A serum-free cryopreservation solution and its use in the cryopreservation of cells

Abstract

The application provides a serum-free cryopreservation solution and application thereof in cell cryopreservation. The serum-free cryopreservation solution comprises a first additive, a saccharide, an inorganic salt, a protein and a second additive; in the serum-free cryopreservation solution, the mass concentration of the first additive is 0.2-15% (w / v), the concentration of the saccharide is 2-15 mg / mL, the mass concentration of the inorganic salt is 0.7-1.2% (w / v), the concentration of the protein is 0.05-0.5 g / mL, and the concentration of the second additive is 0.5-5 mg / mL. The serum-free cryopreservation solution provided by the application ensures that the cell still has high survival rate, high activity and functional integrity after cryopreservation through the combined action of the above components, the composition of the cryopreservation solution is clear, and no animal-derived serum needs to be added, so that the immunogenicity risk, pathogen contamination and batch difference caused by the animal-derived serum are avoided, and the safety of the cryopreservation solution is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of cell preservation technology, specifically to a serum-free cryopreservation solution and its application in cell cryopreservation. Background Technology

[0002] Cell cryopreservation enables cell therapy, regenerative medicine, disease model construction, and the establishment of biobanks. Cell cryopreservation primarily involves placing cells in a deep cryogenic environment (such as -196°C liquid nitrogen), virtually halting their metabolic activity, thus enabling long-term preservation. Cryopreserved cells can be thawed and used when needed, which is crucial for ensuring the stability of cell product quality and the timeliness of therapeutic applications. Currently, cell cryopreservation technology is widely used in the preservation of immune cells (such as CAR-T and NK cells), stem cells (such as mesenchymal stem cells), and other primary cells.

[0003] Successful cell cryopreservation relies heavily on cryopreservation solutions, which aim to protect cells from multiple forms of damage during the cooling and thawing process. Current cryopreservation solution formulations primarily involve adding fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) to a basal culture medium. DMSO acts as a permeable cryoprotectant, providing physical protection by lowering the cell's freezing point and reducing intracellular ice crystal formation, while the serum provides nutrients, growth factors, and some protein protection. However, this type of cryopreservation solution has the following limitations: First, the introduction of animal-derived serum (mainly FBS) introduces significant safety risks and batch-to-batch variations. Its composition is complex and uncertain, potentially containing foreign proteins, viruses, mycoplasma, and other pathogens. This not only poses potential risks of immunogenicity and pathogen transmission but may also unpredictably interfere with cell differentiation. Second, DMSO exhibits clear toxicity to cells at effective concentrations, causing protein denaturation and membrane damage. Furthermore, the sudden change in osmotic pressure it induces is itself a stressor, all of which can lead to low cell viability, loss of function, or apoptosis in cryopreserved cells.

[0004] Therefore, there is an urgent need to develop a safe and effective cryopreservation solution that can effectively address the multiple damages that occur during the entire process of cell freezing and thawing. This is of great significance for promoting the industrialization and clinical application of cell therapy. Summary of the Invention

[0005] To address the safety risks associated with the use of animal-derived serum in existing cell cryopreservation solutions and the toxicity of DMSO to cells, this invention provides a serum-free cryopreservation solution and its application in cell cryopreservation.

[0006] According to a first aspect of the present invention, a serum-free cryopreservation solution is provided, the serum-free cryopreservation solution comprising a first additive, sugars, inorganic salts, proteins, and a second additive; In this serum-free cryopreservation solution, the mass concentration of the first additive is 0.2-15% (w / v), the concentration of sugars is 2-15 mg / mL, the mass concentration of inorganic salts is 0.7-1.2% (w / v), the concentration of protein is 0.05-0.5 g / mL, and the concentration of the second additive is 0.5-5 mg / mL. The first additive includes at least one of dimethyl sulfoxide (DMSO), glycerol, dextran, and trehalose; Sugars include at least one of dextran, glucose, sucrose, and fructose; The second additive includes insulin.

[0007] The serum-free cryopreservation solution provided by this invention combines a first additive, sugars, inorganic salts, proteins, and a second additive, controlling the concentration of each component within the aforementioned range. This enables the construction of a multi-dimensional synergistic protection system during cell cryopreservation. Firstly, the first additive, as a permeable preservative, can rapidly enter the cell interior to lower the freezing point of the cell interior (i.e., the cytoplasmic solution). Sugars provide energy to the cells and stabilize the osmotic pressure. The first additive can synergistically work with the non-permeable hyperosmotic environment formed by sugars outside the cells to jointly regulate the cell dehydration process, minimizing physical damage to cells caused by the formation and growth of intracellular ice crystals during cryopreservation. Secondly, insulin in the second additive, as a metabolic regulator, can effectively alleviate the energy metabolism crisis of cells under low-temperature stress, reducing the risk of inhibited intracellular adenosine triphosphate (ATP) synthesis due to suppressed mitochondrial function and cellular stress during cryopreservation. Furthermore, insulin can activate cell survival signaling pathways (such as... PI3K / Akt enhances the cell's resistance to stress, maintaining high activity during cryopreservation and thawing. It also maintains the cell's metabolic balance, reduces apoptosis and necrosis caused by cryopreservation, and significantly improves the survival rate and functional recovery ability of cryopreserved cells. Insulin and inorganic salts used to maintain the physiological ionic environment during cell cryopreservation can synergistically enhance the cell's anti-apoptotic ability and stabilize the cell membrane potential, which is conducive to the rapid recovery of function of cryopreserved cells after thawing. Thus, the second additive and inorganic salts ensure that cryopreserved cells still have high cell activity through physical protection and synergistic regulation of biological signals. Thirdly, proteins act as carrier stabilizing molecules, which is conducive to stabilizing the cell membrane structure and function at low temperature and reducing oxidative stress. Moreover, proteins can reversibly bind to the first additive and cell stress products, which can synergistically buffer chemical toxicity (derived from DMSO in the first additive) and stabilize the active ingredients in serum-free cryopreservation solution, including insulin, thereby improving the biocompatibility and stability of the entire serum-free cryopreservation solution system. In summary, the serum-free cryopreservation solution provided by this invention ensures that cells maintain high viability, high activity, and functional integrity after cryopreservation through the synergistic effect of the aforementioned components. Furthermore, the serum-free cryopreservation solution provided by this invention has a clearly defined composition and does not require the addition of animal-derived serum, fundamentally avoiding the risks of immunogenicity, pathogen contamination, and batch-to-batch variability associated with animal-derived serum components, significantly improving the safety of this serum-free cryopreservation solution in actual clinical applications.

[0008] In the above serum-free cryopreservation solution, the mass concentration of dimethyl sulfoxide and glycerol in the first additive is 5%~15% (w / v), and the mass concentration of dextran and trehalose is 0.2~2% (w / v).

[0009] Preferably, in the above-mentioned serum-free cryopreservation solution, the mass concentration of the first additive is 7-9% (w / v), the concentration of sugars is 4-11 mg / mL, the mass concentration of inorganic salts is 0.8-1.0% (w / v), the concentration of protein is 0.1-0.3 g / mL, and the concentration of the second additive is 1-2 mg / mL.

[0010] Optimizing the concentration of each active substance in the serum-free cryopreservation solution can further enhance the synergistic effect between the components, thereby further improving the cell survival rate, cell activity, and functional integrity after cryopreservation.

[0011] Preferably, the inorganic salt includes at least one of sodium chloride, disodium hydrogen phosphate, sodium lactate, potassium chloride, sodium acetate, magnesium chloride, calcium chloride, magnesium sulfate, sodium bisulfite, and sodium gluconate.

[0012] Using the above materials as inorganic salt components in serum-free cryopreservation solution can effectively simulate the extracellular fluid environment. Together with insulin in the second additive, it can maintain the transmembrane ion gradient and enzyme activity of cells, providing an ion basis for cell survival signal transduction, thereby enabling cryopreserved cells to maintain high activity.

[0013] Preferably, the protein includes at least one of human serum albumin and bovine serum albumin.

[0014] Human serum albumin and / or bovine serum albumin are used as protein components in serum-free cell cryopreservation solutions. Human serum albumin and bovine serum albumin have superior carrier functions and endogenous antioxidant capacity, which can form a highly efficient complement to the ion balance of inorganic salts and the signal regulation of insulin, together creating a stable cryopreservation microenvironment for cells that is closer to their physiological state.

[0015] Preferably, the inorganic salts include sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride.

[0016] Preferably, the protein is human serum albumin.

[0017] Preferably, the first additive includes dimethyl sulfoxide and dextran; Preferably, the sugars include dextran and glucose.

[0018] Preferably, the serum-free cryopreservation solution further includes a third additive; in the serum-free cryopreservation solution, the concentration of the third additive is 0.5~100 mg / L; the third additive includes at least one of glutathione, vitamin C, and vitamin E.

[0019] Cells are prone to generating reactive oxygen species (ROS) during freeze-thaw cycles. ROS can attack cell membranes, causing cell damage or death, and oxidize key amino acid residues on the side chains of proteins in cells, leading to protein misfolding, cross-linking, aggregation, or degradation. This results in cytoskeleton protein dysfunction and affects cell morphology and adhesion / migration ability after thawing.

[0020] By introducing the aforementioned third additive into the serum-free cryopreservation solution, firstly, the third additive can provide an antioxidant defense system for cryopreserved cells and work synergistically with the endogenous antioxidant capacity of proteins to efficiently remove ROS generated during cell freeze-thaw cycles. Secondly, the third additive can reduce oxidative damage to cells during cryopreservation and interact with insulin to reduce the risk of apoptosis caused by oxidative stress. Thus, through these two effects, the cell viability of cryopreserved cells is improved, enabling them to retain high proliferative activity after thawing.

[0021] Preferably, the third additive includes vitamin C and vitamin E.

[0022] Preferably, in the serum-free cryopreservation solution, the concentration of vitamin C is 55-65 mg / L and the concentration of vitamin E is 5-15 mg / L.

[0023] By combining vitamin C and vitamin E and using them as a third additive in serum-free cryopreservation solution, and controlling their concentrations within the aforementioned range, it is possible to ensure that the third additive effectively scavenge free radicals (ROS) and also to better integrate with other components in the serum-free cryopreservation solution (especially proteins and inorganic salts) to achieve their intended effects. This helps to avoid ineffective reactions or the generation of new impurities, thus ensuring the long-term stability and safety of the entire serum-free cryopreservation solution system.

[0024] According to a second aspect of the present invention, the application of the above-described serum-free cryopreservation solution in the cryopreservation of cells is provided.

[0025] Preferably, the cells include at least one of natural killer cells (NK cells), mesenchymal stem cells (MSC cells), human leukemia cells, and T cells (Jurkat cells).

[0026] Preferably, the human leukemia cells include K562 cells (human chronic myeloid leukemia cell line).

[0027] Preferably, the T cells include Jurkat cells (human peripheral blood leukemia T cells).

[0028] Applying the serum-free cryopreservation solution provided by this invention to cell cryopreservation can reduce the risk of cell damage or apoptosis during the cryopreservation process, and enable cells to maintain high survival rate, high activity and functional integrity after cryopreservation. Attached Figure Description

[0029] Figure 1 The image shows the morphological observation results of NK cells after cryopreservation and thawing culture using the serum-free cryopreservation solution provided in Example 1.

[0030] Figure 2 The image shows the cell morphology observation results after MSC cells were cryopreserved and thawed using the serum-free cryopreservation solution provided in Example 1.

[0031] Figure 3 This image shows the cell morphology observation results of K562 cells after cryopreservation and thawing culture using the serum-free cryopreservation solution provided in Example 1.

[0032] Figure 4 This image shows the morphological observation results of Jurkat cells after cryopreservation and thawing culture using the serum-free cryopreservation solution provided in Example 1. Detailed Implementation

[0033] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Examples 1-3 Examples 1-3 provide a serum-free cryopreservation solution, the specific components of which are shown in Table 1. The serum-free cryopreservation solution includes: 5-15% (w / v) of the first additive, 2-15 mg / mL of sugars, 0.7-1.2% (w / v) of inorganic salts, 0.05-0.5 g / mL of protein, and 0.5-5 mg / mL of the second additive. The serum-free cryopreservation solutions provided in Examples 1-3 are prepared by adding a first additive, sugars, proteins, and a second additive to an inorganic salt solution with a concentration of 0.7-1.2% (w / v) as a base, and mixing them thoroughly.

[0035] Table 1. Specific components of serum-free cryopreservation solutions in Examples 1-3

[0036] Examples 4-5 Examples 4 and 5 provide a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the concentrations of each component in the serum-free cryopreservation solution are different. The specific components are shown in Table 2.

[0037] Table 2. Specific components of the serum-free cryopreservation solutions in Examples 4-5

[0038] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Examples 4 and 5 are strictly consistent with those in Example 1.

[0039] Example 6 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the first additive used is dextran, and the concentration of dextran in the serum-free cryopreservation solution is 2% (w / v).

[0040] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0041] Example 7 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the first additive used includes DMSO and dextran, and the concentration of DMSO in the serum-free cryopreservation solution is 5% (w / v) and the concentration of dextran is 2% (w / v).

[0042] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0043] Example 8 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride are used as inorganic salts. In the serum-free cryopreservation solution, the concentrations of sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride are 0.5% (w / v), 0.1% (w / v), 0.1% (w / v), 0.1% (w / v), and 0.1% (w / v), respectively.

[0044] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0045] Example 9 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that sodium chloride, disodium hydrogen phosphate, sodium acetate, potassium chloride, and calcium chloride are used as inorganic salts. Furthermore, in the serum-free cryopreservation solution, the concentrations of sodium chloride, disodium hydrogen phosphate, sodium acetate, potassium chloride, and calcium chloride are 0.5% (w / v), 0.1% (w / v), 0.1% (w / v), 0.1% (w / v), and 0.1% (w / v), respectively.

[0046] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0047] Example 10 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the sugars used include sucrose and glucose, and the concentration of sucrose in the serum-free cryopreservation solution is 2 mg / mL and the concentration of glucose is 2 mg / mL.

[0048] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0049] Example 11 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the sugars used include dextran and glucose, and the concentration of dextran in the serum-free cryopreservation solution is 2 mg / mL and the concentration of glucose is 2 mg / mL.

[0050] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0051] Example 12 This embodiment provides a serum-free cryopreservation solution. The difference in composition compared to Example 1 is that the protein used is bovine serum albumin.

[0052] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0053] Example 13 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the serum-free cryopreservation solution also includes a third additive, glutathione, and the concentration of glutathione in the serum-free cryopreservation solution is 70 mg / mL.

[0054] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0055] Example 14 This embodiment provides a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the serum-free cryopreservation solution also includes a third additive, vitamin C and vitamin E. Furthermore, in the serum-free cryopreservation solution, the concentration of vitamin C is 55 mg / mL and the concentration of vitamin E is 5 mg / mL.

[0056] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0057] Example 15 A serum-free cryopreservation solution comprising: 5% (w / v) DMSO, 2% (w / v) dextran, 2 mg / mL dextran, 2 mg / mL glucose, 0.5% (w / v) sodium chloride, 0.1% (w / v) sodium gluconate, 0.1% (w / v) sodium acetate, 0.1% (w / v) potassium chloride, 0.1% (w / v) magnesium chloride, 0.05 g / mL human serum albumin, and 2 mg / mL insulin.

[0058] Comparative Example 1 This comparative example provides a serum-free cryopreservation solution, which differs from Example 1 in that it does not contain the second additive, insulin.

[0059] Apart from the differences mentioned above, the materials, formulation ratios, and preparation procedures used in this comparative example are strictly consistent with those in Example 1.

[0060] Comparative Examples 2-3 Comparative Examples 2 and 3 provide a serum-free cryopreservation solution. Compared with Example 1, the difference in composition is that the concentrations of each component in the serum-free cryopreservation solution are different, and the specific components are shown in Table 3.

[0061] Table 3. Specific components of serum-free cryopreservation solutions from Comparative Examples 2-3

[0062] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Comparative Examples 2 and 3 were strictly consistent with those in Example 1.

[0063] Test case In this test case, the cells were resuspended in the serum-free cryopreservation solutions provided in Examples 1-15 and Comparative Examples 1-3 and added to cryovials at a rate of 1 mL / tube. The cryovials were then transferred to a programmed cooling box and placed in a -80°C freezer for 24-72 hours. After that, the cryovials were removed and transferred to a liquid nitrogen tank for storage for more than one month. After the cryovials were removed from the liquid nitrogen tank and the cells were thawed, the cell viability was tested. The results are shown in Table 4. Cell viability (%) = number of live cells / (number of live cells + number of dead cells) × 100%.

[0064] Table 4 Cell viability after cryopreservation and thawing

[0065] Cells were resuspended in the serum-free cryopreservation solutions provided in Examples 1-15 and Comparative Examples 1-3, then incubated at -80°C for 24-72 hours and frozen in liquid nitrogen for more than one month. Cell viability results are shown in Table 4. Simultaneously, NK cells, MSC cells, K562 cells, and Jurkat cells were cryopreserved, thawed, and cultured using the serum-free cryopreservation solution provided in Example 1, and cell morphology was observed. Results are shown below. Figures 1-4 As shown, Figure 1 , 2 Figures 3 and 4 show the cell morphology observation results after NK cells, MSC cells, K562 cells, and Jurkat cells were cryopreserved for more than one month using the serum-free cryopreservation solution provided in Example 1 and then thawed and cultured.

[0066] As shown in Table 4: Compared to Comparative Examples 1-3, the serum-free cryopreservation solutions provided in Examples 1-15 contained 0.2-15% (w / v) of the first additive, 2-15 mg / mL of carbohydrates, 0.7-1.2% (w / v) of inorganic salts, 0.05-0.5 g / mL of protein, and 0.5-5 mg / mL of the second additive. After cryopreservation and thawing of NK cells using the serum-free cryopreservation solutions provided in Examples 1-15, the cell viability was significantly higher than that in Comparative Examples 1-3. Furthermore, combined with… Figure 1 , 2 As can be seen from points 3 and 4, when NK cells, MSC cells, K562 cells, and Jurkat cells are cryopreserved for more than one month using the serum-free cryopreservation solution provided in Example 1, the cells can produce normally and maintain good cell morphology after being thawed and cultured.

[0067] Compared with Example 1, the serum-free cryopreservation solutions provided in Examples 4 and 5 include 7-9% (w / v) of the first additive, 4-11 mg / mL of sugars, 0.8-1.0% (w / v) of inorganic salts, 0.1-0.3 g / mL of protein, and 1-2 mg / mL of the second additive. By comparing the cell viability data of Examples 1, 4, and 5, it can be seen that further controlling the concentration of each component in the serum-free cryopreservation solution within the above range can further improve the cell viability after cryopreservation.

[0068] By comparing the cell viability data of Examples 1, 6, and 7, it can be seen that using DMSO and dextran as the first additives has a synergistic effect, even at low concentrations, thereby improving the cell viability after cryopreservation.

[0069] Comparison of cell viability data from Examples 1, 8, and 9 shows that the types of inorganic salts in the serum-free cryopreservation solution affect cell viability after cryopreservation; comparison of cell viability data from Examples 1, 10, and 11 shows that the types of sugars in the serum-free cryopreservation solution affect cell viability after cryopreservation; comparison of cell viability data from Examples 1 and 12 shows that the types of proteins in the serum-free cryopreservation solution affect cell viability after cryopreservation.

[0070] Compared with Example 1, the serum-free cryopreservation solutions provided in Examples 13-14 also contain a third additive (glutathione, or vitamin C + vitamin E). After NK cells were cryopreserved and thawed using the serum-free cryopreservation solutions provided in Examples 13-14, the cell viability was higher than that in Example 1.

[0071] In Examples 1-15, the cell viability was highest after NK cells were cryopreserved and thawed using the serum-free cryopreservation solution provided in Example 15.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A serum-free cryopreservation solution, characterized in that, The serum-free cryopreservation solution includes a first additive, sugars, inorganic salts, proteins, and a second additive. In the serum-free cryopreservation solution, the mass concentration of the first additive is 0.2-15% (w / v), the concentration of the sugar is 2-15 mg / mL, the mass concentration of the inorganic salt is 0.7-1.2% (w / v), the concentration of the protein is 0.05-0.5 g / mL, and the concentration of the second additive is 0.5-5 mg / mL. The first additive includes at least one of dimethyl sulfoxide, glycerol, dextran, and trehalose; The sugars include at least one of dextran, glucose, sucrose, and fructose; The second additive includes insulin.

2. The serum-free cryopreservation solution as described in claim 1, characterized in that: In the serum-free cryopreservation solution, the mass concentration of the first additive is 7-9% (w / v), the concentration of the sugar is 4-11 mg / mL, the mass concentration of the inorganic salt is 0.8-1.0% (w / v), the concentration of the protein is 0.1-0.3 g / mL, and the concentration of the second additive is 1-2 mg / mL.

3. The serum-free cryopreservation solution as described in claim 1, characterized in that: The inorganic salt includes at least one of sodium chloride, disodium hydrogen phosphate, sodium lactate, potassium chloride, sodium acetate, magnesium chloride, calcium chloride, magnesium sulfate, sodium bisulfite, and sodium gluconate. The protein includes at least one of human serum albumin and bovine serum albumin.

4. The serum-free cryopreservation solution as described in claim 3, characterized in that: The inorganic salts include sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and magnesium chloride, and the protein is human serum albumin.

5. The serum-free cryopreservation solution as described in claim 1, characterized in that: The first additive includes dimethyl sulfoxide and dextran; The sugars include dextran and glucose.

6. The serum-free cryopreservation solution as described in claim 1, characterized in that: The serum-free cryopreservation solution also includes a third additive; In the serum-free cryopreservation solution, the concentration of the third additive is 0.5~100 mg / L; The third additive includes at least one of glutathione, vitamin C, and vitamin E.

7. The serum-free cryopreservation solution as described in claim 6, characterized in that: The third additive includes vitamin C and vitamin E.

8. The serum-free cryopreservation solution as described in claim 7, characterized in that: In the serum-free cryopreservation solution, the concentration of vitamin C is 55-65 mg / L, and the concentration of vitamin E is 5-15 mg / L.

9. The application of the serum-free cryopreservation solution as described in any one of claims 1 to 8 in the cryopreservation of cells.

10. The application of the serum-free cryopreservation solution as described in claim 9 in cell cryopreservation, characterized in that: The cells include at least one of natural killer cells, mesenchymal stem cells, human leukemia cells, and T cells.

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