A cell preservation solution, a preparation method thereof, a preparation thereof, and a cell preservation method

Abstract

The present application relates to the technical field of biology, and discloses a cell preservation solution, a preparation method and preparation thereof, and a cell preservation method. The cell preservation solution contains 1-10 mL compound electrolyte glucose injection, 1-10 mL human blood albumin injection, 1-10 mL compound amino acid injection, 10-20 uM salvianolic acid B, 20-60 uM rhodioside, and 1-10 uM ginkgo biloba bilobal, and the compound electrolyte injection is supplemented. The cell preservation solution can improve the biological activity of cells and prolong the preservation time of cells in multiple ways by adding traditional Chinese medicine extracts as antioxidants, membrane stabilizers and antithrombotics in response to the excess of free radicals in the low-temperature preservation process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a cell preservation solution and its preparation method, formulation, and cell preservation method. Background Technology

[0002] With the development of the cell and gene therapy (CGTs) industry, the clinical applications of functional cells are increasing. Clinical applications of functional cells often require "ready-to-use" cell preparations, such as using mesenchymal stem cells (MSCs) to repair acute injury and for immunomodulatory therapy.

[0003] Traditional cell cryopreservation methods for direct clinical use have the following limitations: Complex resuscitation process: Clinical use requires liquid nitrogen transport of frozen cells, on-site resuscitation, and even cell washing to form the final injection solution, increasing the risk of contamination, as well as time and equipment costs; Freeze-thaw damage risk: Ice crystal formation and changes in osmotic pressure can lead to cell membrane rupture or loss of function; DMSO toxicity issues: Excessive DMSO concentration in the cryopreservation solution may cause adverse reactions in patients.

[0004] The following limitations exist in the process of washing and preparing infusion solutions after cell resuscitation: Under storage conditions of 2-8℃, the cell viability in the cell preservation solution can only be maintained above 90% for 6-8 hours, posing a significant challenge to the clinical application and long-distance transportation of functional cells due to time constraints. As described in invention patent CN110199987A, the preservation solution is physiological saline with the addition of 0.1%–0.5% sodium hyaluronate and 0.2%–0.4% albumin, which can achieve a cell viability of over 80% for mesenchymal stem cells within 24 hours. However, this invention has a short cell preservation time, making it unsuitable for long-distance cell transportation and use; moreover, the cell viability is low, only 80%. A low cell viability not only affects the efficacy of cell therapy but also leads to adverse reactions in the human body due to the large number of dead cells.

[0005] Decreased cell viability leads to a poorer cell condition and can cause cell aggregation and precipitation, resulting in poor suspension stability and cell clumping during subsequent infusion, and even the risk of thrombosis. Prolonged storage at low temperatures can also lead to decreased expression of cell surface markers (such as CD73 and CD90) and impaired paracrine function, ultimately resulting in loss of function. Summary of the Invention

[0006] To address the aforementioned technical problems in cell preservation, this invention provides a cell preservation solution, its preparation method, formulation, and cell preservation method, which prolong preservation time and maintain cell functional activity.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a cell protection solution, each 100ml of which contains 1-10mL of compound electrolyte glucose injection, 1-10mL of human serum albumin injection, 1-10mL of compound amino acid injection, 10-20μM salvianolic acid B, 20-60μM rhodioloside, 1-10μM ginkgo biloba extract, and the compound electrolyte injection is used to make up the difference.

[0009] In a preferred embodiment, the cell protection solution also contains 10-50 μM hesperidin and / or 20-50 μM total saponins of Panax notoginseng.

[0010] In this invention, the compound amino acid injection is compound amino acid injection 18AA-II.

[0011] In this invention, the cell protection solution has a pH of 6-8 and an osmolar concentration of 270-380 OSmol.

[0012] The present invention also provides a method for preparing the above-mentioned cell protection solution, wherein the salvianolic acid B, the rhodioloside and the ginkgo biflavonoids are respectively prepared into solutions, and the resulting solutions are mixed with the compound electrolyte injection, the compound electrolyte glucose injection, the human serum albumin injection and the compound amino acid injection in proportion to the final concentration.

[0013] As one implementation method, the salvianolic acid B, the rhodioloside, the ginkgo biflavonoids, the hesperidin and the total saponins of Panax notoginseng are each prepared into a solution, and the resulting solution is mixed with the compound electrolyte injection, the compound electrolyte glucose injection, the human serum albumin injection and the compound amino acid injection in proportion to the final concentration.

[0014] The present invention also provides a cell protection solution formulation, wherein the cell protection solution formulation is a lyophilized powder or concentrate of the above-mentioned cell protection solution or ice crystals in a frozen state.

[0015] The present invention also provides a cell preparation comprising cells and the above-described cell protection solution.

[0016] In this invention, the cells include one or more of somatic cells, stem cells, and immune cells.

[0017] The present invention also provides a method for preserving cells, comprising the following steps: resuspending the cells in the above-mentioned cell preservation solution and storing them at 2-8°C.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This invention's cell preservation solution addresses the excessive free radicals (such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) during cryopreservation. It establishes an antioxidant defense system at its core, using free radical scavenging and antioxidation as entry points. The solution adds salvianolic acid B as an antioxidant to rapidly scavenge free radicals in the cytoplasm, stabilize mitochondria, directly inhibit the Caspase cascade, and quickly block apoptosis. It also adds ginkgo biloba extract as a membrane stabilizer, utilizing its lipid solubility to scavenge lipid peroxidation free radicals while activating the Nrf2 / ARE pathway, upregulating the cell's own antioxidant enzyme (SOD), and enhancing cellular oxidative stress resistance. Finally, it adds rhodioloside as a cell homeostatic agent, activating the PI3K / Akt and ERK survival pathways to improve cell viability and regulating the AMPK pathway to maintain cellular homeostasis. These three components synergistically enhance cellular antioxidant capacity, maintain biological activity, and extend cell preservation time. This invention further adds hesperidin as a RORα / γ agonist to maintain cellular homeostasis, improve mitochondrial energy supply, and enhance stress resistance during cell preservation; it also adds total saponins of Panax notoginseng as a calcium homeostasis regulator to maintain intracellular calcium ion homeostasis. Simultaneously, the saponin components bind to membrane cholesterol to further enhance membrane stability, ensuring cell membrane integrity and regulating osmotic pressure. Both hesperidin and total saponins of Panax notoginseng work synergistically to reduce the risk of thrombosis during clinical use. Experiments show that after cells are preserved in the cell preservation solution of this invention at 2-8℃ for 120 hours, there are no significant changes in pH and osmotic pressure. Cell viability reaches 90% and can be maintained for more than 120 hours. Cell surface markers (such as CD73 and CD90) expression does not decrease, cell function is intact, cell reattachment and growth are good, proliferation is normal, and cell quality is unaffected.

[0020] The herbal extracts selected in this invention have the following advantages compared to synthetic or other natural antioxidants: ① Complex composition: This invention uses the main components of multiple herbal extracts, which work synergistically through different mechanisms (tanshinone B scavenging cytoplasmic free radicals, ginkgo biloba flavonoids scavenging lipid free radicals, and rhodioloside regulating cellular homeostasis) to form a more comprehensive antioxidant network; ② Multi-faceted combined effects: The antithrombotic ability of hesperidin combined with the vasodilatory effect of total saponins of Panax notoginseng in this invention results in a stronger antithrombotic ability; ③ Low side effects: Herbal antioxidants have high safety with long-term use, while some synthetic antioxidants (such as BHA / BHT) may have potential hepatotoxicity or carcinogenic controversies. The combination of components in this invention focuses more on regulating metabolism and restoring homeostasis, which is more in line with the biological laws of cells. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the detection process of cells after preservation in cell preservation solution according to the present invention.

[0022] Figure 2The effect of hesperidin on platelet aggregation in different groups in Experiment Example 8. Detailed Implementation

[0023] This invention provides a cell protection solution, wherein each 100ml of cell protection solution contains 1-10 mL of compound electrolyte glucose injection, 1-10 mL of human serum albumin injection, 1-10 mL of compound amino acid injection, 10-20 μM of salvianolic acid B, 20-60 μM of rhodioloside, 1-10 μM of ginkgo biloba extract, and the compound electrolyte injection is used to make up the difference.

[0024] In a preferred embodiment, the cell protection solution of the present invention also contains 10-50 μM hesperidin and / or 20-50 μM total saponins of Panax notoginseng.

[0025] Early techniques directly used physiological saline or basal culture medium (such as DMEM) to preserve MSCs at 4°C, but low-temperature metabolic inhibition led to ATP depletion and ion pump dysregulation, resulting in massive cell apoptosis within 24 hours. The cell preservation solution of this invention adds compound electrolyte glucose injection and compound amino acid injection as energy substrates to delay ATP depletion and maintain energy metabolism; the addition of compound electrolyte injection regulates osmotic pressure, electrolyte balance, energy supply, and acid-base homeostasis, maximizing the maintenance of cell morphological integrity, structural stability, and biological activity. Preferably, each 100ml of the cell preservation solution of this invention contains 3-8mL of compound electrolyte glucose injection and 3-8mL of compound amino acid injection, with the compound electrolyte injection supplementing the remainder; more preferably, each 100ml of the cell preservation solution contains 5mL of compound electrolyte glucose injection, 5mL of compound amino acid injection, and 84.5mL of compound electrolyte injection. The compound amino acid injection is preferably compound amino acid injection 18AA-II. This invention does not particularly limit the source of the compound electrolyte glucose injection, compound amino acid injection, and compound electrolyte injection. All of them can be commercially available products, or compound electrolyte glucose solution, compound amino acid solution, and / or compound electrolyte solution prepared in accordance with the national standards of the above-mentioned injections.

[0026] The cell protection solution of this invention contains human serum albumin injection. Human serum albumin, as an anti-apoptotic component, inhibits the initiation of the caspase pathway, thereby slowing down the process of cell apoptosis. As one embodiment, each 100 ml of the cell protection solution preferably contains 3-8 mL of human serum albumin injection, more preferably 5 mL. This invention does not particularly limit the source of the human serum albumin injection; commercially available products or human serum albumin solutions prepared according to the national standards for the aforementioned injection solutions can be used.

[0027] This invention reveals that during cell preservation, free radical damage significantly reduces cell viability and leads to apoptosis. Excessive free radicals (such as reactive oxygen species (ROS) and reactive nitrogen species (RNS)) can damage cells through multiple pathways: ① Lipid peroxidation: Free radicals attack unsaturated fatty acids in the cell membrane, triggering a chain reaction that disrupts membrane fluidity and integrity, affecting cell signal transduction and substance transport, leading to cell membrane rupture and cell death; ② Protein oxidation: Free radicals oxidize amino acids (such as thiol groups) in protein side chains, causing protein denaturation, enzyme inactivation, or abnormal aggregation, resulting in functional impairment and cell death; ③ DNA damage: Free radicals attack nucleic acid bases (such as guanine oxidized to 8-oxoguanine) or cause chain breaks, inducing mutations and genomic instability, thus leading to cell death; ④ Mitochondrial dysfunction: Free radicals directly damage mitochondrial DNA (mtDNA) and the electron transport chain (ETC), leading to reduced ATP synthesis and increased ROS leakage, creating a vicious cycle. ⑤ Apoptosis and necrosis: Excessive oxidative stress can activate pro-apoptotic factors such as p53 and Bax, or directly induce necrotizing cell death, accelerating tissue degeneration.

[0028] Therefore, this invention selects five components (tanshinone B, rhodioloside, ginkgo biloba flavonoids, hesperidin, and total saponins of Panax notoginseng) based on their unique and complementary cell biological mechanisms of action, precisely targeting key nodes of the aforementioned damage pathways, and constructing a "multi-target, multi-level, and dynamically synergistic" cell protection network to achieve high viability during cell preservation. ① Tanshinone B, as a potent water-soluble polyphenol, rapidly clears intracytoplasmic ROS in the initial stage of damage, providing antioxidant protection, while stabilizing mitochondria, directly inhibiting the Caspase cascade reaction, and suppressing apoptosis; ② Rhodioloside activates the PI3K / Akt pathway. After AKt activation, it phosphorylates the transcription factor Nrf2. This phosphorylation can stabilize Nrf2, inhibit its binding to the negative regulatory protein Keap1, and promote the transport of Nrf2 into the cell nucleus, improving cell homeostasis and cell viability; ③ The addition of ginkgo biloba flavonoids acts synchronously on the Nrf2 pathway, further releasing Nrf2 from the inhibition of Keap1. The two work together to cause Nrf2 to accumulate in large quantities and continuously in the cell nucleus, and bind to antioxidant response elements (AREs), driving the transcription of antioxidant proteins and building an endogenous antioxidant system. Ginkgo biloba extract, due to its strong lipid solubility, specifically embeds into and stabilizes the cell membrane and organelle membranes. ④ The addition of hesperidin, through the AMPK pathway, directly phosphorylates and activates peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), promoting mitochondrial DNA replication and electron transport chain protein synthesis, repairing and adding mitochondria, while positively regulating the convergence of Nrf2 with the aforementioned endogenous antioxidant system. In addition, PGC-1α activation can enhance the expression of mitochondrial antioxidant enzymes (such as SOD2). ⑤ The addition of Panax notoginseng saponins, whose saponin structure interacts with cell membrane cholesterol, enhances membrane stability and works synergistically with ginkgo biloba extract to improve the integrity of the cell membrane and organelle membranes. At the same time, Panax notoginseng saponins can inhibit calcium ion influx induced during cell preservation and improve the endoplasmic reticulum stress capacity. In summary, the active ingredients in the cell protection solution of this invention, through the aforementioned pathway, construct a cell protection system in which each component exerts its own efficacy while simultaneously exhibiting highly efficient synergistic effects among the components. All these effects ultimately manifest in the endogenous antioxidant system within cells, the scavenging of exogenous ROS, and the multidimensional improvement of cellular homeostasis and stress resistance. This invention also reveals that adding tangeretin to the cell protection solution can serve as a substitute for low-molecular-weight heparin calcium, and combined with the vasodilatory capacity of Panax notoginseng saponins, it can ensure clinical infusion and prevent thrombosis. The salvianolic acid B, tangeretin, ginkgo biloba extract, rhodioloside, and Panax notoginseng saponins used in this invention can all be commercially available products. In the specific embodiments of this invention, all of the above-mentioned pharmaceutical components were purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0029] In one embodiment, the concentration of salvianolic acid B in the cell protection solution of the present invention can be selected from any concentration among 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, and 20 μM, or a concentration between any two of the above numerical ranges. In the concentration range of 0–10 μM, salvianolic acid B is insufficient to scavenge free radicals, exhibits weak antioxidant capacity, and does not significantly improve cell viability. In the concentration range of 10–20 μM, as the concentration of salvianolic acid B gradually increases, cell viability significantly improves, and the proportion of apoptosis decreases. In the concentration range of 20–50 μM, as the concentration of salvianolic acid B gradually increases, cell viability shows a decreasing trend, and the proportion of apoptosis increases. This may be because excessively high concentrations of salvianolic acid B exceed the cellular metabolic range, producing cytotoxicity, and high concentrations of phenolic substances may actually generate negative feedback regulation, forming a pro-oxidative effect.

[0030] As one embodiment, the concentration of rhodioloside in the cell protection solution of the present invention can be selected from any concentration among 20μM, 25μM, 30μM, 35μM, 40μM, 45μM, 50μM, 55μM, and 60μM, or concentrations between any two of the above numerical ranges. Rhodioloside in the 0-20μM concentration range is insufficient to interfere with cell signaling pathways, failing to initiate cell stress resistance, and cell viability is not significantly improved. In the 20-60μM range, cell viability is significantly positively increased, and the apoptosis rate is significantly decreased. In the 60-100μM range, cell viability is significantly decreased, and the apoptosis rate is significantly increased. This may be because high concentrations of rhodioloside far exceed the cell's metabolic capacity and transform protective stress into lethal stress.

[0031] As one embodiment, the concentration of ginkgo biloba flavonoids in the cell protection solution of the present invention can be selected from any concentration among 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, and 10μM, or a concentration between any two of the above numerical ranges. Without the addition of ginkgo biloba flavonoids, cell viability is increased, but the difference is not significant, indicating that the lipid antioxidant capacity of ginkgo biloba flavonoids and the synergistic effect with other components of the present invention are important. In the 1~10μM range, ginkgo biloba flavonoids significantly improve cell viability and reduce the proportion of apoptotic cells. Ginkgo biloba flavonoids have poor water solubility and are prone to precipitation at concentrations above 10μM. Furthermore, excessive addition significantly reduces cell viability, possibly because high concentrations of biloba flavonoid molecules affect membrane integrity and induce auto-oxidation, transforming the antioxidant into a pro-oxidant, further exacerbating cell death.

[0032] As one embodiment, the concentration of norepinephrine in the cell protection solution of the present invention can be selected from any concentration among 10μM, 15μM, 20μM, 25μM, 30μM, 35μM, 40μM, 45μM, and 50μM, or a concentration between any two of the above numerical ranges. Norepinephrine in the 0-10μM range is insufficient to effectively activate key signaling pathways such as AMPK, Nrf2, and SIRT1, and fails to significantly affect cell metabolism and antioxidant capacity, with no significant change in cell viability. At 10μM to 50μM, cell viability and cell proliferation efficiency are significantly improved. Above 50μM, cell viability and proliferation are significantly reduced, possibly because high concentrations of norepinephrine excessively activate signaling pathways, leading to negative feedback in the signaling pathways and affecting normal cell metabolism.

[0033] As one embodiment, the concentration of total saponins from Panax notoginseng in the cell protection solution of the present invention can be selected from any concentration among 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM, or a concentration between any two of the above numerical ranges. In the 0-20 μM range, total saponins from Panax notoginseng show no significant change in cell viability, possibly because their effects on cell membrane stability, calcium homeostasis regulation, and endoplasmic reticulum stress inhibition are insufficient. In the 20-50 μM range, total saponins from Panax notoginseng significantly increase cell viability and cell proliferation efficiency. Above 50 μM, nobiletin significantly decreases cell viability and proliferation, possibly because high concentrations of total saponins from Panax notoginseng excessively bind to membrane cholesterol, leading to membrane rupture and cell death.

[0034] The cell preservation solution of the present invention provides a stable pH suitable for cell preservation. As one embodiment, the pH of the cell preservation solution of the present invention is between 6 and 8, more preferably between 6.5 and 7.5. The cell preservation solution of the present invention maintains the normal osmotic pressure required for cell preservation. As one embodiment, the osmolar concentration of the cell preservation solution of the present invention is 270-380 5 mol, preferably 390-350 5 mol.

[0035] The method for preparing the cell protection solution of the present invention includes preparing solutions of the above-mentioned salvianolic acid B, the rhodioloside, the total saponins of Panax notoginseng, the hesperidin and the ginkgo biloba flavonoids respectively, and mixing the resulting solutions with the compound electrolyte injection, the compound electrolyte glucose injection, the human serum albumin injection and the compound amino acid injection in proportion to the final concentration.

[0036] This invention also provides a cell protection solution formulation, which prepares the above-mentioned cell protection solution into lyophilized powder, concentrated solution, or cryopreserved ice crystals, maximizing the retention of the stability of the components and cell protection activity of the protection solution, while adapting to the storage, transportation, and handling requirements of different application scenarios. The lyophilized powder removes moisture through vacuum freeze-drying. The solid powder state of the lyophilized powder avoids chemical reactions and microbial growth in the dissolved state of the components, resulting in a long shelf life. The concentrated solution has reduced water activity at high concentrations, making it difficult for microorganisms to multiply, and the component concentration gradient is stable, avoiding ion diffusion and precipitation at low concentrations, resulting in a long shelf life. Cryopreserved ice crystals are frozen into solid ice crystals at -80℃ or liquid nitrogen (-196℃), where the molecular motion of the components is stopped, completely inhibiting degradation reactions, making them suitable for long-term storage. Cryopreserved ice crystals can be directly placed in a resuscitation device for synchronous resuscitation with cells, avoiding the need for additional protective solution, making them suitable for the long-term cryopreservation of fragile cells (such as stem cells and platelets).

[0037] This invention also provides a cell preparation comprising cells and the aforementioned cell protection solution. Cells in the cell protection solution can maintain cell viability and ensure cell function for an extended period. The cells of this invention are animal cells, including but not limited to one or more of somatic cells, stem cells, and immune cells. The somatic cells include various tissue cells of the body, such as epithelial tissue cells, connective tissue cells, muscle tissue cells, and nerve tissue cells. The epithelial tissue cells include, but are not limited to, epidermal cells, keratinocytes, intestinal columnar epithelial cells, respiratory ciliated epithelial cells, gastric parietal gland epithelial cells, sweat gland cells, salivary gland cells, pancreatic secretory cells, human umbilical vein endothelial cells, and vascular smooth muscle endothelial cells. The connective tissue cells include, but are not limited to, fibroblasts, macrophages, mast cells, adipocytes, chondrocytes, osteocytes, erythrocytes, leukocytes (neutrophils, lymphocytes, etc.), and platelets. The muscle tissue cells include, but are not limited to, skeletal muscle cells, cardiomyocytes, and smooth muscle cells. The nerve tissue cells include, but are not limited to, astrocytes, oligodendrocytes, and microglia. The stem cells mentioned include, but are not limited to, embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and induced pluripotent stem cells (iPS cells). The immune cells mentioned include, but are not limited to, T cells, B cells, macrophages, granulocytes, NK cells, CAR-T cells, DC cells, and CIK cells. The cell preservation solution in the cell preparation of this invention can maximize the extension of cell preservation time, maintain cell homeostasis, and preserve functional activity.

[0038] This invention also provides a method for cell preservation, comprising resuspending cells in the cell preservation solution described above and storing them at 2-8°C. The cell preservation method of this invention maintains a cell viability of 90% for more than 120 hours; after 120 hours of storage, the expression of cell surface markers (such as CD73, CD90, and CD105) does not decrease, cell function remains intact, cell reattachment and growth are good, proliferation is normal, and cell quality is unaffected.

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The technical solution of the present invention includes, but is not limited to, the following embodiments.

[0040] In the following examples, the compound electrolyte glucose injection and the compound electrolyte injection are manufactured by Sichuan Kelun Pharmaceutical Co., Ltd., with drug approval numbers H20063443 and H20113476, respectively. The compound amino acid injection (18AA-II) is manufactured by Fresenius Kabi (China) Investment Co., Ltd., with drug approval number H10980029. The human serum albumin injection is manufactured by Octapharma AG (Austria), with drug approval number SJ20160037.

[0041] In the following examples, each traditional Chinese medicine component was sourced from Chengdu Mansite Biotechnology Co., Ltd., and was prepared and stored as follows: each component was prepared as a 1mL 10mM solution.

[0042] Table 1. Sources, preparation methods, and preservation techniques of each traditional Chinese medicine component.

[0043] Example 1

[0044] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 10μM salvianolic acid B, 20μM rhodioloside and 1μM ginkgo biflavonoids.

[0045] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0046] Example 2

[0047] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside and 10μM ginkgo biloba flavonoids.

[0048] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0049] Example 3

[0050] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids and 20μM total saponins of Panax notoginseng.

[0051] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0052] Example 4

[0053] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids and 50μM total saponins of Panax notoginseng.

[0054] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0055] Example 5

[0056] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids and 10μM tangeretin.

[0057] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0058] Example 6

[0059] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids and 30μM tangeretin.

[0060] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0061] Example 7

[0062] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids and 50μM tangeretin.

[0063] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0064] Example 8

[0065] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5amL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 10μM salvianolic acid B, 20μM rhodioloside, 20μM total saponins of Panax notoginseng, 10μM tangeretin and 1μM ginkgo biloba flavonoids.

[0066] The above components are mixed in proportion to prepare the cell protection solution at the above final concentration.

[0067] Example 9

[0068] A cell protection solution, wherein the raw material components per 100ml of cell protection solution are as follows: 84.5mL compound electrolyte injection, 5mL compound electrolyte glucose injection, 5mL human serum albumin injection, 5mL compound amino acid injection (18AA-II), 20μM salvianolic acid B, 60μM rhodioloside, 10μM ginkgo biloba flavonoids, 50μM tangeretin and 50μM total saponins of Panax notoginseng.

[0069] Comparative Example 1

[0070] 100mL of compound electrolyte injection solution was used as a cell protection solution.

[0071] Comparative Example 2

[0072] 84.5 mL of compound electrolyte injection, 5 mL of compound electrolyte glucose injection, 5 mL of human serum albumin injection, and 5 mL of compound amino acid injection (18AA-II) were mixed to prepare a cell protection solution.

[0073] Comparative Example 3

[0074] Compared with Example 2, except for the following differences, everything else is the same as Example 2.

[0075] It does not contain ginkgo biflavonoids (Comparative Example 3-1).

[0076] It does not contain salvianolic acid B (Comparative Example 3-2);

[0077] It does not contain rhodioloside (Comparative Example 3-3);

[0078] It does not contain salvianolic acid B and ginkgo biflavonoids (comparative examples 3-4).

[0079] It does not contain salvianolic acid B and rhodioloside (comparative examples 3-5);

[0080] It does not contain ginkgo biflavonoids and rhodioloside (comparative examples 3-6).

[0081] Example 10

[0082] A cell preparation method is as follows:

[0083] 1. Cell observation: After 3 days of culture, P5 human umbilical cord mesenchymal stem cells were observed under a microscope to grow in a spindle-shaped and whorled pattern. The color and clarity of the cell culture supernatant were normal, and the cell confluence was ≥90%.

[0084] 2. Cell digestion: Discard the supernatant from each T75 flask. After discarding the supernatant, add 10 mL of PBS for washing. After washing, discard the PBS. Add 3 mL of TryPLE digestion solution to each T75 flask for digestion.

[0085] 3. Digestion termination: Observe obvious cell detachment. At this time, digestion is complete. Add 9 mL of PBS containing 2% human serum albumin to each T75 culture flask to terminate digestion.

[0086] 4. Cell collection: Collect the cell suspension and centrifuge at 400g for 5 minutes;

[0087] 5. Cell washing: After centrifugation, discard the supernatant and add 20 mL of compound electrolyte injection containing 5% human serum albumin to each T75 flask; centrifuge at 400g for 5 min to wash.

[0088] 6. Cell counting: After centrifugation, discard the supernatant, add 20 mL of compound electrolyte injection containing 5% human serum albumin to each T75 flask, and take samples for counting; according to the counting results, group the cell suspensions and centrifuge at 400g for 5 min.

[0089] 7. After centrifugation, discard the supernatant and resuspend the corresponding cell pellets using the cell protection solution formulations in Examples 1-7 and Comparative Examples 1-3, respectively. Mix well by pipetting and transfer to a storage temperature of 2-8°C.

[0090] Experiment 1: Cell Count Detection

[0091] The cell preparations prepared with different cell protection solutions in Example 10 were stored at 2-8°C for 24h, 72h, 96h, and 120h, and the cell viability was detected.

[0092] Cell samples from various formulations were collected at different time points. After mixing by pipetting, 20 μL of cell suspension was transferred to a 1.5 mL EP tube. Then, 20 μL of AO / PI staining solution was added to the 1.5 mL EP tube, and the mixture was pipetted and mixed. 20 μL of the mixed cell suspension was then transferred to a cell counting chamber, inserted into the cell counter, and the counting was initiated. The instrument automatically displayed the cell count and viability detection results, as shown below:

[0093] Table 2 Effects of different cell preservation solutions on cell viability

[0094] Results analysis:

[0095] Comparative Example 1 and Comparative Example 2 provided basic protection: Comparative Example 1 only provided the most basic osmotic pressure and ion balance, without nutrition and activity protection, simulating cell damage in a purely physiological environment, and had the lowest cell viability at 120h (8.74%); Comparative Example 2 added energy (glucose), protein synthesis raw materials (amino acids) and colloidal osmotic pressure support (albumin), providing the material basis for cell survival and repair, and significantly increased cell viability to 47.28% at 120h.

[0096] The protective effect of a single component in the cell protection solution of this invention: Based on Comparative Example 2, the cell viability increased with the addition of each component, demonstrating the protective effect of each component in this invention. Comparative Example 3-1 showed a cell viability of 78.32% after 120 hours, lacking the membrane-targeted antioxidant and strong Nrf2 pathway activation effects of Ginkgo biloba extract, indicating incomplete protection. Comparative Example 3-2 showed a cell viability of 72.47% after 120 hours, lacking the potent direct antioxidant and rapid anti-apoptotic (Caspase inhibition) effects of Tanshinone B, indicating insufficient protection against acute oxidative stress. Comparative Example 3-3 showed a cell viability of 70.16% after 120 hours, lacking the activation and metabolic regulation effects of the Rhodiola rosea glycoside core survival signaling pathway (PI3K / Akt), indicating that the cell's anti-stress capacity was not fully activated.

[0097] The two-component synergistic effect of the cell preservation solution of the present invention: Comparative Examples 3-4 to 3-6, the removal of any two components significantly reduced cell viability, indicating that the three components of the present invention are highly complementary in terms of antioxidant capacity and cell viability enhancement during cell preservation. Furthermore, any combination of two components cannot cover all key pathways of cryopreservation damage, and there are obvious shortcomings.

[0098] The cell protection solution of this invention has a synergistic effect of three components: In Examples 1 and 2, when the three components coexist, a synergistic effect is achieved between the components. Tanshinone B directly and rapidly removes free radicals in the cytoplasm, Ginkgo biloba flavonoids remove lipid free radicals and protect membrane integrity, and Rhodiola rosea glycosides activate cell survival pathways and enhance cell stress response. The three components work together to achieve a cell viability of up to 90.63% after 120 hours.

[0099] The protective effects of single components of hesperidin and total saponins of Panax notoginseng: The cell viability of Example 4 was slightly improved compared with Example 2, mainly due to the membrane stabilization and calcium homeostasis regulation of total saponins of Panax notoginseng, but lacked the metabolism and homeostasis regulation of hesperidin; The cell viability of Example 7 was also slightly improved compared with Example 2, mainly due to the metabolism and homeostasis regulation of hesperidin, but lacked the membrane structure anchoring function of total saponins of Panax notoginseng.

[0100] The five components of the cell protection solution of this invention exhibit synergistic effects: In Examples 8 and 9, when the five components coexist, salvianolic acid B, ginkgo biloba flavonoids, and rhodioloside form the core of antioxidant and cell homeostasis enhancement; the addition of hesperidin and total saponins of Panax notoginseng further produces synergistic and additive effects, with cell viability increasing to a maximum of 96.06% after 120 hours. Furthermore, hesperidin and total saponins of Panax notoginseng also serve as the main antithrombotic components in this invention.

[0101] Experiment Example 2: Detection of Cell Surface Markers

[0102] The cell preparations prepared with different cell protection solutions in Example 10 were stored at 2-8°C, and cell surface markers were detected after 0h, 24h, 72h, and 120h.

[0103] Cell samples from each formulation were collected at different time points, mixed thoroughly by pipetting, and then analyzed by flow cytometry at a density of 1×10⁻⁶. 6 Cells / tube, a total of 6 tubes, were used for flow cytometry antibody labeling. 5 μl of fluorescently labeled antibody was added to each tube for IgG, CD73, and CD105, and 2 μl of fluorescently labeled antibody was added to each tube for CD90. After adding the antibody, the cells were kept at room temperature in the dark for at least 30 minutes. Centrifugation was then performed, and the supernatant was discarded. 500 μl of PBS was added to each tube to wash the cells, and the supernatant was discarded after centrifugation at 400g for 5 minutes. This step was repeated twice. After the final supernatant was discarded, any remaining liquid at the bottom of the tube was aspirated, and 500 μl of PBS was added to each tube before flow cytometry analysis. The statistical results are as follows:

[0104] Table 3. Effects of different cell protection solutions on cell surface markers

[0105] Based on the cell flow cytometry results, no flow cytometry was performed on Comparative Example 1 (72h and 120h) and Comparative Example 2 (120h) because the cell viability was below 50%. However, it can be seen that the expression of cell surface markers (such as CD73, CD90, and CD105) did not decrease at any node in each group of the Example groups, thus confirming that the cell function was normal.

[0106] Experiment 3: pH Detection

[0107] Different cell preservation solutions in the examples and comparative examples were stored at 2-8°C, and 5ml was taken out with a syringe at different time points each time for pH value detection.

[0108] The pH meter was used for testing. After the instrument was powered on and calibrated, the electrode was placed in the sample solution to begin measurement. Once the electrode output stabilized, the display showed the pH value of the sample solution. The pH test results are as follows:

[0109] Table 4. pH values ​​of different cell preservation solutions at different times

[0110] pH tests were performed on each formulation at various time points, and there were no significant changes in the results among the groups, proving that the formulations of this invention are suitable for cell preservation.

[0111] Experiment Example 4: Osmotic Pressure Detection

[0112] Take 60µl of samples from different cell preservation solutions in the examples and comparative examples, stored at 2-8℃, and inject them into test tubes (ensuring no visible air bubbles). Use an osmotic pressure analyzer to detect the osmotic pressure changes of each cell preservation solution at different time periods.

[0113] Push the test tube into the support to the stop position, ensuring the temperature probe is fully immersed in the sample within the test tube. The test begins; the handle moves automatically and gently downwards, steadily inserting the temperature probe (test tube) into the cooling tank. The instrument displays the real-time temperature of the sample. When the sample temperature reaches the supercooled temperature (-7℃), the probe descends to initiate crystal formation, and the instrument displays the osmolality value of the sample. After the test, the handle rises, and the instrument automatically provides the osmolality value, freezing point value, and osmolality ratio of the sample. The osmolality test results are as follows:

[0114] Table 5 Osmotic pressure of different cell preservation solutions at different times

[0115] Osmolarity was measured at various time points for each formulation. In the comparative examples and other groups, osmolarity gradually increased with the increase of formulation components. From the perspective of cell preservation, the osmolarity of this invention does not affect cell quality.

[0116] Experiment 5: Detection of Cell Adhesion Efficiency

[0117] Cell samples from Examples 1-9 and Comparative Examples 1 and 2 were collected after 120 hours of incubation. After cell counting, 1×10⁶ cells were collected based on the cell count results. 5 Cells were centrifuged at 400g for 5 minutes. After centrifugation, the supernatant was discarded, and 2.5 mL of complete culture medium was added to each group to resuspend the cell pellet. The pellet was mixed by pipetting and seeded into 6-well plates. After seeding, the cells were shaken well and observed under a microscope to ensure they were evenly distributed across the bottom of the culture flask. The 6-well plates were then placed horizontally in a CO2 incubator at 37°C with 5% CO2. The cell adhesion efficiency was observed the next day.

[0118] Cell re-adhesion assay results showed that after re-seeding and culturing cell samples from each formulation for 120 hours, fewer cells adhered in Comparative Examples 1 and 2, likely due to low cell viability and impaired cell function during preservation. In contrast, in Examples 1-9, cell re-adhesion and growth were excellent, with normal proliferation, indicating that cell quality was not affected during preservation.

[0119] Experiment Example 6: Detection of Cell SOD Values

[0120] Cell preparations from Comparative Example 2 and Example 9, preserved in their cell preservation solutions for 0h, 24h, 72h, and 120h, were analyzed for SOD values ​​using the WST-8 assay. The results are as follows:

[0121] Table 6. SOD activity at different time points in different cell preparations

[0122] The results of SOD activity detection in the cell preparations showed that: Comparative Example 2's cell preservation solution contained no traditional Chinese medicine extracts; its SOD activity briefly increased at 24 hours, then continuously decreased between 24 and 120 hours, reaching its lowest point after 120 hours. In contrast, the cell preservation solution of Example 9 showed a continuous increase in SOD activity from 0 to 72 hours, reaching its maximum value; from 72 to 120 hours, SOD activity gradually decreased, but the SOD activity value at 120 hours was significantly higher than that of Comparative Example 2. This indicates that the antioxidants in the cell preservation solution of this invention played a positive role, effectively scavenging free radicals and inhibiting lipid peroxidation.

[0123] Experimental Example 7: Antithrombotic Function of Total Saponins from Panax notoginseng

[0124] Panax notoginseng saponins exert their thrombotic effect by regulating vasodilation. Prostaglandin I2 is a potent vasodilator and platelet aggregation inhibitor. Therefore, this experiment aims to demonstrate whether Panax notoginseng saponins can promote the secretion of prostaglandin I2 by vascular endothelial cells, thus proving that Panax notoginseng saponins inhibit thrombosis by regulating vasodilation.

[0125] Human umbilical vein endothelial cells (HUVECs) from passage P4 were resuscitated and cultured at 37°C and 5% CO2 using ECM supplemented with 10% fetal bovine serum. After 72 hours of culture, the cells were digested and cultured at a rate of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 6-well plates and cultured at 37°C with 5% CO2. When the cell confluence reached 80%, the original culture medium was replaced with basal medium, and the cells were cultured for another 12 hours.

[0126] After culture, the cells were divided into four groups: negative group, positive group, 20 μM group, and 50 μM group. The culture medium for the negative group was left untreated; bradykinin (a prostaglandin I2 stimulant) was added to the culture medium for the positive group; the culture medium for the 20 μM group was replaced with the cell preservation solution from Example 3, which contained 20 μM total saponins of Panax notoginseng; and the culture medium for the 50 μM group was replaced with the cell preservation solution from Example 4, which contained 50 μM total saponins of Panax notoginseng.

[0127] Each group was cultured for another 12 hours. After the culture was completed, the supernatant of each group was collected and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was then used for ELISA detection of PGI2 stable metabolites (6-keto-prostaglandin F1α).

[0128] Table 7. Antithrombotic function of total saponins from Panax notoginseng

[0129] Table 7 shows that the concentration of 6-keto-PGF1α in the positive group was significantly higher than that in the negative group, proving that the endothelial cells functioned normally in this experiment and responded significantly to the stimulant. The concentrations of 6-keto-PGF1α in the 20μM and 50μM groups were significantly higher than those in the negative group, indicating that 20-50μM Panax notoginseng total saponins have a strong ability to induce PGI2 secretion, and thus possess the ability to prevent thrombosis by regulating vasodilation under the action of this pathway.

[0130] Experimental Example 8: Antithrombotic effect of nobiletin

[0131] Noriheptacorlina has antithrombotic effects. During thrombosis, platelet aggregation and release form a positive feedback loop that accelerates thrombosis. Therefore, this experiment uses reducing platelet aggregation as the starting point to reduce thrombosis in order to elucidate the antithrombotic effect of noriheptacorlina.

[0132] Fresh blood was collected from volunteers, and an anticoagulant was added. The blood was centrifuged at 300g for 5 minutes, and the supernatant was collected to obtain platelet-rich plasma. The platelet-rich plasma was divided into four groups: negative group, positive group, 10μM group, 30μM group, and 50μM group.

[0133] Negative group: Platelet-rich plasma was added, and no other treatment was performed;

[0134] Positive group: Platelet-rich plasma was supplemented with 10 μM ADP (platelet aggregation inducer).

[0135] 10μM group: Platelet-rich plasma and cell protection solution from Example 5 were added, containing 10μM hesperidin, and incubated at 37°C for 10 min, followed by the addition of 10μM ADP (platelet aggregation inducer).

[0136] 30μM group: Platelet-rich plasma and cell protection solution from Example 6 were added, containing 30μM hesperidin, incubated at 37°C for 10 min, and 10μM ADP (platelet aggregation inducer) was added.

[0137] 50 μM group: Platelet-rich plasma and cell protection solution from Example 7 were added, containing 50 μM hesperidin; incubated at 37°C for 10 min, and 10 μM ADP (platelet aggregation inducer) was added.

[0138] Detecting platelet aggregation in each group, such as Figure 2 As shown in the diagram. The negative group (bottom curve) is a horizontal straight line, with the aggregation rate consistently maintained at the baseline level of 0-2%, indicating that the sample is normal and there is no spontaneous aggregation. The positive group (dark gray curve) shows rapid aggregation within 1 minute after the addition of ADP, reaching a plateau phase in 2-4 minutes, indicating that the sample is normal and platelet aggregation under induced conditions is normal. The 10μM group (orange curve) shows a significantly reduced slope, a delayed plateau phase, and a significantly lower aggregation rate than the positive group. The 30μM group (light gray curve) shows greater inhibition of both the aggregation plateau phase and aggregation rate compared to the 10μM group; the 50μM group (green curve) shows almost complete inhibition, with only a slight initial aggregation reaction, demonstrating a significant inhibitory effect. The above data fully demonstrate that linalool can effectively inhibit platelet aggregation, thereby producing an antithrombotic effect.

[0139] Experimental Examples 7 and 8 demonstrate that both hesperidin and total saponins of Panax notoginseng have antithrombotic abilities. However, their mechanisms of action differ. Hesperidin focuses more on reducing platelet aggregation, while total saponins of Panax notoginseng focus more on regulating vasodilation. The two work synergistically to further enhance the antithrombotic ability of the present invention.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cell protection solution, characterized in that, Each 100ml of cell protection solution contains 1-10 mL of compound electrolyte glucose injection, 1-10 mL of human serum albumin injection, 1-10 mL of compound amino acid injection, 10-20 μM of salvianolic acid B, 20-60 μM of rhodioloside, 1-10 μM of ginkgo biloba extract, and is supplemented with compound electrolyte injection.

2. The cell protection solution according to claim 1, characterized in that, The cell protection solution also contains 10-50 μM hesperidin and / or 20-50 μM total saponins of Panax notoginseng.

3. The cell protection solution according to claim 1 or 2, characterized in that, The compound amino acid injection is Compound Amino Acid Injection 18AA-II.

4. The cell protection solution according to claim 1 or 2, characterized in that, The cell protection solution has a pH of 6-8 and an osmolar concentration of 270-380 OSmol.

5. The method for preparing the cell protection solution according to any one of claims 1 to 4, characterized in that, The salvianolic acid B, rhodioloside, and ginkgo biflavonoids were each prepared into solutions. The resulting solutions were then mixed with the compound electrolyte injection, the compound electrolyte glucose injection, the human serum albumin injection, and the compound amino acid injection in proportion to the final concentration.

6. The preparation method according to claim 5, characterized in that, The salvianolic acid B, rhodioloside, ginkgo biflavonoids, tangeretin and total saponins of Panax notoginseng were each prepared into solutions. The resulting solutions were then mixed with the compound electrolyte injection, the compound electrolyte glucose injection, the human serum albumin injection and the compound amino acid injection in proportion to the final concentration.

7. A cell protection solution formulation, characterized in that, The cell protection solution formulation is a lyophilized powder or concentrate of the cell protection solution according to any one of claims 1 to 4, or ice crystals in a frozen state.

8. A cell preparation, characterized in that, Includes cells and the cell protection solution as described in any one of claims 1 to 4.

9. The cell preparation according to claim 8, characterized in that, The cells include one or more of somatic cells, stem cells, and immune cells.

10. A method for preserving cells, characterized in that, The procedure includes the following steps: resuspending the cells in the cell protection solution described in any one of claims 1 to 4 and storing them at 2-8°C.

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