Cryopreservation liquid composition and cryopreservation method thereof

Abstract

The invention relates to the field of cell cryopreservation, in particular to a cryopreservation solution combination and a cryopreservation method thereof.The cryopreservation solution combination comprises a cryopreservation solution A and a cryopreservation solution B. The cryopreservation solution A comprises NAD + or precursor molecules thereof or proline; the cryopreservation solution B comprises any one or more of L-carnitine, methyl cellulose, synanthrin, betaine, polyvinyl alcohol (PVA), trehalose or dextran. Meanwhile, the invention provides a cryopreservation method using the cryopreservation liquid combination, the apoptosis phenomenon of cells after resuscitation can be remarkably reduced, the dryness of the cells is maintained, the high activity and high survival rate of the resuscitated cells are ensured, and the cryopreservation liquid combination is particularly suitable for cryopreserved single blood sampling.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation, specifically to a cryopreservation solution combination and its cryopreservation method, which is particularly suitable for cryopreserving single-collected blood. Background Technology

[0002] Cell cryopreservation technology is an important component of cell therapy and immunotherapy. Especially in the field of immunocellular therapy, cells such as T cells, NK cells, and γδT cells have demonstrated significant therapeutic potential in clinical practice. However, existing cell cryopreservation technologies face significant challenges in long-term preservation and thawing, such as delayed apoptosis and decreased cell viability, which severely impact their clinical efficacy.

[0003] Currently, commonly used cell cryopreservation solutions can protect cells to some extent, but existing cryopreservation methods still result in significant delayed apoptosis after cell thawing. This delayed apoptosis leads to low cell viability after thawing, frequent cell clumping and lysis, and decreased cell function, severely impacting clinical application. In particular, there is currently no cryopreservation solution specifically designed for apheresis blood samples.

[0004] Apheresis refers to blood collected using a blood component separator, containing primarily mononuclear cells, along with a small number of red blood cells, granulocytes, and plasma components. Unlike PBMCs, apheresis blood is a composite component and does not require cell separation, making it more suitable for large-scale culture of target cells in practical applications. Immune cells in apheresis samples have broad application prospects in disease diagnosis, drug analysis, antibody research, and cell therapy. Therefore, there is an urgent need to develop a cryopreservation solution and method for high-quality preservation of immune cells in apheresis samples. Summary of the Invention

[0005] To address the aforementioned technical problems, the inventors now provide a cryopreservation solution combination and its cryopreservation method, which can solve the problem that cryopreservation of cells, especially cryopreservation of single blood samples, may lead to apoptosis of immune cells after thawing in the prior art.

[0006] The specific technical solution is as follows:

[0007] A cryopreservation solution combination includes cryopreservation solution A and cryopreservation solution B, wherein cryopreservation solution A includes NAD+ or its precursor molecule or proline, and cryopreservation solution B includes a cryoprotectant.

[0008] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide ribonucleoside (NR), or a food- or pharmaceutically acceptable salt, derivative, or prodrug thereof.

[0009] In some specific implementations, the NAD+ precursor is preferably nicotinamide ribonucleoside (NR) or nicotinamide mononucleotide (NMN).

[0010] The cryopreservation solution B contains a cryoprotectant and can use cryopreservation solutions known in the art. In some embodiments, the cryopreservation solution is CS10 (Biolife, USA) or PBMC cryopreservation solution (Cycion Biotech, CS-PM-D1).

[0011] In some embodiments, the cryopreservation solution B includes any one or more of the following components: L-carnitine, methylcellulose, inulin, betaine, polyvinyl alcohol (PVA), trehalose, or dextran; preferably, the cryopreservation solution B includes a combination of L-carnitine, methylcellulose, and inulin, or a combination of betaine, PVA, trehalose, and dextran.

[0012] In some specific embodiments, the cryopreservation solution B also contains dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose, and HSA.

[0013] In some specific implementations, cryopreservation solution A and cryopreservation solution B also contain a solvent selected from compound electrolyte injection or DPBS.

[0014] In some specific embodiments, the cryopreservation solution combination includes cryopreservation solution A and cryopreservation solution B, wherein cryopreservation solution A contains NAD+ or its precursor molecules, and cryopreservation solution B contains DMSO, L-carnitine, methylcellulose, sodium carboxymethylcellulose, inulin, and HSA.

[0015] In some other specific embodiments, cryopreservation solution A contains proline, and cryopreservation solution B contains DMSO, betaine, PVA, sodium carboxymethyl cellulose, trehalose, dextran, and HSA.

[0016] In some specific implementations, the content of each component in the cryopreservation solution combination is as follows:

[0017] Cryopreservation solution A: NAD+ or its precursor molecules at 0.1-0.7 mg / mL, proline at 0.01-0.1 g / mL;

[0018] Cryopreservation solution B: DMSO 5%-15%, L-carnitine 0.01-0.05 g / mL, methylcellulose 0.01-0.05 g / mL, sodium carboxymethyl cellulose 0.005-0.015 g / mL, inulin 0.05-0.15 g / mL, HSA 0.005-0.015 g / mL, betaine 0.01-0.05 g / mL, PVA 0.005-0.015 g / mL, trehalose 0.05-0.15 g / mL, dextran 0.05-0.15 g / mL.

[0019] Another object of the present invention is to provide a cell cryopreservation method, comprising: treating cells with cryopreservation solution A, and cryopreserving the treated cells with cryopreservation solution B; preferably, it comprises the following steps:

[0020] 1) The cryopreservation method described above uses the aforementioned cryopreservation solution A to resuspend cells, followed by incubation and cooling;

[0021] 2) After centrifugation, resuspend the cells in the aforementioned cryopreservation solution B, cool, and then freeze.

[0022] In some implementations, the method has one or more of the following features:

[0023] ① The incubation time is 1-5 hours, and the temperature is 35-45℃, preferably 40℃ for 3 hours;

[0024] ② The cooling time is 5-30 minutes and the temperature is 1-5°C. Preferably, the cooling time is 10 minutes and the cooling temperature is 4°C.

[0025] ③ The density of the resuspended cells is 1×10⁻⁶. 6 -5×10 7 cells / mL, preferably 3×10⁻⁶ 6 -2×10 7 cells / mL.

[0026] In some implementations, the cells are peripheral blood mononuclear cells or cells derived from apheresis.

[0027] Another object of the present invention is to provide a cell obtained by cryopreservation and thawing using the above method, which is selected from any one or more of lymphocytes, dendritic cells, monocytes, macrophages, granulocytes or mast cells, preferably lymphocytes, and more preferably T lymphocytes.

[0028] The beneficial effects of this invention are as follows:

[0029] The cryopreservation solution uses all pharmacopoeia components to ensure its safety and efficacy. Combined with the cryopreservation method, it can significantly reduce cell apoptosis after thawing and maintain cell stemness, ensuring high cell activity and high survival rate after thawing. The method is simple, convenient and quick to operate, and is suitable for clinical and research applications, especially for cryopreserving single-collection blood samples. Attached Figure Description

[0030] Figure 1 This shows the resuscitation viability of white blood cells in apheresis samples;

[0031] Figure 2 The results show the T cell viability of the resuscitated apheresis samples.

[0032] Figure 3 The proliferation status of the sorted T cells was shown;

[0033] Figure 4 It is a T-cell typing after resuscitation. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0035] One objective of this invention is to provide a cryopreservation solution combination, the cryopreservation solution comprising cryopreservation solution A and cryopreservation solution B, wherein cryopreservation solution A contains NAD+ or its precursor molecule or proline, and cryopreservation solution B contains a cryoprotectant.

[0036] The term "NAD+ precursor" refers to any small molecule that leads to an increase in NAD+, and the small molecule can exist in reduced or non-reduced forms.

[0037] NAD+ is a cofactor that plays a central role in metabolism, and its levels decline with age. NAD+ helps repair DNA, regulate immune cell transmission, provide cellular energy, and reduce aging. Adding NAD+ during the in vitro preparation of immune cells provides sufficient energy for immune cell proliferation, increasing cell growth and reducing cellular aging indicators. NAD+ precursors are compounds that are chemically converted into NAD+.

[0038] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide ribonucleoside (NR), or a food- or pharmaceutically acceptable salt, derivative, or prodrug thereof.

[0039] In some specific implementations, the NAD+ precursor is preferably nicotinamide ribonucleoside (NR) or nicotinamide mononucleotide (NMN).

[0040] The cryopreservation solution B can be any cryopreservation solution known in the art. In some embodiments, the cryopreservation solution is CS10 (Biolife, USA) or PBMC cryopreservation solution (Cycio Biotech, CS-PM-D1).

[0041] The CS10 was purchased from Biolife in the United States. It is a clinical-grade, ready-to-use cryopreservation solution, pre-formulated with 10% DMSO, a penetrating cryoprotectant that helps reduce damage caused by ice formation.

[0042] The PBMC cryopreservation solution (Saiqiao Biotechnology, CS-PM-D1) contains a self-developed ice control protectant + 10% USP grade DMSO, and is free of serum, protein, and animal-derived components.

[0043] In some embodiments, the cryopreservation solution B includes any one or more of the following components: L-carnitine, methylcellulose, inulin, betaine, polyvinyl alcohol (PVA), trehalose, or dextran; preferably, the cryopreservation solution B includes a combination of L-carnitine, methylcellulose, and inulin, or a combination of betaine, PVA, trehalose, and dextran.

[0044] In some specific embodiments, the cryopreservation solution B also contains dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose, and HSA.

[0045] In some specific implementations, cryopreservation solution A and cryopreservation solution B also contain a solvent selected from compound electrolyte injection or DPBS.

[0046] In some specific embodiments, the cryopreservation solution combination includes cryopreservation solution A and cryopreservation solution B, wherein cryopreservation solution A contains NAD+ or its precursor molecules, and cryopreservation solution B contains DMSO, L-carnitine, methylcellulose, sodium carboxymethylcellulose, inulin, and HSA.

[0047] In some other specific embodiments, cryopreservation solution A contains proline, and cryopreservation solution B contains DMSO, betaine, PVA, sodium carboxymethyl cellulose, trehalose, dextran, and HSA.

[0048] In some specific implementations, the content of each component in the cryopreservation solution combination is as follows:

[0049] Cryopreservation solution A: NAD+ or its precursor molecules at 0.1-0.7 mg / mL, for example 0.1 mg / mL, 0.13 mg / mL, 0.15 mg / mL, 0.17 mg / mL, 0.2 mg / mL, 0.26 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.6 mg / mL or 0.7 mg / mL.

[0050] Proline is 0.01-0.1 g / mL, for example 0.01 g / mL, 0.03 g / mL, 0.05 g / mL, 0.07 g / mL or 0.1 g / mL.

[0051] Cryopreservation solution B: DMSO at 5%-15%, for example 5%, 7%, 10%, 12% or 15%.

[0052] L-carnitine is present in concentrations of 0.01-0.05 g / mL, such as 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, or 0.05 g / mL.

[0053] Methylcellulose is 0.01-0.05 g / mL, for example 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL or 0.05 g / mL.

[0054] Sodium carboxymethyl cellulose is available in concentrations of 0.005-0.015 g / mL, such as 0.005 g / mL, 0.007 g / mL, 0.01 g / mL, 0.012 g / mL, or 0.015 g / mL.

[0055] Inulin is present in concentrations of 0.05-0.15 g / mL, such as 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, 0.12 g / mL, or 0.15 g / mL.

[0056] HSA concentration is 0.005-0.015 g / mL, for example, 0.005 g / mL, 0.007 g / mL, 0.01 g / mL, 0.012 g / mL or 0.015 g / mL.

[0057] The betaine concentration is 0.01-0.05 g / mL, for example 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL or 0.05 g / mL.

[0058] PVA is 0.005-0.015 g / mL, for example 0.005 g / mL, 0.007 g / mL, 0.01 g / mL, 0.012 g / mL or 0.015 g / mL.

[0059] Trehalose is 0.05-0.15 g / mL, for example 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, 0.12 g / mL or 0.15 g / mL.

[0060] The dextran concentration is 0.05-0.15 g / mL, for example 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, 0.12 g / mL or 0.15 g / mL.

[0061] A second objective of this invention is to provide a cell cryopreservation method, comprising: treating cells with cryopreservation solution A, and cryopreserving the treated cells with cryopreservation solution B; preferably, it includes the following steps:

[0062] 1) The cryopreservation method described above uses the aforementioned cell cryopreservation solution A to resuspend cells, followed by incubation and cooling;

[0063] 2) After centrifugation, resuspend the cells in the aforementioned cell cryopreservation solution B, cool, and then cryopreserve.

[0064] In some implementations, the method has one or more of the following features:

[0065] ① The incubation time is 1-5 hours, and the temperature is 35-45℃, preferably 40℃ for 3 hours;

[0066] ② The cooling time is 5-30 minutes and the temperature is 1-5°C. Preferably, the cooling time is 10 minutes and the cooling temperature is 4°C.

[0067] ③ The density of the resuspended cells is 1×106-5×107 cells / mL, preferably 3×106-2×107 cells / mL.

[0068] In some implementations, the cells are peripheral blood mononuclear cells or cells derived from apheresis.

[0069] The term "collected blood" refers to blood collected using a blood component separator, which mainly consists of mononuclear cells, along with a small number of red blood cells, granulocytes, and plasma components. Unlike PBMCs, collected blood is a composite component and does not require cell separation, making it more suitable for large-scale culture of target cells in practical applications.

[0070] The third objective of this invention is to provide a cell that is obtained by cryopreservation and thawing using the above-described method, and is selected from any one or more of lymphocytes, dendritic cells, monocytes, macrophages, granulocytes, or mast cells, preferably lymphocytes, and more preferably T lymphocytes.

[0071] The cryopreservation process involves cooling the temperature to -80°C and then storing the sample in liquid nitrogen. For details on the cooling process, please refer to Chinese Patent CN202410960509.4, Example 3, the entire contents of which are incorporated herein by reference.

[0072] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Unless otherwise specified, the raw materials or processing techniques used in the following embodiments are conventional commercially available raw materials or conventional processing techniques in the art.

[0073] 1. Source of blood samples

[0074] Blood samples were purchased from MyoShun (Shanghai) Biotechnology Co., Ltd., and were separated using a Terumo Spectra Opitia blood cell separator (apheresis machine).

[0075] 2. Thawing of cryopreserved cells

[0076] Prepare a DPBS solution containing 0.5% HSA and 1 mM EDTA (referred to as "washing buffer"), preheated to 37°C. Remove the frozen apheresis blood preparations from the liquid nitrogen container and quickly place them in a water bath preheated to 37°C, shaking rapidly until the ice is just completely thawed. Transfer the thawed cell suspension to pre-prepared centrifuge tubes, count a small number of cells, add washing buffer at more than twice the volume of the cell suspension, centrifuge at 400g for 5 minutes at room temperature, and discard the supernatant.

[0077] 3. Post-resuscitation cell treatment

[0078] Cells were resuspended at 1×10⁶ cells in AIM-V medium containing 0.5% HSA (referred to as "sorting medium"). 8 Cells / mL. Add 300 U / mL nuclease (Merck). Add CD4 magnetic beads and CD8 magnetic beads [1×10⁻⁶] according to the cell volume. 7 [2 μL (Miltenyi) per cell], incubated at room temperature for 30 min. After incubation, repeat the process at 1 × 10⁻⁶ cells / cell. 7 Add 1 mL of sorting buffer to the cells, mix well, and centrifuge at 300 g for 10 min at room temperature.

[0079] 4. T cell sorting

[0080] Discard the upper part of the solution, each 1×10 8 Add 500 μL of sorting buffer to the cells, resuspend them, and thoroughly disperse them. Select and install an MS / LS sorting column (Miltenyi) according to the cell volume. Rinse the column with 3 mL of sorting buffer. When the liquid in the column is almost completely drained, slowly add the cell suspension into the column. After the solution has passed through the column, add 3 mL of sorting buffer to wash the column, repeating once. Remove the sorting column from the separator and place it on a suitable collection tube. Add 5 mL of sorting buffer and quickly push the plunger into the column to rapidly wash the target cells. After counting a small number of cells, centrifuge at 300 g for 10 min at room temperature.

[0081] 5. T cell activation and culture

[0082] Discard the supernatant and adjust the cell culture density to 2.86 × 10⁶ cells / year using AIM-V medium (referred to as "complete medium") containing IL-7 / IL-15 and 5% serum substitute. 6cells / mL. Activation culture was performed by adding the appropriate amount of activator [TransAct volume is 1 / 35 of the cell volume (Miltenyi)] according to the cell volume. The culture flasks were placed in an incubator and activated for 2 days at a temperature of 37℃±1.0℃ and a CO2 concentration of 5.0%±0.2%.

[0083] After taking a small number of cells for counting, adjust the cell culture density to 1×10⁶ using complete culture medium. 6 cells / mL. Place the culture flask in an incubator at a temperature of 37℃±1.0℃ and a CO2 concentration of 5.0%±0.2%, and repeat the above operation every 2 days for a total of 9 days.

[0084] Example

[0085] Example 1: Cryopreservation solution formulation

[0086] Formula 1:

[0087] Cryopreservation solution A1: 0.0026g NR (nicotinamide ribose) dissolved in 10mL of compound electrolyte injection solution (Hebei Tiancheng).

[0088] Cryopreservation solution B1: 10 mL of cryopreservation solution B is prepared by mixing 1 mL DMSO, 0.2 g L-carnitine, 0.2 g methylcellulose, 0.1 g sodium carboxymethylcellulose, 0.8 g inulin, 0.1 g HSA and 9 mL compound electrolyte injection (Hebei Tiancheng).

[0089] Formula 2:

[0090] Cryopreservation solution A2: 0.0013g NR (nicotinamide ribose) dissolved in 10mL of compound electrolyte injection solution (Hebei Tiancheng).

[0091] Cryopreservation solution B1: 10 mL of cryopreservation solution B is prepared by mixing 1 mL DMSO, 0.2 g L-carnitine, 0.2 g methylcellulose, 0.1 g sodium carboxymethylcellulose, 0.8 g inulin, 0.1 g HSA and 9 mL compound electrolyte injection (Hebei Tiancheng).

[0092] Formula 3

[0093] Cryopreservation solution A3: 0.005g NMN (β-nicotinamide mononucleotide) dissolved in 10mL of compound electrolyte injection solution (Hebei Tiancheng).

[0094] Cryopreservation solution B1: 10 mL of cryopreservation solution B is prepared by mixing 1 mL DMSO, 0.2 g L-carnitine, 0.2 g methylcellulose, 0.1 g sodium carboxymethylcellulose, 0.8 g inulin, 0.1 g HSA and 9 mL compound electrolyte injection (Hebei Tiancheng).

[0095] Formula 4:

[0096] Cryopreservation solution A4: 0.003g NMN (β-nicotinamide mononucleotide) dissolved in 10mL of compound electrolyte injection solution (Hebei Tiancheng).

[0097] Cryopreservation solution B1: 10 mL of cryopreservation solution B is prepared by mixing 1 mL DMSO, 0.2 g L-carnitine, 0.2 g methylcellulose, 0.1 g sodium carboxymethylcellulose, 0.8 g inulin, 0.1 g HSA and 9 mL compound electrolyte injection (Hebei Tiancheng).

[0098] Formula 5:

[0099] Cryopreservation solution A5: 0.5g proline dissolved in 10mL DPBS (Gibco)

[0100] Cryopreservation solution B2: 10 mL of cryopreservation solution B is prepared by mixing 1 mL DMSO, 0.2 g betaine, 0.1 g PVA, 0.08 g sodium carboxymethyl cellulose, 0.7 g trehalose, 0.6 g dextran, 0.1 g HSA and 9 mL compound electrolyte injection (Hebei Tiancheng).

[0101] Formula 6:

[0102] Cryopreservation solution A1: 0.0026g NR (nicotinamide ribose) dissolved in 10mL of compound electrolyte injection solution (Hebei Tiancheng).

[0103] Cryopreservation solution B3: CS10 cryopreservation solution (BioLife Solutions)

[0104] Formula 7:

[0105] Cryopreservation solution A5: 0.5g proline dissolved in 10mL DPBS (Gibco)

[0107] Cryopreservation solution B3: CS10 cryopreservation solution (BioLife Solutions)

[0108] Example 2: Cryopreservation effects of different cryopreservation solution formulations

[0109] Centrifuge the apheresis blood sample at 400g for 5 minutes, discard the supernatant, and resuspend the sample in cryopreservation solution A (formulas 1-7). The resuspended cell density should be 3 × 10⁻⁶ cells / mL. 6 Cells / mL were incubated at 40℃ for 3 hours, cooled at 4℃ for 10 minutes, centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in cryopreservation solution B (formulas 1-7) at a resuspended cell density of 2×10⁻⁶ cells / mL. 7Cells / mL were collected and incubated at 4°C for 10-30 min, then transferred to cryovials and cooled to -80°C before being placed in liquid nitrogen. After one month, cells were selected as T cells according to method 2-5 and then activated for culture.

[0110] Comparative Example 1: Single-sample blood collection was centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in CS10 cryopreservation buffer (BioLife Solutions) at a cell density of 2×10⁻⁶. 7 / mL, incubated at 4℃ for 10 min, transferred to cryovials, cooled to -80℃, and then placed in liquid nitrogen. After one month, cells were selected as T cells according to method 2-5 and then activated for culture.

[0111] The experimental results are shown in Figures 1-4 ,in, Figure 1 The survival rate of leukocytes in apheresis samples was higher than that of Comparative Example 1, demonstrating that the cryopreservation solution and cryopreservation method of this invention can maintain good cell viability.

[0112] Figure 2 The T cell viability of the resuscitated apheresis blood samples was significantly higher than that of the control group 1 in formulations 1-5, demonstrating that the cryopreservation solution and cryopreservation method of this invention have a better preservation effect on T cells.

[0113] Figure 3 The proliferation of the sorted T cells was shown to be significantly higher in formulations 1, 5, 6 and 7 than in comparative example 1, demonstrating that the cryopreservation solution and cryopreservation method of the present invention can effectively improve the proliferation rate of T cells.

[0114] The above experiments demonstrate that formulations 1-5 provided by the invention can maintain good cell viability in cell cryopreservation, have a better preservation effect on T cells, and can also improve the T cell proliferation rate. Furthermore, formulations 6 and 7 demonstrate that the cryopreservation methods provided by the invention can still be effective when used alone, improving the T cell proliferation rate.

[0115] Furthermore, the proportion of TCM cells in the T cells sorted after cryopreservation and thawing using Formula 1 and Formula 5 was significantly higher than that in Comparative Formula 1, demonstrating that the cryopreservation solution and cryopreservation method of the present invention can improve the proportion of TCM cells.

[0116] In summary, this invention can significantly improve the recovery effect of cryopreserved cells and has broad market prospects.

Claims

1. A cryopreservation solution combination, characterized in that, The cryopreservation solution combination includes cryopreservation solution A and cryopreservation solution B, wherein cryopreservation solution A includes NAD+ or its precursor molecule or proline, wherein the NAD+ precursor is selected from any one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide ribonucleoside (NR), or its food- or pharmaceutically acceptable salts, derivatives or prodrugs; and cryopreservation solution B includes a cryoprotectant.

2. The cryopreservation solution combination as described in claim 1, characterized in that, The cryopreservation solution B includes any one or more of the following components: L-carnitine, methylcellulose, inulin, betaine, polyvinyl alcohol (PVA), trehalose, or dextran; preferably, the cryopreservation solution B includes a combination of L-carnitine, methylcellulose, and inulin, or a combination of betaine, PVA, trehalose, and dextran.

3. The cryopreservation solution combination as described in claim 1 or 2, characterized in that, The cryopreservation solution B also contains dimethyl sulfoxide (DMSO), sodium carboxymethyl cellulose and HSA.

4. The cryopreservation solution combination according to any one of claims 1-3, characterized in that, Cryopreservation solution A and cryopreservation solution B also contain solvents selected from compound electrolyte injection or DPBS.

5. The cryopreservation solution combination according to any one of claims 1-4, characterized in that, The cryopreservation solution combination includes cryopreservation solution A and cryopreservation solution B. Cryopreservation solution A contains NAD+ or its precursor molecules, and cryopreservation solution B contains DMSO, L-carnitine, methylcellulose, sodium carboxymethylcellulose, inulin, and HSA. Alternatively, cryopreservation solution A contains proline, and cryopreservation solution B contains DMSO, betaine, PVA, sodium carboxymethyl cellulose, trehalose, dextran, and HSA.

6. The cryopreservation solution combination as described in claim 5, characterized in that, The content of each component is as follows: Cryopreservation solution A: NAD+ or its precursor molecules at 0.1-0.7 mg / mL, proline at 0.01-0.1 g / mL; Cryopreservation solution B: DMSO 5%-15%, L-carnitine 0.01-0.05 g / mL, methylcellulose 0.01-0.05 g / mL, sodium carboxymethyl cellulose 0.005-0.015 g / mL, inulin 0.05-0.15 g / mL, HSA 0.005-0.015 g / mL, betaine 0.01-0.05 g / mL, PVA 0.005-0.015 g / mL, trehalose 0.05-0.15 g / mL, dextran 0.05-0.15 g / mL.

7. A method for cell cryopreservation, characterized in that, include: The process involves treating cells with cryopreservation solution A and then cryopreserving the treated cells with cryopreservation solution B; preferably, it includes the following steps: 1) The cryopreservation method uses cryopreservation solution A according to any one of claims 1-6 to resuspend cells, and then cools them after incubation; 2) After centrifugation, the cells are resuspended in cryopreservation solution B as described in any one of claims 1-6, cooled, and then cryopreserved.

8. The cryopreservation method as described in claim 7, characterized in that, The method has one or more of the following features: ① The incubation time is 1-5 hours, and the temperature is 35-45℃, preferably 40℃ for 3 hours; ② The cooling time is 5-30 minutes and the temperature is 1-5°C. Preferably, the cooling time is 10 minutes and the cooling temperature is 4°C. ③ The density of the resuspended cells is 1×10⁻⁶. 6 -5×10 7 cells / mL, preferably 3×10⁻⁶ 6 -2×10 7 cells / mL.

9. The cryopreservation method as described in claim 7 or 8, characterized in that, The cells are peripheral blood mononuclear cells or cells derived from apheresis.

10. A cell, characterized in that, The cells are obtained by cryopreservation and thawing using the method described in any one of claims 7-9, and are selected from any one or more of lymphocytes, dendritic cells, monocytes, macrophages, granulocytes or mast cells, preferably lymphocytes, and more preferably T lymphocytes.

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