Novel cryopreservation protective agent and preparation method thereof

Abstract

The novel cryopreservation protective agent comprises the following components: an ice crystal inhibitor, a natural deep eutectic solvent, an impermeable protective agent, a cell membrane stabilizer, an antioxidant, an isoosmotic adjusting agent, a buffer system and a solvent. The preparation method of the novel cryopreservation protective agent comprises the following steps: S1, preparing an environment and a solvent; s2, preparing a natural deep eutectic solvent, accurately weighing choline and L-proline according to a molar ratio of 1: 1, adding 20% of water for injection, and stirring at 60 DEG C until a clear, transparent and uniform liquid natural deep eutectic solvent is formed; s3, dissolving and mixing; s4, adjusting the pH value; s5, degerming and sub-packaging; and S6, quality control. The invention relates to a clinical-grade (elution-free) human cell cryopreservation protective agent which is completely free of DMSO (dimethylsulfoxide) and free of animal-derived components. After resuscitation, cleaning is not needed, the liquid can be directly infused back to the human body, the clinical operation process is greatly simplified, and the safety is improved.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation technology, specifically to a novel cryopreservation protectant and its preparation method. Background Technology

[0002] Cell science, as one of the core branches of life sciences, has profound and far-reaching significance. It reveals the microscopic basis of life, providing crucial theoretical and experimental evidence for understanding complex biological phenomena, disease mechanisms, and promoting the development of biotechnology. It enables us to clearly understand how life is organized and operates at the cellular level. By studying cell proliferation, differentiation, apoptosis, and their regulatory mechanisms, scientists have gradually understood how organisms form different tissues and organs and maintain their normal physiological functions. The occurrence and development of many diseases are closely related to abnormalities in cell structure and function. Studying cells, such as gene mutations, abnormal protein expression, and disordered cell signaling, provides new ideas and methods for disease diagnosis, treatment, and prevention. Dr. Zhang Hongkai, President of the International Cell Rehabilitation Association of America, believes that humans suffer from only one disease: "cell disease," and has proposed the rudiments of cell medicine theory. Academician Zhang Xuemin of the Chinese Academy of Sciences has called for attention to "cell medicine," that is, medical science that achieves disease treatment through cell repair, cell modification, and cell regulation.

[0003] In conclusion, cell research and application have immeasurable value to life sciences and clinical medicine. They not only unveil the mysteries of life but also provide powerful impetus and support for the development of human health. However, the preparation processes of stem cells, immune cells, and advanced CAR-T cells have always faced an insurmountable obstacle: cell formulations require customization, only one formulation can be produced per batch, the preparation cycle is long, and quality control results are delayed. Meanwhile, small-batch, multi-batch preparation leads to a geometric increase in costs for personnel, equipment, instruments, materials, validation, and environmental maintenance, hindering the rapid development of the cell industry and the progress of clinical trials. This has resulted in the continuous development of universal off-the-shelf formulation processes for even the most advanced CAR-T cell technology.

[0004] Tracing the Defects in Traditional "Fresh Preparations" Processes:

[0005] Dimethyl sulfoxide (DMSO), which is widely used in traditional formulations, has clear cytotoxicity and can cause intracellular protein denaturation and membrane structure damage. After clinical reinfusion, it often leads to adverse events such as nausea, hypertension, and allergic reactions in patients.

[0006] The use of animal-derived serum (such as FBS) carries the risk of contamination by exogenous factors such as viruses and mycoplasma, with significant batch-to-batch variations, severely hindering the standardization of formulations. Although some commercially available serum-free cryopreservation solutions (such as Ezes CryoCell®) have attempted to address these issues, their formulations still contain a certain concentration of DMSO, failing to fundamentally resolve the problem of residual toxicity.

[0007] At the process level, existing cryopreservation solutions require multiple centrifugation and washing processes after thawing to remove DMSO. This process not only results in an average cell loss of 15–20%, but also significantly increases the risk of microbial contamination (with an incidence rate of up to 8.7%).

[0008] Therefore, developing a production process for readily available cell preparations that integrates non-toxic, wash-free cryopreservation preservatives has become an urgent need to promote cell therapy from the laboratory to industrial applications. Summary of the Invention

[0009] The present invention aims to solve the above-mentioned technical problems by providing a novel cryoprotectant and its preparation method. The cryoprotectant is non-toxic and requires no washing.

[0010] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0011] A novel cryoprotectant comprising the following components:

[0012] Ice crystal inhibitor, comprising 0.01-0.05% by mass of the protective agent;

[0013] Natural deep eutectic solvent, accounting for 5-15% by mass of the protective agent;

[0014] Non-permeable protective agent, accounting for 1.37-2.05% of the total protective agent by mass;

[0015] Cell membrane stabilizers, comprising 0.05-0.2% by weight of the protectant;

[0016] Antioxidants, accounting for 0.031-0.154% by mass of the protective agent;

[0017] An isotonic conditioner, comprising 0.6-0.8% by mass of the protective agent;

[0018] The buffer system comprises 0.238-0.596% of the protective agent by mass.

[0019] The solvent accounts for 81.15-92.701% of the protective agent by mass.

[0020] The ice crystal inhibitor is a biomimetic ice-controlling peptide, the non-permeable protective agent is trehalose, the cell membrane stabilizer is fucoidan, the antioxidant is glutathione, the isotonic regulator is sodium chloride, the buffer system is HEPES buffer, and the solvent is water for injection.

[0021] The natural deep eutectic solvent is a choline-proline natural ionic liquid, the biomimetic ice-controlling peptide is an antifreeze protein functional peptide, and the antioxidant is reduced glutathione.

[0022] A method for preparing a novel cryoprotectant includes the following steps:

[0023] S1 environment and solvent: In a B+A cleanroom environment, water for injection is used as the solvent.

[0024] S2 Natural Deep Eutectic Solvent Pre-preparation: Accurately weigh choline and L-proline in a molar ratio of 1:1, add 20% water for injection, and stir at 60°C until a clear and transparent homogeneous liquid natural deep eutectic solvent is formed.

[0025] S3 Dissolution and Mixing: Take 70% water for injection, and add precisely weighed trehalose, fucoidan, HEPES buffer, sodium chloride, and reduced glutathione in sequence while stirring. Continue stirring until completely dissolved. Then add natural deep eutectic solvent and stir evenly. Next, add biomimetic cryoprotectant and stir at low speed to fully dissolve it. After fully dissolving, add the remaining water for injection and stir evenly to obtain the cryoprotectant.

[0026] S4 pH adjustment: Adjust the pH of the cryoprotectant to a suitable value using dilute sodium hydroxide solution or dilute hydrochloric acid solution.

[0027] It also includes the following steps:

[0028] S5 sterilization and dispensing: Sterilization is achieved by filtration using a 0.22μm polyethersulfone membrane. Under Class A laminar flow protection, the filtrate is aseptically dispensed into sterile, pyrogen-free containers.

[0029] S6 quality control involves conducting sterility checks, endotoxin tests, osmolality tests, and pH tests on each batch of products, and only those that pass the tests are released.

[0030] In step S3, 70% of the water used for injection is used, and the temperature of the water used for injection is controlled at 25-30℃.

[0031] In step S4, the appropriate pH value is 7.2 ± 0.1.

[0032] In the cryoprotectant prepared in step S3, the biomimetic cryoprotectant accounts for 0.01-0.05% of the cryoprotectant by mass, the natural deep eutectic solvent accounts for 5-15% of the cryoprotectant by mass, trehalose accounts for 1.37-2.05% of the cryoprotectant by mass, fucoidan accounts for 0.05-0.2% of the cryoprotectant by mass, glutathione accounts for 0.031-0.154% of the cryoprotectant by mass, sodium chloride accounts for 0.6-0.8% of the cryoprotectant by mass, HEPES buffer accounts for 0.238-0.596% of the cryoprotectant by mass, and water for injection accounts for 81.15-92.701% of the cryoprotectant by mass.

[0033] The technical effects achievable by this invention are as follows: This invention is a clinical-grade (wash-free) human cell cryopreservation protectant that is completely free of DMSO and animal-derived components; the core protective components of this cryopreservation protectant are biomimetic ice-controlling peptides (functional peptides derived from polar fish antifreeze proteins) and natural deep eutectic solvents (choline-proline natural ionic liquid), replacing traditional DMSO and serum; the bacterial endotoxin level is <0.03 EU / mL, and it can be mixed with cells for cryopreservation. After thawing, no washing is required, and it can be directly reinfused into the human body, greatly simplifying the clinical operation process and improving safety.

[0034] The present invention will be further described in detail below with reference to specific embodiments: Detailed Implementation

[0035] The present invention will now be described in further detail.

[0036] A novel cryoprotectant, characterized by comprising the following components:

[0037] Ice crystal inhibitor, comprising 0.01-0.05% by mass of the protective agent;

[0038] Natural deep eutectic solvent, accounting for 5-15% by mass of the protective agent;

[0039] Non-permeable protective agent, accounting for 1.37-2.05% of the total protective agent by mass;

[0040] Cell membrane stabilizers, comprising 0.05-0.2% by weight of the protectant;

[0041] Antioxidants, accounting for 0.031-0.154% by mass of the protective agent;

[0042] An isotonic conditioner, comprising 0.6-0.8% by mass of the protective agent;

[0043] The buffer system comprises 0.238-0.596% of the protective agent by mass.

[0044] The solvent accounts for 81.15-92.701% of the protective agent by mass.

[0045] Specifically, the ice crystal inhibitor is a biomimetic ice-controlling peptide, the non-permeable protectant is trehalose, the cell membrane stabilizer is fucoidan, the antioxidant is glutathione, the isotonic regulator is sodium chloride, the buffer system is HEPES buffer, and the solvent is water for injection.

[0046] More specifically, the natural deep eutectic solvent is a choline-proline natural ionic liquid, the biomimetic ice-controlling peptide is an antifreeze protein functional peptide, and the antioxidant is reduced glutathione.

[0047] A method for preparing a novel cryoprotectant includes the following steps:

[0048] S1 environment and solvent: In a B+A cleanroom environment, water for injection is used as the solvent.

[0049] S2 Natural Deep Eutectic Solvent Pre-preparation: Accurately weigh choline and L-proline in a molar ratio of 1:1, add 20% water for injection, and stir at 60°C until a clear and transparent homogeneous liquid natural deep eutectic solvent is formed.

[0050] S3 Dissolution and Mixing: Take 70% water for injection (temperature controlled at 25-30℃), and add precisely weighed trehalose, fucoidan, HEPES buffer, sodium chloride, and reduced glutathione in sequence while stirring. Continue stirring until completely dissolved. Then add natural deep eutectic solvent and stir evenly. Next, add biomimetic cryoprotectant and stir at low speed to fully dissolve it. After fully dissolving, add the remaining water for injection and stir evenly to obtain the cryoprotectant.

[0051] S4 pH adjustment: Adjust the pH of the cryoprotectant to a suitable value using dilute sodium hydroxide solution or dilute hydrochloric acid solution. The suitable pH value is 7.2±0.1.

[0052] It also includes the following steps:

[0053] S5 sterilization and dispensing: Sterilization is achieved by filtration using a 0.22μm polyethersulfone membrane. Under Class A laminar flow protection, the filtrate is aseptically dispensed into sterile, pyrogen-free containers.

[0054] S6 quality control involves conducting sterility checks, endotoxin tests, osmolality tests, and pH tests on each batch of products, and only those that pass the tests are released.

[0055] In the cryoprotectant prepared in step S3, the biomimetic cryoprotectant accounts for 0.01-0.05% of the cryoprotectant by mass, the natural deep eutectic solvent accounts for 5-15% of the cryoprotectant by mass, trehalose accounts for 1.37-2.05% of the cryoprotectant by mass, fucoidan accounts for 0.05-0.2% of the cryoprotectant by mass, glutathione accounts for 0.031-0.154% of the cryoprotectant by mass, sodium chloride accounts for 0.6-0.8% of the cryoprotectant by mass, HEPES buffer accounts for 0.238-0.596% of the cryoprotectant by mass, and water for injection accounts for 81.15-92.701% of the cryoprotectant by mass.

[0056] The biomimetic ice-controlling peptide of this invention is chemically synthesized (solid-phase synthesis method). Its sequence is derived from the functional region of Antarctic cod antifreeze protein (AFPIII). It can specifically adsorb onto the surface of ice crystals, inhibiting the growth and recrystallization of ice crystals, thereby reducing the mechanical damage to cells caused by ice crystals during freezing from the source. This mechanism is different from the simple permeation protection or vitrification formation of traditional cryoprotectants, and provides a new way to physically inhibit ice crystal formation.

[0057] A natural deep eutectic solvent is formed by mixing choline (plant-derived or chemically synthesized) and L-proline (fermentation-extracted) in a 1:1 molar ratio, adding 20% ​​water for injection, and stirring at 60°C to form a homogeneous liquid. Choline-proline natural ionic liquid (Choline-Proline NADES) is used as the main permeability protectant in clinical-grade cell cryopreservation solutions. This solvent, composed of naturally occurring choline and proline, has excellent vitrification ability, high biocompatibility, and low toxicity. It can effectively penetrate cells, protect intracellular structures, and completely replace DMSO.

[0058] A multi-layered synergistic protection system is composed of biomimetic ice-controlling peptides, natural deep eutectic solvent (NADES), trehalose, and fucoidan.

[0059] a) Biomimetic ice-controlling peptides inhibit ice crystal formation;

[0060] b) Natural deep eutectic solvents (NADES) provide intracellular protection and vitrification;

[0061] c) Trehalose provides extracellular vitrification and membrane stability;

[0062] d) Fucoidan enhances membrane stability and inhibits apoptosis.

[0063] This system achieves more comprehensive and efficient protection of cells through the synergistic effect of multiple targets and mechanisms.

[0064] This invention adds reduced glutathione as a dedicated antioxidant to specifically reduce oxidative stress damage during the recovery process. This is an aspect that is not adequately considered in many existing cryopreservation solutions (including Ezes CryoCell) formulations, and helps to better maintain cell function, especially for metabolically active immune cells and stem cells.

[0065] The following are the sources of the constituent substances:

[0066]

[0067] The following is the composition of the cryoprotectant formulation:

[0068]

[0069] This invention uses natural deep eutectic solvent (NADES) as its foundation, replacing toxic DMSO and simultaneously serving as both a permeability protectant and a deep eutectic solvent, achieving highly efficient intracellular protection and detoxification. NADES works synergistically with trehalose to form a protective glassy state outside the cell, constituting a comprehensive vitrification cryopreservation system. Sodium chloride and HEPES buffers work together to maintain the stability of the physicochemical environment of the cryopreservation solution (isotonic and pH stable), which is fundamental for cell survival. Biomimetic ice-controlling peptides actively intervene in ice crystal morphology at the microscopic physical level, reducing damage at its source. Fucoidan and glutathione provide protection at the biochemical level, targeting the two key pathways of cryopreservation damage: cell membrane integrity and oxidative stress, respectively.

[0070] The fundamental reason why this invention achieves "wash-free" results is:

[0071] a) All ingredients are derived from natural or highly biocompatible substances, and DMSO, the most toxic substance in traditional formulas, has been removed;

[0072] b) The concentrations of each component are set between the lower limit of the effective concentration and the upper limit of the safety concentration. While protecting the cells, the final product (cell + cryopreservation solution mixture) can be directly used for reinfusion, avoiding cell loss and secondary damage risks caused by the elution process.

[0073] Formulation design represents an important direction for the development of cell cryopreservation technology from "laboratory research" to "clinical application".

[0074] Example 1:

[0075] Cryoprotectant formulation composition (by mass percentage):

[0076] Bionic ice-controlling peptide: 0.05%;

[0077] Natural deep eutectic solvent: 15%;

[0078] Trehalose: 2.05%;

[0079] Fucoidan: 0.2%;

[0080] Glutathione: 0.154%;

[0081] Sodium chloride: 0.8%;

[0082] HEPES buffer: 0.596%;

[0083] Water for injection: 81.15%.

[0084] Experimental data:

[0085] I. Cell viability assay (trypan blue rejection method)

[0086] Experimental subjects: human umbilical cord blood mesenchymal stem cells (P5 generation) and human peripheral blood mononuclear cells (PBMCs, including immune cell populations).

[0087] Experimental methods:

[0088] Experimental group: Cells were mixed with the cryoprotectant of Example 1 at a volume ratio of 1:1, and then cryopreserved in liquid nitrogen for 7 days after gradient cooling. During thawing, the cells were rapidly thawed in a water bath at 37°C and directly reinfused into the simulation (no elution required, diluted with an equal volume of physiological saline).

[0089] Control group: Cells were stored in commercial cryopreservation solution containing 10% DMSO (elution required), following the same cryopreservation-thawing procedure, and washed three times with PBS after thawing.

[0090] Test results:

[0091]

[0092] II. Cytotoxicity Detection (CCK-8 Assay)

[0093] Experimental method: After resuscitation, cells were seeded in 96-well plates (1×10⁻⁶ cells / wells). 4 Cells / well), after culturing for 24h, 48h, and 72h, CCK-8 reagent was added, and the absorbance value at 450nm (OD value) was measured to calculate the relative proliferation rate (with fresh, unfrozen cells as 100%).

[0094] Test results:

[0095]

[0096] III. Hemolytic toxicity test (in vitro hemolysis test)

[0097] Experimental method: Fresh venous blood was collected from healthy individuals, and a 2% red blood cell suspension was prepared. The suspension was mixed with the cryoprotectant from Example 1 at a volume ratio of 1:1 and incubated at 37°C for 1 hour. After centrifugation, the absorbance of the supernatant at 545 nm was measured, and the hemolysis rate was calculated (positive control: distilled water, hemolysis rate 100%; negative control: physiological saline, hemolysis rate < 5%).

[0098] Test results: The hemolysis rate was 1.2±0.3%, which is far below the safety threshold of 5%, meeting the requirements for biocompatibility and non-hemolytic toxicity.

[0099] IV. Acute toxicity test in animals (tail vein injection in mice)

[0100] Experimental methods:

[0101] Twenty SPF-grade ICR mice (half male and half female, weighing 20±2g) were injected via tail vein with the cryopreservative from Example 1 (dose 5mL / kg, equivalent to 10 times the clinical human dose). The mice were observed for 7 consecutive days, and their behavior, weight, and mortality were recorded.

[0102] Test results:

[0103] No mice died within 7 days, and their behavior was normal (no abnormalities in diet, activity, or mental state). The weight gain rate was 8.5±1.2% (no significant difference compared with the saline control group, P>0.05). After dissection, no gross pathological damage was found in the major organs such as the heart, liver, spleen, lungs, and kidneys. HE staining of tissue sections showed that the organ structures were normal, with no inflammatory infiltration or cell necrosis.

[0104] Example 2:

[0105] Cryoprotectant formulation composition (by mass percentage):

[0106] Bionic ice-controlling peptide: 0.01%;

[0107] Natural deep eutectic solvent: 5%;

[0108] Trehalose: 1.37%;

[0109] Fucoidan: 0.05%;

[0110] Glutathione: 0.031%;

[0111] Sodium chloride: 0.6%;

[0112] HEPES buffer: 0.238%;

[0113] Water for injection: 92.701%.

[0114] Experimental data:

[0115] I. Cell viability assay (trypan blue rejection method)

[0116] Experimental subjects: human umbilical cord blood mesenchymal stem cells (P5 generation) and human peripheral blood mononuclear cells (PBMCs).

[0117] Experimental method: Same as Example 1 (frozen for 7 days, then directly reinfused after thawing to simulate the process; the control group was a commercial cryopreservation solution containing 10% DMSO).

[0118] Test results:

[0119]

[0120] II. Cytotoxicity Detection (CCK-8 Assay)

[0121] Experimental method: Same as Example 1

[0122] Test results:

[0123]

[0124] III. Hemolytic toxicity test (in vitro hemolysis test)

[0125] Experimental method: Same as Example 1

[0126] Test results: The hemolysis rate was 0.9±0.2%, which is below the 5% safety threshold, and there was no hemolytic toxicity.

[0127] IV. Acute toxicity test in animals (tail vein injection in mice)

[0128] Experimental method: Same as Example 1 (injection dose 5 mL / kg)

[0129] Test results: No mice died within 7 days, their behavior and weight were normal (weight gain rate 7.8±1.5%), the pathological sections of major organs were normal, and there were no acute toxic reactions.

[0130] Example 3:

[0131] Cryoprotectant formulation composition (by mass percentage):

[0132] Bionic ice-controlling peptide: 0.03%;

[0133] Natural deep eutectic solvent: 10%;

[0134] Trehalose: 1.95%;

[0135] Fucoidan: 0.12%;

[0136] Glutathione: 0.105%;

[0137] Sodium chloride: 0.7%;

[0138] HEPES buffer: 0.405%;

[0139] Water for injection: 86.69%.

[0140] Experimental data:

[0141] I. Cell viability assay (trypan blue rejection method)

[0142] Experimental subjects: human umbilical cord blood mesenchymal stem cells (P5 generation) and human peripheral blood mononuclear cells (PBMCs).

[0143] Experimental method: Same as Example 1 (frozen for 7 days, then directly reinfused after thawing to simulate the process; the control group was a commercial cryopreservation solution containing 10% DMSO).

[0144] Test results:

[0145]

[0146] II. Cytotoxicity Detection (CCK-8 Assay)

[0147] Experimental method: Same as Example 1

[0148] Test results:

[0149]

[0150] III. Hemolytic toxicity test (in vitro hemolysis test)

[0151] Experimental method: Same as Example 1

[0152] Test results: The hemolysis rate was 0.9±0.2%, which is below the 5% safety threshold, and there was no hemolytic toxicity.

[0153] IV. Acute toxicity test in animals (tail vein injection in mice)

[0154] Experimental method: Same as Example 1 (injection dose 5 mL / kg)

[0155] Test results: No mice died within 7 days, their behavior and weight were normal (weight gain rate 7.8±1.5%), the pathological sections of major organs were normal, and there were no acute toxic reactions.

[0156] Explanation of the experiments in the above embodiments:

[0157] The origin of the "Toxicity Level 1 (Non-toxic)" designation is based on the internationally accepted standard (ISO 10993-5). The experiment uses the CCK-8 assay to detect the "relative cell proliferation rate," and then compares it to the standard to determine the level. The entire process has a clear "detection logic" and "judgment rules," and it is not arbitrary. Let's break it down in simple terms:

[0158] First, clarify the criteria for determining toxicity levels (ISO 10993-5).

[0159] This is the international mandatory standard for testing the cytotoxicity of biological materials (such as cryopreservation solutions that are to be directly reinfused into the human body). All biocompatible materials used in the human body are judged according to this standard to determine their toxicity level.

[0160] The core logic is: the closer the cell's proliferation ability is to that of a normal cell after it comes into contact with the material, the lower its toxicity.

[0161] A clearly defined toxicity classification (the key factor is the "relative proliferation rate"):

[0162]

[0163] For cryopreservation solutions to be directly reinfused into the human body, they must meet the "Level 1 (non-toxic)" standard to be acceptable—this is also a core indicator that the patent department focuses on.

[0164] II. How was the "Toxicity Level 1" detected in the experiment?

[0165] The CCK-8 assay was used (a commonly used, authoritative, and easy-to-operate method for cytotoxicity detection). The entire detection process and data logic are as follows, corresponding to the previous experimental design:

[0166] 1. Detection principle (in layman's terms)

[0167] CCK-8 reagent contains a "chromogenic substance." When cells are alive and can proliferate normally, they produce a "dehydrogenase." This enzyme can react with CCK-8 to produce blue "formazan" (the intensity of the color is positively correlated with cell activity and number).

[0168] Measure the absorbance (OD value) of the blue light using an instrument: The higher the OD value, the stronger the cell activity and the better the proliferation, and the less toxic the cryopreservation solution is to the cells.

[0169] 2. Detection steps (already performed in the experiment, but the logic for toxicity determination was not clearly defined)

[0170] Step 1: Prepare the "reference baseline" - take fresh, unfrozen cells (the same type as the ones you have frozen, such as mesenchymal stem cells or PBMCs), and seed them into a 96-well plate. This is the "100% viability control group" (assuming that fresh cells have no toxic effects and a proliferation rate of 100%).

[0171] Step 2: Treating the experimental and control groups —

[0172] Experimental group: Cryopreserve cells in your cryopreservation solution → Thaw (without elution, simulating direct reinfusion) → Seed cells into 96-well plates;

[0173] Control group: Cryopreserved using standard DMSO cryopreservation solution → thawed (eluted) → seeded cells;

[0174] Step 3: Culture + Detection – Culture for 24h, 48h, and 72h respectively (to observe whether the cells are affected by toxicity at different time points). Add CCK-8 reagent at each time point and measure the OD value.

[0175] Step 4: Calculate the "relative growth rate" (core data) —

[0176] Relative proliferation rate (%) = (OD value of experimental group ÷ OD value of fresh cell group) × 100%

[0177] (This means: after cryopreservation, what percentage of the cell proliferation capacity is compared to fresh cells?)

[0178] 3. Why can the data determine that it is "Level 1 non-toxic"?

[0179] Based on the experimental data from the three sets of embodiments, let's take any one set as an example (such as Embodiment 1):

[0180] Human umbilical cord blood mesenchymal stem cells (24h): The relative proliferation rate of the experimental group was 95.7±3.2% → much higher than 80% (Grade 1 standard);

[0181] Human PBMCs (72h): The relative proliferation rate in the experimental group was 97.8±2.6%, which was also much higher than 80%;

[0182] At other time points and in the other two sets of examples (e.g., the mesenchymal stem cell proliferation rate of Example 2 at 72h was 96.8±2.7%, and the PBMC proliferation rate of Example 3 at 48h was 94.6±3.0%), all were ≥91%, which meets the Level 1 standard of "≥80%".

[0183] In short: After cryopreservation, the cell proliferation capacity is almost the same as that of fresh cells, so according to ISO 10993-5 standard, it is directly judged as "Level 1 (non-toxic)".

[0184] III. Supplement: Why can the standard be met even if "no washing is required"?

[0185] The core advantage of cryopreservation solution is that it can be directly reinfused without elution. Therefore, this was taken into consideration in the experimental design. The experimental group did not wash out the solution after recovery and directly simulated the reinfusion scenario. The test was conducted on the toxicity of the cryopreservation solution components when they coexist with the cells.

[0186] Data shows that even without elution, the relative proliferation rate is still ≥91%, indicating that the components in the cryopreservation solution (bionic ice-controlling peptides, natural deep eutectic solvents, etc.) are themselves non-toxic to cells and will not inhibit cell proliferation due to residues, fully meeting the safety requirements of "direct reinfusion".

[0187] In summary: The "proliferative capacity of cryopreserved cells" was measured using the CCK-8 assay, and the calculated "relative proliferation rate" was all above 91%, far exceeding the requirement of "Grade 1 non-toxic (≥80%)" in the international standard (ISO 10993-5). Therefore, it can be directly judged as Grade 1 - the whole process is based on standards and supported by data.

Claims

1. A novel cryoprotectant, characterized in that, Includes the following ingredients: Ice crystal inhibitor, comprising 0.01-0.05% by mass of the protective agent; Natural deep eutectic solvent, accounting for 5-15% by mass of the protective agent; Non-permeable protective agent, accounting for 1.37-2.05% of the total protective agent by mass; Cell membrane stabilizers, comprising 0.05-0.2% by weight of the protectant; Antioxidants, accounting for 0.031-0.154% by mass of the protective agent; An isotonic conditioner, comprising 0.6-0.8% by mass of the protective agent; The buffer system comprises 0.238-0.596% of the protective agent by mass. The solvent accounts for 81.15-92.701% of the protective agent by mass.

2. The novel cryoprotectant according to claim 1, characterized in that: The ice crystal inhibitor is a biomimetic ice-controlling peptide, the non-permeable protective agent is trehalose, the cell membrane stabilizer is fucoidan, the antioxidant is glutathione, the isotonic regulator is sodium chloride, the buffer system is HEPES buffer, and the solvent is water for injection.

3. The novel cryoprotectant according to claim 2, characterized in that: The natural deep eutectic solvent is a choline-proline natural ionic liquid, the biomimetic ice-controlling peptide is an antifreeze protein functional peptide, and the antioxidant is reduced glutathione.

4. A method for preparing a novel cryoprotectant, characterized in that, Includes the following steps: S1 environment and solvent: In a B+A cleanroom environment, water for injection is used as the solvent. S2 Natural Deep Eutectic Solvent Pre-preparation: Accurately weigh choline and L-proline in a molar ratio of 1:1, add 20% water for injection, and stir at 60°C until a clear and transparent homogeneous liquid natural deep eutectic solvent is formed. S3 Dissolution and Mixing: Take 70% water for injection, and add precisely weighed trehalose, fucoidan, HEPES buffer, sodium chloride, and reduced glutathione in sequence while stirring. Continue stirring until completely dissolved. Then add natural deep eutectic solvent and stir evenly. Next, add biomimetic cryoprotectant and stir at low speed to fully dissolve it. After fully dissolving, add the remaining water for injection and stir evenly to obtain the cryoprotectant. S4 pH adjustment: Adjust the pH of the cryoprotectant to a suitable value using dilute sodium hydroxide solution or dilute hydrochloric acid solution.

5. The method for preparing a novel cryoprotectant according to claim 4, characterized in that, It also includes the following steps: S5 sterilization and dispensing: Sterilization is achieved by filtration using a 0.22μm polyethersulfone membrane. Under Class A laminar flow protection, the filtrate is aseptically dispensed into sterile, pyrogen-free containers. S6 quality control involves conducting sterility checks, endotoxin tests, osmolality tests, and pH tests on each batch of products, and only those that pass the tests are released.

6. The method for preparing a novel cryoprotectant according to claim 4, characterized in that: In step S3, 70% of the water used for injection is used, and the temperature of the water used for injection is controlled at 25-30℃.

7. The method for preparing a novel cryoprotectant according to claim 4, characterized in that: In step S4, the appropriate pH value is 7.2 ± 0.

1.

8. The method for preparing a novel cryoprotectant according to claim 4, characterized in that: In the cryoprotectant prepared in step S3, the biomimetic cryoprotectant accounts for 0.01-0.05% of the cryoprotectant by mass, the natural deep eutectic solvent accounts for 5-15% of the cryoprotectant by mass, trehalose accounts for 1.37-2.05% of the cryoprotectant by mass, fucoidan accounts for 0.05-0.2% of the cryoprotectant by mass, glutathione accounts for 0.031-0.154% of the cryoprotectant by mass, sodium chloride accounts for 0.6-0.8% of the cryoprotectant by mass, HEPES buffer accounts for 0.238-0.596% of the cryoprotectant by mass, and water for injection accounts for 81.15-92.701% of the cryoprotectant by mass.

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