A wide temperature range protection solution suitable for somatic cells and a preparation method and application thereof

Abstract

This invention discloses a wide-temperature-range protective solution suitable for somatic cells, its preparation method, and its applications, belonging to the fields of biotechnology and cell preservation. The protective solution comprises an osmotic pressure regulating system, an antioxidant protection system, a nutrient supply agent, a pH-stabilizing buffer pair, a cell membrane protectant and strengthener, a preservative and antibacterial agent, and an anticoagulant. Through the synergistic effect of these multiple systems, the protective solution can provide a stable environment for cells within a wide temperature range of -196 to 25°C, effectively resisting temperature fluctuations that may occur during transportation, maintaining cell viability, and significantly extending cell preservation time. It is suitable for the transportation and short-term preservation of cell therapy products.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cell preservation, specifically to a wide-temperature-range preservation solution suitable for somatic cells, its preparation method, and its application. Background Technology

[0002] In cell research, medicine, and other fields, the preservation of somatic cells is crucial. Traditional cell preservation methods have many limitations. While cryopreservation technology (liquid nitrogen -196°C or deep cryogenic freezer -80°C) is the mainstream method, the equipment procurement and operating costs are high, and the freezing and thawing processes are prone to cell damage due to ice crystal formation, reducing cell viability. Furthermore, cryopreserved cells are limited in applications such as long-distance transportation and emergency medical rescue, making it difficult to meet immediate needs. Cryogenic cell transport places stringent requirements on transport conditions, equipment, and duration, necessitating the continuous maintenance of a low-temperature environment, which increases cost and complexity. At room temperature, cell survival time is short, and their activity and function are easily affected. Existing protective reagents still fall short in balancing room-temperature preservation effectiveness and adaptability to multiple temperature zones. Studies have shown that cell membrane fluidity, metabolic activity, and the structure and function of biomolecules change under different temperature conditions. High temperatures may lead to protein denaturation and decreased cell membrane stability; low temperatures, on the other hand, easily trigger intracellular ice crystal formation, damaging the cell membrane and organelles. Therefore, there is an urgent need to develop a protective solution that can adapt to both refrigerated environments of 2–8°C and ambient environments of 10–25°C, reduces processing steps, has low cytotoxicity, and meets the standards for medical raw materials and excipients, thus facilitating the preservation and transportation of cell-based drugs. Summary of the Invention The purpose of this invention is to overcome the shortcomings of the prior art and provide a protective solution with clearly defined components, good stability, and the ability to effectively maintain the activity and function of somatic cells in a wide temperature range (-196~25℃).

[0003] Another object of the present invention is to provide a method for preparing the above-mentioned protective liquid.

[0004] Another object of the present invention is to provide the use of the above-mentioned protective solution in the preparation of formulations for preserving somatic cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a wide-temperature-range protective solution suitable for somatic cells, comprising the following components by weight-volume percentage (w / v): a) Osmotic pressure regulation system: containing 2%–8% trehalose, 1%–5% mannitol and 0.5%–1.5% sucrose; b) Antioxidant protection system: Contains 0.1%–3% vitamin C and 0.1%–1% glutathione; c) Nutritional supplements: containing 0.5%–3% of an amino acid mixture, 2.0%–3.5% of glucose, 0.2%–0.5% of glutamine, and 0.1%–1% of B vitamins; d) pH-stabilizing buffer pair: containing 0.2%–3% HEPES, 0.3%–0.6% disodium hydrogen phosphate, 0.2%–0.4% potassium dihydrogen phosphate and 0.1%–1% sodium bicarbonate; e) Cell membrane protectors and enhancers: containing 0.05%–0.2% hyaluronic acid, 0.1%–1% cholesterol, and 0.05%–0.5% phosphatidylcholine; f) Preservative and antibacterial agent: 0.01% to 0.1% sodium benzoate; g) Anticoagulant: Low molecular weight heparin sodium, concentration ≤10 U / mL; The remainder is solvent, which is physiological saline or a buffer system based on DPBS (Durbeco phosphate buffered saline) or HBSS (Hankes balanced salt solution).

[0006] Preferably, in the osmotic pressure regulating system, the concentration of trehalose is 3% to 6%, the concentration of mannitol is 2% to 4%, and the concentration of sucrose is 0.8% to 1.2%.

[0007] Preferably, in the antioxidant protection system, the concentration of vitamin C is 0.5% to 2%, and the concentration of glutathione is 0.2% to 0.8%.

[0008] Preferably, in the nutritional supplement, the amino acid mixture contains all essential and non-essential amino acids; the B vitamins include one or more of vitamins B1, B2, B3, B5, B6, B7, B9, and B12.

[0009] Preferably, in the cell membrane protectant and strengthener, the concentration of hyaluronic acid is 0.08% to 0.15%, the concentration of cholesterol is 0.3% to 0.7%, and the concentration of phosphatidylcholine is 0.1% to 0.3%.

[0010] Preferably, the concentration of the anticoagulant, low molecular weight heparin sodium, is 5–8 U / mL.

[0011] Preferably, the pH value of the protective solution is 7.2 to 7.4.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned wide-temperature-range protective liquid, comprising the following steps: (1) Dissolve glucose, sucrose, potassium dihydrogen phosphate, disodium hydrogen phosphate, HEPES and sodium bicarbonate in part of DPBS or HBSS solvent and stir until completely dissolved to form a basic buffer solution. (2) Dissolve vitamin C and glutathione in a small amount of deionized water, then add them to the basic buffer solution obtained in step (1) and stir until homogeneous; (3) Add trehalose, mannitol, amino acid mixture, glutamine and B vitamins to the mixed solution obtained in step (2) in sequence, and stir until completely dissolved; (4) Gently heat the solution obtained in step (3) to 50°C, add cholesterol and phosphatidylcholine, stir until completely dissolved, cool to room temperature, add hyaluronic acid, and stir thoroughly to disperse it evenly. (5) Add sodium benzoate and low molecular weight heparin sodium to the solution obtained in step (4), and make up the balance with DPBS or HBSS. Adjust the pH to 7.2 to 7.4 precisely using dilute hydrochloric acid or sodium hydroxide solution. (6) Filter the protective solution obtained in step (5) to sterilize it (e.g., using a 0.22 μm filter membrane) and dispense it.

[0013] Thirdly, the present invention provides the use of the above-mentioned wide-temperature-range protective liquid in the preparation of formulations for preserving somatic cells in a temperature range of -196 to 25°C.

[0014] Preferably, the cells include adherent cells (such as mesenchymal stem cells, fibroblasts, or epithelial cells) and suspension cells (such as T cells, NK cells, and DC cells).

[0015] The beneficial effects of this invention are as follows: Wide temperature range stability: Through the synergistic effect of multiple components, this protective solution provides a stable environment for cells within a wide temperature range of -196 to 25°C, effectively resisting temperature fluctuations that may occur during transportation and significantly extending cell preservation time. At low temperatures of 2–8°C, it reduces the risk of ice crystal damage; at room temperature (10–25°C), cell survival time is extended to more than 7 days with over 80% activity maintained; and at higher temperatures of 30–37°C, it alleviates protein denaturation, facilitating cell transportation and emergency use.

[0016] Multiple protection mechanisms: Osmotic pressure balance: Trehalose, mannitol and sucrose work together to precisely regulate osmotic pressure within the physiological range, preventing cell shrinkage or rupture.

[0017] Antioxidant damage: Vitamin C and glutathione form a highly effective antioxidant system that eliminates free radicals generated by cell metabolism and the environment, reducing oxidative stress damage.

[0018] Nutritional and energy support: Glucose provides energy, amino acid mixtures and glutamine provide the substrates required for anabolic metabolism, and B vitamins participate in energy metabolism as coenzymes, together maintaining basic cellular metabolism and delaying apoptosis.

[0019] pH stability: HEPES, phosphate and sodium bicarbonate form a multiple buffer system that can effectively maintain pH stability at different temperatures and avoid acidosis or alkalosis.

[0020] Membrane structure stability: Cholesterol and phosphatidylcholine can be used to repair and stabilize cell membrane structure, while hyaluronic acid forms a hydration protective layer around the cell, reducing mechanical damage.

[0021] Safety and usability: All ingredients are biocompatible or approved pharmaceutical excipients. Low molecular weight heparin prevents cell aggregation, and sodium benzoate inhibits microbial growth, ensuring the safety and usability of the product.

[0022] The preparation process is simple: the preparation method provided by this invention has clear steps, mild conditions, and is easy to scale up for production.

[0023] In this invention, the term "normal temperature" refers to an ambient temperature of 10°C to 25°C, including but not limited to any integer temperature value within this range and any value between them. Attached Figure Description

[0024] Figure 1 Microscopic images of human mesenchymal stem cells after 7 days of preservation at room temperature using the traditional protective solution in Example 1 of this invention; Figure 2 Microscopic images of NK cells after being preserved at -196°C for 24 hours using the traditional protective solution in Example 1 of this invention; Figure 3 Microscopic images of mouse mononuclear macrophages after one month of cryopreservation using Example 1 of the present invention and conventional cryopreservation solution; Figure 4 These are microscopic images of NK cells after one month of cryopreservation at -80°C using the traditional cryoprotectant solution of Example 1 of this invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0026] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0027] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0028] In this invention, the term "room temperature" refers to an ambient temperature between 10°C and 25°C. It should be understood that this range is intended to cover any value from 10°C to 25°C, including but not limited to any integer temperature value within this range and any value between them. In the following embodiments, the value in parentheses after "room temperature" refers to the temperature value or range when the specific experiment was conducted, and does not define a temperature range of room temperature.

[0029] The following table shows the source and specifications of some of the main raw materials in the examples.

[0030]

[0031] Table 1. Sources and specifications of some main raw materials in the embodiments. Examples 1-3 Examples 1-3 provide a wide-temperature-range protective solution suitable for somatic cells. The composition of the protective solution in each example is shown in Table 2.

[0032] Table 2. Components and content of protective liquid in Examples 1-3

[0033] The preparation method is as follows: (1) Take about 800 mL of DPBS pre-cooled at 4℃ into a sterile beaker and place it on a magnetic stirrer; (2) While stirring, add HEPES, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, sucrose and glucose in sequence, and stir until completely dissolved to obtain the basic buffer solution; (3) Dissolve vitamin C and glutathione in 10 mL of sterile deionized water respectively. After they are completely dissolved, slowly add them to the basic buffer solution in step 2 and mix well. (4) Add trehalose, mannitol, amino acid mixture, glutamine and compound vitamin B powder to the above solution in sequence, and stir continuously until the solution is clear and transparent; (5) Place the beaker in a 50°C water bath and heat gently. Add cholesterol and phosphatidylcholine while stirring. Maintain the temperature and stir for about 30 minutes until they are completely dissolved and dispersed. Then remove the solution and cool it to room temperature (25°C). (6) At room temperature (25℃), slowly add hyaluronic acid and increase the stirring speed, stirring for 1 hour to make it fully and evenly dispersed; (7) Add sodium benzoate and low molecular weight heparin sodium, and stir until dissolved; (8) Add DPBS to bring the volume to 1000 mL; (9) Finely adjust the pH of the solution to 7.3 using 1M NaOH or 1M HCl; (10) The prepared protective solution was filtered and sterilized using a 0.22 μm polyethersulfone (PES) filter membrane; (11) Dispense into sterile containers under aseptic conditions, seal, and store at 2-8°C away from light for later use.

[0034] Comparative Example 1 The osmotic pressure regulation system was replaced with 9% DMSO, and the rest was the same as in Example 1.

[0035] Comparative Example 2 The nutrient supply was replaced with an equal volume of RPMI-1640 culture medium, and the rest was the same as in Example 1.

[0036] Comparative Example 3 Trehalose was removed from the osmotic pressure regulation system, leaving only 6% mannitol and 3% sucrose, with the rest being the same as in Example 1.

[0037] Comparative Example 4 Glutathione was removed from the antioxidant protection system, leaving 2.2% vitamin C.

[0038] Comparative Example 5 The pH buffer does not contain sodium bicarbonate; its amount is replaced with disodium bicarbonate, and the rest is the same as in Example 1.

[0039] Comparative Example 6 The cell membrane protectant and enhancer does not contain hyaluronic acid, but contains 0.756% cholesterol and 0.324% phosphatidylcholine, and the rest is the same as in Example 1.

[0040] Comparative Example 7 In the anticoagulant, sodium heparin was replaced with EDTA, and the rest was the same as in Example 1.

[0041] Example 1: Somatic cell protection effect test at room temperature Experimental materials: human mesenchymal stem cells, protective solutions for Examples 1-4 and Comparative Examples 1-7, conventional room temperature preservation solutions (commercial room temperature cell preservation solutions), cell culture plates, etc. Experimental steps: hUC-MSCs in the logarithmic growth phase were collected by digestion and centrifugation, and washed twice with PBS. Adjust the cell density to 1×10 6 Cells / mL, for later use. Mix the cell suspension with Examples 1-4, Comparative Examples 1-7, and conventional room temperature preservation solution at a 1:1 (volume ratio), and gently pipette to ensure homogeneity; the final cell density is 5 × 10⁻⁶ cells / mL. 5cells / mL; each group was divided into 3 replicates, with 200 μL of the mixture added to each well of a 96-well plate. The culture plate was placed at room temperature (22 ± 2℃), avoiding light and vibration; samples were taken on days 1, 3, 5, and 7 for the following assays: CCK-8 assay: Add 20 μL of CCK-8 reagent to each well, incubate for 2 hours, and then measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate the relative cell viability (with day 0 as 100%). Morphological observation: Morphological observation was performed on day 7. Using an inverted phase-contrast microscope, three fields of view were selected for each replicate in each group, observed under 4× and 10× objectives. The results of the relative cell viability experiment are shown in Table 3. The results show that the protective solutions of Examples 1-4 of this invention exhibit excellent protective effects on human mesenchymal stem cells (hUC-MSCs) at room temperature (22±2℃). As can be seen from the data in Table 3, the cell viability of Example 1 on days 1, 3, 5, and 7 were 97%, 93%, 87%, and 82%, respectively. Other Examples (2-4) also showed similar high viability, with the viability remaining above 79% on day 7, indicating that the cells maintained high viability and morphological integrity within 7 days. In contrast, the cell viability of the traditional room temperature preservation solution group was only 43% on day 7, and cell viability significantly decreased to below 55% from day 5 onwards, with a large number of cells aging and undergoing apoptosis.

[0042] Comparative Example 1 showed a cell survival rate of 57% on day 7, which was much lower than that of Example 1, indicating that the osmotic pressure regulation system (containing trehalose, mannitol and sucrose) is crucial for maintaining cell osmotic balance and that DMSO may not be an effective substitute.

[0043] Comparative Example 2 showed a cell survival rate of 71% on day 7, which was higher than that of the conventional preservation solution but lower than that of Example 1, indicating that the special nutrient supply can more effectively provide the energy and metabolic support required by the cells.

[0044] The cell survival rate of Comparative Example 3 on day 7 was 76%, which was lower than that of Example 1, indicating that trehalose, as an osmotic protectant, can enhance cell resistance to stress, and its absence leads to a decrease in protective effect.

[0045] Comparative Example 4 showed a cell survival rate of 61% on day 7, which was lower than that of Example 1, indicating that the synergistic antioxidant effect of glutathione and vitamin C is crucial for reducing oxidative damage, and that vitamin C alone is insufficient.

[0046] Comparative Example 5 showed a cell survival rate of 73% on day 7, which was lower than that of Example 1, indicating that sodium bicarbonate is superior in maintaining pH stability and can better support the cellular metabolic environment.

[0047] Comparative Example 6 and phosphatidylcholine have a synergistic effect, which is lower than that of Example 1, and have a positive effect on strengthening cell membrane structure and reducing mechanical damage.

[0048] Comparative Example 7 showed a cell survival rate of 66% on day 7, which was lower than that of Example 1, indicating that sodium heparin is superior to EDTA in preventing cell aggregation and anticoagulation, and can better maintain cell suspension stability.

[0049] Morphological observation results (see) Figure 1 Under an inverted phase-contrast microscope, the cells in the preservation solution group of Examples 1-4 of this invention were observed to be regularly spindle-shaped, with plump cytoplasm, good refractive properties, and clear outlines. Most cells adhered firmly to the wall, and only a few cells were observed to be rounded or loose, easily detached with slight shaking; floating cells (<2%) were also observed. The field of view was clean, with occasional small fragments, estimated to account for about 1-2%. The cells were in good condition. In contrast, the cells in the traditional preservation solution group showed increased cell fragmentation and morphological damage.

[0050] In summary, the protective solution of this invention significantly improves cell survival rate and morphological integrity at room temperature through the synergistic effect of multiple systems (including osmotic pressure regulation, nutrient supply, antioxidant protection, pH buffering, cell membrane protection and strengthening, and anticoagulation), and each component is indispensable. Comparative results further confirm the rationality and superiority of the formulation of this invention.

[0051] Table 3 Results of somatic cell protection effect test at room temperature

[0052] Example 2: Test of the preservation effect of somatic cells (multi-temperature) in a wide temperature range Experimental materials: NK cells, cryoprotectant solutions for Examples 1-4 and Comparative Examples 1-7, conventional (ultra) cryoprotectant control, flow cytometer, etc. Experimental methods: NK cells in the logarithmic growth phase of in vitro expansion culture were collected by centrifugation, resuspended in PBS, and the cell density was adjusted to 1×10⁶. 7 Prepare NK cell suspensions at a ratio of 1:1 (volume ratio) with the cryoprotectants of Examples 1-4 and Comparative Examples 1-7, and conventional (ultra) cryoprotectants. Gently pipette to ensure the cells are fully suspended in the cryoprotectant system, achieving a final cell density of 5 × 10⁻⁶ cells / mL. 6 Cells / mL. The cell-protection solution mixtures from each group were aliquoted into cryovials and stored at four different temperatures (-196℃ (liquid nitrogen), -20℃, 4℃, and 25℃) for 24 hours. After 24 hours, the samples under each temperature condition were thawed (the -196℃ and -20℃ groups required rapid thawing in a 37℃ water bath), and cell viability was accurately detected using an AOPI dual-fluorescence counter.

[0053] Morphological observation: Cell suspensions from each group (Examples 1-4, Comparative Examples 1-7, and the conventional cryoprotectant group) were collected after being cryopreserved in liquid nitrogen (-196℃) for 24 hours and then thawed. Fresh NK cells in the logarithmic growth phase, which were not cryopreserved, served as normal controls. The cell suspensions were mixed with AOPI at an appropriate ratio. 20 μL of the stained cell suspension was added to a clean glass slide, and a coverslip was gently placed on top to avoid air bubbles. At least three non-overlapping fields of view were selected from each sample group for observation using an inverted microscope.

[0054] The results are shown in Table 4 and... Figure 2 The results showed that the protective solutions of Examples 1-4 of the present invention exhibited excellent protective effects on NK cells in a wide temperature range from ultra-low temperature (-196℃) to room temperature (25℃).

[0055] Excellent wide temperature range adaptability: In Example 1, cell viability rates at -196℃, -20℃, 4℃, and 25℃ were as high as 96%, 94%, 87%, and 83%, respectively. This indicates that this protective solution can not only effectively cope with ice crystal damage and osmotic stress caused by ultra-low temperature freezing, but also significantly prolong cell survival time under refrigeration and room temperature conditions by maintaining cell metabolic balance and inhibiting apoptosis, achieving a breakthrough in the application of "one protective solution for multiple temperature scenarios".

[0056] Significantly superior to traditional cryoprotectants: While traditional (ultra) cryoprotectants maintain a 78% cell survival rate at -196°C, their protective effect decreases sharply with increasing temperature, especially at room temperature (25°C), where cell survival is only 27%, almost completely inactivated. In contrast, the cryoprotectant of this invention exhibits a significant protective advantage at all temperatures, particularly in non-cryoprotective environments.

[0057] Compared with Comparative Examples 1 and 3 and Example 1: The survival rate was significantly reduced at -20°C and -196°C, indicating that a complete osmotic pressure regulation system (containing trehalose, mannitol, and sucrose) is crucial for mitigating osmotic pressure shocks and ice crystal damage during freezing / thawing.

[0058] Compared with Example 1, Comparative Example 2 showed a decrease in survival rate at all temperatures, indicating that cells still require basic nutrients to maintain energy metabolism under non-low temperature conditions, and a lack of nutrients will lead to cell exhaustion.

[0059] Compared with Example 1, Comparative Example 4 showed a significant decrease in survival rate, indicating that oxidative damage is a major factor leading to cell death under various temperature stresses, and the synergistic antioxidant effect of glutathione and vitamin C is indispensable.

[0060] Compared with Example 1, Comparative Example 5 showed a significant decrease in survival rate, especially at 4°C and 25°C, indicating that a stable pH environment is fundamental to maintaining cell viability, and the sodium bicarbonate system played a key role in this process.

[0061] Compared with the Example, Comparative Example 6 showed a sharp drop in survival rate at -20°C and 4°C, demonstrating that sodium hyaluronate, in synergy with cholesterol and phosphatidylcholine, can greatly enhance the stability of cell membranes under low temperature and mechanical stress.

[0062] Compared with Example 1, Comparative Example 7 showed a significant decrease in cell viability, indicating that heparin sodium is more effective in preventing cell aggregation and maintaining cell monodispersity, which is beneficial for uniform cell protection and subsequent recovery.

[0063] Morphological observation results (see) Figure 2 The results of observation under an inverted microscope further corroborated the survival rate data. After being stored at -196℃ for 24 hours, the NK cells in Example 1 group showed no abnormal morphology such as shrinkage, swelling, or rupture; the cells were evenly dispersed with only a small amount of aggregation (<2%), and there was little cell debris in the field of view. In contrast, the cells in the traditional protectant group exhibited typical apoptotic and necrotic characteristics such as cell membrane rupture, vacuolation, increased cell aggregation, and increased debris.

[0064] Table 4. Protective effect on NK cells at different temperatures

[0065] Example 3: Protection Function Test Experimental materials: mouse mononuclear macrophages, cryopreservation solutions of Examples 1-4 and Comparative Examples 1-7, conventional cryopreservation solutions (control), cryopreservatives, liquid nitrogen tanks, etc. Experimental Methods: Mouse mononuclear macrophages in the logarithmic growth phase were collected after digestion and centrifugation, and the cell pellet was resuspended and washed once with PBS. The cell pellet was thoroughly mixed with the cryopreservation solutions of Examples 1-4 and Comparative Examples 1-7, as well as conventional cryopreservation solutions, and the final cell density was adjusted to 1×10⁻⁶ cells / year. 7 Cells / mL were measured to ensure uniform cell suspension in each cryopreservation solution. The mixed cell suspension was aliquoted into sterile cryovials and placed in a programmed cooling chamber. The chamber was then placed in an ultra-low temperature freezer at -80°C, allowing the cells to cool slowly at a rate of approximately -1°C / min. After 24 hours, the cryovials were rapidly transferred to liquid nitrogen (-196°C) for long-term storage for one month. After one month, the cryovials were removed from the liquid nitrogen and immediately placed in a 37°C water bath, gently agitated continuously to thaw completely within one minute. The thawed cell suspension was transferred to centrifuge tubes containing complete culture medium, centrifuged to remove the cryopreservation solution components, and the cells were resuspended in fresh, preheated complete culture medium.

[0066] Testing indicators: Cell viability assay: A portion of the resuspended cells were used to detect the real-time cell viability using an AOPI dual fluorescence counter.

[0067] Cell proliferation assay: Remaining cells were seeded at the same density in 96-well plates and cultured under standard conditions (37℃, 5% CO2) for 24 or 48 hours. Cell proliferation activity was then assessed using the CCK-8 assay. Absorbance (OD value) was measured at 450 nm using a microplate reader; a higher OD value indicates stronger proliferation capacity of surviving cells. Morphological observation: Consistent with the CCK-8 assay time points, observations were performed at 24 and 48 hours of culture. After resuscitation and centrifugation, the cell suspension was seeded at the same density as in the CCK-8 experiment into 6-well plates containing sterile coverslips. Cells were incubated at 37°C in a 5% CO2 incubator. The 6-well plates were removed from the incubator at 24 and 48 hours. Cells were gently washed twice with PBS to remove dead cells and serum debris. An appropriate amount of paraformaldehyde fixative was added, and the cells were fixed at room temperature (25°C) for 15-20 minutes. The fixative was discarded, and the cells were washed three times with PBS and stained with trypan blue solution for 2-3 minutes. After a rapid rinse with PBS, cell morphology was observed using an inverted microscope.

[0068] The cell viability and proliferation results are shown in Table 5. The results show that the cryoprotectants of Examples 1-4 of this invention exhibited excellent performance after one month of liquid nitrogen cryopreservation. Example 1 showed the most outstanding effect, with a cell viability rate as high as 94%, and its proliferation capacity (OD value 1.27) was the highest among all groups. This indicates that this cryoprotectant not only effectively protects cells from ice crystal damage and osmotic pressure shock during cryopreservation / thawing, but also maximizes the preservation of physiological activity after cell thawing, laying a solid foundation for its subsequent in vitro culture or in vivo application.

[0069] Advantages compared to traditional cryopreservation solutions: The cell viability rate of the traditional DMSO-based cryopreservation solution group was 86%, and the proliferation OD value was 1.16, both significantly lower than those in Example 1. This indicates that the cryopreservation solution formulation of the present invention is comprehensively superior to traditional methods in terms of protective effect and functional maintenance.

[0070] Comparative Examples 3 and 4: The cell viability rates of these two groups (85% and 84%) decreased significantly, further confirming the stabilizing effect of trehalose on cell membranes and proteins during freezing, and the crucial role of glutathione in resisting oxidative stress damage during cryopreservation and thawing.

[0071] Comparative Example 5: Both the survival rate (85%) and proliferation capacity (1.14) were low, indicating that a stable pH buffer system is indispensable for maintaining intracellular and extracellular acid-base balance and preventing damage such as acidosis under long-term cryopreservation conditions.

[0072] Comparative Examples 1 and 6: Their cell viability (90%, 90%) was acceptable, but their proliferation capacity (1.18, 1.20) did not reach the optimal level. This indicates that a complete osmolarity regulation system and cell membrane protection system play an important role in promoting the rapid recovery of normal metabolic and proliferative functions after cell resuscitation.

[0073] Comparative Example 2 and Comparative Example 7: The survival rate (91%) and proliferation capacity (1.21) of Comparative Example 2 were relatively better, indicating that the dependence of cells on nutrients may be lower under cryopreservation conditions than that under ambient temperature conditions; the survival rate (87%) and proliferation capacity (1.15) of Comparative Example 7 were lower, suggesting that heparin sodium is better than EDTA in preventing cell aggregation and avoiding cryopreservation damage caused by uneven local concentrations.

[0074] Morphological observation results (see) Figure 3 The results showed that the cells in the embodiment of this invention adhered quickly, were plump and typically round or oval in shape, spread out, with clear boundaries, protruding pseudopodia, and uniformly translucent cytoplasm. Their morphology was similar to that of normal cells that had not been frozen. A very small number of cells showed apoptotic characteristics such as shrinkage, increased brightness, and disappearance of pseudopodia (<2%). After 24 and 48 hours of observation, cell density increased and showed a good trend of uniform growth.

[0075] The above results demonstrate that the cryoprotectant of this invention not only ensures extremely high cell survival rates in long-term cryopreservation scenarios, but also effectively maintains the proliferative potential of cells after thawing. This is attributed to the synergistic effect of the various systems in its composite formulation, providing cells with comprehensive protection throughout the entire process from freezing and storage to thawing and thawing, and its overall performance is significantly superior to traditional cryopreservation solutions.

[0076] Table 5 Protection Function Test

[0077] Example 4: Verification of freezing effects at different temperatures Experimental materials: NK cells, preservation solutions for Examples 1-4 and Comparative Examples 1-7, and conventional cryopreservation solutions. Experimental methods: NK cells in the logarithmic growth phase were collected by centrifugation, and the cell pellet was thoroughly mixed with equal volumes of cryopreservation solutions from Examples 1-4, Comparative Examples 1-7, and conventional cryopreservation solutions, respectively, to adjust the final cell density to 1×10⁻⁶. 7 cells / mL. Dispense the well-mixed cell suspension into sterile cryovials at 1 mL volumes per tube.

[0078] Each group of protective fluids is set to the following two temperature conditions: -20℃ slow freezing group: Place the cryovials directly into the -20℃ freezer for storage.

[0079] -80℃ Programmable Freezing Group: Place the cryovials in the programmable cooling box, and then transfer them to an -80℃ ultra-low temperature freezer for storage.

[0080] All cells were cryopreserved for one month under both of the above temperature conditions. After the cryopreservation period, the cryovials were quickly removed and thawed.

[0081] Cell viability assay: The thawed cell suspension was diluted with preheated complete culture medium, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in PBS and detected using an AOPI dual-fluorescence counter.

[0082] Trypan blue staining observation: 10 μL of the recovered and washed cell suspension was mixed with an equal volume of trypan blue staining solution. After standing for 3 minutes, the cells were observed and counted under an optical microscope using a hemocytometer. Live cells were colorless and transparent, plump, and had smooth edges; dead cells were stained blue, and appeared swollen or shrunken. The trypan blue rejection rate was calculated to verify the results against those obtained using an AOPI dual-fluorescence counter.

[0083] The results are shown in Table 6 and... Figure 4 The results showed that after one month of cryopreservation at -20℃ and -80℃, the cell viability of the protective solutions of Examples 1-4 of this invention remained at a high level of 81% to 85%, and the results were highly consistent among the groups. This indicates that the protective solutions of this invention have good temperature adaptability and can provide reliable protection under different freezing conditions. The protective effect at -80℃ is generally better than that at -20℃: Comparing the data of the same group, it can be found that the cell viability at -80℃ is equal to or slightly higher than that at -20℃ in all groups. This is because -20℃ is in the "recrystallization zone" of water, where ice crystals grow slowly and become larger, causing continuous mechanical damage to the cells; while the low temperature environment of -80℃ can "fix" the cell state more quickly, reducing the time and risk of ice crystal growth, thus providing better protection.

[0084] Comparative Example 3 showed one of the lowest survival rates (77% and 80%) at both temperatures, further highlighting the key role of trehalose in inhibiting ice crystal growth and stabilizing cell membrane structure, especially in the -20°C environment where ice crystal damage is more significant.

[0085] The relatively low survival rates (78%) of Comparative Examples 5 and 7 at -20°C indicate that unsuitable pH environments and cell aggregation problems can amplify the damage to cells under non-ideal freezing conditions (-20°C).

[0086] Although the survival rates of other comparative examples (such as Comparative Examples 1, 2, 4, and 6) were similar to those of the examples, they were generally 1 to 3 percentage points lower. This indicates that the absence or substitution of any component would have a subtle impact on the perfection of the protective effect, demonstrating the synergistic advantages and rigor of the formulation of this invention.

[0087] Comparison with conventional cryopreservation solutions: Conventional cryopreservation solutions also showed good protective effects in this experiment (82% at -20℃, 83% at -80℃), which is related to their content of standard concentrations of DMSO, making them suitable for short- to medium-term cryopreservation. However, the performance of the embodiment group of this invention is comparable to or even better than that of conventional solutions, while avoiding the potential toxicity of high concentrations of DMSO and the cumbersome washing steps required after use, demonstrating better application convenience and safety.

[0088] The above results demonstrate that the cryoprotectant of this invention can provide continuous, stable, and excellent long-term protection for NK cells under both commonly used freezing conditions of -20℃ and -80℃, with effects comparable to or better than traditional DMSO cryopreservation solution. This further expands the application scenarios of this cryoprotectant, making it suitable not only for deep cryogenic storage in liquid nitrogen but also for cryogenic storage and transportation in conventional refrigerators, demonstrating its strong wide-temperature range adaptability and practicality.

[0089] Table 6. Results of freezing at different temperatures

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A wide-temperature-range protective solution suitable for somatic cells, characterized in that, It contains the following components by mass-volume percentage: a) Osmotic pressure regulation system: containing 2%–8% trehalose, 1%–5% mannitol and 0.5%–1.5% sucrose; b) Antioxidant protection system: Contains 0.1%–3% vitamin C and 0.1%–1% glutathione; c) Nutritional supplements: containing 0.5%–3% of an amino acid mixture, 2.0%–3.5% of glucose, 0.2%–0.5% of glutamine, and 0.1%–1% of B vitamins; d) pH-stabilizing buffer pair: containing 0.2%–3% HEPES, 0.3%–0.6% disodium hydrogen phosphate, 0.2%–0.4% potassium dihydrogen phosphate and 0.1%–1% sodium bicarbonate; e) Cell membrane protectors and enhancers: containing 0.05%–0.2% hyaluronic acid, 0.1%–1% cholesterol, and 0.05%–0.5% phosphatidylcholine; f) Preservative and antibacterial agent: 0.01% to 0.1% sodium benzoate; g) Anticoagulant: Low molecular weight heparin sodium, concentration ≤10 U / mL; The remainder is solvent, which is physiological saline or a buffer system based on DPBS or HBSS.

2. The wide-temperature-range protective liquid according to claim 1, characterized in that, In the osmotic pressure regulation system, the concentration of trehalose is 3%–6%, the concentration of mannitol is 2%–4%, and the concentration of sucrose is 0.8%–1.2%.

3. The wide-temperature-range protective liquid according to claim 1, characterized in that, In the antioxidant protection system, the concentration of vitamin C is 0.5% to 2%, and the concentration of glutathione is 0.2% to 0.8%.

4. The wide-temperature-range protective liquid according to claim 1, characterized in that, In the aforementioned nutrient supply agent The amino acid mixture contains all essential and non-essential amino acids for the human body; The B vitamins include one or more of vitamins B1, B2, B3, B6, B9, B12, B4, B7, and B8.

5. The wide-temperature-range protective liquid according to claim 1, characterized in that, In the cell membrane protectant and enhancer, the concentration of hyaluronic acid is 0.08%–0.15%, the concentration of cholesterol is 0.3%–0.7%, and the concentration of phosphatidylcholine is 0.1%–0.3%.

6. The wide-temperature-range protective liquid according to claim 1, characterized in that, The concentration of the anticoagulant, low molecular weight heparin sodium, is 5–8 U / mL.

7. The wide-temperature-range protective liquid according to claim 1, characterized in that, The pH value of the protective solution is 7.2 to 7.

4.

8. A method for preparing a wide-temperature-range protective solution suitable for somatic cells according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Dissolve glucose, sucrose, potassium dihydrogen phosphate, disodium hydrogen phosphate, HEPES and sodium bicarbonate in part of DPBS or HBSS solvent and stir until completely dissolved to form a basic buffer solution. (2) Dissolve vitamin C and glutathione in deionized water, then add them to the basic buffer solution obtained in step (1) and stir until homogeneous; (3) Add trehalose, mannitol, amino acid mixture, glutamine and B vitamins to the mixed solution obtained in step (2) in sequence, and stir until completely dissolved; (4) Heat the solution obtained in step (3) to 50°C, add cholesterol and phosphatidylcholine, stir until completely dissolved, cool and add hyaluronic acid, stir thoroughly to make it evenly dispersed; (5) Add sodium benzoate and low molecular weight heparin sodium to the solution obtained in step (4), and make up the balance with DPBS or HBSS to adjust the pH to 7.2-7.4; (6) The protective solution obtained in step (5) is filtered, sterilized, and dispensed.

9. Use of the wide-temperature-range protective liquid according to any one of claims 1 to 7 in the preparation of formulations for preserving somatic cells in a temperature range of -196 to 25°C.

10. The use according to claim 9, characterized in that, The somatic cells include, but are not limited to, T cells, NK cells, DC cells, mesenchymal stem cells, fibroblasts, or epithelial cells.

Images