Cell culture fluid as well as preparation method and application thereof

Abstract

The invention provides a cell culture solution as well as a preparation method and application thereof. The cell culture fluid comprises a basic culture medium, an auxiliary synergistic component, a microenvironment regulation and control component, a stability regulation and control component and a balling induction component, the auxiliary synergistic component comprises an RHO related kinase inhibitor, an antioxidant, a serum substitute, a cell adhesion enhancer and an apoptosis inhibition factor, the microenvironment regulation component comprises a growth factor, the stability regulation component comprises a surfactant and a buffer agent, and the balling induction component comprises at least one polyglycerol ester. The technical defects that a traditional stent-free system is low in balling efficiency, long in period and insufficient in stability are overcome, and meanwhile high cost, batch difference and clinical safety risks caused by introduction of additional materials of a stent system are avoided; the technical problems of insufficient efficiency, poor uniformity and poor stability generally existing in three-dimensional cell balling culture are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a cell culture medium and a preparation method and application thereof. BACKGROUND

[0002] Three-dimensional spheroid culture of cells as a key means to simulate the in-vivo microenvironment has been widely used in many frontier fields such as tumor development mechanism research, stem cell clinical transformation, and drug screening and evaluation, because it is closer to real physiological conditions in terms of spatial structure, metabolic state, and signal pathway regulation. Existing technologies generally use scaffold-free culture systems or scaffold culture systems to achieve spheroid formation, but both methods have significant shortcomings. Scaffold-free systems rely on low adsorption culture plates or suspension incubators, which avoid the introduction of heterogeneous materials, but lack effective cell aggregation induction mechanisms, often resulting in low spheroid formation efficiency, prolonged spheroid formation period, and unstable spheroids. Scaffold systems rely on materials such as Matrigel and hydrogel, which can provide physical support to some extent, but have high preparation costs, large batch differences, and obvious risks in clinical transformation due to the presence of animal-derived components.

[0003] More critically, in the above systems, the culture medium is usually only a basic nutrient supply medium, and its function is limited to providing nutrients such as glucose, amino acids, and inorganic salts, and it cannot promote cell aggregation or maintain spheroid stability. For example, patent CN116083363A discloses an application scheme for promoting cell spheroid formation by composite hydrogel material. This technology can achieve rapid cell spheroid formation by polyvinyl alcohol / sodium alginate composite hydrogel, but it still relies on external scaffold materials, and the hydrogel is prone to displacement and rupture in dynamic suspension or large-scale reactor environments, leading to cell sphere disintegration, thus naturally limiting its large-scale application. For another example, CN11997953A discloses a method for promoting mesenchymal stem cell spheroid formation by compound CT, but this method relies on high-concentration dimethyl sulfoxide carriers, which have cytotoxicity risks, and the spheroid formation process is slow, making it difficult to achieve uniformity and stability in large-scale applications.

[0004] In summary, existing technologies have not yet provided a technical solution that can effectively induce cell spheroid formation through the design of culture medium components. Therefore, how to develop a special culture medium with clear components, controllable cost, strong compatibility, and high safety to fundamentally solve the technical problems of low spheroid formation efficiency, long period, poor uniformity, and insufficient stability has become a key breakthrough in this field. SUMMARY

[0005] To solve the above problems, according to the first aspect of the present application, a cell culture medium is provided, which comprises a basic culture medium, an auxiliary synergistic component, a microenvironment regulating component, a stability regulating component, and a spheroid induction component. The auxiliary synergistic components include RHO-related kinase inhibitors, antioxidants, serum substitutes, cell adhesion enhancers and apoptosis inhibitors, the microenvironment regulating components include growth factors, the stability regulating components include surfactants and buffers, and the spheroid-inducing components include at least one polyglycerol ester.

[0006] Optionally, the polyglycerol ester is selected from a combination of one or more of polyglyceryl-3-decanoate and polyglyceryl-4-octanoate; When the polyglycerol ester is a mixture of polyglyceryl-3-decanoate and polyglyceryl-4-octanoate, the mass ratio of the polyglyceryl-3-decanoate to the polyglyceryl-4-octanoate is any value in the range of 1: (0.9-1.1); The concentration of the polyglycerol ester is any value in the range of 0.2 mg / mL-0.6 mg / mL.

[0007] Optionally, the RHO-related kinase inhibitor is selected from a combination of one or more of trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride and fasudil; The antioxidants include reduced glutathione and vitamin C; The serum substitutes are selected from a combination of one or more of medical grade human blood albumin, transferrin and insulin; The cell adhesion enhancers are selected from a combination of one or more of laminin, fibronectin and collagen I; The apoptosis inhibitors are selected from a combination of one or more of brain-derived neurotrophic factor, insulin growth factor and vascular endothelial growth factor.

[0008] Optionally, the concentration of the RHO-related kinase inhibitor is any value in the range of 5.0 μM-10.0 μM; The concentration of the reduced glutathione is any value in the range of 0.1 mM-0.2 mM, and the concentration of the vitamin C is any value in the range of 0.03 mM-0.08 mM; The mass volume concentration of the serum substitutes is any value in the range of 0.15%-0.3%; The concentration of the cell adhesion enhancers is any value in the range of 3.0 μg / mL-8.0 μg / mL; The concentration of the apoptosis inhibitors is any value in the range of 1.0 ng / mL-3.0 ng / mL.

[0009] Optionally, the growth factors are selected from a combination of one or more of fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor; The concentration of the growth factor is any value ranging from 1.0 ng / mL to 20.0 ng / mL.

[0010] Optionally, the surfactant is selected from a combination of one or more of Tween-80, Poloxamer 188 and polysorbate-20. The buffer is selected from a combination of one or more of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid and tris(hydroxymethyl)aminomethane. The mass-volume concentration of the surfactant is any value ranging from 0.01% to 0.03%. The concentration of the buffer is any value ranging from 18.0 mM to 22.0 mM.

[0011] Optionally, the basal medium is selected from a combination of one or more of DMEM / F-12, CMRL 1066 and RPMI 1640. When the basal medium is a mixture of DMEM / F-12 and CMRL 1066, the volume ratio of the DMEM / F-12 to the CMRL 1066 is any value ranging from (2.8-3.2):1. The volume of the basal medium is any value ranging from 85% to 92% of the total volume of the cell culture solution.

[0012] According to a second aspect of the present application, a preparation method of the aforementioned cell culture solution is provided, comprising the following steps: Respectively preparing the basal medium, the mother liquor of the sphere-inducing component, the mother liquor of the auxiliary synergistic component, the mother liquor of the microenvironment-regulating component and the mother liquor of the stability-regulating component; Adding each of the mother liquors into the basal medium with a preset temperature according to a preset ratio, and mixing to obtain the cell culture solution.

[0013] Optionally, the step of respectively preparing the basal medium, the mother liquor of the sphere-inducing component, the mother liquor of the auxiliary synergistic component, the mother liquor of the microenvironment-regulating component and the mother liquor of the stability-regulating component further comprises: Loading the microenvironment-regulating component into microspheres, treating to obtain a microsphere mother powder, resuspending the microsphere mother powder in the basal medium at a preset concentration to obtain the mother liquor of the microenvironment-regulating component; The microspheres are polylactic acid-glycolic acid copolymer, and the particle size of the microspheres is any value ranging from 1.0 μm to 5.0 μm.

[0014] According to a third aspect of the present application, the use of the aforementioned cell culture solution is provided, which is used for three-dimensional spheroid culture of tumor cells and stem cells, wherein the tumor cells include H1975 non-small cell lung cancer cell line, Hep human hepatoma cells and MCF-7 human breast cancer cells.

[0015] According to the scheme of the present application, by innovatively introducing spheroid induction components, auxiliary synergistic components, microenvironment regulation components and stability regulation components in the framework of traditional basal medium, multiple functions of nutrient supply, aggregation induction, signal regulation and environmental stability are simultaneously achieved in a single system, and a cell culture solution with systematicity and synergy is constructed.

[0016] Specifically, the polyglycerol ester in the spheroid induction component can directly promote specific aggregation between cells by changing the physicochemical properties of the extracellular environment, and is a core element for driving three-dimensional spheroid formation; the auxiliary synergistic components, including ROH-related kinase inhibitors, antioxidants, serum substitutes, cell adhesion enhancers and apoptosis inhibitors, synergistically act to improve cell survival rate, enhance adhesion ability and inhibit programmed cell death, thereby significantly improving spheroid efficiency and activity maintenance; the microenvironment regulation components achieve sustained and stable signal supply through exogenous growth factors under the action of sustained-release carriers, ensuring the dynamic homeostasis and function maintenance of cells during spheroid formation; the stability regulation components further ensure the consistency and long-term stability of the system in the physical and chemical environment through the combination of surfactants and buffers, avoiding spheroid instability caused by pH fluctuations or component precipitation. The organic combination and synergistic effect of the above components in the unified system enable the culture solution of the present application to efficiently induce cell spheroid formation without a scaffold, and the obtained cell spheroids have the significant advantages of uniform structure, high stability and good functional state.

[0017] Compared with the prior art, the present application not only overcomes the technical defects of low spheroid efficiency, long cycle and insufficient stability of the traditional scaffold-free system, but also avoids the high cost, batch difference and clinical safety risk caused by the introduction of additional materials in the scaffold system, thereby solving the technical problems of insufficient efficiency, poor uniformity and poor stability commonly existing in three-dimensional cell spheroid culture. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A schematic diagram of the preparation method of the cell culture solution in an embodiment of the present application is shown; Figure 2 An inverted microscope image of the cell culture spheroid in Example 1 of the present application is shown. DETAILED DESCRIPTION

[0019] In order to make the above objectives, characteristics and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below with reference to the drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application. In addition, it should be noted that, for the convenience of description, only the parts related to the present application are shown in the drawings, but not all the structures. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0020] The terms "comprising" and "having" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device including a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products or devices.

[0021] Reference to "embodiments" herein means that the specific features, structures or characteristics described in connection with the embodiments can be included in at least one embodiment of the present application. The phrase appears at various places in the specification does not necessarily all refer to the same embodiments, nor is it necessarily independent or alternative embodiments to other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0022] The present application provides a cell culture medium, comprising a basic medium, an auxiliary synergistic component, a microenvironment regulating component, a stability regulating component and a sphere-forming inducing component. The auxiliary synergistic component comprises a RHO-related kinase inhibitor, an antioxidant, a serum substitute, a cell adhesion enhancer and an apoptosis inhibitor. The microenvironment regulating component comprises a growth factor. The stability regulating component comprises a surfactant and a buffer. The sphere-forming inducing component comprises at least one polyglycerol ester.

[0023] According to the scheme of the present application, by innovatively introducing the sphere-forming inducing component, the auxiliary synergistic component, the microenvironment regulating component and the stability regulating component in the framework of the traditional basic medium, the multiple functions of nutrient supply, aggregation induction, signal regulation and environmental stability are realized in a single system, and a cell culture medium with systematicity and synergy is constructed.

[0024] Specifically, the polyglycerol ester in the sphere-inducing component can directly promote specific aggregation between cells by changing the physicochemical properties of the extracellular environment, and is the core element driving the formation of three-dimensional spheres; the auxiliary synergistic component can improve cell survival rate, enhance adhesion ability and inhibit programmed cell death through the synergistic effect of a multi-factor combination including ROH-related kinase inhibitors, antioxidants, serum substitutes, cell adhesion enhancers and apoptosis inhibitors, thereby significantly improving sphere formation efficiency and activity maintenance; the microenvironment regulating component can ensure dynamic homeostasis and functional maintenance of cells during sphere formation by realizing continuous and stable signal supply of exogenous growth factors under the action of a slow-release carrier; the stability regulating component can further ensure the consistency and long-term stability of the system in the physical and chemical environment by the combination of surfactants and buffers, thereby avoiding sphere instability caused by pH fluctuation or component precipitation. The organic combination and synergistic effect of the above components in the unified system enable the culture solution of the present application to efficiently induce cell sphere formation under scaffold-free conditions, and the obtained cell spheres have the significant advantages of uniform structure, high stability and good functional state.

[0025] Compared with the prior art, the present application not only overcomes the technical defects of low sphere formation efficiency, long cycle and poor stability of the traditional scaffold-free system, but also avoids the high cost, batch difference and clinical safety risk caused by the introduction of additional materials in the scaffold system, thereby solving the technical problems of insufficient efficiency, poor uniformity and poor stability commonly existing in three-dimensional cell sphere culture.

[0026] In one embodiment, the polyglycerol ester is selected from a combination of one or more of polyglyceryl-3-decanoate and polyglyceryl-4-octanoate. Polyglyceryl-3-decanoate and polyglyceryl-4-octanoate are derived from decanoic acid and octanoic acid, respectively. Both decanoic acid and octanoic acid are medium-chain fatty acids with good biocompatibility and safety. When forming polyesters with glycerol, they can retain more hydroxyl active groups, and these structures can specifically bind to the cadherin on the cell membrane surface, thereby significantly promoting cell-cell interaction and aggregation. In addition, the differences in chain length and branching degree between polyglyceryl-3-decanoate and polyglyceryl-4-octanoate endow them with different hydrophilic / hydrophobic balance properties. The combination of the two can enhance the interaction with the cell membrane while maintaining good solubility, making the spheroid formation process more efficient and stable. In one embodiment, when the polyglycerol ester is a mixture of polyglyceryl-3-decanoate and polyglyceryl-4-octanoate, the mass ratio of polyglyceryl-3-decanoate to polyglyceryl-4-octanoate is any value within the range of 1 : (0.9-1.1), for example, it can be 1 :0.9, 1 :1.0, or 1 :1.1. The selection of this ratio range is based on the complementary effect of the two in hydrophobicity and hydrophilicity, which can form the best interfacial activity state during cell spheroid formation. If the ratio is less than 0.9, polyglyceryl-4-octanoate is insufficient, which will result in an increase in the overall hydrophilicity of the molecular system, weakening the interaction with the hydrophobic region of the cell membrane, reducing the spheroid formation efficiency, and if the ratio is higher than 1.1, polyglyceryl-3-decanoate is excessive, the hydrophobicity of the system is too strong, which may cause cell membrane disturbance or reduce solution stability, causing cell stress and even apoptosis. In one embodiment, the concentration of polyglycerol ester is any value within the range of 0.2 mg / mL-0.6 mg / mL, for example, it can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, or 0.6 mg / mL. When the concentration of polyglycerol ester is less than 0.2 mg / mL, the number of polyglycerol ester molecules is insufficient to cover enough cell surface adhesion proteins, resulting in a significant decrease in spheroid induction efficiency, and when the concentration is higher than 0.6 mg / mL, excessive polyglycerol ester will increase the colloidal viscosity of the solution and interfere with the integrity of the cell membrane, which may lead to a decrease in cell activity and even cell death. Therefore, the selection of the concentration of polyglycerol ester within this range can achieve efficient spheroid formation in the shortest period of time and maintain high cell viability and spheroid stability.

[0027] In one embodiment, the method for preparing polyglycerol ester comprises the following steps: First, the glycerol and fatty acid are mixed in a molar ratio of 1:(1.1-1.3), and an acidic catalyst is added in a mass fraction of 0.2%-0.4% therein, for example, the molar ratio of glycerol and fatty acid can be 1:1.1, 1:1.2 or 1:1.3. Controlling the molar ratio to be 1:(1.1-1.3) can ensure sufficient reaction and maintain a suitable product structure, thereby obtaining a polyglycerol ester with the best cell induction effect. The mass fraction of the acidic catalyst may, for example, be 0.2%, 0.3% or 0.4%. The range is selected to ensure efficient esterification reaction while avoiding side reactions. Then, the reaction is carried out at a temperature of 110°C-130°C under normal pressure for 3-4 hours. Subsequently, the temperature is raised to 140°C-160°C, and the reaction is continued under vacuum for 6-8 hours. After the reaction is completed, the resulting product is washed and dried to obtain the polyglycerol ester. The first stage of the reaction at normal pressure and moderate temperature can effectively start the esterification and avoid rapid consumption of raw materials. The second stage of the reaction under higher temperature and vacuum conditions can accelerate the removal of water and promote the polymerization reaction to a higher degree of polymerization. Under the above conditions, the polyglycerol ester obtained not only has a moderate degree of polymerization and good water solubility, but also avoids the generation of by-products caused by excessive reaction, thereby obtaining a polyglycerol ester with a stable structure and ideal induction effect.

[0028] In one embodiment, the fatty acid can be decanoic acid or octanoic acid. In one embodiment, the acidic catalyst is p-toluenesulfonic acid. The selection of the fatty acid is based on its moderate carbon chain length, which can balance the biocompatibility and the hydrophobicity adjustment of the molecular structure, thereby enhancing the interaction between the polyglycerol ester and the cell membrane surface during the sphere formation process. The mechanism of the catalyst is to enhance the nucleophilicity and electrophilicity of the protonated hydroxyl and carboxyl groups, thereby promoting the smooth progress of the esterification reaction. In one embodiment, the conditions for the washing treatment are as follows: using a mixture of anhydrous ethanol and n-hexane in a volume ratio of 1:1 to wash 4-6 times to remove unreacted glycerol and fatty acid.

[0029] In one embodiment, the auxiliary synergistic components include RHO-related kinase inhibitors, antioxidants, serum substitutes, cell adhesion enhancers and apoptosis inhibitors. These components collectively constitute an auxiliary synergistic system, which improves the survival rate and sphere formation efficiency of cells through comprehensive regulation of multiple pathways and mechanisms. Specifically, the RHO-related kinase inhibitors reduce the cytoskeleton tension and apoptosis rate, the antioxidants scavenge free radicals and maintain cellular homeostasis, the serum substitutes provide nutrition and osmotic pressure balance, the cell adhesion enhancers promote cell contact and aggregation, and the apoptosis inhibitors delay programmed death through signal pathways. These components cooperate with each other to enable the cells not only to quickly aggregate during sphere formation, but also to maintain high activity and functional stability, thereby significantly improving the overall culture effect.

[0030] In one embodiment, the RHO-associated kinase inhibitor is selected from the group consisting of a combination of one or more of trans-4-[(R)-1-aminoethyl]-N-(4-pyridinyl)cyclohexanecarboxamide dihydrochloride and fasudil, preferably trans-4-[(R)-1-aminoethyl]-N-(4-pyridinyl)cyclohexanecarboxamide dihydrochloride. Such inhibitors can significantly reduce the apoptosis rate of cells in the initial stage of digestion, suspension and spheroid formation by inhibiting the RHO / ROCK signaling pathway, reducing cytoskeleton contraction and stress response. At the same time, by improving the flexibility of the cell membrane and the stability of cell-cell contact, it promotes the rapid aggregation of cells into regular spheroids. In one embodiment, the concentration of the RHO-associated kinase inhibitor is any value between 5.0 μΜ and 10.0 μΜ, for example, it can be 5.0 μΜ, 6.0 μΜ, 7.0 μΜ, 8.0 μΜ, 9.0 μΜ or 10.0 μΜ. When the concentration is lower than 5.0 μΜ, the inhibition is insufficient and cannot significantly reduce the apoptosis rate of cells, and when the concentration is higher than 10.0 μΜ, the inhibition is too strong and can interfere with the normal cytoskeleton dynamics and division process of cells, leading to dysfunction and even death. Controlling within this range can effectively block the apoptosis signal while maintaining the normal physiological function of cells, thereby achieving the purpose of improving the spheroid formation efficiency and stability.

[0031] In one embodiment, the antioxidants include reduced glutathione and vitamin C. These two antioxidants work together to scavenge free radicals and maintain the redox balance in cells through different mechanisms. Reduced glutathione, as the main endogenous reducing agent in cells, can effectively protect the integrity of cell proteins and membrane structures by reacting with active oxygen free radicals through its thiol group. Vitamin C can directly scavenge superoxide anions and hydroxyl radicals through electron donor action, while promoting collagen synthesis and extracellular matrix stability. The combination of the two can significantly reduce cell apoptosis and necrosis caused by oxidative stress during cell spheroid culture, ensuring the stability and uniformity of cell spheroids. In one embodiment, the concentration of reduced glutathione is any value between 0.1 mM and 0.2 mM, for example, it can be 0.1 mM, 0.15 mM or 0.2 mM. When the concentration is lower than 0.1 mM, the free radical scavenging effect is insufficient and the cells are prone to oxidative damage, and when the concentration is higher than 0.2 mM, the excessive reducing environment can interfere with signal transduction in cells, leading to decreased proliferation capacity. In one embodiment, the concentration of vitamin C is any value between 0.03 mM and 0.08 mM, for example, it can be 0.03 mM, 0.04 mM, 0.055 mM, 0.06 mM, 0.07 mM or 0.08 mM. When the concentration is lower than 0.03 mM, the ability of vitamin C to scavenge active oxygen is insufficient and it cannot effectively protect cells, and when the concentration is higher than 0.08 mM, vitamin C itself can be oxidized, generating free radicals and causing secondary damage.

[0032] In one embodiment, the serum substitute is selected from a combination of one or more of medical grade human serum albumin, transferrin and insulin, preferably medical grade human serum albumin. The serum substitute is mainly used to provide nutrition and maintain osmotic pressure balance in the culture medium, avoiding batch difference and immunogenicity risk caused by the use of animal-derived serum. Among them, medical grade human serum albumin can effectively bind and stabilize small molecule factors due to its high purity and good biocompatibility, thereby maintaining the stability of the culture medium and significantly improving the survival rate and uniformity of the cell spheroid process. In one embodiment, when the serum substitute is preferably medical grade human serum albumin, the material needs to be treated by double virus inactivation. The treatment process includes: inactivating lipid envelope viruses at 60°C±0.5°C for 10h, and then removing non-lipid envelope viruses by using polyether sulfone membrane with pore size of 20nm-50nm for cross-flow filtration. The human serum albumin obtained by this process has a purity of ≥98% and does not contain preservatives, antibiotics and exogenous proteins. The treatment process is to ensure the biosafety of the serum substitute in the clinical conversion application, avoid the pollution of potential pathogens, and at the same time improve the stability and consistency of the product, so as to ensure the reliability of the culture medium in large-scale application. In one embodiment, the mass concentration of the serum substitute is any value between 0.15% and 0.3%, for example, it can be 0.15%, 0.2%, 0.225%, 0.25% or 0.3%. When the concentration is lower than 0.15%, the nutrition supply is insufficient, and the cell survival rate decreases, while when the concentration is higher than 0.3%, the viscosity of the culture medium increases, affecting the interaction between cells, and even possibly leading to uneven spheroid formation.

[0033] In one embodiment, the cell adhesion enhancer is selected from a combination of one or more of laminin, fibronectin and collagen I, preferably laminin. The cell adhesion enhancer promotes the mutual recognition and combination between cells by simulating the extracellular matrix components, thereby accelerating the aggregation of cells into spheroids. Laminin has a stronger cell adhesion promotion effect and can significantly improve the tightness and stability of spheroids due to its specific binding with integrin receptors. In one embodiment, the concentration of the cell adhesion enhancer is any value between 3.0μg / mL and 8.0μg / mL, for example, it can be 3.0μg / mL, 4.0μg / mL, 5.5μg / mL, 6.0μg / mL, 7.0μg / mL or 8.0μg / mL. When the concentration is lower than 3.0μg / mL, the cell aggregation efficiency is insufficient, and the spheroid formation process is prolonged, while when the concentration is higher than 8.0μg / mL, excessive adhesion proteins may cause abnormal aggregation or form irregular clumps, affecting the uniformity of the spheroids.

[0034] In one embodiment, the apoptosis inhibitor is selected from a combination of one or more of brain-derived neurotrophic factor, insulin growth factor and vascular endothelial growth factor, preferably brain-derived neurotrophic factor. The apoptosis inhibitor effectively delays the process of programmed cell death by activating anti-apoptotic signaling pathways and promoting the expression of cell survival-related genes. Brain-derived neurotrophic factor is preferred in the present application because it can significantly enhance the survival signal of cells through the TrkB receptor, improve the tolerance of cells in suspension culture and sphere formation, and thus improve the integrity and activity of the spheres. In one embodiment, the concentration of the apoptosis inhibitor is any value ranging from 1.0 ng / mL to 3.0 ng / mL, for example, it can be 1.0 ng / mL, 2.0 ng / mL or 3.0 ng / mL. When the concentration is lower than 1.0 ng / mL, the effect of inhibiting apoptosis is not obvious, and cells are prone to die in large quantities during culture. When the concentration is higher than 3.0 ng / mL, over-activation of the signaling pathway can interfere with normal cell differentiation and metabolism, leading to abnormal function.

[0035] In one embodiment, the microenvironment regulating component includes growth factors, for example, a combination of one or more of fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor. The above-mentioned growth factors synergistically act through different signaling pathways to provide sustained microenvironment support for cell sphere formation. Specifically, fibroblast growth factor-2 can promote cell proliferation and angiogenesis, insulin-like growth factor-1 can improve cell survival rate and metabolic activity by activating the PI3K / AKT pathway, transforming growth factor-β1 plays an important role in maintaining cell stemness and regulating cell differentiation within the sphere, and epidermal growth factor promotes cell growth and aggregation by stimulating the EGFR signaling pathway. The combination of the four growth factors can construct a stable and dynamic microenvironment during three-dimensional sphere culture of cells, achieving the dual goals of efficient sphere formation and function maintenance. In one embodiment, the concentration of the growth factor is any value ranging from 1.0 ng / mL to 20.0 ng / mL, for example, it can be 1.0 ng / mL, 3.0 ng / mL, 5.0 ng / mL, 8.0 ng / mL, 10.0 ng / mL, 13.0 ng / mL, 15.0 ng / mL, 18.0 ng / mL or 20.0 ng / mL. When the concentration is lower than 1.0 ng / mL, the stimulation of the growth factor is insufficient, and the efficiency of cell sphere formation is low. When the concentration is higher than 20.0 ng / mL, abnormal signal over-activation can cause cell differentiation imbalance or excessive proliferation. Therefore, by controlling the concentration within this range, the cells can obtain moderate and stable signal support during sphere formation, thereby achieving efficient and uniform sphere formation.

[0036] In one embodiment, the microenvironment regulating components are preferably fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor, wherein the concentration of fibroblast growth factor-2 is 15.0 ng / mL, the concentration of insulin-like growth factor-1 is 8.0 ng / mL, the concentration of transforming growth factor-β1 is 3.0 ng / mL and the concentration of epidermal growth factor is 10.0 ng / mL. This preferred combination can provide precise signal division during the process of sphere formation: fibroblast growth factor-2 ensures efficient cell expansion, insulin-like growth factor-1 maintains cell survival, transforming growth factor-β1 regulates cell fate, and epidermal growth factor enhances cell adhesion and aggregation. The synergistic effect of the four can reconstitute a physiological microenvironment in the culture medium, thereby significantly improving sphere formation efficiency, compacting and stabilizing the structure of the spheres, and better simulating the physiological state in vivo.

[0037] In one embodiment, the stability regulating components include surfactants and buffers. The role of such components is to maintain the stability of the physicochemical properties of the culture medium, wherein the surfactants can improve the dispersibility and solubility of poorly soluble components by reducing the surface tension, thereby avoiding the precipitation or aggregation of sphere formation inducing components or growth factors in the system. The buffers can adjust and stabilize the pH value of the culture medium, thereby avoiding cell damage due to fluctuations in environmental pH. The combined use of the two types of substances can ensure that the culture medium maintains a stable environment throughout the sphere formation period, providing reliable support for cell growth.

[0038] In one embodiment, the surfactant is selected from a combination of one or more of Tween-80, Poloxamer 188 and polysorbate-20, preferably Tween-80. The surfactant in the culture medium not only improves the solubility of the hydrophobic substances, but also reduces the risk of non-specific adsorption during cell balling by improving the uniformity of the liquid phase. Tween-80 is preferred as the surfactant because it is pharmacopoeia-registered, has low toxicity and side effects, and has been widely used in biological and pharmaceutical preparations, ensuring the stability and clinical convertibility of the culture medium. In one embodiment, the mass concentration of the surfactant is any value between 0.01% and 0.03%, for example, it can be 0.01%, 0.02% or 0.03%. When the concentration is lower than 0.01%, it is difficult to play a sufficient solubilizing role, which may cause the precipitation of core ingredients such as polyglycerol esters, and when the concentration is higher than 0.03%, the surfactant may damage the stability of the cell membrane, causing cell damage. In one embodiment, the buffer is selected from a combination of one or more of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid and tris(hydroxymethyl)aminomethane, preferably 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The role of the buffer in the culture medium is to maintain the pH stability of the system, avoiding the fluctuation of the acid-base degree caused by the accumulation of lactic acid or the dissolution of carbon dioxide in the cell metabolism process. In one embodiment, the concentration of the buffer is any value between 18.0mM and 22.0mM, for example, it can be 18.0mM, 19.0mM, 20.0mM, 21.0mM or 22.0mM. When the concentration is lower than 18.0mM, the buffering capacity is insufficient, which may cause the culture medium to be significantly acidified during the balling period, thereby affecting the cell activity, and when the concentration is higher than 22.0mM, the increased osmotic pressure may cause stress to the cells, affecting the proliferation.

[0039] In one embodiment, the basal medium is selected from one or more combinations of DMEM / F-12, CMRL 1066, and RPMI 1640, preferably a mixture of DMEM / F-12 and CMRL 1066. The basal medium provides cells with essential carbon sources, amino acids, and inorganic salts, ensuring their metabolic and energy needs are met. DMEM / F-12 offers comprehensive nutrition and supports the growth of various cell lines, while CMRL 1066 excels in maintaining high-density cell culture and long-term survival. The combination of these two media can balance rapid cell growth and long-term maintenance, thus providing a more ideal support environment for cell formation. When the basal medium is a mixture of DMEM / F-12 and CMRL 1066, the volume ratio of DMEM / F-12 to CMRL 1066 is any value within the range of (2.8-3.2):1, for example, 2.8:1, 2.9:1, 3.0:1, 3.1:1, or 3.2:1. This ratio range was optimized through experiments on cell spheroidization efficiency and survival rate. When the ratio is below 2.8:1, the proportion of CMRL 1066 is too high, which may cause an excess of some nutrients and affect the uniformity of the spheroids. When the ratio is above 3.2:1, the proportion of DMEM / F-12 is too high, and the cells are prone to metabolic imbalance during long-term culture. Therefore, this ratio range can achieve a dynamic balance of nutrient supply, ensuring that the cell spheroidization process is rapid and stable.

[0040] In one embodiment, the volume of the basal culture medium is any value between 85% and 92% of the total volume of the cell culture medium, for example, 85%, 86%, 87%, 88.5%, 90%, 91%, or 92%. When the proportion is below 85%, the relative concentration of auxiliary components is too high, which may irritate the cells. When the proportion is above 92%, the auxiliary components are insufficient and cannot fully exert their role in promoting cell spheroidization. Therefore, this range can ensure a reasonable ratio of basic nutrients and functional components, achieving a synergistic balance between nutritional support and functional regulation during cell spheroidization. In one embodiment, a penicillin-streptomycin solution is also added to the basal culture medium, accounting for 1% of the total cell culture medium, i.e., a final penicillin concentration of 100 U / mL and a final streptomycin concentration of 100 μg / mL. The purpose of this step is to avoid bacterial contamination interfering with cell spheroidization culture. The above concentrations can effectively inhibit the growth of most common bacteria without having a toxic effect on eukaryotic cells, thereby ensuring the sterility and stability of the culture environment.

[0041] Figure 1 A schematic diagram illustrating a method for preparing cell culture medium according to one embodiment of the present invention is shown. Figure 1 As shown, the preparation method includes the following steps: S1, respectively, prepare the base medium, the mother liquor of the sphere-inducing component, the mother liquor of the auxiliary synergistic component, the mother liquor of the microenvironment regulating component and the mother liquor of the stability regulating component.

[0042] S2, add each mother liquor to the base medium with a preset temperature according to a preset ratio, and mix to obtain a cell culture solution.

[0043] In step S1, the following steps are also included: S101, mix the selected base medium components in proportion, add 1% penicillin-streptomycin solution, and preheat in a 37℃ constant temperature water bath for 30 min to match the cell culture temperature and avoid low temperature stimulation.

[0044] S102, weigh the polyglycerol ester powder, dissolve it in sterile ultrapure water, add a surfactant, and magnetically stir at 37℃ for 3h until completely dissolved to prepare the mother liquor of the sphere-inducing component. After filtration sterilization, store at 4℃.

[0045] S103, use dimethyl sulfoxide to prepare the RHO-related kinase inhibitor, which is sterilized by filtration and stored at -20℃. Use sterile ultrapure water to prepare antioxidants, cell adhesion enhancers and apoptosis inhibitors, which are sterilized by filtration and stored for use to obtain the mother liquor of the auxiliary synergistic component.

[0046] S104, load the microenvironment regulating component into microspheres, process to obtain microsphere mother powder, resuspend the microsphere mother powder in the base medium at a preset concentration to obtain the mother liquor of the microenvironment regulating component.

[0047] S105, use sterile ultrapure water to prepare the buffer, which is sterilized by filtration and stored at 4℃.

[0048] In step S104, the growth factors can be stably released in a sustained-release form throughout the culture period by loading the growth factors on the microspheres, avoiding the signal fluctuation caused by rapid degradation or inactivation in a short time under the traditional addition mode, to realize the continuous and effective effect of the growth factors, maintain the signal balance of the cells in the process of sphere formation, and make the obtained cell spheres more uniform and stable. In an embodiment, the microspheres are polylactic acid-glycolic acid copolymer, and the particle size of the microspheres is any value in the range of 1.0 μm-5.0 μm, for example, 1.0 μm, 2.0 μm, 3.0 μm, 4.0 μm or 5.0 μm. The polylactic acid-glycolic acid copolymer is widely used as a sustained-release carrier due to its excellent biodegradability and biocompatibility. Controlling the particle size in this range can ensure uniform distribution of the microspheres in the extracellular matrix and achieve a stable release rate. In an embodiment, the release rate of the growth factors can be regulated by adjusting the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer to ensure that the concentration of the growth factors is stably maintained in the effective range during the culture period. In an embodiment, the molar ratio of lactic acid to glycolic acid is any value in the range of (50-75):(25-50), for example, 50:25, 50:37.5, 50:50, 75:25, 75:37.5 or 75:50. When the proportion of lactic acid increases, the hydrophobicity of the polymer increases and the degradation rate slows down. When the proportion of glycolic acid increases, the hydrophilicity increases and the degradation rate accelerates. In an embodiment, the sustained release rate of fibroblast growth factor-2 is 1 ng / (mL·d)-2 ng / (mL·d), the release rate of insulin-like growth factor-1 is 0.5 ng / (mL·d)-1 ng / (mL·d), the release rate of transforming growth factor-β1 is 0.3 ng / (mL·d)-0.5 ng / (mL·d), and the release rate of epidermal growth factor is 0.8 ng / (mL·d)-1.2 ng / (mL·d). This rate range can ensure that the growth factors continuously play a role throughout the sphere formation period, avoid the situation that the signal is too low to induce sphere formation or too high to cause abnormal differentiation, and realize the dynamic balance of the growth factor signal.

[0049] In an embodiment, step S104 further comprises: preparing a dichloromethane solution of polylactic acid-glycolic acid copolymer with a mass fraction of 8%-10% as an oil phase, and dissolving each growth factor in sterile ultrapure water containing 0.1%-0.3% human serum albumin as an aqueous phase. Then, the oil phase and the aqueous phase are mixed in a volume ratio of (3-4):1, and an emulsion is formed after ultrasonic emulsification. Subsequently, the emulsion is stirred in a 0.1% polyvinyl alcohol solution for 4-5 h to solidify the microspheres, and the microspheres are collected after centrifugation, washed and freeze-dried to obtain the microsphere mother powder. This step realizes efficient encapsulation of the growth factors in the microspheres by the emulsion-evaporation method, uses human serum albumin to improve the stability of the factors, and finally realizes the sustained-release effect.

[0050] In one embodiment, the cell culture solution is used for three-dimensional spheroid culture of tumor cells and stem cells, wherein the tumor cells include H1975 non-small cell lung cancer cell line, Hep human hepatoma cells and MCF-7 human breast cancer cells. The culture solution induces cell aggregation into spheroids by polyglycerol ester, promotes cell activity and inhibits apoptosis by auxiliary synergistic components, maintains signal balance by microenvironment regulating components, and keeps the system stable by stability regulating components, so as to promote the formation of highly uniform and stable cell spheroids. By designing the composition of the culture solution, a physiological microenvironment can be reconstructed, the use of external scaffolds or toxic carriers is avoided, the spheroid efficiency and stability are improved, and the risk of clinical application is significantly reduced, so that the culture solution is suitable for three-dimensional culture of tumor cells and stem cells which are highly dependent on microenvironment.

[0051] Embodiment 1 The embodiment 1 of the present application provides a cell culture solution, which comprises: DMEM / F-12 and CMRL 1066 as the basic culture medium; Polyglycerol-3-decanoate as the spheroid induction component; Trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, reduced glutathione, vitamin C, medical grade human albumin, laminin and brain-derived neurotrophic factor as auxiliary synergistic components; Fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor as microenvironment regulating components; Tween-80 and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 3-(N-morpholino)propanesulfonic acid as stability regulating components.

[0052] The embodiment 1 of the present application further provides a preparation method of the above-mentioned cell culture solution, which comprises the following steps: (1) Mix DMEM / F-12 and CMRL 1066 at a volume ratio of 3:1, add 1% penicillin-streptomycin solution by volume, and preheat at 37℃ for 30 min to obtain the basic culture medium.

[0053] (2) Dissolve polyglycerol-3-decanoate in a 0.02% Tween-80 solution by mass volume concentration to prepare a 10mg / mL stock solution, which is sterilized by 0.22μm filter membrane and stored at 4℃. When used, add the basic culture medium to make the final concentration in the culture solution 0.4mg / mL.

[0054] (3) Trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride is dissolved in dimethyl sulfoxide solution to prepare a 10 mM solution. When used, the base medium is added to make the final concentration in the culture solution 8 μM. Reduced glutathione is prepared as a 100 mM solution in ultrapure water, and the final concentration when used is 0.15 mM. Vitamin C is prepared as a 50 mM solution in ultrapure water, and the final concentration when used is 0.05 mM. Commercially available medical-grade human albumin solution with a mass concentration of 20% is used, and the final concentration when used is 0.2%. Laminin is prepared as a 100 μg / mL solution in ultrapure water, and the final concentration when used is 5 μg / mL. Brain-derived neurotrophic factor is prepared as a 100 ng / mL solution in ultrapure water containing 0.1% human albumin, and the final concentration when used is 2 ng / mL.

[0055] (4) PLA-PEG (50:50) is used as the microsphere material. PLA-PEG is dissolved in dichloromethane solution to prepare a 10% solution as the oil phase. Fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor are dissolved in sterile ultrapure water containing 0.1% human albumin as the water phase. Then the oil phase and the water phase are mixed in a volume ratio of 3:1, and emulsified by ultrasonic emulsification. The microspheres are solidified by stirring in 0.1% polyvinyl alcohol solution for 4 h, and then collected by centrifugation. After being washed three times with sterile ultrapure water, the microspheres are freeze-dried to obtain the growth factor-loaded PLA-PEG microsphere mother powder. Before use, the microsphere mother powder is resuspended in the base medium to a final concentration. The final concentrations are: fibroblast growth factor-2, 15.0 ng / mL; insulin-like growth factor-1, 8.0 ng / mL; transforming growth factor-β1, 3.0 ng / mL; and epidermal growth factor, 10.0 ng / mL.

[0056] (5) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is prepared as a 1 M solution in ultrapure water, and the final concentration when used is 20 mM.

[0057] (6) The solutions in steps (2) to (5) are sequentially added to 100 mL of the base medium to obtain the cell culture solution.

[0058] The embodiment 1 of the present application also provides a cell spheroid culture method, comprising the following steps: (1) Select the logarithmic growth phase of H1975 tumor cells, discard the old culture medium, and wash twice with phosphate buffer. Then add digestive enzymes and incubate at 37°C for 2 min. Add 2 times the volume of phosphate buffer to terminate digestion, and then blow thoroughly to a single cell suspension.

[0059] (2) Filter the single cell suspension through a 40 μm cell screen to remove undigested cell clumps, then mix 10 μL of the filtered suspension with 10 μL of 0.4% trypan blue dye solution, and count on a cell counting plate to ensure that the cell viability is ≥90%.

[0060] (3) Resuspend the cells in the cell culture solution prepared above to a concentration of 1.5 x 10 5 / mL, and inoculate in a low-adsorption 6-well plate, with 2.5 mL of cell suspension per well. Then place the culture plate in a 37°C, 5% CO2 incubator while on a horizontal shaker.

[0061] (4) Perform half-volume medium replacement every half hour, i.e. remove 1.25 mL of old medium and add 1.25 mL of fresh medium, and observe and test at 24 h, 48 h, and 72 h of culture, respectively.

[0062] Example 2 Example 2 differs from Example 1 only in that polyglyceryl-4-octanoate at a final concentration of 0.4 mg / mL is used as the sphere-forming inducing component. The cell culture time is 72 h.

[0063] Example 3 Example 3 differs from Example 1 only in that a mixture of polyglyceryl-3-decanoate and polyglyceryl-4-octanoate at a final concentration of 0.4 mg / mL is used as the sphere-forming inducing component. The mass ratio of polyglyceryl-3-decanoate to polyglyceryl-4-octanoate is 1:1. The cell culture time is 72 h.

[0064] Example 4 Example 4 differs from Example 1 only in that the cultured cells are MCF-7 human breast cancer cells. The cell culture time is 72 h.

[0065] Comparative Example 1 Comparative Example 1 differs from Example 1 only in that no sphere-forming inducing component is added to the cell culture medium. The cell culture time is 72 h.

[0066] Comparative Example 2 Comparative Example 2 differs from Example 1 only in that in step (4), fibroblast growth factor-2 at a final concentration of 15.0 ng / mL, insulin-like growth factor-1 at a final concentration of 8.0 ng / mL, transforming growth factor-β1 at a final concentration of 3.0 ng / mL, and epidermal growth factor at a final concentration of 10.0 ng / mL are directly added, without using microsphere loading. The cell culture time is 72 h.

[0067] Comparative Example 3 Comparative Example 3 differs from Example 4 only in that a commercial culture solution is used for cell culture, which is StemCell TM MammoCult TM . The cell culture time is 72 h.

[0068] In the Examples and Comparative Examples of the present application, after a certain period of cell culture, an inverted microscope is used to image and record the cell morphology in the culture wells, and the obtained images are imported into the image processing software ImageJ for quantitative analysis. The quantitative analysis includes three indicators: spheroidization rate, average diameter of spheroids, and average circularity of spheroids, wherein: spheroidization rate = (1 - number of non-spheroidized single cells / total number of cells inoculated) x 100%, circularity = 4π x area / perimeter 2 , and circularity ≥ 0.85 is a regular spheroid. Secondly, after a certain period of cell culture, the cells are fluorescently labeled by live-dead staining, and then imaged under a confocal microscope, and the obtained images are imported into the image processing software ImageJ for analysis of the percentage of viable cells in each spheroid. The effective cell spheroids with a diameter ≥ 50 μm and a circularity ≥ 0.85 are selected by image screening, and the proportion of viable cells in each spheroid is counted. Among them, the viable spheroid rate = (number of spheroids with a viable cell proportion ≥ 80% / total number of effective cell spheroids) x 100%.

[0069] Figure 2 The inverted microscope image of the cell culture spheroids in Example 1 of the present application is shown. As Figure 2 shown, after 24 h of culture in the culture solution system of the present application, H1975 cells can quickly aggregate and form morphologically complete three-dimensional spheroids under low adsorption conditions. As can be seen from the image, the obtained spheroids have clear outlines, regular boundaries, and are approximately circular in shape, with densely arranged internal cells, and no obvious cavities or loose areas, with a spheroid diameter of about 100 μm. This result fully demonstrates that the spheroid induction component designed in the present application can effectively drive specific aggregation between cells in a short time. Figure 2 The results directly show that the culture solution of the present application can significantly improve the cell aggregation efficiency and quickly form regular, dense and uniform spheroids in the early stage of spheroid formation, overcoming the technical defects of long spheroid formation period, irregular morphology and unstable structure in the prior art.

[0070] The following Table 1 shows the data of the related indexes of the cells after the cell balling culture in Example 1 of the present application.

[0071] The following Table 1 shows the data of the related indexes of the cells after the cell balling culture in Example 1 of the present application. As shown in Table 1, with the prolongation of the culture time, the cell balling rate gradually increased from 78.5% at 24h to 92.1% at 72h, and the average diameter increased from 125.0 μm to 185.0 μm, while the average circularity remained above 0.85, and the proportion of the spheroids with the circularity ≥0.85 also increased from 91.2% to 96.3%. The results showed that the culture solution of the present application could effectively induce the cell aggregation in the early stage, and continuously maintain the stability and expansion of the spheroids in the subsequent culture process. This is because the spheroid induction component polyglycerol ester promotes the cell-cell interaction by regulating the extracellular interfacial activity. Meanwhile, the auxiliary synergistic component improves the cell survival rate and aggregation capacity. In addition, the microenvironment regulation component continuously supplies the growth factors through the sustained-release carrier to ensure the signal stability. In addition, the stability regulation component maintains the physical and chemical balance of the solution. The synergistic effect of the four components ensures that the cells can quickly ball in the scaffold-free system and maintain high regularity and uniformity, thereby significantly overcoming the defects of low balling efficiency and unstable structure in the prior art.

[0072] The following Table 2 shows the data of the related indexes of the cells after the cell balling culture in Examples 2-3 and Comparative Examples 1-2 of the present application.

[0073] Table 2 above shows the statistics of the cell balling index in the culture of 72h in the examples 2-3 and the comparative examples 1-2 of the present application by ImageJ. As shown in Table 2, the balling rates of the examples 2 and 3 are 88.7% and 91.5% respectively, the average roundness is 0.89 and 0.91 respectively, and the proportion of the ball with roundness ≥0.85 is more than 93%, which is significantly better than the balling performance of the comparative examples 1 and 2. In contrast, the balling rate of the comparative example 1 is only 45.2% in the absence of the balling induction component, the average roundness is 0.72, and the proportion of the regular ball is less than 40%, which shows that the simple reliance on the basic culture condition cannot effectively drive the cell balling. The comparative example 2 adds the growth factor, but does not use the microsphere slow-release mode, the balling rate is only 76.3%, the average roundness is 0.82, and the proportion of the regular ball is 82.5%, which is significantly lower than the examples of the present application. This shows that the simple external growth factor is difficult to maintain a stable signal environment, resulting in loose ball structure and poor uniformity. Therefore, the present application promotes the specific aggregation of the cells by the interfacial activity of the polyglycerol ester, and realizes the long-acting and stable release of the growth factor by the slow-release mechanism of the PLGA microsphere, which not only improves the balling efficiency, but also guarantees the regularity and stability of the ball, and solves the problems of slow cell balling, unstable signal and uneven structure in the prior art.

[0074] Table 3 below shows the related index data of the cell balling culture in the example 4 and the comparative example 3 of the present application.

[0075] Table 3 above shows the statistics of the cell balling index in the culture of 72h in the examples 4 and 3 of the present application by ImageJ. As shown in Table 3, the balling rate of the example 4 in the culture of the MCF-7 human breast cancer cells is 91.2%, the average diameter is 180.0μm, the average roundness is 0.90, and the live ball rate is as high as 93.5%, which is significantly better than the comparative example 3. The comparative example 3 uses the commercial StemCell TM MammoCult TMThe culture solution has a spheroidization rate of only 85.4%, an average diameter of 165.0 μm, an average roundness of 0.84, and a viable spheroid rate of 88.7%, which is significantly inferior to the present application in terms of spheroidization efficiency, structural regularity and activity maintenance. The reason for this difference is that the commercial culture solution is mainly used as a nutrition supply system and lacks special design for cell aggregation and three-dimensional structure maintenance, while the present application significantly enhances the aggregation ability and survival stability of cells in a suspension environment by introducing the spheroidization induction effect of polyglycerol ester, the cell protection effect of auxiliary synergistic components and the dynamic signal regulation effect of microsphere slow-release growth factors, thereby achieving higher spheroidization efficiency and spheroid integrity. This result further proves that the culture solution of the present application is not only suitable for non-small cell lung cancer cells, but also suitable for different types of tumor cells such as breast cancer cells, and shows superior broad-spectrum applicability and clinical transformation potential.

[0076] In summary, the present application innovatively introduces spheroidization induction components, auxiliary synergistic components, microenvironment regulation components and stability regulation components on the basis of traditional basal culture medium, thereby simultaneously achieving multiple functions of nutrition supply, aggregation induction, signal regulation and environmental stability in a single system, and constructing a cell culture solution with systematicity and synergy. The cell culture solution provided by the present application can rapidly induce spheroidization, shorten the spheroidization time, improve the spheroidization rate, and the quality of the spheroids obtained by culture is excellent and the function is stable.

[0077] The above are only a plurality of specific embodiments of the present application, and any improvement made on the basis of the concept of the present application is considered to be within the protection scope of the present application.

Claims

1. A cell culture medium, characterized in that, This includes basal culture medium, auxiliary synergists, microenvironment regulators, stability regulators, and sphere-forming inducing components; The synergistic components include RHO-related kinase inhibitors, antioxidants, serum substitutes, cell adhesion enhancers, and apoptosis inhibitors; the microenvironment regulation components include growth factors; the stability regulation components include surfactants and buffers; and the spheroidization induction components include at least one polyglycerol ester.

2. The cell culture medium according to claim 1, characterized in that, The polyglycerol ester is selected from one or more combinations of polyglycerol-3-decanoate and polyglycerol-4-octanoate; When the polyglycerol ester is a mixture of polyglycerol-3-decanoate and polyglycerol-4-octanoate, the mass ratio of the polyglycerol-3-decanoate to the polyglycerol-4-octanoate is any value in the range of 1:(0.9-1.1); The concentration of the polyglycerol ester is any value between 0.2 mg / mL and 0.6 mg / mL.

3. The cell culture medium according to claim 1 or 2, characterized in that, The RHO-related kinase inhibitor is selected from one or more combinations of trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride and fasudil; The antioxidants include reduced glutathione and vitamin C; The serum substitute is selected from one or more combinations of medical-grade human serum albumin, transferrin, and insulin; The cell adhesion enhancer is selected from one or more combinations of laminin, fibronectin and collagen I; The apoptosis inhibitor is selected from one or more combinations of brain-derived neurotrophic factor, insulin-like growth factor, and vascular endothelial growth factor.

4. The cell culture medium according to claim 3, characterized in that, The concentration of the RHO-related kinase inhibitor is any value between 5.0 μM and 10.0 μM; The concentration of the reduced glutathione is any value between 0.1 mM and 0.2 mM, and the concentration of the vitamin C is any value between 0.03 mM and 0.08 mM. The mass-volume concentration of the serum substitute is any value between 0.15% and 0.3%; The concentration of the cell adhesion enhancer is any value between 3.0 μg / mL and 8.0 μg / mL; The concentration of the apoptosis inhibitor is any value between 1.0 ng / mL and 3.0 ng / mL.

5. The cell culture medium according to claim 4, characterized in that, The growth factor is selected from one or more combinations of fibroblast growth factor-2, insulin-like growth factor-1, transforming growth factor-β1 and epidermal growth factor. The concentration of the growth factor is any value between 1.0 ng / mL and 20.0 ng / mL.

6. The cell culture medium according to claim 5, characterized in that, The surfactant is selected from one or more combinations of Tween-80, poloxamer 188 and polysorbate-20; The buffer is selected from one or more combinations of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholinyl)propanesulfonic acid, and tris(hydroxymethyl)aminomethane; The mass-volume concentration of the surfactant is any value between 0.01% and 0.03%. The concentration of the buffer is any value between 18.0 mM and 22.0 mM.

7. The cell culture medium according to claim 6, characterized in that, The basal culture medium is selected from one or more combinations of DMEM / F-12, CMRL 1066 and RPMI 1640; When the basal culture medium is a mixture of DMEM / F-12 and CMRL 1066, the volume ratio of DMEM / F-12 to CMRL1066 is any value within the range of (2.8-3.2):1; The volume of the basal culture medium is any value between 85% and 92% of the total volume of the cell culture medium.

8. A method for preparing a cell culture medium according to any one of claims 1-7, characterized in that, Includes the following steps: Prepare the basal culture medium, the stock solution of the spheroidizing induction component, the stock solution of the auxiliary synergistic component, the stock solution of the microenvironment regulation component, and the stock solution of the stability regulation component, respectively. Each of the mother liquors is added to the basal culture medium at a preset temperature according to a preset ratio, and the mixture is stirred to obtain the cell culture medium.

9. The preparation method according to claim 8, characterized in that, The steps of preparing the basal culture medium, the stock solution of the spheroidizing induction component, the stock solution of the auxiliary synergistic component, the stock solution of the microenvironment regulation component, and the stock solution of the stability regulation component respectively further include: The microenvironment regulation component is loaded into microspheres and processed to obtain microsphere master powder. The microsphere master powder is then resuspended in the basic culture medium at a preset concentration to obtain the mother liquor of the microenvironment regulation component. The microspheres are polylactic acid-glycolic acid copolymers, and the particle size of the microspheres is any value between 1.0 μm and 5.0 μm.

10. The application of a cell culture medium as described in any one of claims 1-7, characterized in that, The cell culture medium is used for three-dimensional spheroidization culture of tumor cells and stem cells, including H1975 non-small cell lung cancer cell line, Hep human liver cancer cells and MCF-7 human breast cancer cells.

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