A cell preservation composition for long-distance transportation of cells and a preparation process and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a cell preservation composition beneficial to long-distance transportation of cells and a preparation process and application thereof, the composition realizes high survival rate and functional stability of cells in long-distance transportation and short-term storage through synergistic effect of specific active ingredients and serum-free substitutes, combined with buffer system and cytokine support.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a cell preservation composition that facilitates long-distance cell transport, its preparation process, and its application. Background Technology

[0002] As a core material in biomedical research, cell therapy, and regenerative medicine, maintaining cell viability during long-distance transport is a key bottleneck restricting the clinical translation and application of related technologies. With the rapid development of cell therapies (such as CAR-T and stem cell therapy), tissue engineering, and biopharmaceuticals, the demand for cross-regional cell transport is increasing. However, existing long-distance cell transport technologies and preservation methods have significant limitations, necessitating the development of novel cell preservation compositions to improve cell viability and functional stability during transport.

[0003] Currently, long-distance cell transport mainly relies on three methods, but all have significant shortcomings: First, room temperature transport (20-25℃) involves transporting cells in culture flasks filled with fresh culture medium, suitable for short distances. Second, dry ice (-78.5℃) or liquid nitrogen (-196℃) transport inhibits cell metabolism through low temperatures, theoretically preserving cell viability for extended periods. However, dry ice is volatile and is prohibited for air transport; liquid nitrogen transport requires specialized small liquid nitrogen tanks, which are costly and pose a risk of leakage due to turbulence, making them unsuitable for long-distance transport. Furthermore, some studies have attempted to maintain the cell environment using thermostatic transport devices, but these are limited by power supply and insulation performance, hindering large-scale application.

[0004] Besides the transportation method, the performance of cell preservation solutions directly affects cell viability and functional stability after transport. Traditional cell preservation solutions have the following key drawbacks, such as: insufficient safety of components. Commonly used preservation solutions contain 10%-20% dimethyl sulfoxide (DMSO) or glycerol as cryoprotectants. Although this can lower the freezing point and reduce ice crystal damage, DMSO is cytotoxic (high concentrations can cause cell membrane damage and mitochondrial dysfunction) and may trigger immunogenic reactions in patients. Secondly, existing preservation solutions only focus on inhibiting ice crystal formation or maintaining osmotic pressure, without designing a complex protective mechanism against the multiple damages during long-distance transportation, making it difficult to comprehensively guarantee cell viability.

[0005] In the prior art, some patent documents have attempted to improve cell transport and preservation effects by optimizing the composition. For example, Chinese patent application CN112752505A (substantive examination, composition for transport and / or cryopreservation of cells and / or tissues) discloses a cell transport composition. This composition is mainly composed of basic salts (such as NaCl, KCl) and nutrients (glucose, alanine-glutamine), with 0.05 μM naringenin and 0.1 μM Trolox™ stock solution added as antioxidants. The addition of DMSO mainly serves as a solvent for poorly soluble active ingredients, its core function being to solve the solubility problem of naringenin and Trolox™ stock solution in aqueous solution, with a final DMSO concentration ≤0.0005% (v / v). The cryopreservation composition further incorporates 5% DMSO, 5% dextran 40, and 1% sucrose as cryoprotectants. This approach improves cell transport survival rate and reduces some toxicity risks by introducing antioxidants. However, CN112752505A still has the following limitations: First, the cryopreservation composition still relies on high concentrations of DMSO, which can inhibit ice crystal formation, but the risk of cytotoxicity is not completely eliminated; Second, the active protective ingredients only focus on anti-oxidation (naringenin, Trolox™ stock solution), and do not design a complex protective mechanism for multi-factor damage such as cell adhesion damage caused by mechanical vibration and accumulation of metabolic waste.

[0006] In summary, while existing technologies have made some progress in the field of cell preservation compositions, there is still a need to develop novel cell preservation compositions that are low in toxicity, multifunctional, serum-free, and pH-stable to meet the clinical-grade requirements for long-distance cell transport and cryopreservation. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a cell preservation composition that facilitates long-distance cell transport, along with its preparation process and application. This addresses issues such as the reliance of existing cell preservation solutions on highly toxic DMSO, the lack of a comprehensive protective mechanism against multiple damages during long-distance transport, and the cytotoxicity caused by high concentrations of DMSO during cryopreservation. The present invention provides a low-toxicity, serum-free, and pH-stable cell preservation composition to achieve high cell survival rates and functional stability during long-distance transport and short-term storage.

[0008] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a cell preservation composition that facilitates long-distance cell transport, comprising:

[0010] Basic salts and buffering components, nutritional metabolism support components, active protective components, serum-free alternative components, and cytokine support components;

[0011] The active protective ingredient is a combination of 0.08-0.12 μM kaempferol, 0.12-0.18 μM Trolox™ stock solution, and 45-55 mM betaine.

[0012] The serum-free substitute is PEG-8000, and the amount of the serum-free substitute added is 0.4-0.6% by mass-volume ratio, that is, 0.4-0.6 grams of PEG-8000 are added per 100 ml of solution;

[0013] The solvent is water.

[0014] Preferably, the basic salts and buffer components include: NaCl 80-90 mM, KCl 3.0-4.0 mM, CaCl2 0.06-0.10 mM, MgCl2 hexahydrate 0.50-0.60 mM, NaHCO3 4-6 mM, anhydrous Na2HPO4 11-13 mM, and HEPES 23-27 mM;

[0015] More preferably, the basic salts and buffer components include: NaCl 85-88 mM, KCl 3.2-3.8 mM, CaCl2 0.07-0.09 mM, MgCl2 hexahydrate 0.52-0.58 mM, HEPES 24-26 mM, NaHCO3 4.5-5.5 mM, and anhydrous Na2HPO4 11-12 mM.

[0016] Preferably, the nutritional metabolic support components include: D-(+)-glucose 11-13 mM and alanyl-glutamine 2.0-3.0 mM.

[0017] More preferably, the nutritional metabolic support components include: D-(+)-glucose 11.5-12.5 mM and alanyl-glutamine 2.2-2.8 mM.

[0018] Preferably, the cytokine support component is ITS-X premix, added at 1% (v / v, i.e., 1 mL of ITS-X premix is ​​added to 100 mL of the composition). The ITS-X premix contains 9-11 μg / mL insulin, 4-6 μg / mL transferrin, and 28-32 nM sodium selenite. More preferably, the ITS-X premix contains 10 μg / mL insulin, 5.5 μg / mL transferrin, and 30 nM sodium selenite.

[0019] Preferably, the active protective ingredient further includes 5-10 mM of trehalose to inhibit cell membrane damage caused by mechanical vibration.

[0020] Preferably, the osmotic pressure of the cell preservation composition that facilitates long-distance cell transport is 290-310 mOsm / kg.

[0021] In a second aspect, the present invention further provides a method for preparing the composition provided in the first aspect, comprising the following steps:

[0022] S1. Preparation of basic salt concentrate:

[0023] NaCl, KCl, CaCl2, MgCl2 hexahydrate, anhydrous Na2HPO4, and HEPES were dissolved in pyrogen-free raw water to prepare a concentrated solution, which was then filtered through a 0.22 μm filter membrane to remove bacteria, yielding a basic salt concentrate.

[0024] S2. Preparation of basic working solution:

[0025] Take the basic salt concentrate prepared by S1, dilute it with pyrogen-free raw water, and then add NaHCO3, D-(+)-glucose, alanyl-glutamine and PEG-8000. Stir to dissolve and obtain the basic working solution.

[0026] S3. Add active ingredients:

[0027] Since kaempferol is a flavonoid compound with extremely low solubility in water, it is necessary to prepare a kaempferol stock solution before use. The method is as follows: dissolve kaempferol in pyrogen-free raw water containing 0.1% DMSO to prepare a 1 mM kaempferol stock solution, and filter it through a 0.22 μm filter membrane for sterilization before use.

[0028] Then, take the basic working solution, add betaine and trehalose, then add ITS-X premix, and then add kaempferol stock solution and Trolox™ stock solution under light-protected conditions;

[0029] S4. Adjust volume and perform secondary sterilization:

[0030] Add calorific raw water to the final volume, filter through a 0.22 μm filter membrane, dispense into light-proof containers, and store at 4°C.

[0031] Thirdly, the present invention further provides applications of the aforementioned cell preservation compositions that facilitate long-distance cell transport, for example:

[0032] Cryogenic perfusion preservation of ex vivo organs;

[0033] Transportation of immune cell therapy products;

[0034] Short-term storage of tumor biopsy samples.

[0035] Alternatively, the aforementioned cell preservation compositions that facilitate long-distance cell transport may be used in the preservation and transport of human hair follicle cells, urine-derived cells, CAR-T cells, or induced pluripotent stem cells (iPSCs).

[0036] Furthermore, the present invention also provides a long-distance cell transport kit comprising the composition provided in the first aspect.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] In the composition of this invention, kaempferol and Trolox™ stock solution form a complementary antioxidant chain, significantly reducing oxidative damage to cells caused by the accumulation of reactive oxygen species (ROS) during transport; betaine maintains intracellular osmotic balance, and trehalose effectively inhibits cell membrane structural damage caused by mechanical vibration; the two work synergistically to enhance cellular environmental adaptability; in addition, PEG-8000 blocks abnormal cell adhesion through steric hindrance, preventing nutrient uptake obstacles caused by aggregation during transport. Compared to existing solutions that rely on high concentrations of DMSO or a single protective mechanism, the composition of this invention provides a safer and more efficient solution for long-distance cell transport through its serum-free, low-toxicity component design and multiple synergistic protection strategies. Detailed Implementation

[0039] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and are therefore only examples and should not be used to limit the scope of protection of the present invention. Example

[0040] The present invention aims to provide a cell preservation composition that facilitates long-distance cell transport and a method for preparing the same. Specifically, the composition is formulated as follows:

[0041] Basic salts and buffer components: NaCl 86 mM, KCl 3.5 mM, CaCl2 0.08 mM, MgCl2 hexahydrate 0.55 mM, NaHCO3 5 mM, anhydrous Na2HPO4 12 mM, HEPES (purchased from Sigma-Aldrich H4034) 25 mM;

[0042] Nutritional and metabolic support ingredients: D-(+)-glucose 12 mM, alanyl-glutamine 2.5 mM;

[0043] Active protective ingredients: Kaempferol 0.1 μM, Trolox™ stock solution 0.15 μM, Betaine 50 mM, Trehalose 8 mM;

[0044] Serum-free alternative: PEG-8000 0.5% (w / v);

[0045] Cytokine support components: 1% ITS-X premix (v / v, i.e., 1 mL of ITS-X premix added to 100 mL of composition), which contains 10 μg / mL insulin, 5.5 μg / mL transferrin, and 30 nM sodium selenite.

[0046] The composition is prepared as follows:

[0047] S1. Preparation of basic salt concentrate:

[0048] NaCl, KCl, CaCl2, MgCl2 hexahydrate, anhydrous Na2HPO4, and HEPES were dissolved in pyrogen-free raw water in a certain proportion to prepare a concentrated solution. The solution was then filtered through a 0.22 μm filter membrane to remove bacteria, resulting in a basic salt concentrate.

[0049] S2. Preparation of basic working solution:

[0050] Take the base salt concentrate obtained from S1, dilute it with pyrogen-free raw water, and then add NaHCO3, D-(+)-glucose, alanyl-glutamine and PEG-8000, and stir until completely dissolved.

[0051] S3. Add active ingredients:

[0052] Since kaempferol is a flavonoid compound with extremely low solubility in water, a kaempferol stock solution is first prepared: the method is to dissolve kaempferol in pyrogen-free raw water containing 0.1% DMSO to prepare a 1 mM kaempferol stock solution, which is then filtered through a 0.22 μm filter membrane for sterilization before use.

[0053] Then take the basic working solution and add trehalose, betaine and ITS-X premix to it; under light-protected conditions, add kaempferol stock solution (1 mM, containing 0.1% DMSO) and Trolox™ stock solution, and mix gently.

[0054] S4. Adjust volume and perform secondary sterilization:

[0055] Add pyrogen-free raw water to the final volume, filter through a 0.22 μm filter membrane for sterilization, dispense into light-proof sterile containers, and store at 4°C for later use.

[0056] Among them, betaine was purchased from Shandong Ruihong Biotechnology; ITS-X premix was from Thermo Fisher Scientific (Gibco); Trolox™ stock solution was from Beijing Bio-Laibo Technology Co., Ltd.; and kaempferol was from Thermo Fisher Scientific (Gibco). Example

[0057] The purpose of this invention is to provide a cell preservation composition that facilitates long-distance cell transport and its preparation method. Specifically, the composition formulation is as follows (reagents are the same as in the examples):

[0058] Basic salts and buffer components: NaCl 88 mM, KCl 3.2 mM, CaCl2 0.09 mM, MgCl2 hexahydrate 0.52 mM, NaHCO3 5.2 mM, anhydrous Na2HPO4 11.5 mM, HEPES 24 mM;

[0059] Nutritional and metabolic support ingredients: D-(+)-glucose 11.8 mM, alanyl-glutamine 2.6 mM;

[0060] Active protective ingredients: Kaempferol 0.12 μM, Trolox™ stock solution 0.13 μM, Betaine 48 mM, Trehalose 7 mM;

[0061] Serum-free alternative: PEG-8000 0.6% (w / v);

[0062] Cytokine support components: ITS-X premix 1% (v / v), containing insulin 10 μg / mL, transferrin 5.5 μg / mL, and sodium selenite 30 nM.

[0063] The composition is prepared as follows:

[0064] S1. Preparation of basic salt concentrate:

[0065] NaCl, KCl, CaCl2, MgCl2 hexahydrate, anhydrous Na2HPO4, and HEPES were dissolved in pyrogen-free raw water to prepare a concentrated solution, which was then filtered through a 0.22 μm filter membrane for sterilization to obtain a basic salt concentrate.

[0066] S2. Preparation of basic working solution:

[0067] Take the basic salt concentrate obtained from S1, dilute it with pyrogen-free raw water, add NaHCO3, D-(+)-glucose, alanyl-glutamine and PEG-8000, stir to dissolve, and obtain the basic working solution.

[0068] S3. Add active ingredients:

[0069] Prepare the kaempferol stock solution according to Example 1, then take the basic working solution prepared in S2, and add trehalose, betaine and ITS-X premix solution to it; add the kaempferol stock solution and Trolox™ stock solution under light-protected conditions, and mix gently.

[0070] S4. Adjust volume and perform secondary sterilization:

[0071] Add pyrogen-free raw water to the final volume, filter through a 0.22 μm filter membrane for sterilization, dispense into light-proof containers, and store at 4°C. Example

[0072] The present invention aims to provide a cell preservation composition that facilitates long-distance cell transport and a method for preparing the same. Specifically, the composition is formulated as follows:

[0073] Basic salts and buffer components: NaCl 85 mM, KCl 3.8 mM, CaCl2 0.07 mM, MgCl2 hexahydrate 0.58 mM, NaHCO3 4.8 mM, anhydrous Na2HPO4 12.5 mM, HEPES 26 mM;

[0074] Nutritional and metabolic support ingredients: D-(+)-glucose 12.2 mM, alanyl-glutamine 2.4 mM;

[0075] Active protective ingredients: Kaempferol 0.09 μM, Trolox™ stock solution 0.16 μM, Betaine 52 mM, Trehalose 9 mM;

[0076] Serum-free alternative: PEG-8000 0.4% (w / v);

[0077] Cytokine support component: ITS-X premix 1% (v / v).

[0078] The composition is prepared as follows:

[0079] S1. Preparation of basic salt concentrate:

[0080] NaCl, KCl, CaCl2, MgCl2 hexahydrate, anhydrous Na2HPO4, and HEPES were dissolved in pyrogen-free raw water to prepare a 10× concentrate. The concentrate was then filtered through a 0.22 μm filter membrane to remove bacteria, yielding a basic salt concentrate.

[0081] S2. Preparation of basic working solution:

[0082] Take the basic salt concentrate obtained from S1, dilute it with pyrogen-free raw water, and then add NaHCO3, D-(+)-glucose, alanyl-glutamine and PEG-8000. Stir to dissolve and obtain the basic working solution.

[0083] S3. Add active ingredients:

[0084] Prepare the kaempferol stock solution according to Example 1, then take the basic working solution prepared in S2, and add trehalose, betaine and ITS-X premix solution to it; add the kaempferol stock solution and Trolox™ stock solution under light-protected conditions, and mix gently.

[0085] S4. Adjust volume and perform secondary sterilization:

[0086] Add pyrogen-free raw water to the final volume, filter through a 0.22 μm filter membrane for sterilization, dispense into light-proof containers, and store at 4°C.

[0087] The purpose of this comparative example is to verify the protective mechanism of the active component in this invention on cell transport and the effect of PEG-8000, as follows:

[0088] 1.1 Experimental Design

[0089] Baseline: The composition of Example 1 (kaempferol 0.1 μM + Trolox™ stock solution 0.15 μM + betaine 50 mM + PEG-8000 0.5% + trehalose 8 mM + other basic ingredients).

[0090] Comparative groups: a total of 8 groups, each group changed only 1 key variable, and the remaining components and preparation methods were completely consistent with Example 1. The key variable settings for each group are shown in Table 1.

[0091] Table 1. Experimental groupings and key variable settings for each group

[0092] Group Variable settings Group 1-1 Kaempferol missing Group 1-2 Naringenin (0.1 μM) as a substitute for kaempferol Group 1-3 Quercetin (0.1 μM) as a substitute for kaempferol Groups 1-4 Trolox™ stock solution missing Groups 1-5 lacking betaine Groups 1-6 PEG-8000 missing Groups 1-7 PEG-8000 concentration 0.2% Groups 1-8 PEG-8000 concentration 0.8%

[0093] 1.2 Experimental methods and key detection indicators

[0094] 1.2.1 Cell origin

[0095] Human hair follicle cells (commercially available, passaged to the 3rd generation to ensure good condition) were collected in the logarithmic growth phase. Cells were digested with trypsin, centrifuged (1000 rpm, 5 min), washed twice with PBS, resuspended in the respective combination mixtures, and adjusted to a density of 1×10⁻⁶ cells / mL. 6 cells / mL (survival rate >95% confirmed by trypan blue staining).

[0096] 1.2.2 Simulated Transportation Conditions

[0097] The experiment was conducted using the following methods:

[0098] Simulated transport container: 5 mL sterile centrifuge tubes (each tube contains 3 mL of cell suspension, ensuring consistent liquid level).

[0099] Temperature control: Placed in a 4℃ constant temperature transport box (model: Thermo Scientific TSX-150) to simulate 72 hours of long-distance transport.

[0100] Vibration simulation: The transport box has a built-in horizontal shaking table (50 rpm) to simulate mechanical vibration during transportation.

[0101] 1.2.3 The following indicators shall be tested after transportation:

[0102] Cell viability: Trypan blue staining was used to count the percentage of unstained live cells in 200 cells (number of live cells / total number of cells × 100%), and the average value was taken after 3 replicates.

[0103] ROS level: Intracellular fluorescence intensity (AU) was detected by flow cytometry using the DCFH-DA fluorescent probe (excitation wavelength 488 nm, emission wavelength 525 nm) to reflect the degree of oxidative damage.

[0104] LDH release: The activity (U / L) of lactate dehydrogenase (LDH) in the supernatant was detected by colorimetry (450 nm wavelength). The higher the LDH release, the more severe the damage to membrane integrity.

[0105] Cell aggregation rate: The percentage of clumps with ≥5 cells in the field of view under an optical microscope (200×), reflecting cell adhesion damage caused by mechanical vibration.

[0106] The experimental results are shown in Table 2:

[0107] Table 2. Experimental results of cells in each experimental group under simulated transport conditions.

[0108] Group Survival rate (%) ROS fluorescence intensity (AU) LDH release (U / L) Aggregation rate (%) Group 1-1 78.2±1.2 132.4±2.5 7.2±0.3 5.3±0.4 Group 1-2 82.1±1.5 118.3±2.1 6.4±0.2 5.1±0.3 Group 1-3 85.3±1.8 102.5±1.9 5.6±0.1 5.0±0.2 Groups 1-4 81.4±1.3 125.1±2.3 6.8±0.2 5.2±0.3 Groups 1-5 75.6±1.1 105.2±1.7 8.1±0.4 5.4±0.5 Groups 1-6 83.7±1.6 103.8±1.8 5.8±0.1 22.4±3.2 Groups 1-7 87.1±1.9 101.9±1.6 5.3±0.2 12.3±2.1 Groups 1-8 84.5±1.7 102.1±1.5 5.5±0.1 4.8±0.2 Example 1 97.2±0.9 76.4±1.2 4.5±0.2 2.2±0.3

[0109] The results above demonstrate that the absence or concentration adjustment of key components significantly affects cell viability. When antioxidants (such as kaempferol and Trolox™ stock solution) are absent, intracellular ROS levels significantly increase (by 20%-30% compared to the baseline group), membrane integrity is impaired (LDH release increases by 10%-40%), ultimately leading to a decrease in cell viability (a reduction of approximately 5%-15%). Among these, the absence of kaempferol has the most significant impact on ROS clearance, while the absence of Trolox™ stock solution also leads to a decrease in cell viability.

[0110] The effect of PEG-8000 concentration changes on cell aggregation rate is particularly prominent: when PEG-8000 is absent, the aggregation rate increases sharply to more than 4 times that of the baseline group (22.4% vs 5%), possibly due to enhanced intercellular adhesion leading to restricted nutrient diffusion; although low concentration (0.2%) or high concentration (0.8%) of PEG-8000 does not completely lose its protective effect, the protective effect is significantly reduced, which is speculated to be due to insufficient steric hindrance (aggregation rate 12.3%) or membrane interference (decreased adhesion rate) resulting in a lower survival rate than the baseline group.

[0111] The purpose of this comparative example is to verify the protective mechanism of the active component in this invention on cell transport, specifically as follows:

[0112] Baseline: The composition of Example 1 (50 mM betaine + 8 mM trehalose + other ingredients).

[0113] Comparative groups: There are 4 groups in total. In each group, only the combination of betaine and trehalose is adjusted (to keep the total osmotic pressure of all groups the same and make up with mannitol). The other components and preparation methods are the same as in Example 1. See Table 3 for details.

[0114] Table 3. Experimental groupings and key variable settings for each group.

[0115] Group Variable settings Group 2-1 Contains only betaine (50 mM), no trehalose. Group 2-2 Contains only trehalose (8 mM), no betaine. Group 2-3 Betaine (45 mM) + Trehalose (8 mM) Group 2-4 Betaine (55 mM) + Trehalose (8 mM)

[0116] Cell preparation and transport conditions were simulated in Comparative Example 1. The following indicators were detected after transport:

[0117] Cell viability: CCK-8 assay (450 nm absorbance, OD value reflects metabolic activity), repeated 3 times and the average value was taken.

[0118] Early apoptosis rate: Annexin V + / PI - Double staining (flow cytometry, Q3 quadrant ratio) reflects the degree of membrane damage.

[0119] Osmotic recovery rate: The ratio (%) of cell volume after transport to the initial volume is calculated using forward scattered light (FSC) before flow cytometry, reflecting the osmotic regulation capacity.

[0120] Membrane protein integrity: Flow cytometry was used to detect the positivity rate (%) of keratin on the surface of hair follicle cells to reflect the stability of cell membrane structure.

[0121] Table 4. Experimental results of the synergistic protective effect of betaine and trehalose.

[0122] Group Survival rate (%) Early apoptosis rate (%) Permeability recovery rate (%) Membrane protein positivity rate (%) Group 2-1 77.3±1.4 12.1±1.2 88.2±2.1 79.5±2.3 Group 2-2 79.4±1.2 11.8±1.1 75.6±1.8 72.5±2.0 Group 2-3 88.2±1.6 7.8±0.9 85.1±2.0 83.2±2.5 Group 2-4 86.4±1.5 8.3±1.0 88.5±2.2 80.2±2.1 Example 1 97.0±1.3 3.1±0.4 97.1±2.5 89.1±2.4

[0123] The above experimental results show that the synergistic effect of betaine and trehalose is a key factor in improving cell viability and functional integrity, as detailed below:

[0124] Regarding cell viability, the cell viability was significantly lower when betaine (group 2-1, 77.3%) or trehalose (group 2-2, 79.4%) were used alone compared to the experimental group using a combination of both.

[0125] Among them, the survival rate of Example 1 (50 mM betaine + 8 mM trehalose) was the highest (97.0%), which was significantly higher than that of Groups 2-3 (45 mM + 8 mM, 88.2%) and Groups 2-4 (55 mM + 8 mM, 86.4%) after adjusting the betaine concentration, indicating that the ratio of 50 mM betaine to 8 mM trehalose can maximize the cell viability maintenance effect.

[0126] The early apoptosis rate results further validated the advantages of the synergistic effect: the early apoptosis rate of Example 1 was only 3.1%, far lower than that of the single-component group (11.8%-12.1%) and other combination groups (7.8%-8.3%), indicating that the combined use of the two components can effectively reduce membrane damage during transport and inhibit early cell apoptosis. The osmotic recovery rate and membrane protein integrity indicators revealed differences in the synergistic protection mechanism: Group 2-1, using betaine alone, had a higher osmotic recovery rate (88.2%), but a lower membrane protein positivity rate (79.5%); Group 2-2, using trehalose alone, had a slightly higher membrane protein positivity rate (72.5%), but a significantly lower osmotic recovery rate (75.6%). Example 1 showed the best performance in both indicators (osmotic recovery rate 97.1%, membrane protein positivity rate 89.1%), suggesting that betaine mainly maintains cell volume homeostasis through osmotic regulation, while trehalose focuses on stabilizing membrane structure, and the two synergistically achieve the dual effects of "osmotic protection + membrane stabilization".

[0127] This experiment demonstrates that the combination of 50 mM betaine and 8 mM trehalose can significantly improve cell survival during transport, inhibit early apoptosis, enhance osmotic regulation, and maintain membrane protein integrity through a synergistic effect. This ratio provides a key basis for optimizing cell transport protectants, and its synergistic mechanism may be related to the complementary effects of maintaining osmotic balance and membrane structural stability.

[0128] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A cell preservation composition that facilitates long-distance cell transport, characterized in that, The composition is a combination of active protective components, serum-free alternative components, basic salts and buffering components, nutritional metabolic support components, and cytokine support components: The active protective ingredient is a combination of kaempferol 0.08-0.12 μM, Trolox™ stock solution 0.12-0.18 μM, betaine 48-52 mM, and trehalose 5-10 mM. The serum-free substitute is PEG-8000, and its addition amount is calculated as 0.4-0.6% by mass-volume ratio; The basic salts and buffer components include: NaCl 80-90mM, KCl 3.0-4.0mM, CaCl2 0.06-0.10mM, MgCl2 hexahydrate 0.50-0.60mM, NaHCO3 4-6mM, anhydrous Na2HPO4 11-13mM, and HEPES 23-27mM; The nutritional metabolic support components include: D-(+)-glucose 11-13 mM and alanyl-glutamine 2.0-3.0 mM; The cytokine support component is ITS-X premix, which contains 9-11 μg / mL insulin, 4-6 μg / mL transferrin, and 28-32 nM sodium selenite. The amount of ITS-X premix added is 1% by volume fraction.

2. The method for preparing the cell preservation composition facilitating long-distance cell transport as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of basic salt concentrate: NaCl, KCl, CaCl2, MgCl2 hexahydrate, anhydrous Na2HPO4, and HEPES were dissolved in pyrogen-free raw water to prepare a concentrated solution, which was then filtered through a 0.22 μm filter membrane to remove bacteria, yielding a basic salt concentrate. S2. Preparation of basic working solution: Take the basic salt concentrate obtained from S1, dilute it with pyrogen-free raw water, and then add NaHCO3, D-(+)-glucose, alanyl-glutamine and PEG-8000. Stir to dissolve and obtain the basic working solution. S3. Add other ingredients: Betaine and trehalose were added to the basic working solution, followed by ITS-X premix solution. Kaempferol stock solution and Trolox™ stock solution were added under light-protected conditions. The kaempferol stock solution was an aqueous solution containing 0.1% DMSO. S4. Adjust volume and perform secondary sterilization: Add calorific raw water to the final volume, filter through a 0.22μm filter membrane, dispense into light-proof containers, and store at 4℃.

3. The application of the cell preservation composition for long-distance cell transport as described in claim 1 in the preservation and transport of human hair follicle cells.