Non-cryopreserved cell preserving fluid and application thereof

Abstract

The invention relates to a non-cryopreserved cell preservation solution and application thereof. The cell preservation solution comprises a basic solution and a core additive, wherein the core additive comprises a ferroptosis inhibitor and a mitochondrial protective agent, based on an intracellular fluid type ionic environment and multi-mechanism synergy, an optimized basic fluid system with high potassium, low sodium and histidine buffering is adopted, the ferroptosis inhibitor and the mitochondrial targeted protective agent are integrated, and by simulating an intracellular environment, stabilizing pH and inhibiting a key death pathway, the activity of the ferroptosis inhibitor is improved; the survival rate and the functional activity of various cells under the low-temperature non-cryopreservation condition are remarkably improved, and the method has important application significance in the field of cell preservation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the field of cell preservation technology, specifically to a non-cryopreservation cell preservation solution and its application. Background Technology

[0002] The rapid development of cell therapy has placed extremely high demands on the quality of ex vivo cells. Traditional cryopreservation methods, due to issues such as ice crystal damage and DMSO toxicity, may result in poor recovery of cell viability and function, and the complex procedures make it difficult to meet the needs of off-the-shelf cell products. HypoThermosol® FRS, as the market's gold standard low-temperature non-cryopreservation solution, provides protection by maintaining intracellular homeostasis, but its protective efficacy still has room for improvement.

[0003] Related research has revealed novel cell death mechanisms under low-temperature stress. Among them, ferroptosis, an iron-dependent lipid peroxidation-driven novel programmed cell death, is one of the key mechanisms of cellular damage. In addition, mitochondrial dysfunction is another core component of low-temperature-induced cell death, leading to the excessive production of reactive oxygen species (ROS) and energy depletion.

[0004] On the other hand, UW and HTK solutions used clinically for static cryopreservation of organs are formulated for organ-level hypoxic injury, but their complex formulations are not optimized for cryopreservation of single-cell suspensions.

[0005] Currently, no preservation solution can simultaneously integrate the three major strategies of "inhibiting ferroptosis", "protecting mitochondria through multiple pathways" and "providing organ-level metabolic support"; existing technologies have also not disclosed a scheme for combining ferroptosis inhibitors with various mitochondrial protectants and synergizing with the effective components of organ preservation solutions for low-temperature non-cryopreservation of cells.

[0006] There are still no effective solutions to the problems that the protective effect of current preservation solutions needs to be improved and that the complex formulations of organ preservation solutions are not specifically designed for cell preservation. Summary of the Invention

[0007] The purpose of this invention is to address at least one deficiency in the prior art by providing a non-cryopreservation cell preservation solution and its application. The base fluid system of the non-cryopreservation cell preservation solution has been deeply optimized and achieves better cryoprotection for various cell types through multi-target and multi-pathway synergistic effects. Moreover, all components of the formula are clearly defined, eliminating the need for oxygen carriers such as perfluorocarbon, effectively avoiding the complexity and potential uncertainties of the composition. This solves the problems in related technologies, such as the need to improve the protective effect of preservation solutions and the complexity of organ preservation solution formulations that are not targeted at cell preservation.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention is to provide a non-cryopreservation cell preservation solution, the cell preservation solution comprising a base solution and a core additive; wherein the core additive comprises a ferroptosis inhibitor and a mitochondrial protectant, the concentration of the ferroptosis inhibitor being 1~1000 μM and the concentration of the mitochondrial protectant being 0.1~50 μM.

[0009] Further, the ferroptosis inhibitor is selected from at least one of deferoxamine (DFO), deferoxone (DFP), ferrostatin-1, ciclopirox olamine (CPX), deferasirox, Fer-1, ATF3-IN-1, MHY1485, and transferrin; when it is ferrostatin-1, the added concentration is 1~100 μM (specifically 10 μM), and when it is DFO / DFP / deferasirox, the added concentration is 0.1~2.0 mM (specifically 0.5 mM).

[0010] Furthermore, the mitochondrial protectant is a mitochondrial-targeting aromatic cationic compound selected from at least one of SUL-109, Mitoquinone, and Skulachev Ion; when it is SUL-109, the concentration range is 1~50 μM (specifically 10~20 μM); when it is Mitoquinone, the concentration range is 0.1~10 μM (specifically 1 μM).

[0011] Furthermore, the base liquid includes an ion system, a buffer system, an osmotic pressure regulating system, and an energy substrate.

[0012] Furthermore, the ion system includes potassium ions and sodium ions, wherein, in non-cryopreservation cell preservation solutions, potassium ions (K ions) + The concentration of sodium ions (Na+) is 80-140 mM, preferably 100-120 mM; + The concentration of ) is 10~40 mM, preferably 20~30 mM.

[0013] Furthermore, the buffer system includes at least one of histidine / histidine hydrochloride buffer pair and phosphate buffer pair, wherein, in the non-cryopreservation cell preservation solution, the concentration of the histidine / histidine hydrochloride buffer pair is 15-50 mM, preferably 25-30 mM; and the concentration of the phosphate buffer pair is 5-25 mM, preferably 15 mM.

[0014] Furthermore, the osmotic pressure adjustment system includes an osmotic pressure support agent and an osmotic pressure regulator, wherein the osmotic pressure of the cell preservation solution is adjusted to 290~320 mOsm / L, preferably 300~310 mOsm / L, using the osmotic pressure regulator.

[0015] Furthermore, in the non-cryopreservation cell preservation solution, the concentration of the energy substrate is 1-10 mM, and the energy substrate includes at least one of adenosine and ketoglutarate.

[0016] Furthermore, adenosine is present at a concentration of 1–10 mM as a precursor for ATP synthesis; ketoglutarate is present at a concentration of 1–5 mM as an intermediate in the tricarboxylic acid cycle.

[0017] Furthermore, the osmotic pressure support agent includes at least one of lacturonic acid, hydroxyethyl starch, and trehalose. Specifically, in non-cryopreservation cell preservation solutions, when lacturonic acid and / or trehalose are used, the concentration of the osmotic pressure support agent is 30-120 mM; when hydroxyethyl starch is used, the amount of the osmotic pressure support agent is 0.5-5% w / v.

[0018] Furthermore, in non-cryopreservation cell solutions, when lacturonic acid is used, the concentration of the osmotic pressure support agent is 50-120 mM; when trehalose is used, the concentration of the osmotic pressure support agent is 30-100 mM. Furthermore, the osmotic pressure regulator includes at least one of glucose, sucrose, and mannitol.

[0019] Furthermore, the osmotic pressure regulator is used to prevent cell edema and provide colloidal osmotic pressure.

[0020] Furthermore, the base solution also includes a membrane stabilizer; wherein, in the non-cryopreservation cell preservation solution, the concentration of the membrane stabilizer is 1-15 mM, and the membrane stabilizer includes at least one of reduced glutathione, N-acetylcysteine, and magnesium ions, wherein, when it is reduced glutathione, its concentration is 1-5 mM, serving as an endogenous antioxidant; when it is N-acetylcysteine, its concentration is 1-50 mM, preferably 5 mM, serving as an antioxidant; and when it is magnesium ions, its concentration is 5-15 mM, serving as an important enzyme cofactor.

[0021] Furthermore, the buffer system also includes HEPES buffer, wherein the concentration of the HEPES buffer is 10~20 mM.

[0022] A second aspect of the present invention is to protect a method for cell and / or tissue preservation, which uses a non-cryopreservation cell preservation solution as described in the first aspect for cryopreservation of cell and / or tissue samples.

[0023] Furthermore, the cell samples are derived from animal and human primary cells, stem cells, and tumor cells, and the tissue samples are derived from animal and human tissue samples. Specifically, the cell samples include T cells, stem cells, and hepatocytes.

[0024] Furthermore, the T cells are primary human T cells, and the stem cells are human mesenchymal stem cells (hMSCs).

[0025] Furthermore, the storage temperature of the non-cryopreservation cell preservation solution is 2℃~8℃, and the cell survival rate is greater than 85% after 7 days of cell preservation at 4℃.

[0026] A third aspect of the invention is to provide an application of a non-cryopreservation cell preservation solution as described in the first aspect or a cell and / or tissue preservation method as described in the second aspect, the application including at least one of the following: application in the preparation of cell product storage formulations, application in the preparation of cell product transport formulations, application in the preparation of cell samples, application in the preparation of tissue product storage formulations, application in the preparation of tissue product transport formulations, and application in the preparation of tissue samples.

[0027] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: The non-cryopreservation cell preservation solution of this invention abandons simple isotonic saline and is specifically designed for cell preservation. It adopts a high-potassium, low-sodium intracellular fluid formulation, which fundamentally reduces the ion pump load and edema risk of cells at low temperatures, providing cells with a stable "dormant" internal environment. The basic system is optimized and is suitable for the preservation of various tissues. By introducing histidine buffer pairs, the buffering capacity of the cell preservation solution far exceeds that of conventional cell preservation solutions (such as HypoThermosol) and is even comparable to organ preservation solutions (such as HTK), maintaining pH stability for a long time. The optimized base solution provides basic homeostasis for cells, and ferroptosis inhibitors and mitochondrial-targeting protectants precisely target key damage pathways on this basis, providing a synergistic effect. By simulating the intracellular fluid ion environment and integrating multiple mechanisms such as ferroptosis inhibition, mitochondrial-targeting protection, and enhanced metabolic support, the survival rate and function of different cells after cryopreservation are significantly improved. Moreover, all components of the cell preservation solution formulation of this invention are clearly defined, eliminating the need for oxygen carriers such as perfluorocarbon, avoiding component complexity and potential uncertainties, and facilitating standardized production and quality control. It has important application significance and value in the transportation and temporary storage of cell therapy products. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the effects of inhibiting ferroptosis, mitochondrial protection, and the synergistic effect of both on MSC cell survival in one embodiment of the present invention. Figure 2 This is a graph showing the effect of different preservation solutions on the survival rate of MSC cells in one embodiment of the present invention; Figure 3 This is a graph showing the change in the overall survival rate of human primary CD3+ T cells over time in different preservation solutions in one embodiment of the present invention. Figure 4 This is a graph showing the change in the total survival rate of human mesenchymal stem cells over time in different preservation solutions in one embodiment of the present invention; Figure 5 This is a graph showing the effect of different preservation solutions on the cytokine secretion capacity of T cells in one embodiment of the present invention; Figure 6 This is a graph showing the cytotoxic efficacy of different preservation solutions in different treatment groups with different effector cell ratios in one embodiment of the present invention; Figure 7 This is a diagram showing the results of immunofluorescence staining of intracellular albumin in different preservation solutions (representative field of view) in one embodiment of the present invention; Figure 8 This is a semi-quantitative analysis result of the relative fluorescence intensity of albumin staining in hepatocytes with different preservation solutions in one embodiment of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0031] Other manufacturers not specifically indicated in the embodiments of this invention can be obtained through commercial purchases.

[0032] In some specific embodiments, the present invention provides a formulation and preparation method for a non-cryopreservation cell preservation solution: 1. Formulation of non-cryopreservation cell preservation solution The above-mentioned non-cryopreservation cell preservation solution is used for cell preservation at low temperatures (2℃ ~ 8℃) and includes a base solution and core additives. The specific composition of the base solution is as follows: (1) Ionic composition: The intracellular liquid formula with high potassium and low sodium is adopted, with potassium ion concentration of 80~140 mM and sodium ion concentration of 10~40 mM, in order to reduce the ion gradient of the cell membrane, reduce energy consumption and cell edema.

[0033] (2) The buffer system adopts a composite buffer system, which includes: ① Histidine / histidine hydrochloride buffer pair, with a concentration of 15~50 mM, provides strong physiological pH buffering capacity and effectively combats hypothermic metabolic acidosis.

[0034] ② Phosphate buffer pairs, with a concentration of 5~25 mM, provide a stable source of inorganic phosphates.

[0035] ③ (Optional) Organic buffers such as HEPES: concentration 10~20 mM, providing additional buffering capacity.

[0036] (3) Osmotic pressure regulation system: ① Osmotic pressure support agent: It adopts non-permeable anionic / macromolecule, selected from at least one of lacturonic acid (50~120 mM), hydroxyethyl starch (HES) (0.5~5% w / v), and trehalose (30~100 mM), which effectively prevents cell edema and provides colloidal osmotic pressure.

[0037] ②Osmotic pressure regulator: Use at least one of glucose, sucrose, mannitol, etc. to precisely adjust the osmotic pressure to 290~320 mOsm / L.

[0038] (4) The energy substrate is selected from at least one of adenosine and ketoglutarate; wherein the preferred concentration of adenosine is 1-10 mM, as a precursor for ATP synthesis; and the preferred concentration of ketoglutarate is 1-5 mM, as an intermediate in the tricarboxylic acid cycle.

[0039] (5) The core functional additives include ferroptosis inhibitors and mitochondrial protectants.

[0040] ① The ferroptosis inhibitor is an effective dose of iron ion chelating agent selected from at least one or more of deferoxamine (DFO), deferoxone (DFP), ferrostatin-1, ciclopirox olamine (CPX), deferasirox, ATF3-IN-1, MHY1485, and transferrin. Preferred concentrations: Ferrostatin-1 (1~100 μM), DFO / DFP / deferasirox (0.1~2.0 mM).

[0041] Ferroplasmosis inhibitors (such as Ferrostatin-1, DFO, and DFP) need to achieve effective inhibition of lipid peroxidation under low-temperature conditions while avoiding interference with normal cellular metabolism. This invention measured cell viability after cryopreservation for 5 or 7 days by adding different doses of ferroplasmosis inhibitors to a basic preservation solution. It was found that concentrations below the preferred range did not completely inhibit lipid peroxidation, resulting in insufficient protection; concentrations above the preferred range may impair iron-dependent enzyme activity, thereby affecting energy metabolism and cell viability.

[0042] ② The mitochondrial protectant is a mitochondrial-targeting aromatic cationic compound selected from at least one of SUL-109, Mitoquinone (MitoQ), and Skulachev Ion (SkQ1), with a preferred concentration of 0.1~50 μM; preferably, the mitochondrial protectant is SUL-109, with a concentration range of 1~50 μM).

[0043] Effective doses of mitochondrial protectants are compounds that can improve mitochondrial function, reduce mitochondrial reactive oxygen species (ROS) production, or alleviate mitochondrial oxidative stress. Mitochondrial-targeting aromatic cations accumulate via mitochondrial membrane potential; low doses are effective, but excessive doses can lead to mitochondrial membrane potential imbalance due to over-accumulation. This invention uses different doses of ferroptosis inhibitors added to a basic preservation solution formulation to cryopreserve cells for 5 or 7 days, after which cell viability is measured. It was found that below the preferred concentration range, it is difficult to significantly alleviate ROS accumulation and energy depletion, while above the preferred concentration range, "excessive membrane potential interference" is likely to occur, even causing mitochondrial dysfunction.

[0044] Some non-cryopreservation cell preservation solutions also contain membrane stabilizers selected from at least one of reduced glutathione, N-acetylcysteine, and magnesium ions; wherein the preferred concentration of reduced glutathione is 1-5 mM, in its reduced form, as an endogenous antioxidant; when N-acetylcysteine ​​is used, its concentration is 1-50 mM, preferably 5 mM, as an antioxidant; and the preferred concentration of magnesium ions is 5-15 mM, as an important enzyme cofactor.

[0045] 2. Preparation method At a low temperature of 4 degrees Celsius, the base solution and the core additive formula are mixed evenly under sterile conditions according to the above formula to ensure that all components are fully dissolved; then filtered, the filtrate is the non-cryopreservation cell preservation solution.

[0046] Non-cryopreservation cell preservation solutions prepared with the above-mentioned component contents can all achieve low-temperature preservation of cells, tissues, or organs.

[0047] The present invention will be described by way of example below.

[0048] Example 1 This embodiment relates to a preferred formulation of a cell preservation solution, the specific formulation of which is shown in Tables 1 and 2.

[0049] Table 1 - Base Fluid Formulation Table 2 - Core Additives and Concentrations Example 2 This embodiment relates to a preferred formulation of a cell preservation solution, the specific formulation of which is shown in Tables 3 and 4.

[0050] Table 3 - Base Fluid Formulation Table 4 - Core Additives and Concentrations Example 3 This embodiment relates to a preferred formulation of a cell preservation solution, the specific formulation of which is shown in Tables 1 and 5.

[0051] The base solution formulation is the same as Table 1 in Example 2.

[0052] Table 5 - Core Additives and Concentrations Example 4 - Synergistic Validation of Ferrapholysis Inhibitors and Mitochondrial Protectants This embodiment uses the base solution formulation of Example 1. By comparing the effects of adding only ferroptosis inhibitors, mitochondrial protectants, and the combined addition of both additives, we can determine whether ferroptosis inhibitors or mitochondrial protectants are added.

[0053] MSCs (including human MSCs and other stem cells, primary cells, tumor cells, and animal and human tissue samples, etc.; in this example, human mesenchymal stem cells (hMSCs)) in the logarithmic growth phase and with a viability >95% were collected. After washing with PBS, they were resuspended in four different preservation solutions (as shown in Table 6 below), and the cell density was adjusted to 1×10⁻⁶. 6 cells / mL. Aliquot 1-2 mL into each tube. Store all cell suspensions statically at 4°C. After 5 days of storage, remove one tube of cells from each group, allow to return to room temperature, and perform a trypan blue rejection assay. Results are as follows: Figure 1 As shown.

[0054] Table 6 - Grouping of studies on inhibition of ferroptosis, mitochondrial protection, and their synergistic effects All experiments were conducted with at least three biological replicates (n=3). Data are presented as mean ± standard deviation. Two-way ANOVA was performed using software such as GraphPad Prism to compare statistical differences between different groups and time points. A p-value < 0.05 was considered statistically significant.

[0055] from Figure 1 It was found that adding ferroptosis inhibitors alone could improve MSC cell survival compared to the basic preservation solution, and adding mitochondrial-targeting protectants alone could also significantly improve MSC cell survival. However, the overall protective effect of the two alone was not as good as the combined regimen. The results suggest that simultaneously blocking two key damage pathways exhibits a significant synergistic effect, and is superior to the application of either component alone in terms of cell survival and functional preservation.

[0056] Example 5 - Investigating the effects of basal solution on cells This embodiment uses the base solution formulation of Example 1 to explore its effects on cells and its advantages in cell preservation compared to conventional base solutions.

[0057] Experimental group: The basic solution formulation is the same as in Table 1 of Example 1, but without ferroptosis inhibitors and mitochondrial protectants.

[0058] Control group: The basal solution was conventional α-MEM medium, without serum.

[0059] Following the same steps as in Example 4, MSC cells (including human MSCs and other stem cells, primary cells, tumor cells, and animal and human tissue samples, etc.; in this example, human mesenchymal stem cells (hMSCs)) in the logarithmic growth phase with >95% viability were collected. After washing with PBS, the cells were resuspended in the basal solution formulation and α-MEM culture medium preservation solution, respectively, and the cell density was adjusted to 1×10⁻⁶ cells / mL. 6 cells / mL. Aliquot 1-2 mL into each tube. Store all cell suspensions statically at 4°C. After 5 days of storage, remove one tube of cells from each group, allow them to return to room temperature, and then perform a trypan blue rejection assay. Results are as follows: Figure 2 As shown.

[0060] from Figure 2 It is evident that using the optimized basal solution alone (high potassium, low sodium + histidine buffer + membrane stabilizer + energy substrate + osmotic pressure regulation system) significantly improved cell survival under non-cryopreservation conditions at 4°C. Compared to conventional basal solutions, the basal solution in Example 1 more effectively maintained cell membrane homeostasis, reduced osmotic edema, and maintained better pH stability and energy supply. Although it lacked ferroptosis inhibition or mitochondrial targeted protection, it still outperformed conventional basal solutions, such as α-MEM, in maintaining cell viability, demonstrating the contribution of the improved basal solution system to cell preservation.

[0061] Example 6 - Investigating the effect of preservation solution on cell viability and activity In this embodiment, the cell preservation solution prepared in Example 1 was used to preserve human primary CD3+ T cells and human mesenchymal stem cells (hMSCs) to investigate the differences in cell survival rate and activity between these two methods and those using the commercial gold standard HypoThermosol® FRS.

[0062] (1) Experimental materials and grouping: The cells used were primary human CD3+ T cells (isolated from PBMCs) and human mesenchymal stem cells (hMSCs, passaged 3-5 times). The cells were collected from clinical research.

[0063] The experimental groups included: the experimental group (optimized formula, i.e., innovative formula), the positive control group, and the negative control group, as shown in Table 7.

[0064] Table 7 - Experimental Groups (2) Experimental steps: Human primary CD3+ T cells and human mesenchymal stem cells (hMSCs) were stored in the above-mentioned preservation solution and tested on days 1, 3, 5, and 7, respectively. The specific steps are as follows: Cells in the logarithmic growth phase with >95% viability were collected, washed with PBS, and resuspended in three different preservation solutions, with the cell density adjusted to 1×10⁶ cells / year. 6 cells / mL. 1-2 mL aliquots were dispensed into each tube. All cell suspensions were stored statically at 4°C. At each preset time point, one tube of cells was removed from each group, allowed to return to room temperature, and then subjected to trypan blue exclusion assay and Annexin V-FITC / PI double staining flow cytometry. The results are shown in Tables 8 and 9.

[0065] All experiments must have at least three biological replicates (n=3). Data are expressed as mean ± standard deviation. Two-way ANOVA was performed using software such as GraphPad Prism to compare statistical differences between different groups and different time points. p < 0.05 was considered statistically significant. Specifically, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0066] Human primary T cells (A) and human mesenchymal stem cells (B) were preserved at 4°C in optimized formulations, HypoThermosol® FRS, and basal culture medium, respectively. Overall cell viability was assessed at specified time points using a trypan blue rejection assay. Results are as follows: Figure 3 , Figure 4 As shown. The data are expressed as mean ± SD (n=3). After two-way ANOVA, compared with the FRS group, *p<0.05, **p<0.01, ***p<0.001.

[0067] Table 8 - Statistical results of flow cytometry on day 7 of human primary T cells Table 9 - Flow cytometry results of human mesenchymal stem cells (hMSCs) on day 7 From Table 8, Table 9, Figure 3 and Figure 4 It is evident that the innovative formulation of this invention significantly outperforms the market gold standard HypoThermosol® FRS in maintaining cell viability and inhibiting apoptosis during 7 days of storage at 4°C. The viability rates of human primary CD3+ T cells and human mesenchymal stem cells (hMSCs) in the optimized formulation of this application were 85% and 88%, respectively. The optimized formulation maintained a higher overall cell viability during long-term cryopreservation, and the advantages of the optimized formulation were particularly evident in the later stages of storage, demonstrating the effectiveness of the optimized formulation in multi-mechanism synergistic protection.

[0068] Example 7 - Verification of the preservation effect of the preservation solution on T cells This embodiment uses the optimized formulation prepared in Example 1 as the cell preservation solution to investigate its preservation effect on T cells. The experimental grouping used is the same as in Example 6, as shown in Table 7; the T cells used are collected from clinical research.

[0069] After being stored at 4°C for 7 days, T cells from each group were revived and seeded into cell culture plates. T cell activators (such as anti-CD3 / CD28 antibody magnetic beads, or PMA / ionomycin) were added to all groups of cells, and the cells were cultured at 37°C and 5% CO2 for 24 hours. The culture supernatant was collected, and the concentration of cytokines in the supernatant was detected using a human IL-2 ELISA kit, strictly following the instructions. The results are as follows: Figure 5 As shown, the data are expressed as mean ± standard deviation (n=3). Two-way ANOVA analysis showed that, compared with the FRS group, *p<0.05, **p<0.01.

[0070] The cytotoxic function was assessed using the traditional LDH release assay. The K562 tumor cell line (K-562-CCL-243, ATCC), a T-cell-sensitive target cell line, was selected. Resuscitated T cells and target cells were co-cultured at a specific effector-to-target ratio (E:Tratio 5:1, 1:1) for 24 hours. The activity of lactate dehydrogenase released into the supernatant was measured at the endpoint using the LDH assay. Results are as follows: Figure 6 As shown, the data are mean ± SD (n=3). Compared with the FRS group, *p<0.05, **p<0.01.

[0071] Human primary T cells were stored at 4°C for 7 days using an optimized formulation, HypoThermosol® FRS, and basal culture medium, respectively, and then activated for 24 hours using anti-CD3 / CD28 antibody. The culture supernatant was collected, and the IL-2 concentration was detected by ELISA. The results are as follows: Figure 5 As shown in the data, the cytokine secretion level of the optimized formulation group (the cell preservation solution selected in Example 1 of this invention) was significantly higher than that of the HypoThermosol® FRS group (FRS), and both were significantly better than the level of serum-free α-MEM-preserved T cells (control). This indicates that the optimized formulation significantly enhances the cytokine secretion capacity of T cells after preservation, demonstrating its superior advantage in functional protection. From Figure 6 As can be seen, after 24 hours of co-culture, the specific lysis rate of target cells was calculated by the LDH release method. At each effector-to-target ratio (E:TRatio), the T cells preserved by the optimized formula showed significantly higher cytotoxic activity than the FRS group. At different effector cell ratios, the optimized formula group showed the best killing efficacy, proving the reliability of the functional advantages of the optimized formula.

[0072] Example 8 - Verification of the preservation solution's effect on hepatocyte preservation This embodiment uses the cell preservation solution prepared according to the cell preservation solution formulation of Example 1 to investigate its preservation effect on hepatocytes (normal human liver cells, LONZA). The experimental grouping used is the same as in Example 6, as shown in Table 7.

[0073] In this embodiment, immunofluorescence staining is used to label albumin synthesized in hepatocytes with green fluorescence. Fluorescence microscopy is then used to visually compare the fluorescence intensity of intracellular albumin and the proportion of positive cells in hepatocytes treated with different preservation solutions, thereby semi-quantitatively assessing the preservation of hepatocyte functional activity. The specific steps are as follows.

[0074] (1) Inoculation: After the hepatocytes in each group are resuscitated, they are inoculated at a certain density (e.g., 5×10⁻⁶). 4 Cells / wells are seeded into 24-well plates pre-placed with cell slides. The cells are then placed in a 37°C incubator and allowed to settle to the surface of the slide (but not fully adhere to the plate) for 1-2 hours. This allows for fixation, ensuring the cells are in a fixed position for easy photography and minimizing deviations caused by differences in adhesion.

[0075] (2) Fixation and cell permeabilization: This includes aspirating the culture medium and gently rinsing once with PBS; adding 4% paraformaldehyde (PFA) and fixing at room temperature for 15 minutes; washing the cells three times with PBS for 5 minutes each time; adding 0.1% Triton X-100 (prepared with PBS) and incubating at room temperature for 15 minutes to increase cell membrane permeability; and washing the cells three times with PBS.

[0076] (3) Blocking and antibody incubation: Add PBS containing 5% BSA and block at room temperature for 30 minutes to reduce non-specific binding.

[0077] (4) Primary antibody incubation: Add mouse anti-human albumin monoclonal antibody diluted with blocking buffer (according to the instructions, such as 1:200) directly until the cells are completely covered, and incubate at room temperature in the dark for 1 hour (or overnight at 4°C). Wash the cells 3 times with PBS for 5 minutes each time; then stain with secondary antibody, add Alexa Fluor 488 labeled goat anti-mouse IgG secondary antibody diluted with blocking buffer (such as 1:500), and incubate at room temperature in the dark for 45 minutes; wash the cells thoroughly with PBS in the dark 3 times for 5 minutes each time.

[0078] (5) Mounting and Observation: Remove the slides from the well plate and mount them with anti-fluorescence quenching mounting medium. Observe using a fluorescence microscope under a 20x or 40x objective lens. Observe the albumin signal using the FITC channel (green). Randomly select at least 5 fields of view for each group. The microscope parameters (exposure time, aperture, gain, etc.) of all groups must be kept completely consistent. Use ImageJ to delineate the cell area, measure the average fluorescence intensity (Mean Gray Value) of each field of view, subtract the background fluorescence to obtain the corrected fluorescence value, and finally perform statistical comparisons between groups. The results are as follows: Figure 7 , Figure 8 As shown.

[0079] from Figure 7 As can be seen, after human primary hepatocytes were preserved under different conditions for 7 days, the anti-albumin immunofluorescence staining results showed that fresh hepatocytes (part A) had the strongest intracytoplasmic albumin signal. Hepatocytes preserved with the optimized formulation of this invention (the cell preservation solution selected in Example 1 of this invention) (part B) showed a bright green fluorescence similar to that of fresh cells, indicating that their protein synthesis function was well protected. Cells preserved with HypoThermosol® FRS (part C) and basal medium (part D) showed a significantly weakened albumin signal, indicating severely impaired cell function. Scale bar = 50 μm.

[0080] Semi-quantitative analysis results of relative fluorescence intensity of albumin staining in hepatocytes are as follows: Figure 8 As shown in the bar chart, the semi-quantitative results of fluorescence image analysis using ImageJ software are presented. Data were normalized to 100% based on the mean fluorescence intensity of the fresh cell group. The results show that the fluorescence intensity of the optimized formulation group was significantly higher than that of the HypoThermosol® FRS group (*p<0.001), confirming the superior functional protective advantage of the optimized cell preservation solution. Data are expressed as mean ± standard deviation (n=15 random fields).

[0081] In summary, this invention prepares a low-temperature non-cryopreservation cell preservation solution based on the intracellular liquid ionic environment and the synergistic effects of multiple mechanisms. This preservation solution uses an optimized base solution system with high potassium, low sodium, and histidine buffer, and integrates ferroptosis inhibitors and mitochondrial-targeting protectants. By simulating the intracellular environment, stabilizing pH, and inhibiting key cell death pathways, it significantly improves the survival rate and functional activity of various primary cells under low-temperature non-cryopreservation conditions. This solves the current pain points faced by cell preservation solutions in cell preservation and has great application potential in the field of cell therapy. It also has important application value in the preparation of cell product storage formulations, cell product transport formulations, and cell sample preparation. The above-mentioned cell preservation solution is also suitable for the preservation of tissue samples.

[0082] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A non-cryopreservation cell preservation solution, characterized in that, The cell preservation solution includes a base solution and core additives; wherein the core additives include ferroptosis inhibitors and mitochondrial protectants, the concentration of the ferroptosis inhibitors is 1~1000 μM, and the concentration of the mitochondrial protectants is 0.1~50 μM.

2. The non-cryopreservation cell preservation solution according to claim 1, characterized in that, The ferroptosis inhibitor is selected from at least one of deferoxamine, deferoxone, ferstatin-1, ciclopirox olamine, deirarosi, Fer-1, ATF3-IN-1, MHY1485, and transferrin.

3. The non-cryopreservation cell preservation solution according to claim 1, characterized in that, The mitochondrial protectant is a mitochondrial-targeting aromatic cationic compound selected from at least one of SUL-109, Mitoquinone, and Skulachev Ion.

4. The non-cryopreservation cell preservation solution according to claim 1, characterized in that, The base solution includes an ion system, a buffer system, an osmotic pressure regulating system, and an energy substrate, and the pH of the base solution is 7-8.

5. The non-cryopreservation cell preservation solution according to claim 4, characterized in that, The ionic system includes potassium ions and sodium ions, wherein, in the non-cryopreservation cell preservation solution, the concentration of potassium ions is 80–140 mM and the concentration of sodium ions is 10–40 mM; and / or The buffer system includes at least one of histidine / histidine hydrochloride buffer pairs and phosphate buffer pairs, wherein, in non-cryopreservation cell preservation solutions, the concentration of the histidine / histidine hydrochloride buffer pair is 15-50 mM; the concentration of the phosphate buffer pair is 5-25 mM; and / or The osmotic pressure regulation system includes an osmotic pressure support agent and an osmotic pressure regulator, wherein the osmotic pressure of the cell preservation solution is adjusted to 290~320 mOsm / L using the osmotic pressure regulator; and / or The energy substrate includes at least one of adenosine and ketoglutarate, wherein the concentration of the energy substrate in the non-cryopreservation cell preservation solution is 1-10 mg.

6. The non-cryopreservation cell preservation solution according to claim 5, characterized in that, The osmotic support agent includes at least one of lacturonic acid, hydroxyethyl starch, and trehalose, wherein, in non-cryopreservation cell preservation solutions, when lacturonic acid and / or trehalose are used, the concentration of the osmotic support agent is 30-120 mM; when hydroxyethyl starch is used, the amount of the osmotic support agent is 0.5-5% w / v; and / or The osmotic pressure regulator includes at least one of glucose, sucrose, and mannitol.

7. The non-cryopreservation cell preservation solution according to claim 5, characterized in that, The base solution also includes a membrane stabilizer; wherein, in non-cryopreservation cell preservation solutions, the concentration of the membrane stabilizer is 1–15 mM; and / or The buffer system also includes HEPES buffer, wherein the concentration of HEPES buffer in non-cryopreservation cell preservation solution is 10-20 mM.

8. A method for preserving cells and / or tissues, characterized in that, Cell and / or tissue samples are cryopreserved using non-cryopreservation cell preservation solutions as described in any one of claims 1 to 7.

9. The cell preservation method according to claim 8, characterized in that, The cell samples are derived from animal and human primary cells, stem cells, and tumor cells, and the tissue samples are derived from animal and human tissue samples.

10. The application of a non-cryopreservation cell preservation solution as described in any one of claims 1 to 8 or a cell and / or tissue preservation method as described in any one of claims 8 to 9, characterized in that, The application includes at least one of the following: application in the preparation of cell products and / or tissue storage formulations, application in the preparation of cell and / or tissue product transport formulations, and application in the preparation of cell and / or tissue samples.

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