A complex fatty acid delivery system for cell culture and its preparation method and application

Abstract

The application provides a composite fatty acid delivery system for cell culture and a preparation method and application thereof, relates to the technical field of cell culture, and comprises lipids, albumin and an antioxidant; the lipids comprise saturated fatty acids and / or unsaturated fatty acids, are not emulsified by means of nonionic surfactants, can improve the dispersion stability of the composite fatty acids in an aqueous phase culture system, and can reduce the potential influence of surfactants on cell adhesion, spreading and proliferation. The lipid concentrate solution of the application can better support the early adhesion and proliferation of MSC. The technical problem that in the prior art, a lipid supplement system is mostly emulsified or solubilized by means of surfactants such as Tween-80 and Pluronic F-68, or fatty acids are pre-dissolved by means of organic solvents such as DMSO, methanol and ethanol, so that aggregation, turbidity or precipitation are prone to occurring after the system is added into an aqueous phase culture system, and the system stability and batch consistency are seriously affected is solved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a complex fatty acid delivery system for cell culture, its preparation method, and its application. Background Technology

[0002] With the development of cell therapy, gene therapy, regenerative medicine, and the biopharmaceutical industry, cell culture systems are facing higher demands for component specificity, batch stability, safety, and quality control. While traditional serum-containing culture media can provide cells with a variety of nutrients and adhesion support components, their sources are complex, their composition is unclear, batch-to-batch variations are significant, and there is a potential risk of exogenous contamination, making it difficult to meet the needs of high-quality cell preparation and clinical translational production. Therefore, serum-free culture media and chemically defined culture media are gradually becoming important development directions in the field of cell culture.

[0003] Under serum-free or serum-reduced culture conditions, cells lack the support of natural lipids, apolipoproteins, and adhesion-related factors found in serum. This is especially true for primary adherent cells and mesenchymal stem cells, which are more difficult to culture. These cells often exhibit decreased adhesion, reduced proliferation rate, restricted cell membrane synthesis, abnormal morphology, poor long-term passage stability, and increased oxidative stress. Lipids, as important components of cell membranes and organelle membranes, participate in energy metabolism, membrane fluidity regulation, lipid raft formation, receptor signal transduction, and cytokine responses, playing a crucial role in cell proliferation and functional maintenance. Therefore, adding an appropriate lipid supplementation system to chemically limited culture media is essential.

[0004] Existing commercial lipid supplements, such as Gibco's Chemically Defined Lipid Concentrate, are typically available as concentrated lipid emulsions. These emulsions provide cell culture systems with essential lipid components such as cholesterol, fatty acids, and tocopherols, reducing or replacing some of the lipid sources in serum. However, these systems often rely on surfactants like Tween 80 and Pluronic F-68 to emulsify or solubilize fatty acids and lipid-soluble components, primarily maintaining a basic lipid supply. They are insufficient for providing physiological lipid delivery support to cells with high lipid requirements. Furthermore, surfactants may interact with cell membranes during long-term culture, affecting membrane stability, membrane protein function, receptor aggregation, and cell signaling. They may also interfere with early adhesion, cell spreading, and extracellular matrix deposition of adherent cells. Additionally, some surfactants and unsaturated fatty acids may oxidize during storage and use, producing peroxidation byproducts and increasing the risk of oxidative stress in the culture system. In addition, existing lipid supplements typically contain low levels of fatty acids, which are insufficient to meet the needs of rapidly expanding cells for membrane lipid synthesis, lipid metabolism, cell adhesion recovery, and long-term passage stability. Directly increasing the concentration of free fatty acids may cause lipotoxicity, leading to endoplasmic reticulum stress, mitochondrial damage, and elevated levels of reactive oxygen species.

[0005] In summary, the key challenge in constructing efficient, chemically-defined lipid supplementation systems is how to improve the effective supply of lipids while reducing the toxicity of free fatty acids and maintaining system stability. From the perspective of lipid transport mechanisms within cells, fatty acids are typically not present in a large free state, but rather are bound, buffered, and delivered through natural carriers such as serum albumin and apolipoproteins. Some technologies have utilized the binding properties between albumin and fatty acids to construct albumin-fatty acid complexes and applied them to drug delivery. However, these technologies primarily focus on drug loading, nanoparticle stability, and in vivo delivery efficiency, and cannot directly address issues such as stable lipid nutrient supply, adherent cell adaptation, lipid oxidation control, and long-term culture consistency in cell culture systems.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a complex fatty acid delivery system for cell culture, in order to solve the problems in existing lipid supplementation systems that mostly rely on surfactants such as Tween-80 and Pluronic F-68 for emulsification or solubilization, or use organic solvents such as DMSO, methanol, and ethanol to pre-dissolve fatty acids. However, fatty acids pre-dissolved in organic solvents are still prone to aggregation, turbidity, or precipitation after being added to the aqueous culture system, resulting in poor system stability and batch-to-batch consistency. In adherent cell culture, there are technical problems such as insufficient compatibility, poor lipid oxidation stability, and insufficient long-term culture consistency.

[0008] A second objective of this invention is to provide a method for preparing the aforementioned complex fatty acid delivery system.

[0009] A third objective of this invention is to provide the application of the above-mentioned complex fatty acid delivery system or the complex fatty acid delivery system prepared by the above-mentioned method in cell culture or in the preparation of products for cell culture.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a complex fatty acid delivery system for cell culture, wherein the system comprises lipids, albumin at a working concentration of 200-1000 mg / L and an antioxidant at a working concentration of 0.1-2 mg / L, based on a 1× working concentration after reconstitution. The lipids include fatty acids, which include saturated fatty acids and / or unsaturated fatty acids.

[0011] Furthermore, the complex fatty acid delivery system does not contain nonionic surfactants; Preferably, the complex fatty acid delivery system does not contain β-cyclodextrin or its derivatives.

[0012] Furthermore, the albumin is recombinant human serum albumin.

[0013] Furthermore, the fatty acids include at least one of oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, arachidonic acid, lauric acid, capric acid, caprylic acid, docosahexaenoic acid, eicosapentaenoic acid, γ-linolenic acid, and their salts or ester derivatives. Preferably, the fatty acids include oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, and arachidonic acid; Preferably, based on 1× working concentration after reconstitution, the working concentrations of oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, palmitic ...

[0014] Furthermore, the lipids also include at least one of phospholipids, sterol lipids, fat-soluble vitamins or their derivatives; Preferably, the phospholipids include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and their derivatives; Preferably, the sterol lipids include one or more of cholesterol, phytosterols, or their derivatives.

[0015] Furthermore, the antioxidant includes at least one of tocopherol, tocopherol acetate, Trolox, and tocopherol phosphate salts, preferably disodium tocopherol phosphate.

[0016] Furthermore, the dosage form of the complex fatty acid delivery system includes liquid formulations or lyophilized formulations; Preferably, when the dosage form of the complex fatty acid delivery system is a lyophilized formulation, the system further includes a stabilizer; the working concentration of the stabilizer is 200~1500 mg / L, calculated as 1× working concentration after reconstitution. Preferably, the stabilizer includes at least one of trehalose, sucrose, fructose or mannitol, with trehalose being the most preferred.

[0017] Secondly, the present invention provides a method for preparing the above-mentioned complex fatty acid delivery system, comprising the following steps: A. Dissolve albumin in water according to the formula amount, adjust the pH to 7.5~7.8, and obtain albumin solution; B. Place the lipids in anhydrous ethanol according to the formula amount, mix well, and then add the antioxidant to obtain a compound fatty acid-antioxidant ethanol premix. C. Slowly and in portions, add the compound fatty acid-antioxidant ethanol premix to the albumin solution to obtain the compound fatty acid delivery system.

[0018] Furthermore, the preparation method does not use DMSO and methanol; Preferably, when the complex fatty acid delivery system is at a working concentration of 100, the volume percentage of anhydrous ethanol in the system is <10%; Preferably, when the complex fatty acid delivery body is a lyophilized formulation, step A includes the addition of trehalose while dissolving albumin in water; Preferably, when the complex fatty acid delivery body is a lyophilized formulation, the method further includes step D, freeze-drying; Preferably, the freeze-drying includes pre-freezing, primary drying, and secondary drying; Preferably, the freeze-drying is carried out under light-protected or low-oxygen conditions; Preferably, the pre-freezing temperature is -20°C to -80°C; Preferably, the primary drying is carried out in a vacuum environment.

[0019] Thirdly, the present invention provides the application of the above-mentioned complex fatty acid delivery system or the complex fatty acid delivery system prepared by the above-mentioned preparation method in cell culture or in the preparation of products for cell culture. Preferably, the product includes reagents and / or culture media; Preferably, the culture medium includes at least one of chemically defined culture medium, serum-free culture medium, serum-reduced culture medium, or cell expansion culture medium; Preferably, the cells include at least one of primary adherent cells, mesenchymal stem cells, fibroblast-like cells, epithelial-like cells, or immune cells; Preferably, the cell culture includes in vitro culture; Preferably, the in vitro culture includes at least one stage of adherence recovery, proliferation and expansion, morphology maintenance, or continuous passage culture.

[0020] This invention provides a complex fatty acid delivery system using albumin as the delivery carrier. It does not rely on nonionic surfactants for emulsification, thus improving the dispersion stability of the complex fatty acids in aqueous culture systems while reducing the potential impact of surfactants on cell adhesion, spreading, and proliferation. The adhesion rate reached 94.58% after 4 hours, and the amplification fold reached 9.61 times after 72 hours, both higher than existing commercial lipid supplements in comparative tests. The lipid concentrate of this invention better supports early MSC adhesion and proliferation. This invention solves the technical problem that existing lipid supplementation systems often rely on surfactants such as Tween-80 and Pluronic F-68 for emulsification or solubilization, or use organic solvents such as DMSO, methanol, and ethanol to pre-dissolve fatty acids, leading to aggregation, turbidity, or precipitation after addition to the aqueous culture system, severely affecting system stability and batch-to-batch consistency. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The figure shows the effect of different lipid supplementation systems provided in the embodiments of the present invention on the adhesion and proliferation performance of MSCs. In this figure, A is the adhesion rate of MSCs in each group after 4 h of seeding, B is the cell expansion fold of MSCs in each group after 72 h of culture, and C is the population doubling time of MSCs in each group. Figure 2 Bright-field micrographs of MSCs cultured for 72 h using different lipid supplementation systems provided in this embodiment of the invention, under 4× field of view. Figure 3 Bright-field micrographs at 4× field of view of MSCs cultured for 24 h and 72 h using different lipid delivery systems according to embodiments of the present invention. Figure 4 The figure shows the effect of different lipid delivery systems on the proliferation performance of MSCs in this embodiment of the invention. In this figure, A represents the cell expansion fold of each group of MSCs after 72 h of culture, and B represents the population doubling time of each group of MSCs. Figure 5 These are bright-field micrographs at 10× field of view of the recombinant human serum albumin without tocopherol phosphate disodium salt group of the present invention and the lyophilized and reconstituted MSCs cultured for 24 h in Example 1. Detailed Implementation

[0023] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0024] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The present invention provides a complex fatty acid delivery system for cell culture, wherein the system comprises lipids, albumin at a working concentration of 200-1000 mg / L and an antioxidant at a working concentration of 0.1-2 mg / L, based on a 1× working concentration after reconstitution. The lipids include fatty acids, which include saturated fatty acids and / or unsaturated fatty acids.

[0027] This invention uses albumin as a complex fatty acid delivery carrier, eliminating the need for emulsification with nonionic surfactants. This improves the dispersion stability of the complex fatty acids in aqueous culture systems while reducing the potential impact of surfactants on cell adhesion, spreading, and proliferation. The adhesion rate reached 94.58% after 4 hours, and the amplification fold reached 9.61 times after 72 hours, both exceeding those of existing commercial lipid supplements in comparative tests. The lipid concentrate of this invention better supports early MSC adhesion and proliferation. It solves the technical problem that existing lipid supplementation systems often rely on surfactants such as Tween-80 and Pluronic F-68 for emulsification or solubilization, or use organic solvents such as DMSO, methanol, and ethanol to pre-dissolve fatty acids, leading to aggregation, turbidity, or precipitation after addition to the aqueous culture system, severely affecting system stability and batch-to-batch consistency.

[0028] In some specific embodiments, the complex fatty acid delivery system does not contain nonionic surfactants. The nonionic surfactants include Tween-80 and / or P188; in some specific embodiments, the complex fatty acid delivery system does not contain β-cyclodextrin or its derivatives.

[0029] In some specific embodiments, the albumin is recombinant human serum albumin, serving as a complex fatty acid delivery carrier.

[0030] In some specific embodiments, the fatty acids include at least one selected from oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, arachidonic acid, lauric acid, capric acid, caprylic acid, docosahexaenoic acid, eicosapentaenoic acid, γ-linolenic acid, and their salts or ester derivatives; in some specific embodiments, the fatty acids include oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, and arachidonic acid; in some specific embodiments, based on 1× working concentration after reconstitution, the working concentration of oleic acid in the system is 0.2~5 mg / L, the working concentration of linoleic acid is 0.2~5 mg / L, the working concentration of palmitic acid is 0.005~2 mg / L, the working concentration of stearic acid is 0.05~1 mg / L, the working concentration of palmitoleic acid is 0.005~0.2 mg / L, and the working concentration of linolenic acid is 0.005~0.2 mg / L. The working concentrations for myristic acid and arachidonic acid are 0.005-0.2 mg / L and 0.001-0.02 mg / L, respectively.

[0031] In some specific embodiments, the lipids further include at least one of phospholipids, sterol lipids, fat-soluble vitamins or their derivatives; in some specific embodiments, the phospholipids include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and their derivatives; in some specific embodiments, the sterol lipids include one or more of cholesterol, phytosterols or their derivatives.

[0032] In some specific embodiments, the antioxidant includes at least one of tocopherol, tocopherol acetate, Trolox, and tocopherol phosphate salts, preferably disodium tocopherol phosphate. Introducing disodium tocopherol phosphate as an antioxidant helps reduce the oxidation risk of unsaturated fatty acids during preparation, storage, and use, and promotes the maintenance of lipid system stability and MSC culture compatibility.

[0033] In some specific embodiments, the dosage form of the complex fatty acid delivery system includes liquid formulations or lyophilized formulations.

[0034] The lyophilized formulation exhibits good storage, transportation, and industrial compatibility. In some specific embodiments, when the dosage form of the complex fatty acid delivery system is a lyophilized formulation, the system further includes a stabilizer; the working concentration of the stabilizer is 200-1500 mg / L, calculated as 1× working concentration after reconstitution; in some specific embodiments, the stabilizer includes at least one of trehalose, sucrose, fructose, or mannitol, preferably trehalose.

[0035] According to another aspect of the present invention, a method for preparing the above-described complex fatty acid delivery system is also provided, comprising the following steps: A. Dissolve albumin in water according to the formula amount, adjust the pH to 7.5~7.8, and obtain albumin solution; B. Place the lipids in anhydrous ethanol according to the formula amount, mix well, and then add the antioxidant to obtain a compound fatty acid-antioxidant ethanol premix. C. Slowly and in portions, add the compound fatty acid-antioxidant ethanol premix to the albumin solution to obtain the compound fatty acid delivery system.

[0036] Ethanol is used to pre-dissolve lipids during the preparation process, but DMSO and methanol are not used. Ethanol is used to improve the initial solubility and uniformity of fatty acids. After adding recombinant human serum albumin solution to fatty acid ethanol solution, the complex fatty acids combine with recombinant human serum albumin to form albumin-combined fatty acid delivery system. The stable dispersion and delivery of fatty acids in the final aqueous system are mainly achieved through the lipid affixation of recombinant human serum albumin.

[0037] The ethanol content in the final use system is much lower than its content in the fatty acid pre-dissolution solution. In some specific embodiments, when the complex fatty acid delivery system is 100 × working concentration, the volume percentage of anhydrous ethanol in the system is <10%. In some specific embodiments, when the complex fatty acid delivery body is a lyophilized formulation, step A includes the addition of trehalose while dissolving albumin in water.

[0038] In some specific embodiments, when the complex fatty acid delivery body is a lyophilized formulation, step D, freeze-drying, is further included; in some specific embodiments, the freeze-drying includes pre-freezing, primary drying, and secondary drying; in some specific embodiments, the freeze-drying is carried out under light-protected or low-oxygen conditions; in some specific embodiments, the pre-freezing temperature is -20°C to -80°C; in some specific embodiments, the primary drying is carried out under vacuum.

[0039] According to another aspect of the present invention, the application of the above-described complex fatty acid delivery system or the complex fatty acid delivery system prepared by the above-described preparation method in cell culture or in the preparation of products for cell culture is also provided. In some specific embodiments, the product includes reagents and / or culture media; in some specific embodiments, the culture media includes at least one of chemically defined culture media, serum-free culture media, serum-reduced culture media, or cell expansion culture media.

[0040] In some specific embodiments, the cells include at least one of primary adherent cells, mesenchymal stem cells, fibroblast-like cells, epithelial-like cells, or immune cells.

[0041] In some specific embodiments, the cell culture includes in vitro culture; in some specific embodiments, the in vitro culture includes at least one stage of adherence recovery, proliferation and expansion, morphology maintenance, or continuous passage culture.

[0042] The present invention will be further illustrated below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] I. Preparation of Recombinant Human Serum Albumin-Complex Fatty Acid Lipid Concentrate The recombinant human serum albumin-complex fatty acid lipid concentrate comprises the components shown in Table 1, based on the final use concentration.

[0044] Table 1. Composition and concentration (mg / L) of recombinant human serum albumin-complex fatty acid lipid concentrate.

[0045] Taking the preparation of 100 mL of the 100× recombinant human serum albumin-complex fatty acid lipid concentrate from Example 1 as an example. The concentrations shown in Table 1 are the final concentrations of each component after the lipid concentrate is diluted to 1× working concentration.

[0046] The specific preparation process is as follows: (1) Preparation of recombinant human serum albumin-trehalose aqueous solution. Take about 70 mL of water for injection or purified water, heat to 30~35℃, add 5 g of recombinant human serum albumin and 5 g of trehalose under continuous stirring, and let them dissolve completely to obtain recombinant human serum albumin-trehalose aqueous solution. Adjust the pH of the system to 7.5~7.8 using 5 M NaOH solution.

[0047] (2) Preparation of compound fatty acid-antioxidant ethanol premix. Take 20 mg of oleic acid, 20 mg of linoleic acid, 20 mg of palmitic acid, 10 mg of stearic acid, 0.2 mg of palmitoleic acid, 0.2 mg of linolenic acid, 0.2 mg of myristic acid, 0.05 mg of arachidonic acid and 5 mg of disodium tocopherol phosphate, add them to an appropriate amount of anhydrous ethanol, and mix by vortexing or stirring to dissolve or disperse them evenly to obtain compound fatty acid-antioxidant ethanol premix.

[0048] (3) Formation of recombinant human serum albumin-complex fatty acid delivery system. Under continuous stirring, the complex fatty acid-antioxidant ethanol premix obtained in step two is slowly and in portions added to the recombinant human serum albumin-trehalose aqueous solution obtained in step one, so that the complex fatty acid and recombinant human serum albumin can fully contact and bind to form a recombinant human serum albumin-complex fatty acid delivery system.

[0049] (4) Volume adjustment and filtration. After the system is mixed evenly, the volume is adjusted to 100 mL with water for injection or purified water. Then the pH is adjusted to 7.0~7.4 and cooled to 2~8 ℃ and filtered through a 0.22 μm filter membrane for sterilization to obtain 100× recombinant human serum albumin-complex fatty acid lipid concentrate.

[0050] The prepared lipid concentrate is free of Tween-80, Pluronic F-68, DMSO and methanol.

[0051] II. Preparation of Recombinant Human Serum Albumin-Complex Fatty Acid Lyophilized Formulation Example 5 The recombinant human serum albumin-complex fatty acid lipid concentrate prepared in Example 1 was dispensed into sterile lyophilization bottles under light-protected conditions. The dispensing volume of each bottle was 1-10 mL, preferably 2-5 mL, and in this example, it was 5 mL. After dispensing, the bottles were partially stoppered and placed in a freeze dryer for lyophilization.

[0052] Freeze-drying includes pre-freezing, primary drying, and secondary drying steps.

[0053] Pre-freezing stage: Cool the dispensed lipid concentrate to -60℃ and keep it for 12 h to allow the system to freeze completely.

[0054] First drying stage: Sublimation drying is carried out under vacuum conditions, with the vacuum degree controlled at 50 Pa, the shelf temperature controlled at -40℃, and the drying time at 48 h.

[0055] Secondary drying stage: Under vacuum conditions, the shelf temperature is raised to 10°C and maintained for 12 hours to further remove bound water and reduce ethanol residue.

[0056] After freeze-drying, the freeze-drying bottle is stoppered and sealed to obtain recombinant human serum albumin-complex fatty acid freeze-dried powder.

[0057] In use, the lyophilized powder is reconstituted with water for injection or purified water to obtain a recombinant human serum albumin-complex fatty acid lipid concentrate or lipid working solution. The reconstituted system can be added to serum-free culture medium, chemically defined culture medium, or serum-reduced culture medium. In this embodiment, the lyophilized powder, after reconstitution, does not affect cell adhesion, proliferation, or morphology maintenance when used for cell culture.

[0058] In this embodiment, trehalose is used as a stabilizer and lyophilization protectant to reduce the risks of recombinant human serum albumin aggregation, lipid precipitation, and system heterogeneity during lyophilization and reconstitution. Disodium tocopherol phosphate is used as an antioxidant to reduce the oxidation risk of unsaturated fatty acids during preparation, lyophilization, storage, and reconstitution. The lyophilized formulation helps reduce the system's moisture content and ethanol residue, improving the product's storage stability, transportation convenience, and industrial applicability.

[0059] Experiment 1: Application of recombinant human serum albumin-complex fatty acids in MSC culture This experiment was used to verify the application effects of the 100× recombinant human serum albumin-complex fatty acid lipid concentrate prepared in Examples 1-3 and the lyophilized reconstituted preparation prepared in Example 5 in mesenchymal stem cell culture.

[0060] Human umbilical cord mesenchymal stem cells (MSCs) in good growth condition were used as experimental cells, and α-MEM + 1% platelet lysate (PL) was used as the basal culture medium.

[0061] The experiment consisted of four groups: Blank control group: α-MEM + 1% PL medium was used, without the addition of lipid supplements; Commercial lipid control group: 1% of a commercial lipid supplement (Gibco) was added to α-MEM + 1% PL medium. TM Chemically Defined Lipid Concentrate (CDLC); Example 1: The lipid concentrate prepared in Example 1 was added to α-MEM + 1% PL medium; Example 2: The lipid concentrate prepared in Example 2 was added to α-MEM + 1% PL medium; Example 3: The lipid concentrate prepared in Example 3 was added to α-MEM + 1% PL medium; Example 5: Add the lyophilized formulation prepared in Example 5 to α-MEM + 1% PL medium for reconstitution; The lipid concentrate was added to the basal culture medium at a ratio of 1:100 to achieve a working concentration of 1× in the final culture system. The lyophilized formulation was reconstituted and added to the culture system at the same working concentration as in Example 1.

[0062] Except for the lipid supplementation system, the culture conditions in each group were kept consistent.

[0063] MSCs were prepared at a rate of 8000 cells / cm². 2Cells were seeded at a density in T25 culture flasks. The cell adhesion rate at 4 h post-inoculation, the cell expansion fold at 72 h post-inoculation, and the population doubling time were measured. Each group was tested three times independently, and the average values ​​were compared.

[0064] The results are as follows Figure 1 As shown, the adhesion rate of the blank control group was 85.25% after 4 hours, the cell expansion fold was 6.76 times after 72 hours, and the doubling time was 26.14 hours; the adhesion rate of the commercial lipid control group was 90.40% after 4 hours, the cell expansion fold was 8.29 times after 72 hours, and the doubling time was 23.61 hours; the adhesion rate of the invention group in Example 1 was 94.58% after 4 hours, the cell expansion fold was 9.61 times after 72 hours, and the doubling time was 22.06 hours; the adhesion rate of the invention group in Example 2 was 93.53% after 4 hours, the cell expansion fold was 9.52 times after 72 hours, and the doubling time was 22.16 hours; the adhesion rate of the invention group in Example 3 was 93.97% after 4 hours, the cell expansion fold was 9.57 times after 72 hours, and the doubling time was 22.10 hours; and the adhesion rate of the lyophilized and reconstituted group in Example 5 was 93.92% after 4 hours, the cell expansion fold was 9.47 times after 72 hours, and the doubling time was 22.21 hours.

[0065] The results showed that, compared with the blank control group, the addition of the lipid concentrate and lyophilized reconstituted formulation of this invention could improve the 4-hour adhesion rate and 72-hour expansion fold of MSCs, and shorten the cell doubling time, which was superior to the effect of adding commercial lipids. This indicates that lipids, as important nutrient components for cell membrane synthesis, membrane structure maintenance, and proliferation metabolism, play a positive role in the expansion of MSCs under low-concentration PL culture conditions. Specifically, the invention group in Example 1 showed better performance than both the blank control group and the commercial lipid control group in terms of 4-hour adhesion rate, 72-hour cell expansion fold, and doubling time, indicating that the recombinant human serum albumin-complex fatty acid lipid concentrate of this invention can better support the early adhesion and proliferation of MSCs. It is speculated that this effect is related to the use of recombinant human serum albumin as a fatty acid delivery carrier in this invention, and the absence of surfactants such as Tween-80 and P188, thereby reducing the potential interference of surfactants on cell membrane state, adhesion process, and cell proliferation.

[0066] The adhesion rate and amplification fold of the freeze-dried and reconstituted group in Example 5 were not significantly different from those in Example 1, and were superior to the blank control group and the commercial lipid control group. This indicates that freeze-drying and reconstitution did not have an adverse effect on the complex fatty acid delivery system provided by the present invention, and that it could still maintain good MSC culture compatibility after freeze-drying and reconstitution.

[0067] Meanwhile, the cell morphology of each group of MSCs was observed under a microscope after 72 h of culture, and the results are as follows: Figure 2As shown, the cells in the blank control group were relatively loosely arranged, and the morphology of some cells was not uniform enough; the cell density and arrangement in the commercial lipid control group were improved compared with the blank control group. In contrast, the cells in the invention groups of Examples 1, 2, and 3 were more tightly arranged and regular, with better overall morphological uniformity, suggesting that the recombinant human serum albumin-complex fatty acid lipid concentrate of the present invention is beneficial to improving the adhesion, spreading, and growth of MSCs. The cell morphology of the freeze-dried and reconstituted group of Example 5 was similar to that of the invention group of Example 1.

[0068] Experiment 2: Comparison of different lipid delivery systems This experiment was used to compare the effects of different lipid delivery systems and auxiliary components on the stability, lyophilization compatibility and MSC culture effect of lipid complexes in Examples 1, 4 and Comparative Examples 1-3.

[0069] In the preliminary experiment, when a complex fatty acid ethanol solution without recombinant human serum albumin was directly added to the culture medium, the system exhibited significant turbidity, lipid aggregation, or precipitation, making it difficult to form a stable and homogeneous culture addition system. Therefore, it was not used as the control group for formal cell culture. This indicates that simple ethanol pre-dissolution can only improve the initial solubility of fatty acids and cannot effectively solve the problem of stable delivery of complex fatty acids in aqueous culture systems; complex fatty acids still need to be bound or loaded with recombinant human serum albumin to form a stable lipid delivery system suitable for cell culture.

[0070] The experiment consisted of five groups: the Tween-80+P188 emulsified group (Comparative Example 1), the fatty acid-free group (Comparative Example 2), the recombinant human serum albumin without trehalose group (Example 4), the recombinant human serum albumin without tocopherol phosphate disodium salt group (Comparative Example 3), and Example 1 group. The types and final concentrations of the complex fatty acids remained consistent across all groups; only the lipid delivery system or auxiliary components were changed, while all other culture conditions remained the same.

[0071] The five lipid systems were added to α-MEM + 1% PL medium and cultured with P4 generation MSCs in good growth condition. Cell morphology and proliferation were observed after 72 h. The results are as follows: Figure 3 and Figure 4As shown, Comparative Example 1 achieved fatty acid dispersion, but its MSC adhesion, morphological uniformity, and proliferation ability were inferior to those of Example 1. Its 72-hour amplification fold was 6.64-fold, and its population doubling time was 26.40 hours, suggesting that surfactants may have a negative impact on MSC adhesion and cell state. Comparative Example 2 showed a 72-hour amplification fold of 6.34-fold and a population doubling time of 27.09 hours, with MSC proliferation ability lower than the groups containing complex fatty acids, indicating that fatty acids are an essential nutrient component for MSC amplification. Example 4 showed a 72-hour amplification fold of 9.17-fold and a population doubling time of 22.54 hours, close to the 9.29-fold and 22.41 hours of Example 1, indicating that under liquid culture conditions, trehalose is not the main functional component directly promoting MSC proliferation; its main function is more focused on lyophilization protection and reconstitution stabilization. While the three comparative groups could support MSC culture under freshly prepared conditions, cell morphology observation revealed lipid oxidation-related abnormalities, manifested as poor cell spindle shape, abnormal edge refractive index, increased cytoplasmic granulation, and the presence of fine particles in the culture medium. Their 72-hour expansion fold was 7.23-fold, and the population doubling time was prolonged to 25.26 hours, indicating that the lack of antioxidants reduced the stability of the complex fatty acid system and affected MSC adhesion, morphological maintenance, and proliferation. This effect may further accumulate during continuous passage.

[0072] In the lyophilization compatibility evaluation, both Comparative Example 1 and Example 4 showed difficulty in forming stable, homogeneous, and resolvable lyophilized formulations after lyophilization, thus making them unsuitable as lyophilized dosage forms. Trehalose needs to be added for these formulations. Comparative Example 3, containing trehalose, allowed for lyophilized resolvability, but severe lipid oxidation was observed after 24 hours of culture following resolvability, resulting in decreased cell state and culture compatibility, a conclusion consistent with pre-lyophilization levels. Example 5, however, showed no significant lipid oxidation-related abnormalities (e.g., ...). Figure 5 (As shown).

[0073] The above results demonstrate that this invention does not simply apply albumin-fatty acid complexes to cell culture, but rather addresses issues such as fatty acid delivery, oxidative stability, lyophilization-reconstitution, and adaptability to adherent cells during the culture process through a combined design. In this invention's system, the complex fatty acids are crucial nutrients supporting cell proliferation; recombinant human serum albumin is a key component for aqueous delivery of the complex fatty acids; trehalose primarily promotes lyophilization and enhances reconstitution stability; and disodium tocopheryl phosphate primarily reduces the oxidative risk of the complex fatty acids, especially unsaturated fatty acids. The combined effect of these components makes each embodiment superior to surfactant-emulsified systems and lipid systems lacking key auxiliary components in terms of MSC culture adaptability and system stability.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A complex fatty acid delivery system for cell culture, characterized in that, Based on a working concentration of 1× after reconstitution, the system comprises lipids, albumin at a working concentration of 200-1000 mg / L, and an antioxidant at a working concentration of 0.1-2 mg / L. The lipids include fatty acids, which include saturated fatty acids and / or unsaturated fatty acids.

2. The complex fatty acid delivery system according to claim 1, characterized in that, The complex fatty acid delivery system does not contain nonionic surfactants; Preferably, the complex fatty acid delivery system does not contain β-cyclodextrin or its derivatives.

3. The complex fatty acid delivery system according to claim 1, characterized in that, The albumin in question is recombinant human serum albumin.

4. The complex fatty acid delivery system according to claim 1, characterized in that, The fatty acids include at least one of oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, arachidonic acid, lauric acid, decanoic acid, caprylic acid, docosahexaenoic acid, eicosapentaenoic acid, γ-linolenic acid, and their salts or ester derivatives. Preferably, the fatty acids include oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, myristic acid, and arachidonic acid; Preferably, based on 1× working concentration after reconstitution, the working concentrations of oleic acid, linoleic acid, palmitic acid, stearic acid, palmitoleic acid, linolenic acid, palmitic ...

5. The complex fatty acid delivery system according to claim 4, characterized in that, The lipids also include at least one of phospholipids, sterol lipids, fat-soluble vitamins or their derivatives; Preferably, the phospholipids include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and their derivatives; Preferably, the sterol lipids include one or more of cholesterol, phytosterols, or their derivatives.

6. The complex fatty acid delivery system according to claim 1, characterized in that, The antioxidant includes at least one of tocopherol, tocopherol acetate, Trolox, and tocopherol phosphate salts, preferably disodium tocopherol phosphate.

7. The complex fatty acid delivery system according to any one of claims 1 to 6, characterized in that, The dosage forms of the complex fatty acid delivery system include liquid formulations or lyophilized formulations; Preferably, when the dosage form of the complex fatty acid delivery system is a lyophilized formulation, the system further includes a stabilizer; the working concentration of the stabilizer is 200~1500 mg / L, calculated as 1× working concentration after reconstitution. Preferably, the stabilizer includes at least one of trehalose, sucrose, fructose or mannitol, with trehalose being the most preferred.

8. A method for preparing the complex fatty acid delivery system according to any one of claims 1 to 7, characterized in that, Includes the following steps: A. Dissolve albumin in water according to the formula amount, adjust the pH to 7.5~7.8, and obtain albumin solution; B. Place the lipids in anhydrous ethanol according to the formula amount, mix well, and then add the antioxidant to obtain a compound fatty acid-antioxidant ethanol premix. C. Slowly and gradually add the compound fatty acid-antioxidant ethanol premix to the albumin solution to obtain the compound fatty acid delivery system.

9. The complex fatty acid delivery system according to claim 8, characterized in that, The preparation method does not use DMSO and methanol; Preferably, when the complex fatty acid delivery system is at a working concentration of 100, the volume percentage of anhydrous ethanol in the system is <10%; Preferably, when the complex fatty acid delivery body is a lyophilized formulation, step A includes the addition of trehalose while dissolving albumin in water; Preferably, when the complex fatty acid delivery body is a lyophilized formulation, the method further includes step D, freeze-drying; Preferably, the freeze-drying includes pre-freezing, primary drying, and secondary drying; Preferably, the freeze-drying is carried out under light-protected or low-oxygen conditions; Preferably, the pre-freezing temperature is -20°C to -80°C; Preferably, the primary drying is carried out in a vacuum environment.

10. The application of the complex fatty acid delivery system according to any one of claims 1 to 7 or the complex fatty acid delivery system prepared by the preparation method according to claim 8 or 9 in cell culture or in the preparation of products for cell culture; Preferably, the product includes reagents and / or culture media; Preferably, the culture medium includes at least one of chemically defined culture medium, serum-free culture medium, serum-reduced culture medium, or cell expansion culture medium; Preferably, the cells include at least one of primary adherent cells, mesenchymal stem cells, fibroblast-like cells, epithelial-like cells, or immune cells; Preferably, the cell culture includes in vitro culture; Preferably, the in vitro culture includes at least one stage of adherence recovery, proliferation and expansion, morphology maintenance, or continuous passage culture.

Images