Immune cell serum-free cryopreservation liquid and preparation method thereof

Abstract

The invention relates to an immune cell serum-free cryopreservation solution and a preparation method thereof, and belongs to the technical field of cell biology and biological pharmacy. According to the cryopreservation liquid, DMSO and DPBS are mixed according to the volume ratio of 10: 90 to form a buffer solution, hydroxyethyl starch, heparin sodium, trehalose, glutamine, HEMA and taurine are added into the buffer solution, a serum-free cryopreservation formula with definite components and high stability is formed, and the preparation method comprises the five steps of accurate component weighing, buffer solution preparation, dissolution and uniform mixing, constant volume and pH adjustment and sterilization and filtration. Wherein the component concentration can be adjusted within a preset interval according to experimental requirements, the cryopreservation liquid is suitable for immune cell preservation experiments under different conditions, the cryopreservation liquid does not contain serum components, the heterologous protein pollution risk can be remarkably reduced, and the survival rate and the biological function retention rate of cryopreserved cells are increased. The cell cryopreservation device is suitable for medium and long term cryopreservation of various immune cells such as CAR-T cells and NK cells, and has good biological safety and clinical application prospects.

Description

Technical Field

[0001] This application relates to the field of general technologies in the biomedical field, and in particular to a serum-free cryopreservation solution for immune cells and a method for preparing the same. Background Technology

[0002] As the basic structural and functional unit of living organisms, cells hold significant research importance in various fields of modern biology. Cell cryopreservation is a common method for preserving and storing cells long-term during cell culture. Its main principle is to place cells in a low-temperature environment to reduce their basal metabolism, inducing a state similar to "hibernation." Cryopreservation solution is an essential solution used in cell cryopreservation.

[0003] Traditional cell cryopreservation and counting methods typically use cryopreservation solutions composed of a specific ratio of cell culture medium (such as 1640, DMEM, etc.), fetal bovine serum, and DMSO (dimethyl sulfoxide). During cryopreservation, the protective effects of DMSO and serum reduce cell damage caused by intracellular fluid crystallization during temperature reduction, which could puncture the cell membrane or organelles. However, because serum is of animal origin, there is a potential risk of animal viruses. Furthermore, the serum composition is complex, and the proportions of its components vary significantly between batches, making quality control difficult and leading to inconsistent results from different batches of cryopreservation experiments. Additionally, traditional cell cryopreservation protocols often require programmed freezing, which is time-consuming, cumbersome, and has limited processing scale.

[0004] During the rise of the immune cell industry, cell therapy products, as an important part of biomedicine, faced significant challenges in ensuring the homogeneity and stability of their cryopreserved components, as well as the safety and stability of their components to meet the needs of clinical research. In the development of immune cell therapy products in recent years, many scholars and scientists have innovatively invented many different types of cryopreservation solutions to optimize traditional cell cryopreservation solutions. Some cryopreservation solutions reduce the proportion of DMSO, thereby reducing the cytotoxicity of DMSO itself, but the cryopreservation effect is often not good. Some cryopreservation solutions replace animal serum in the traditional cryopreservation solution components with plant-derived proteins or serum substitutes. Although this reduces the potential risk of viruses from animal sources, natural proteins or serum substitutes still have problems such as unclear composition and large batch-to-batch differences.

[0005] Regarding the aforementioned technologies, the inventors believe that there are problems such as poor cryopreservation effect, unclear composition of natural proteins or serum substitutes, and excessive batch-to-batch differences. Summary of the Invention

[0006] To address the issues of poor cryopreservation results, unclear composition of natural proteins or serum substitutes, and excessive batch-to-batch variations, this application provides a serum-free cryopreservation solution for immune cells and its preparation method.

[0007] Firstly, the serum-free cryopreservation solution for immune cells provided in this application adopts the following technical solution:

[0008] A serum-free cryopreservation solution for immune cells includes a cryopreservation solution, said cryopreservation solution being a mixture composed of a buffer solution;

[0009] The buffer solution is a mixture of DMSO and DPBS;

[0010] The buffer solution composed of DMSO and DPBS contains hydroxyethyl starch, sodium heparin, trehalose, glutamic acid dipeptide, HEMA80, and taurine.

[0011] By adopting the above technical solution, cryopreservation solution 1 does not contain serum components found in traditional cell cryopreservation solutions, nor does it use similar plant-derived or recombinant proteins. Its composition is clear and stable, ensuring consistent product performance across different batches. Furthermore, the main function of the cell cryopreservation solution of this invention is to protect the cell interior during cryopreservation using DMSO, and to protect the cell exterior using components such as hydroxyethyl starch and trehalose. Simultaneously, the addition of protective agents such as HEMA maintains intracellular and extracellular moisture, thereby significantly reducing damage caused by ice crystals formed during cell cryopreservation. This also increases cell viability after thawing, and the addition of organic solvents makes the external environment of the cells during cryopreservation more stable, effectively improving cell viability and increasing the scale of cell cryopreservation that can be processed.

[0012] Optionally, the buffer solution is composed of a mixture of 10% DMSO and 90% DPBS by volume.

[0013] By adopting the above technical solution, the buffer solution is composed of 10% DMSO and 90% DPBS by volume. While ensuring the cell permeability protection effect, it can significantly reduce the toxicity risk of DMSO to cells, improve the survival rate and functional retention of cells after cryopreservation, and enhance the biosafety and stability of the cryopreservation solution. It is suitable for cell types such as immune cells that are sensitive to the cryopreservation environment.

[0014] Optionally, the buffer solution, composed of a mixture of 10% DMSO and 90% DPBS (by volume), contains 50–150 g / L of hydroxyethyl starch, 1000–3000 U / L of sodium heparin, 10–40 g / L of trehalose, 5–10 g / L of glutamic-peptide, 0.01–1.00 g / L of HEMA80, and 0.1–0.5 g / L of taurine.

[0015] By adopting the above technical solution, adding hydroxyethyl starch, sodium heparin, trehalose, glutamic acid dipeptide, HEMA80, and taurine to a buffer solution composed of 10% DMSO and 90% DPBS can synergistically enhance the protective effect of cells during cryopreservation. Hydroxyethyl starch and trehalose provide physical barriers and osmotic protection, sodium heparin prevents blood clotting, glutamic acid dipeptide and taurine alleviate oxidative damage to cells, and HEMA80 reduces ice crystal formation, thereby significantly improving the viability and functional retention rate of cryopreserved cells.

[0016] Secondly, the method for preparing serum-free cryopreservation solution for immune cells provided in this application adopts the following technical solution:

[0017] A method for preparing a serum-free cryopreservation solution for immune cells includes the following steps:

[0018] Step 1: Accurately weigh the components

[0019] 40g of hydroxyethyl starch, 50g of sodium heparin, 60g of trehalose, 70g of glutamic acid dipeptide, 80g of HEMA and 90g of taurine were accurately weighed and placed according to the corresponding weighing values ​​listed in Appendix 1, and each group was placed separately.

[0020] Step 2: Prepare buffer solution 10

[0021] The solution used to dissolve the components in step one was prepared by mixing 10% DMSO and 90% DPBS by volume.

[0022] Step 3: The accurately weighed components from Step 1 are dissolved and mixed with a dissolving solution to form a cryopreservation solution semi-finished product. Each group of cryopreservation solution semi-finished product is placed separately after being dissolved and mixed.

[0023] Step 4: After dissolving and mixing all the components, the semi-finished cryopreservation solution is brought to a final volume of 500 mL with the dissolving solution, and the pH is adjusted to 7.1-7.4.

[0024] Step 5: Place the dissolved solutions from Step 4 into separate biosafety cabinets and sterilize them by filtration using a 0.22μm filter or a 0.22μm Nysso filter to obtain cryopreservation solutions for each experimental group.

[0025] By adopting the above technical solution, the key components in step one, such as hydroxyethyl starch, sodium heparin, trehalose, glutamic acid dipeptide, HEMA80, and taurine, were accurately weighed and grouped for management, ensuring the accuracy of the concentration of each component and the repeatability of the experiment, laying the foundation for the subsequent stable synergistic protective effect. In step two, a buffer solution with a volume ratio of 10% DMSO and 90% DPBS was selected to effectively balance the potential toxicity of DMSO while providing osmotic protection, improve the biocompatibility and safety of the cell cryopreservation solution, and DPBS, as a carrier for the isotonic environment, can maintain the osmotic pressure balance inside and outside the cell, which helps the cell maintain structural stability under low temperature conditions.

[0026] In steps three and four, the gradual dissolution and mixing, individual volume adjustment and precise pH adjustment not only ensured the full dissolution and uniform distribution of each functional component, but also avoided the reaction interference between components, maintained the physicochemical stability of the cryopreservation solution system. At the same time, the final pH adjustment to 7.1-7.4 is closer to the physiological environment, which helps to maintain the appropriate state of cell metabolism during cryopreservation and thawing.

[0027] Finally, step five introduces a 0.22μm filtration sterilization process to ensure that the cryopreserved solution is stored and used under sterile conditions, which greatly reduces the risk of microbial contamination and meets the strict requirements of clinical and laboratory sterility.

[0028] Optionally, the volume fraction of DMSO is 10% and the volume fraction of DPBS is 90%, and the solution formed by mixing the two is used for the dissolution of each component.

[0029] By adopting the above technical solution, a solution is formed by mixing 10% DMSO and 90% DPBS to ensure good cell permeability protection while significantly reducing the toxic side effects of DMSO on cells. DPBS provides an isotonic environment, which helps maintain cell stability. The combination of the two as a dissolution medium for each component can improve the dissolution efficiency of the components and the biocompatibility of the system, providing a strong guarantee for the stability of the cryopreservation solution and cell viability.

[0030] Optionally, during the dissolution process, a magnetic stirrer is used to stir at 4°C for no less than 30 minutes to ensure that all components are fully dissolved.

[0031] By adopting the above technical solution, using a magnetic stirrer to continuously stir for no less than 30 minutes at 4℃ can effectively improve the dissolution efficiency and uniformity of each component, ensure that the components are fully dissolved and stable in the low-temperature environment, avoid precipitation or inactivation caused by uneven local concentration, and the low-temperature stirring process helps to reduce the degradation of heat-sensitive components, maintain the stability and functional integrity of the active ingredients in the cryopreservation solution, and improve the overall quality and use effect of the cryopreservation solution.

[0032] Optionally, the pH value of the solution can be measured using pH test paper or a pH meter before the volume adjustment operation. If necessary, the pH can be adjusted by slowly adding 1M NaOH or 1M HCl solution, and the final pH can be controlled at 7.2±0.1.

[0033] By adopting the above technical solution, the pH value of the solution is measured using pH test paper or pH meter before the volume adjustment operation, and the final pH is controlled at 7.2±0.1 by slowly adding 1M NaOH or 1M HCl. This effectively ensures that the cryopreservation solution is close to the physiological environment, which is conducive to maintaining the stability of cell membrane structure and function, reducing cell stress response and survival rate decline caused by pH deviation, improving the biocompatibility and applicability of the cryopreservation solution, and providing a safer and more stable microenvironment for cell cryopreservation and thawing.

[0034] Optionally, a 0.22μm capsule filter is used in the sterilization filtration step, and a constant pressure is maintained in the range of 0.5 to 1.0 bar during filtration to ensure filtration efficiency and liquid flow.

[0035] By adopting the above technical solution, using a 0.22μm capsule filter in the sterilization filtration step and controlling the filtration pressure within the range of 0.5 to 1.0 bar, microbial contamination can be effectively removed, ensuring the sterility of the cryopreservation solution. Constant pressure control helps maintain a stable liquid flow rate, avoiding filter clogging or component loss due to pressure fluctuations, ensuring the complete retention of key active ingredients, improving the safety and stability of the filtration process, and providing strong support for subsequent cryopreservation solution storage and clinical applications.

[0036] Optionally, the filtered cryopreservation solution is stored in a light-proof, sealed, sterile glass bottle and kept at -20°C or lower for a period of not less than 12 months.

[0037] By adopting the above technical solution, the filtered cryopreservation solution is sealed in a sterile glass bottle away from light and stored at -20℃ or lower. This effectively prevents the degradation of active ingredients by light, reduces the risk of oxidation and microbial contamination, ensures the stability and bioactivity of the cryopreservation solution, and slows down the degradation process of the components, so that the shelf life can be stable for more than 12 months, meeting the needs of long-term use in scientific research and clinical practice, and improving the practicality and reliability of the product.

[0038] Optionally, the concentrations of each component in the cryopreservation solution of each experimental group are gradient-proportioned according to the ranges set in Appendix 1, through a single-factor variable design, in order to optimize the survival rate and functional retention rate of immune cells after cryopreservation.

[0039] By adopting the above technical solution, the single-factor variable gradient ratio of the cryopreservation solution in each experimental group is carried out according to the concentration range set in Appendix 1. This allows for a systematic evaluation of the impact of each component on the cryopreservation effect of immune cells, thereby screening out the optimal ratio scheme. This helps to improve the survival rate and functional retention rate of cells after cryopreservation, enhance the scientific nature and specificity of the cryopreservation solution, and provide more efficient and reliable technical support for the long-term preservation and subsequent application of immune cells.

[0040] In summary, this application includes at least one of the following beneficial technical effects:

[0041] 1. The serum-free cryopreservation solution for immune cells contains no serum or natural protein substitutes. It uses synthetic chemical components with clear and controllable composition, effectively solving the problems of unclear composition and large batch-to-batch differences in traditional cryopreservation solutions, ensuring the uniformity and reproducibility of the product in large-scale production and application.

[0042] 2. The cryopreservation solution achieves intracellular protection through DMSO, and combined with extracellular protective components such as hydroxyethyl starch and trehalose, it synergistically reduces the mechanical damage of ice crystals to cell structure, effectively maintains cell integrity, and significantly enhances cell viability during cryopreservation and activity after thawing.

[0043] 3. The buffer solution is a mixture of 10% DMSO and 90% DPBS, which takes into account both osmotic protection and toxicity control. The pH value is adjusted to 7.2±0.1, which is close to the physiological environment, helps to maintain normal cell metabolism and function, and enhances the biocompatibility of the cryopreservation solution.

[0044] 4. Stir for an extended period at 4°C using a magnetic stirrer to ensure thorough and uniform dissolution of all components, while reducing the degradation of heat-sensitive components, maintaining the stability of the cryopreservation solution, and improving the quality of the finished product;

[0045] 5. A 0.22μm capsule filter is used for constant pressure filtration, which effectively removes microbial contamination, maintains the integrity of active ingredients, ensures the sterility of the cryopreservation solution, and meets the safety requirements for experimental and clinical use;

[0046] 6. The finished cryopreservation solution is stored in sterile glass bottles in a light-proof and sealed environment at -20°C or lower. This effectively inhibits photo-oxidative degradation and extends the shelf life to more than 12 months, meeting the needs of long-term storage and transportation. Attached Figure Description

[0047] Figure 1 yes Figure 1 This is a schematic diagram of a serum-free cryopreservation solution for immune cells according to an embodiment of this application.

[0048] Figure 2 This is a schematic diagram of the components of a serum-free cryopreservation solution for immune cells according to an embodiment of this application.

[0049] Figure 3 This is a schematic diagram of experimental data after one month of cryopreservation of a serum-free cryopreservation solution for immune cells, according to an embodiment of this application.

[0050] Figure labeling: 100, cryopreservation solution; 10, buffer solution; 20, DMSO; 30, DPBS; 40, hydroxyethyl starch; 50, sodium heparin; 60, trehalose; 70, glutamic acid dipeptide; 80, HEMA; 90, taurine. Detailed Implementation

[0051] The following is in conjunction with the appendix Figure 1-2 This application will be described in further detail.

[0052] This application discloses a serum-free cryopreservation solution for immune cells. (Refer to...) Figure 1 This includes cryopreservation solution 100, which is a mixture composed of buffer solution 10; buffer solution 10 is a mixture of DMSO (20) and DPBS (30);

[0053] Buffer 10, composed of DMSO (20) and DPBS (30), contains hydroxyethyl starch 40, heparin sodium 50, trehalose 60, glutamic acid dipeptide 70, HEMA 80, and taurine 90. Buffer 10 is composed of 10% DMSO and 90% DPBS by volume. The amount of hydroxyethyl starch 40 in the buffer 10 is 50-150 g / L, the amount of heparin sodium 50 is 1000-3000 U / L, the amount of trehalose 60 is 10-40 g / L, the amount of glutamic acid dipeptide 70 is 5-10 g / L, the amount of HEMA 80 is 0.01-1.00 g / L, and the amount of taurine 90 is 0.1-0.5 g / L, based on the basic composition of cryopreservation solution 100, uses DMSO (20) and DPBS (30) mixed at a volume ratio of 10% and 90% to form buffer solution 10 as the base solution carrier of the entire system, playing a dual key role. DMSO, as a permeable cryoprotectant, can effectively penetrate into the cell interior, preventing excessive water leakage and organelle damage during freezing, reducing intracellular ice crystal formation, and helping to maintain the integrity of the cell membrane structure; DPBS is an isotonic salt solution that can maintain the osmotic pressure stability of the external environment of the cell, preventing the cell from swelling or shrinking due to changes in osmotic pressure, thereby improving the overall cryopreservation environment. Physiological compatibility and buffering capacity: Multiple functional components are further introduced into the buffer system, including hydroxyethyl starch 40, heparin sodium 50, trehalose 60, glutamic acid dipeptide 70, HEMA 80, and taurine 90. Each component exerts its unique synergistic effect within a set concentration range, jointly enhancing the cell's survival and functional retention during cryopreservation. Hydroxyethyl starch 40, at a concentration range of 50–150 g / L, primarily provides an external physical barrier effect, slowing the rate of water migration around cells, reducing the risk of extracellular ice crystal formation, and simultaneously increasing the solution viscosity, which is conducive to forming a stable microenvironment and inhibiting ice crystal growth and aggregation. Heparin sodium 50, at concentrations of 1000–3000 U / L, has excellent anticoagulant properties, making it particularly suitable for handling blood components mixed in with immune cells. It effectively prevents cell damage caused by blood agglutination during cryopreservation and thawing, enhancing the applicability of cryopreservation solutions, especially valuable in the actual cryopreservation of cells after sampling. Trehalose 60, in the range of 10–40 g / L, is a typical non-reducing disaccharide with excellent membrane protection, stabilizing protein structures and the phospholipid bilayer, preventing protein denaturation and membrane damage during low temperatures and drying. Trehalose plays a crucial role in cell freezing. It can form a glassy state, replacing water to stabilize organelle structures, and is one of the widely studied cell-protective sugars; glutamic-amyl dipeptide 70 (5-10 g / L), as a highly stable low-molecular-weight antioxidant peptide, can exert anti-free radical and anti-oxidative damage effects during cryopreservation stress, reducing the damage of ROS (reactive oxygen species) accumulation to cell membranes, mitochondria, and nucleic acids, and helping to maintain cell activity and metabolic function during freezing and thawing; HEMA80 (0.01-1.00 g / L) is a hydrophilic polymer monomer that can significantly inhibit ice crystal formation and block ice crystal expansion channels at appropriate concentrations, thereby reducing mechanical damage to cells. At the same time, HEMA80 can also stabilize the thermodynamic state of the extracellular fluid, enhancing the overall physical protective effect of the cryopreservation solution; taurine 90 (0.1-0.5 g / L) can also be used to protect cells.As an amino acid derivative with multiple physiological functions, 5 g / L plays a role in cell protection, including membrane stabilization, antioxidation, and osmotic pressure buffering. It is particularly advantageous in dealing with cryo-induced oxidative stress and can also help maintain mitochondrial function and promote the recovery of cellular metabolic activity after cell thawing.

[0054] Based on the combined functions and proportions of the components, Cryopreservation Solution 100 successfully achieves multiple protections for immune cells during cryopreservation without relying on any animal-derived components (such as serum, albumin, or natural protein substitutes). These protections include cell membrane protection, osmotic pressure balance, ice crystal inhibition, oxidative damage prevention and control, and anticoagulation synergy. It boasts advantages such as clearly defined components, high controllability, high batch-to-batch consistency, and excellent biosafety. Furthermore, the cryopreservation solution is suitable for various immune cell types, including T cells, NK cells, DC cells, and CAR-T cells, and is particularly suitable for widespread application in immunotherapy, research sample preservation, and the construction of clinical cell banks.

[0055] The implementation principle of this application embodiment is as follows: Dimethyl sulfoxide (DMSO) with a volume fraction of 10% is added to the cryopreservation solution. As a classic cryoprotectant, DMSO can penetrate the cell membrane and enter the cell, forming a hydrogen bond network with water molecules, lowering the freezing point of intracellular water and inhibiting ice crystal formation. This effectively reduces physical damage to the cell membrane and organelles caused by intracellular ice crystals, protecting the cells and maintaining their integrity during freezing and thawing. Simultaneously, the concentration of DMSO is controlled at 10% to ensure its protective effect while avoiding toxic side effects on cells after thawing. A 90% volume fraction of DPBS (phosphate buffered saline) is used as the main solvent, which helps maintain the isotonic state of the extracellular environment, preventing cell swelling or shrinkage caused by abnormal osmotic pressure of the cryopreservation solution. DPBS contains a certain concentration of phosphate buffer pairs, which can stabilize the cryopreservation solution at pH 7.1–7.4. The presence of hydroxyethyl starch (HES) and trehalose in solution primarily serves an extracellular protective function. HES, a macromolecular material, increases the viscosity of the cryopreservation solution, slows water molecule migration and heat conduction, reduces ice crystal growth, and forms a barrier structure on the cell surface, effectively reducing mechanical damage to the cell membrane caused by sudden temperature drops. Trehalose, as a non-reducing disaccharide, can replace water molecules in the cell membrane under low-temperature conditions and releases a protective membrane structure during thawing. This slows down the damage to the phospholipid layer caused by freezing. The addition of sodium heparin as an anticoagulant in the cryopreservation solution effectively inhibits the risk of coagulation caused by residual blood during cell collection and cryopreservation. Heparin can prevent plasma coagulation by inhibiting the activity of coagulation factors, ensuring smooth operation during cell separation, cryopreservation, and thawing, and is particularly suitable for immune cells extracted from peripheral blood, such as PBMCs. The freezing process generates a large amount of ROS (reactive oxygen species), triggering cellular oxidative stress, membrane lipid peroxidation, and mitochondrial dysfunction. Glutamate, as a natural small-molecule antioxidant peptide, can scavenge ROS. OS (Oxygen Defibrillation) repairs oxidative damage and enhances the cell's resilience during cryopreservation. Taurine regulates intracellular calcium ion concentration, stabilizes cell membrane structure, and synergistically combats oxidation with glutamic acid dipeptide, reducing cell stress responses during freeze-thaw cycles. The addition of HEMA80 (hydroxyethyl methacrylate) to the cryopreservation solution helps regulate the glass transition temperature, reduces the formation and expansion of micro-ice crystals, effectively lowering the risk of cell membrane rupture due to ice crystal growth. HEMA-like hydrophilic polymers can also form cross-linked network structures with water, inhibiting the formation of regular crystals from water molecules and improving the stability of the cryopreservation solution. Qualitative and viscoelastic properties help maintain the physical stability of the extracellular microenvironment. Traditional cryopreservation solutions often rely on biologically derived components such as fetal bovine serum (FBS) or human serum albumin, which not only suffers from large batch-to-batch variations and uncertain composition but also may introduce pathogen contamination and immune risks. This embodiment uses only serum-free, non-protein, and clearly defined chemical components. Through the synergistic effect of the physical and chemical properties of each component, it ensures biosafety and facilitates process standardization and product quality control, making it particularly suitable for high-standard applications such as immunotherapy, GMP production, and long-term cell bank storage.

[0056] This application also discloses a method for preparing serum-free cryopreservation solution for immune cells, comprising the following steps:

[0057] Step 1: Accurately weigh the components

[0058] Reference Figure 2 The 40g of p-hydroxyethyl starch, 50g of sodium heparin, 60g of trehalose, 70g of glutamic acid dipeptide, 80g of HEMA and 90g of taurine in each group should be accurately weighed and placed according to the corresponding weighing values ​​listed in Appendix 1, and each group should be placed separately.

[0059] Step 2: Prepare buffer solution 10

[0060] The solution used to dissolve the components in step one was prepared by mixing 10% DMSO and 90% DPBS by volume.

[0061] Step 3: The accurately weighed components from Step 1 are dissolved and mixed with a dissolving solution to form a cryopreservation solution semi-finished product. Each group of cryopreservation solution semi-finished product is placed separately after being dissolved and mixed.

[0062] Step 4: After dissolving and mixing all the components, the semi-finished cryopreservation solution is brought to a final volume of 500 mL with the dissolving solution, and the pH is adjusted to 7.1-7.4.

[0063] Step 5: Place the dissolved solutions from Step 4 into separate biosafety cabinets and sterilize them by filtration using a 0.22μm filter or a 0.22μm Nysso filter to obtain the cryopreservation solutions for each experimental group.

[0064] Experiment 1: NK cells cultured to day 14-15 were used as experimental materials. The viable cell density at cryopreservation was 2.5 × 10^7 cells / mL. Cells were cryopreserved for 1 month, 3 months, 6 months, and 12 months, and then thawed. AO / PI fluorescence counting was performed, and the average viability and recovery rate of each group were calculated. Cell morphology was observed under a microscope after 72 hours of culture. Appendix Table 1 shows the grouping for Experiment 1.

[0065] Appendix 1: Grouping Table for Experiment 1

[0066]

[0067]

[0068] Traditional control group: Mix 70% 1640 culture medium, 20% fetal bovine serum and 10% DMSO in a biosafety cabinet.

[0069] Note: No trehalose was added to experimental group four, but an additional 13.16g of glucose of the same molar mass was added.

[0070] Conclusion of Experiment 1: The detailed results of the viability and recovery rate of NK cells after cryopreservation and thawing in Experiment 1 are shown in Appendix Table 2.

[0071] Appendix Table 2: Survival rate and recovery rate of NK cells frozen for 1 month and 3 months (batch 20221023)

[0072]

[0073] Microscopic images of the product after thawing from cryopreservation for one month are shown below. Figure 3 As shown in the image.

[0074] The results show that: 1. The cryopreservation effect of the traditional control group cryopreservation solution varies. Its recovery rate and average cell viability after one month of cryopreservation are slightly better than the experimental group, but the recovery rate and average cell viability after three months of cryopreservation are lower than some experimental groups; 2. Considering the results of the ten groups, hydroxyethyl starch plays a good protective role in cell cryopreservation (experimental groups one, two, three, and eleven); 3. Trehalose, as a sugar, can effectively protect the activity of cell membranes and membrane protein molecules under low temperature conditions. With increasing trehalose concentration, the cell viability after cryopreservation and thawing increases, but with increasing trehalose concentration... The effect of trehalose on cells gradually approaches saturation, and the optimal concentration of trehalose is 20 g / L (experimental groups II, IV, V, and VI); 4. Sodium heparin can effectively inhibit the clumping of NK cells, especially for high-density cryopreserved cells, sodium heparin can significantly improve the viability of NK cells after thawing (experimental groups II, IX, and X); 5. Taurine and HEMA can both improve cell tolerance and affect the viability of cells after thawing. When both taurine and HEMA are lacking, the cell viability decreases significantly (experimental groups II, VII, and VIII); Based on the experimental results, the formulation of experimental group II was selected as the experimental group for subsequent experiments.

[0075] Conclusion of Experiment 2: The results of cryopreservation and thawing in Experiment 2 are detailed in Appendix Table 3.

[0076] Appendix 3: Average viability and average recovery rate of NK cells from batch 20221118, after one month of cryopreservation using cryopreservation solutions from different manufacturers.

[0077]

[0078]

[0079] The results show that the cell cryopreservation solution obtained by the formulation of experimental group two in this invention has good stability and uniformity. After NK cells are cryopreserved for 1 month, the cryopreservation solution of the traditional control group still has a significant advantage. However, with the increase of cryopreservation time, after 3 months of cryopreservation, the cell cryopreservation solution obtained by the formulation of experimental group two has a higher cell viability and average cell recovery rate after thawing. The cell cryopreservation solution in this scheme has better cryopreservation stability and cryopreservation effect compared with other products.

[0080] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A serum-free cryopreservation solution for immune cells, characterized in that: Includes a cryopreservation solution (100), which is a mixture composed of a buffer solution (10); The buffer solution (10) is a mixture of DMSO (20) and DPBS (30); The buffer (10) consisting of DMSO (20) and DPBS (30) contains hydroxyethyl starch (40), sodium heparin (50), trehalose (60), glutamic acid dipeptide (70), HEMA80 and taurine (90).

2. The serum-free cryopreservation solution for immune cells according to claim 1, characterized in that: The buffer (10) is composed of 10% DMSO and 90% DPBS by volume.

3. The serum-free cryopreservation solution for immune cells according to claim 2, characterized in that: The buffer solution (10), which is composed of 10% DMSO and 90% DPBS by volume, contains 50-150 g / L of hydroxyethyl starch (40), 1000-3000 U / L of sodium heparin (50), 10-40 g / L of trehalose (60), 5-10 g / L of glutamic acid dipeptide (70), 0.01-1.00 g / L of HEMA (80), and 0.1-0.5 g / L of taurine (90).

4. A method for preparing a serum-free cryopreservation solution for immune cells, characterized in that, Includes the following steps: Step 1: Accurately weigh the components Hydroxyethyl starch (40), sodium heparin (50), trehalose (60), glutamic acid dipeptide (70), HEMA (80) and taurine (90) were accurately weighed according to the corresponding weighing numbers listed in Appendix 1 and placed separately for each group. Step 2: Prepare buffer solution (10) The solution used to dissolve the components in step one was prepared by mixing 10% DMSO and 90% DPBS by volume. Step 3: The accurately weighed components from Step 1 are dissolved and mixed with a dissolving solution to form a cryopreservation solution semi-finished product. Each group of cryopreservation solution semi-finished product is placed separately after being dissolved and mixed. Step 4: After dissolving and mixing all the components, the semi-finished cryopreservation solution is brought to a final volume of 500 mL with the dissolving solution, and the pH is adjusted to 7.1-7.

4. Step 5: Place the dissolved solutions from Step 4 into separate biosafety cabinets and sterilize them by filtration using a 0.22μm filter or a 0.22μm Nysso filter to obtain the cryopreservation solutions for each experimental group.

5. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: The DMSO has a volume fraction of 10%, and the DPBS has a volume fraction of 90%. The solution formed by mixing the two is used for dissolving each component.

6. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: During the dissolution process, a magnetic stirrer is used to stir at 4°C for no less than 30 minutes to ensure that all components are fully dissolved.

7. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: Before making up the volume, use pH test paper or a pH meter to measure the pH value of the solution. If necessary, adjust the pH by slowly adding 1M NaOH or 1M HCl solution. Finally, control the pH at 7.2±0.

1.

8. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: The sterilization filtration step uses a 0.22μm capsule filter, and the pressure is maintained at a constant range of 0.5 to 1.0 bar during filtration to ensure filtration efficiency and liquid flow.

9. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: The filtered cryopreservation solution is stored in light-proof, sealed, sterile glass bottles and kept at -20°C or lower for a period of not less than 12 months.

10. The method for preparing serum-free cryopreservation solution for immune cells according to claim 4, characterized in that: The concentrations of each component in the cryopreservation solution of each experimental group were determined according to the ranges set in Appendix 1, and a gradient ratio was implemented through a single-factor variable design to optimize the survival rate and functional retention rate of immune cells after cryopreservation.

Images