example and Example 2 is that the cell membrane strengthening agent is 0.75% cholesterol, and the rest is the same as Example 2.

[0037] Comparative Example 3 The difference between this comparative example and Example 2 is that the cell membrane strengthening agent is 0.75% phosphatidylcholine, and the rest is the same as Example 2.

[0038] Comparative Example 4 The difference between this comparative example and Example 2 is that the cell membrane strengthener is not contained, and the rest is the same as Example 2.

[0039] Comparative Example 5 The difference between this comparative example and Example 2 is that the pH stabilizing buffer is isotonic phosphate buffered saline (PBS), and the rest is the same as Example 2.

[0040] Comparative Example 6 The difference between this comparative example and Example 2 is that the antioxidant protection system is 15.5% human serum albumin, and the rest is the same as Example 2.

[0041] Comparative Example 7 This comparative example differs from Example 2 in that physiological saline is used instead of the cell culture medium, and the rest is the same as Example 2.

[0042] Comparative Example 8 Experiment on cryopreservation of T cells with protective agents of Comparative Examples 1-7 at ultra-low temperature Peripheral blood was collected from donors, and T cells were isolated using magnetic bead sorting. The isolated T cells were randomly divided into seven groups. These groups were mixed with the cryopreservatives prepared in Comparative Examples 1-7 at a 1:1 volume ratio, suspending the T cells in the cryopreservative at a concentration of 1×10^7 cells / mL. The cells were stored at ultra-low temperatures (-196°C) for one month and 12 months before being revived.

[0043] After cell recovery, the cell survival rate was detected using the AOPI method. The results are shown in Table 4.

[0044] Table 4 Cell survival rate of T cells after ultra-low temperature freezing with preservatives in Comparative Examples 1-7

[0045] Comparison of the results of Comparative Examples 1-7 with those of the Examples shows that the combination of the components of the T cell preservative of the present invention has a synergistic effect, and the absence or change of any component, such as the osmotic pressure regulation system, cell membrane strength, pH stabilization buffer, antioxidant protection system, and nutritional supplements, will have a significant effect on T cell survival rate.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A cryopreservative for blood T cells, characterized in that: Calculated by weight volume percentage (w / v), it includes the following components: a. Osmotic pressure adjustment system: 5%-10% glycerol and 10%-20% mannitol; b. Antioxidant protection system: 10% - 20% human serum albumin and 0.1% -1% glutathione; c. Cell membrane strengthener: 0.1% - 1% cholesterol and 0.05% - 0.5% phosphatidylcholine; d. pH stabilizing buffer pair: 0.5% - 3% HEPES and 0.1% - 1% sodium bicarbonate; e. Nutrient supply agent: balance, cell culture medium.

2. The blood T cell-specific cryopreservative according to claim 1, characterized in that: The mass volume percentage of glycerol in the osmotic pressure regulating system is 6%-8%, and the mass volume percentage of mannitol is 15%-18%.

3. The blood T cell-specific cryopreservative according to claim 1, characterized in that: The weight volume percentage of human serum albumin in the antioxidant protection system is 15%-18%, and the weight volume percentage of glutathione is 0.5%-0.8%.

4. The blood T cell-specific cryopreservative according to claim 1, characterized in that: The mass volume percentage of HEPES in the pH stabilizing buffer is 1%-2%, and the mass volume percentage of sodium bicarbonate is 0.3%-0.6%.

5. The blood T cell-specific cryopreservative according to claim 1, characterized in that: The mass volume percentage of cholesterol in the cell membrane strengthening agent is 0.5-0.8%, and the mass volume percentage of phosphatidylcholine is 0.1-0.4%.

6. The blood T cell-specific cryopreservative according to claim 1, characterized in that: The cell culture medium is selected from any one of RPMI 1640, DMEM / F12, and IMDM.

7. Use of the blood T cell-specific cryopreservative according to any one of claims 1 to 6 in the preparation of a T cell preparation for ultra-low temperature preservation; the ultra-low temperature is -80°C to -196°C.

8. Use of the blood T cell-specific cryopreservative according to any one of claims 1 to 6 in preserving T cells.

9. A cryopreserved blood T cell preparation, characterized in that: The method comprises blood T cells and the ultra-low temperature preservation agent for blood T cells according to any one of claims 1 to 6, wherein the ultra-low temperature is -80°C to -196°C.

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