A low-toxicity complex cryopreservation solution for clinical infusion-grade NK cells, a cryopreservation method and application thereof

Abstract

The application discloses a clinical infusion grade NK cell low-toxicity complex cryopreservation solution, a cryopreservation method and application, and after resuscitation, the NK cell can be directly infused without centrifugal washing. The special static / low-frequency alternating magnetic field is matched with the low-toxicity cryopreservation solution, and the two form a synergistic protection system, which can further reduce the freezing and thawing ice crystal damage and inhibit the oxidative stress and apoptosis. In the low DMSO system, the resuscitation activity, the phenotype stability and the in-vitro killing function of the NK cell are further improved. The technical short board that the intracellular ice crystal is easily generated under the low-concentration osmotic protective agent and the cell function slowly decays in long-term liquid nitrogen storage is solved. The application has the advantages of being free of animal source, low toxicity, high efficiency, simple preparation process, high patent barrier and the like, and is suitable for the large-scale cryopreservation and treatment application of the clinical grade NK cell.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy product preparation and cryopreservation technology, specifically relating to a clinical infusion-grade NK cell low-toxicity composite cryopreservation solution, cryopreservation method and application. Background Technology

[0002] Natural killer cells (NK cells), as core effector cells of the innate immune system, possess both non-specific broad-spectrum anti-tumor and anti-infective activities. They do not require MHC-restricted recognition of target cells and have extremely high application value in clinical fields such as hematologic malignancies, immunotherapy for solid tumors, antiviral infections, and immune reconstitution after hematopoietic stem cell transplantation. After large-scale in vitro expansion, NK cells require cryopreservation for long-term storage, batch release testing, and cross-regional clinical infusion. The composition and performance of the cryopreservation solution directly determine the survival rate, phenotypic stability, biological function, and clinical efficacy of NK cells after thawing.

[0003] Existing commercially available and literature-reported cryopreservation solutions generally suffer from the following core defects: First, their protection systems are singular, often covering only one or two protective mechanisms, failing to comprehensively resist multiple damages during freeze-thaw cycles, including mechanical damage from ice crystals, oxidative stress damage, nucleic acid breakage, and aggregation and adhesion caused by loss of cell surface charge. Second, the addition of dimethyl sulfoxide (DMSO) is generally as high as 10%, and high concentrations of DMSO have significant cytotoxicity, easily leading to abnormal NK cell differentiation, apoptosis, necrosis, and loss of surface markers. Furthermore, clinical infusion of cell products with high DMSO content can easily cause allergic reactions, neurotoxicity, and liver and kidney damage in patients. Third, most cryopreservation solutions use cell culture medium as a solvent, requiring multiple centrifugation and washing after thawing to remove culture medium components before infusion, which is cumbersome and prone to cell loss and contamination risks. Fourth, few studies have combined natural active monomers with traditional cryoprotectants; relying solely on chemically synthesized excipients is insufficient to further improve cryopreservation efficacy. Summary of the Invention

[0004] To address the problems in the existing technology, the present invention aims to provide a clinical infusion-grade low-toxicity composite cryopreservation solution for NK cells, a cryopreservation method and application. It can be directly infused without washing, has a low DMSO content, covers four major protection mechanisms, and is equipped with a specific magnetic field-assisted cryopreservation process, which can achieve dual protection of chemical protection and physical magnetic field, and the cryopreservation protection effect is particularly excellent.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A clinically infusion-grade low-toxicity compound cryopreservation solution for NK cells comprises the following components: trehalose 2%–4% by volume, dimethyl sulfoxide 1%–4% by volume, hydroxyethyl starch 3%–6% by volume, dextran 5%–10% by volume, PEG6000 5%–10% by volume, recombinant human serum albumin 0.5%–2% by volume, α-1,4-glucan 2%–4% by volume, chlorogenic acid 1.0%–1.5% by volume, tanshinone IIA 5–20 μM, and the remainder being physiological saline.

[0007] Preferably, it is composed of the following components: 3% by weight and volume of trehalose, 2% by weight and volume of dimethyl sulfoxide, 4.5% by weight and volume of hydroxyethyl starch, 7.5% by weight and volume of dextran, 7.5% by weight and volume of PEG6000, 1.0% by weight and volume of recombinant human serum albumin, 3.5% by weight and volume of α-1,4-glucan, 1.5% by weight and volume of chlorogenic acid, 10 μM molar concentration of tanshinone IIA, and the balance being physiological saline.

[0008] This invention also provides a method for preparing the above-mentioned clinical infusion-grade NK cell low-toxicity composite cryopreservation solution, comprising the following steps:

[0009] In a Class 100 cleanroom environment, a portion of physiological saline was taken and trehalose, hydroxyethyl starch, dextran, PEG6000, recombinant human serum albumin, and α-1,4-glucan were added sequentially. The mixture was stirred at 100-150 rpm for 30-60 minutes at room temperature until all components were completely dissolved and the solution was clear and free of precipitate, thus obtaining mixture A.

[0010] S2 slowly adds dimethyl sulfoxide dropwise to mixture A while stirring continuously. After the addition is complete, continue stirring for 10 minutes. Then add chlorogenic acid powder and tanshinone IIA mother liquor and continue stirring for 15 minutes to obtain mixture B.

[0011] S3 adds the remaining physiological saline to mixture B to the target volume, mixes thoroughly, and then filters under positive pressure through a 0.22 μm organic solvent-resistant filter membrane for sterilization.

[0012] This invention also provides a method for cryopreservation of NK cells using the aforementioned clinical infusion-grade low-toxicity composite cryopreservation solution in conjunction with magnetic field, comprising: resuspending logarithmically growing NK cells in pre-cooled cryopreservation solution to a cell density of 1×10⁻⁶. 6 ~1×10 7 The sample is stored in liquid nitrogen at a density of 1 / mL. After being cooled to -80℃ under magnetic field conditions, it is transferred to liquid nitrogen for storage. The magnetic field is a static magnetic field of 0.3~1.0T or a low-frequency alternating magnetic field of 10~50Hz, and the cooling rate is maintained at -1℃ / min.

[0013] Preferably, the programmed cooling under magnetic field conditions includes: placing the cryopreservation tube in a programmed cooling device with a built-in magnetic field coil / external permanent magnet, and applying a constant magnetic field throughout the process.

[0014] More preferably, the magnetic field is a 0.6T static magnetic field.

[0015] This invention also provides the application of the above-mentioned clinical infusion-grade NK cell low-toxicity composite cryopreservation solution or the above-mentioned method in the preparation of clinical infusion-grade NK cell therapeutic products.

[0016] Preferably, during resuscitation, the cryopreservation tubes are rapidly thawed using a 37°C water bath, and the cell suspension is directly diluted with physiological saline for clinical infusion without the need for centrifugation or washing.

[0017] The beneficial effects of this invention are as follows:

[0018] A novel cryopreservation solution with excellent freezing performance is provided, which has the following advantages:

[0019] (i) A comprehensive, synergistic protective system integrating "anti-ice crystal damage, cell function protection, nucleic acid integrity protection, and cell membrane charge stability" was constructed. The functions of each component are as follows:

[0020] Anti-ice crystal damage system: Intracellular permeable protectant: dimethyl sulfoxide, which can penetrate the cell membrane and enter the cell, lowering the freezing point of the intracellular liquid and reducing the formation of intracellular ice crystals; Extracellular non-permeable protectant: hydroxyethyl starch, dextran, and α-1,4-glucan, which work together to form a dense hydration protective film outside the cell, regulating the osmotic pressure of the cryopreservation system, inhibiting ice crystal nucleation and recrystallization, and avoiding mechanical puncture damage to the cell membrane caused by ice crystals.

[0021] Cell function protection system: Trehalose, a small-molecule, non-permeable protective agent, stabilizes the phospholipid bilayer structure of the cell membrane through hydrogen bonding, preventing membrane protein denaturation and structural damage, while also protecting the activity of intracellular proteins and enzymes. PEG6000, a high-molecular-weight polymer, can form an osmotic pressure gradient outside the cell, reducing cell dehydration and shrinkage, and also has a certain cell membrane stabilizing effect. Recombinant human serum albumin: a multifunctional protective agent that can bind to and remove toxic substances generated during freeze-thaw cycles, maintaining cell membrane integrity while providing essential nutritional support to the cells.

[0022] Nucleic acid integrity protection system: Tanshinone IIA: a natural small molecule active substance with strong antioxidant and anti-apoptotic effects. It can reduce DMSO-induced apoptosis by inhibiting the mitochondrial apoptosis pathway, while clearing intracellular reactive oxygen species (ROS), reducing DNA strand breaks caused by oxidative stress, and protecting the integrity of the cell genome.

[0023] Cell membrane charge stabilization system: Recombinant human serum albumin: Its molecular surface carries a large number of negative charges, which can bind to positively charged sites on the surface of NK cells through electrostatic interactions, maintaining the negative charge density of the cell surface and preventing cell aggregation and adhesion due to charge neutralization. Chlorogenic acid: A natural phenolic compound with significant free radical scavenging ability. Its molecular surface also carries a negative charge, which can synergistically work with recombinant human serum albumin to maintain cell surface charge balance, further reducing cell aggregation rate.

[0024] (ii) Animal-free, ultra-low DMSO design significantly improves safety: The amount of DMSO used is reduced to 1%~4%, which is only 1 / 10~1 / 3 of the traditional formula. Combined with the synergistic detoxification effect of natural antioxidants, it significantly reduces the risk of cytotoxicity and adverse reactions during clinical infusion. All components are free of animal-derived ingredients and meet the requirements of the "Guidelines for Quality Management of Cell Therapy Products".

[0025] (iii) Convenience and safety of clinical infusion: All components are clinically infusion-grade raw materials with osmotic pressure completely isotonic with human plasma; no centrifugation or washing is required after resuscitation, and it can be directly diluted and infused, greatly simplifying the clinical operation process and reducing cell loss and contamination risks.

[0026] Based on the aforementioned clinical infusion-grade low-toxicity composite cryopreservation solution for NK cells, this invention further innovates by introducing a low-temperature magnetic field-assisted cryopreservation process. A dedicated static / low-frequency alternating magnetic field is combined with this low-toxicity cryopreservation solution, forming a synergistic protective system that, together with the chemical components, creates a five-layered protective layer.

[0027] Water is a diamagnetic substance. A static magnetic field of 0.3~1T (or a low-frequency alternating magnetic field of 10~50Hz) can change the hydrogen bond association state of water molecules, increase the supercooling of the system, induce the simultaneous formation of a large number of micron-sized ice crystals, and inhibit the growth of large-sized sharp ice crystals. In the low-temperature range, the magnetic field accelerates the outward permeation of intracellular free water, reduces intracellular water retention, and further reduces the probability of intracellular ice formation under low DMSO conditions. It also works synergistically with polysaccharide extracellular protectants to block mechanical puncture damage.

[0028] Magnetic fields can induce the directional and orderly arrangement of cell membrane phospholipid molecules, enhance the toughness of the membrane structure, and strengthen the fixation effect of trehalose on membrane proteins; reduce the shedding of functional receptors (NKG2D, CD56) on the membrane surface during cryopreservation, and stabilize the expression of functional molecules on the surface of NK cells.

[0029] Magnetic fields can stabilize mitochondrial transmembrane potential, reduce electron leakage and ROS bursts, amplify the scavenging capacity of tanshinone IIA and chlorogenic acid free radicals, inhibit caspase apoptosis pathway activation, reduce DNA breakage and late-stage apoptosis rates after cryopreservation, and improve genome integrity under long-term liquid nitrogen storage.

[0030] The uniform external magnetic field weakens the electrostatic adsorption effect between cells. Combined with the negative charge protection of albumin and chlorogenic acid, the cell suspension has better dispersibility after resuscitation, and the cell aggregation rate decreases by more than 60%, reducing the problems of infusion blockage and decreased in vivo colonization efficiency caused by cell clumps.

[0031] This invention utilizes a multi-component synergistic cryopreservation system to achieve NK cell resuscitation viability ≥99% after long-term liquid nitrogen storage, with a proliferation rate exceeding 4.0-fold after 72 hours of resuscitation and CD3⁻CD56⁺ NK cell phenotype retention ≥99%. These cells exhibit significant killing effects on target cells, fully meeting the requirements for clinical treatment and basic research. With the addition of a magnetic field-assisted process, various indicators are further optimized: immediate resuscitation viability after 30 days of liquid nitrogen cryopreservation increases by 2%–3%; proliferation rate increases by 0.4–0.8-fold after 72 hours of resuscitation; long-term stability of the CD3⁻CD56⁺ phenotype improves by 1%–3%; and equivalence-to-target killing activity increases by 7%–13%. Functional decline is significantly slowed after 12 months of long-term storage, further meeting the quality control requirements for long-term sample retention and bulk stockpiling of cell therapy products.

[0032] Furthermore, the preparation process of this invention is simple, requiring no complex equipment or reaction conditions. Preparation can be completed at room temperature, raw materials are readily available and costs are controllable, batch-to-batch stability is good, and clinical-grade large-scale production is possible. The finished product can be stored for short periods at 4°C, making it convenient to use without the need for immediate preparation. The magnetic field-assisted process only requires adding permanent magnets or electromagnetic coils to existing programmed cooling equipment, without altering the cryopreservation solution preparation process or increasing reagent and consumable costs. It only slightly increases equipment investment, making it suitable for standardized, batch cryopreservation production in cell banks. Attached Figure Description

[0033] Figure 1 The results show the changes in NK cell viability at different times after thawing under the influence of the six cryopreservation formulations in Example 2.

[0034] Figure 2 This is a graph showing the results of detecting changes in the proliferation capacity of NK cells after thawing under the influence of the six cryopreservation formulations in Example 2;

[0035] Figure 3 The graph shows the results of detecting the killing activity of NK cells against K562 target cells treated with the six cryopreservation formulations in Example 2.

[0036] Figure 4 This is a flow cytometry result of the CD3⁻CD56⁺ phenotype of NK cells before cryopreservation in Example 2;

[0037] Figure 5 This is a flow cytometry result of the phenotype of NK cells in the CS10 group 30 min after resuscitation in Example 2;

[0038] Figure 6This is a flow cytometry result of the phenotype of NK cells in the CS10 group 72 h after resuscitation in Example 2;

[0039] Figure 7 This is a flow cytometry result of the phenotype of NK cells in the optimal experimental group 30 min after resuscitation in Example 2;

[0040] Figure 8 This is a flow cytometry result of the phenotype of NK cells in the optimal experimental group 72 h after resuscitation in Example 2;

[0041] Figure 9 This is a graph showing the results of the viability change of NK cells after long-term cryopreservation in Example 2;

[0042] Figure 10A This is a graph showing the cell viability results after thawing following cryopreservation under a magnetic field in Example 3;

[0043] Figure 10B This is a graph showing the intracellular ROS oxidative stress levels of cells thawed after being cryopreserved under a magnetic field in Example 3.

[0044] Figure 10C This is a graph showing the phenotypic results of CD3⁻CD56⁺ cells thawed after being cryopreserved under a magnetic field in Example 3.

[0045] Figure 10D This is a diagram showing the killing effect of cells thawed after being cryopreserved under a magnetic field in Example 3 on K562 cells. Detailed Implementation

[0046] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0047] Example 1

[0048] 1.1 Experimental Materials

[0049] Cell line: Human peripheral blood-derived NK cells: obtained by in vitro induction and expansion of peripheral blood mononuclear cells (PBMCs), K562 human chronic myeloid leukemia cells;

[0050] Reagents: Physiological saline (pharmaceutical grade), trehalose (cell culture grade), dimethyl sulfoxide (DMSO, cell culture grade), hydroxyethyl starch (pharmaceutical grade), dextran (pharmaceutical grade), polyethylene glycol 6000 (PEG6000, cell culture grade), recombinant human serum albumin (pharmaceutical grade, animal-free), α-1,4-glucan (molecular weight 10-100kDa, analytical grade), chlorogenic acid (purity ≥98%, analytical grade), tanshinone IIA (purity ≥98%, analytical grade), acridine orange / propidium iodide (AO / PI) staining solution, calcein AM, APC-labeled anti-human CD3 antibody, PE-labeled anti-human CD56 antibody, 0.22 μm organic solvent resistant filter membrane (PES / PTFE / nylon membrane), sterile cell cryopreservation tubes (2 mL).

[0051] 1.2 Experimental Methods

[0052] A 9-factor, 3-level orthogonal experimental design was used to optimize the concentration of each component in the cryopreservation solution. The factors and levels are shown in Table 1.

[0053] Table 1 Orthogonal Experiment Table

[0054]

[0055] This embodiment discloses an animal-free low-DMSO cryopreservation solution for NK cells, which is the optimal formula obtained by orthogonal experiment screening. It can effectively improve the viability, proliferation capacity and killing activity of NK cells after cryopreservation and thawing. It is suitable for the cryopreservation of clinical-grade NK cells. After 27 sets of orthogonal experiments and range analysis, the optimal combination of each factor was determined to be A2B2C2D2E2F2G2H2I2, as shown in Table 2.

[0056] Table 2. Experimental Results and Analysis of Orthogonal Structure

[0057]

[0058] The optimal formulation of this invention:

[0059] The preparation method comprises the following steps: trehalose 3%, dimethyl sulfoxide 2%, hydroxyethyl starch 4.5%, dextran 7.5%, PEG6000 7.5%, recombinant human serum albumin 1.0%, α-1,4-glucan 3.5%, chlorogenic acid 1.25%, tanshinone IIA 10 μM, and the balance being physiological saline.

[0060] In a Class 100 cleanroom environment, 60 mL of physiological saline was placed in a sterile, pre-cooled container. 3 g of trehalose, 4.5 g of hydroxyethyl starch, 7.5 g of dextran, 7.5 g of PEG6000, 1.0 g of recombinant human serum albumin, and 3.5 g of α-1,4-glucan were added sequentially. The mixture was stirred at room temperature for 45 min until completely dissolved. 2 mL of dimethyl sulfoxide was slowly added dropwise, and the mixture was stirred for 10 min. 1.25 g of chlorogenic acid powder was added, followed by tanshinone IIA stock solution, adjusting the final concentration to 10 μM. The mixture was stirred for another 15 min. Physiological saline was added to bring the total volume to 100 mL. The mixture was then filtered through a 0.22 μm organic solvent-resistant membrane (PES / PTFE / nylon membrane) for sterilization, aliquoted, and stored at 4°C protected from light.

[0061] The detailed steps for cryopreservation and thawing are as follows:

[0062] (1) Steps for cryopreserving NK cells:

[0063] S1: Collect NK cells that have been expanded to the logarithmic growth phase in vitro, centrifuge at 300 g for 5 min, discard the supernatant, wash the cells once with sterile PBS, centrifuge again and discard the supernatant.

[0064] S2: Pre-freezing solution, at a cell density of 1×10⁻⁶ 6 -1×10 7 For cells per mL, resuspend the cell pellet in pre-cooled cryopreservation solution, gently pipette to mix, and dispense 1-1.5 mL into sterile cell cryopreservation tubes.

[0065] S3: Place the cryovials in a programmed cooling box and cool them to -80°C at a rate of -1°C / min. After standing for 8-12 hours, quickly transfer them to a liquid nitrogen tank for long-term storage in the liquid phase environment.

[0066] (2) NK cell resuscitation steps:

[0067] S1: Remove the cell cryopreservation tube from the liquid nitrogen tank and quickly place it in a 37°C constant temperature water bath. Gently shake the cryopreservation tube to allow the cryopreservation solution to thaw completely within 1-2 minutes.

[0068] S2: Transfer the thawed cell suspension directly to a sterile infusion bag, add physiological saline to dilute to the required volume, and it can be used directly for clinical intravenous infusion without centrifugation or washing to remove cryopreservation components, thus completing cell resuscitation.

[0069] Cryopreservation validation: P2 generation NK cells from the same batch were cryopreserved using the above-mentioned optimal formula. After 30 days of liquid nitrogen storage, the cells were thawed and the immediate cell viability reached 99.6%, which is superior to the commercially available CS10 cryopreservation solution (92.3%). It can effectively maintain the proliferation capacity, cell purity and cytotoxic activity of NK cells, and has no animal-derived components, meeting the requirements for clinical application.

[0070] Example 2 Functional Identification System of NK Cell Cryopreservation Solution

[0071] To verify the safety and efficacy of the cryopreservation solution of the present invention in clinical application, a four-in-one functional identification system of "resuscitation survival rate, proliferation capacity, phenotypic stability, and preservation of cytotoxic function" was established. The five formulation groups with the highest survival rate in the orthogonal experiment (corresponding to test numbers 15, 16, 17, 18, and 26 in Table 2) were selected, and CS10 cryopreservation solution was used as the control group. The proliferation capacity, phenotypic stability, and cytotoxic function of the above six cryopreservation solutions were comprehensively identified.

[0072] (1) Cell resuscitation viability assessment

[0073] The acridine orange / propidium iodide (AO / PI) fluorescence staining method was used, and the Countstar counter was used to detect the immediate viability of NK cells after resuscitation. A viability of ≥90% was used as the qualified standard.

[0074] Take cryopreserved NK cells stored in liquid nitrogen for 30 days, and perform cell thawing according to the aforementioned thawing steps. Wash the cells twice with PBS and adjust the cell concentration to 1×10⁻⁶. 6 Cells / mL; Take 10 μL of cell suspension and mix it with 10 μL of AO / PI staining solution. Add the stained cell suspension to the corresponding channel of the Countstar counter. The instrument will automatically identify the number of live and dead cells and calculate and read the cell viability.

[0075] The test results show that: from Figure 1 As can be seen, the optimal experimental group (3% trehalose + 2% DMSO + 4.5% hydroxyethyl starch, 7.5% dextran, 7.5% PEG6000, 1.0% recombinant human serum albumin, 3.5% α-1,4-glucan, 1.25% chlorogenic acid, 10 μM tanshinone IIA) had cell viability of 99.60%, 96.49%, 96.56%, and 97.64% at 30 min, 24 h, 48 h, and 72 h after thawing, respectively, maintaining a high level above 96% throughout the process. Moreover, the decline in viability was much lower than that of the control group and other formulation groups, indicating that this formulation can significantly reduce the acute damage to NK cells during the cryopreservation and thawing process.

[0076] (2) Identification of cell proliferation capacity

[0077] The proliferation capacity of NK cells after resuscitation was detected by CCK-8 assay and cell counting method, and the qualification standard was a proliferation ≥3.0 times at 72 h after resuscitation.

[0078] Harvest NK cells after resuscitation and adjust the cell concentration to 1×10⁻⁶. 6Cells were seeded at a density of 1 / mL into 24-well plates, and NK cell complete culture medium was added. The plates were then incubated at 37°C in a 5% CO2 incubator. Cell counts were taken every 24 hours, and the plates were continuously incubated for 72 hours. Cell counts were recorded, and the fold increase was calculated. At the same time, CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader to assess cell proliferation activity.

[0079] The test results show that: from Figure 2 As can be seen, the optimal experimental group (3% trehalose + 2% DMSO + 4.5% hydroxyethyl starch, 7.5% dextran, 7.5% PEG6000, 1.0% recombinant human serum albumin, 3.5% α-1,4-glucan, 1.25% chlorogenic acid, 10 μM tanshinone IIA) showed a cell density that increased from the initial 1×10⁻⁶ cells / mL after resuscitation. 6 The concentration of cells / mL recovered rapidly and remained stable at 0.95 × 10⁻⁶ for 24 hours. 6 The concentration of cells / mL increased to 2.32 × 10⁻⁶ after 48 hours, entering the exponential growth phase. 6 The concentration of cells / mL reached 4.69 × 10⁻⁶ at 72 h and 96 h, respectively. 6 Cells / mL (4.69-fold amplification) and 5.49 × 10⁻⁶ 6 The cell density / mL (5.49-fold amplification) was the highest among all groups, indicating that this cryopreservation solution can preserve the proliferative potential of NK cells to the greatest extent while protecting the high viability of cells. It is superior to the traditional CS10 cryopreservation solution and other orthogonal formulations, and has a better clinical-grade NK cell cryopreservation protection effect.

[0080] (3) Identification of cell phenotype stability

[0081] The CD3-CD56+ phenotype positivity rate of NK cells after resuscitation was detected by flow cytometry. The retention rate of CD3-CD56+ phenotype could be maintained above 99% at 30 min and 72 h after resuscitation, and the cells could significantly exert a killing effect on target cells as the qualified standard.

[0082] Take NK cells that have been cultured for 72 h after resuscitation, wash twice with PBS, centrifuge at 300 g for 5 min, discard the supernatant, and adjust the cell concentration to 1×10⁻⁶. 6 Cell suspension was obtained at a density of 100 μL / mL. 2 μL of APC-labeled anti-CD3 antibody and 2 μL of PE-labeled anti-CD56 antibody were added to each tube, and the cells were incubated in the dark on ice for 30 min. The cells were washed twice with 1 mL of PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL of PBS and analyzed by flow cytometry. Fresh, unfrozen NK cells were used as a blank control. Viable cell populations were selected, and the proportion of CD3⁻CD56⁺ double-positive cells was analyzed.

[0083] The test results show that: from Figure 4 , Figure 5 and Figure 6 The data shows that before cryopreservation, the CD3⁻CD56⁺ cell count was 96.67%. Figure 5 and Figure 6 The results show that after 30 min and 72 h of CS10 cryopreservation, the percentages of CD3⁻CD56⁺ cells were 96.24% and 96.44%, respectively. Figure 7 and Figure 8 The results show that the optimal experimental group had 96.02% and 98.02% CD3⁻CD56⁺ cells after cryopreservation and thawing for 30 min and 72 h, respectively. Neither the CS10 cryopreservation solution nor the optimal experimental group in this application had a significant impact on the NK cell phenotype after cryopreservation and thawing; the phenotype remained essentially the same as before cryopreservation. This indicates that the cryopreservation solution of this invention can effectively protect the characteristic phenotype of NK cells and is more conducive to maintaining the phenotypic stability and dominance of NK cells after thawing.

[0084] (4) Identification of cell killing function preservation

[0085] The cytotoxic activity of NK cells after resuscitation was detected by the calcein release assay. K562 cells were used as the target cells, with an effector-to-target ratio of 4:1. After co-culturing for 4 hours, the retention rate of NK cell cytotoxic function against K562 target cells ≥50% was considered as the qualified standard.

[0086] Take K562 target cells in the logarithmic growth phase and adjust the concentration to 1×10⁻⁶. 6 Add calcein-AM working solution to a final concentration of 5 μmol / L, incubate at 37°C in the dark for 30 min, add complete culture medium to terminate staining, centrifuge, wash and resuspend; seed the labeled target cells into 96-well plates, 5 × 10⁶ cells per well. 3 Add NK cells that have been revived and cultured for 72 h, and set effector-to-target ratios of 0.5:1, 1:1, 2:1, and 4:1, with 3 replicates per group. Also set up wells for natural release of target cells and wells for maximum release of target cells. After culturing at 37℃ and 5% CO2 for 4 h, centrifuge and collect the supernatant. Detect the fluorescence intensity at 485 nm and 530 nm using an ELISA reader, and calculate the killing rate and the retention rate of killing function.

[0087] The formula for calculating the lethality rate is: Lethality rate (%) = (Fluorescence value of the experimental group - Fluorescence value of the naturally released group) / (Fluorescence value of the maximum release group - Fluorescence value of the naturally released group) × 100%

[0088] The test results show that: from Figure 3As can be seen, under effector-to-target ratios of 0.5:1, 1:1, 2:1, and 4:1, the cytotoxic activity of NK cells in each group increased with increasing effector-to-target ratio, exhibiting a dose-dependent effect. Among them, the optimal experimental group (3% trehalose + 2% DMSO + 4.5% hydroxyethyl starch, 7.5% dextran, 7.5% PEG6000, 1.0% recombinant human serum albumin, 3.5% α-1,4-glucan, 1.25% chlorogenic acid, 10 μM tanshinone IIA) showed the highest cytotoxicity at all effector-to-target ratios. Even at a 4:1 effector-to-target ratio 72 hours after resuscitation, the cytotoxicity still reached 75%, significantly higher than the CS10 control group and other orthogonal formulation groups. This indicates that after cryopreservation and resuscitation, the NK cells in the optimal formulation group of this invention exhibited the strongest cytotoxic activity at all effector-to-target ratios, and the increase in cytotoxicity with increasing effector-to-target ratio was significantly better than that of the control group and other formulation groups. This indicates that the cryopreservation solution can maximize the protection of the cytotoxic function of NK cells during cryopreservation and thawing, preventing damage to their killing activity and preserving key functional advantages for the anti-tumor effect of clinical-grade NK cells.

[0089] (5) Stability assessment of cryopreservation solution

[0090] The survival rate of NK cells after cryopreservation in cryopreservation solutions with different storage times was detected by using a Countstar fluorescence cell counter combined with AO / PI staining, thus verifying the storage stability and long-term cryopreservation protection capability of the cryopreservation solution of the present invention.

[0091] The optimal cryopreservation solution, freshly prepared in Example 1, was aliquoted according to the aforementioned preparation method, cooled to -80°C using a programmed freezing device, and then stored in liquid nitrogen for long-term preservation. NK cells were retrieved after 1, 2, 3, 6, and 12 months of storage in liquid nitrogen, rapidly thawed according to standard procedures, and cell viability was assessed. Cells were then allowed to equilibrate at room temperature for 30 minutes, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL; 10 μL of cell suspension was mixed with 10 μL of AO / PI staining solution, and the cell resuscitation viability was detected using a Countstar fluorescence cell counter. At the same time, CS10 cryopreservation solution (cells stored under the same conditions and from the same batch) was used as a control group for simultaneous detection.

[0092] The cell viability before cryopreservation was 98.92%. NK cells were placed in liquid nitrogen for 1 month, 2 months, 3 months, 6 months, and 12 months, and then recovered and their viability was tested. The results are as follows: Figure 9 As shown, both the optimal cryopreservation solution of Example 1 and the commercially available cryopreservation solution CS10 (control group) showed good long-term protection for NK cells, with no significant difference. Even though the DMSO content in the CS10 cryopreservation solution was as high as 10%, while the DMSO content in the present invention patent was only 2%, it still provided good protection for NK cells.

[0093] Example 3: Standalone Validation Test of Magnetic Field Assisted Cryopreservation Process

[0094] This embodiment independently verifies the effect of different magnetic field parameters combined with the optimal low DMSO cryopreservation solution of the present invention on the cryopreservation effect of NK cells, clarifies the differences in the effect of the optimal magnetic field parameter range and the time of magnetic field application, and compares four groups: no magnetic field, weak magnetic field, optimal magnetic field, and ultra-strong magnetic field, to quantify the improvement of NK cell resuscitation survival rate, oxidative stress level, killing function, and long-term storage stability by the magnetic field.

[0095] 3.1 Experimental Materials

[0096] Cells: Human peripheral blood amplified NK cells and target cells K562.

[0097] Cryopreservation solution: The optimal cryopreservation solution of this invention (3% trehalose + 2% DMSO + 4.5% hydroxyethyl starch, 7.5% dextran, 7.5% PEG6000, 1.0% recombinant human serum albumin, 3.5% α-1,4-glucan, 1.25% chlorogenic acid, 10 μM tanshinone IIA);

[0098] Magnetic field equipment: Adjustable static magnetic field programmed cooling module (magnetic field strength 0~1.5T continuously adjustable), magnetic field strength detector;

[0099] Detection reagents: AO / PI viability staining, ROS fluorescence detection kit, CD3 / CD56 flow cytometry antibody, calcein AM killing assay kit.

[0100] 3.2 Experimental Group Design

[0101] All groups used the optimal cryopreservation solution of this invention and a cell cryopreservation density of 1×10⁻⁶. 7 Standard temperature program cooling at 1 mL / mL and -1℃ / min, differentiated only by magnetic field conditions, with a total of 4 parallel experimental groups:

[0102] Group A (blank control group): No magnetic field throughout;

[0103] Group B (weak magnetic field group): 0.1T static magnetic field throughout;

[0104] Group C (Medium Magnetic Field Group): 0.6T static magnetic field throughout;

[0105] Group D (Ultra-high magnetic field group): 1.2T static magnetic field throughout;

[0106] Each group had 3 biological replicates, and after cryopreservation, the samples were stored in liquid nitrogen for 30 days and then uniformly revived for testing.

[0107] 3.3 Magnetic Field Operation Procedure

[0108] The steps for centrifuging NK cells, resuspending them in pre-frozen storage solution, and aliquoting them into cryovials are the same as the standard cryopreservation procedure described above. The cryovials are evenly placed in the magnetic field chamber of the programmed cooling instrument to ensure that the magnetic field strength of all cryovials is uniform and without gradient difference. After the instrument reaches room temperature equilibrium for 5 minutes, the magnetic field module is turned on and the programmed cooling is started simultaneously, with the magnetic field output continuously throughout the process. After cooling to -80℃, the magnetic field is turned off first, and then the cryovials are removed and transferred to liquid nitrogen for long-term storage.

[0109] 3.4 Test Results

[0110] Cell viability, intracellular ROS oxidative stress level, CD3-CD56+ cell phenotype, and in vitro killing level were measured after cryopreservation and thawing. Figure 10A The results of cell viability testing after cell resuscitation showed that the 0.1T weak magnetic field had little effect on cell viability (only the optimal formula of this patent and CS10 cryopreservation solution increased cell viability by <0.3%). The 0.6T magnetic field increased cell viability by 2% compared to the group without a magnetic field. The 1.2T ultra-high magnetic field, due to its excessive intensity, led to aggravated cell damage and a significant decrease in cell viability. Figure 10B The results showed that the intracellular ROS oxidative stress level (the lower the ROS fluorescence intensity value, the less oxidative damage) was measured. Under a 0.6T magnetic field, the ROS fluorescence intensity decreased by 39.72% (this patented formula) and 33.95% (CS10 cryopreservation solution) compared with the group without a magnetic field. Under a high magnetic field, oxidative stress increased significantly and induced apoptosis. Figure 10C The results of CD3-CD56+ cell phenotype detection after cell resuscitation showed that NK cell characteristic phenotype was best preserved under a 0.6T magnetic field and membrane receptor shedding was significantly reduced, while cell surface antigens were lost in large quantities under a 1.2T ultra-high magnetic field and NK cell function was impaired. Figure 10D The results showed that the killing effect of NK cells on K562 cells after cell resuscitation was better maintained under a 0.6T magnetic field, and the killing efficiency was increased by 11.9% (this patented formula) and 9.0% (CS10 cryopreservation solution) compared with the group without a magnetic field.

[0111] The 0.6T full-process magnetic field, combined with the 2% low DMSO composite cryopreservation solution of this invention, can synergistically reduce intracellular ROS, stabilize cell membranes and NK functional surface antigens, and inhibit mechanical damage from ice crystals. The short-term recovery survival rate and in vitro killing ability are significantly better than the cryopreservation solution without magnetic field process. The magnetic field is a purely physical auxiliary means, does not change the composition of the cryopreservation solution, does not require the addition of additional chemical reagents, and does not increase the risk of clinical infusion toxicity. It is suitable for standardized and large-scale cryopreservation production of clinical-grade NK cells.

[0112] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. A clinically infusion-grade, low-toxicity compound cryopreservation solution for NK cells, comprising the following components: The composition is as follows: trehalose 2%–4% by volume, dimethyl sulfoxide 1%–4% by volume, hydroxyethyl starch 3%–6% by volume, dextran 5%–10% by volume, PEG6000 5%–10% by volume, recombinant human serum albumin 0.5%–2% by volume, α-1,4-glucan 2%–4% by volume, chlorogenic acid 1.0%–1.5% by volume, tanshinone IIA 5–20 μM, with the remainder being physiological saline.

2. The clinical infusion-grade NK cell low-toxicity composite cryopreservation solution according to claim 1, characterized in that, It consists of the following components: trehalose 3% by volume, dimethyl sulfoxide 2% by volume, hydroxyethyl starch 4.5% by volume, dextran 7.5% by volume, PEG6000 7.5% by volume, recombinant human serum albumin 1.0% by volume, α-1,4-glucan 3.5% by volume, chlorogenic acid 1.25% by volume, tanshinone IIA 10 μM, and the balance being physiological saline.

3. The preparation method of the clinical infusion-grade NK cell low-toxicity composite cryopreservation solution according to claim 1, comprising the following steps: In a Class 100 cleanroom environment, a portion of physiological saline was taken and trehalose, hydroxyethyl starch, dextran, PEG6000, recombinant human serum albumin, and α-1,4-glucan were added sequentially. The mixture was stirred at 100-150 rpm for 30-60 minutes at room temperature until all components were completely dissolved and the solution was clear and free of precipitate, thus obtaining mixture A. S2 slowly adds dimethyl sulfoxide dropwise to mixture A while continuously stirring. After the addition is complete, continue stirring for 10 minutes. Then add chlorogenic acid mother liquor and tanshinone IIA mother liquor and continue stirring for 15 minutes to obtain mixture B. S3 adds the remaining physiological saline to mixture B to the target volume, mixes thoroughly, and then filters under positive pressure through a 0.22 μm organic solvent-resistant filter membrane for sterilization.

4. A method for cryopreservation of clinically infusion-grade NK cells with low toxicity composite cryopreservation solution and magnetic field as described in claim 1, comprising: Logarithmic growth phase NK cells were resuspended in pre-chilled cryopreservation solution to a cell density of 1×10⁻⁶. 6 ~1×10 7 The sample is stored in liquid nitrogen at a density of 1 / mL. After being cooled to -80℃ under magnetic field conditions, it is transferred to liquid nitrogen for storage. The magnetic field is a static magnetic field of 0.3~1.0T or a low-frequency alternating magnetic field of 10~50Hz, and the cooling rate is maintained at -1℃ / min.

5. The method according to claim 4, characterized in that, Programmed cooling under magnetic field conditions includes placing the cryovials in a programmed cooling device with a built-in magnetic field coil / external permanent magnet and applying a constant magnetic field throughout the process.

6. The method according to claim 5, characterized in that, The magnetic field used is a 0.6T static magnetic field.

7. The use of the clinical infusion-grade NK cell low-toxicity compound cryopreservation solution according to claim 1 or 2, or the method according to any one of claims 4 to 6, in the preparation of clinical infusion-grade NK cell therapy products.

8. The application according to claim 7, characterized in that, During resuscitation, cryopreservation tubes were rapidly thawed using a 37°C water bath, and the cell suspension was directly diluted with physiological saline for clinical infusion without the need for centrifugation or washing.

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