A mitochondrial function optimization additive for cell culture, a preparation method and application thereof

Abstract

The application provides a kind of mitochondrial function optimization additive for cell culture, preparation method and application, belong to cell culture technical field.The additive includes core active ingredient, auxiliary component and carrier, and the core active ingredient includes D-Arg-Cha-Lys-Phe-NH2 Polypeptide, ubiquinone-10 phosphate, reduced glutathione and alpha-lipoic acid.The application forms a multi-target core active system through the synergy of the four, realizes the triple synergistic protection of stabilizing mitochondrial cardiolipin, maintaining mitochondrial morphology and membrane potential, and reducing mitochondrial ROS, which can significantly improve cell culture quality.When applied, the mitochondrial cardiolipin content can be increased by more than 50%, the mitochondrial abnormality ratio can be reduced to less than 12%, the membrane potential positive cell ratio can be increased to more than 86%, the mitochondrial ROS level can be reduced by more than 40%, and the cell proliferation rate can be increased by more than 25%, which is suitable for in vitro culture of various eukaryotic cells, and the preparation process is simple and has no cytotoxicity.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and relates to a mitochondrial function optimization additive for cell culture, its preparation method, and its application. Background Technology

[0002] Mitochondria are the energy factories of eukaryotic cells, and their functional integrity directly determines the cell's survival, proliferation, and differentiation capabilities. Mitochondrial cardiolipin is a unique tetraacylated diphosphatidylglycerol lipid, mainly located in the inner mitochondrial membrane. It is a key substance for maintaining the structural integrity of the inner mitochondrial membrane, cristae formation, and the assembly of respiratory chain complexes into a supercomplex. It also participates in core physiological processes such as mitochondrial electron transport and oxidative phosphorylation, and is crucial for mitochondrial functional homeostasis.

[0003] During in vitro cell culture, mitochondria often face various stresses such as nutrient deficiency, oxidative stress, and mechanical shearing forces, which easily lead to the oxidative degradation and decreased content of mitochondrial cardiolipin. This, in turn, causes abnormal mitochondrial morphology (such as swelling, cristae breakage, and fragmentation), membrane depolarization, and electron transport chain dysfunction, ultimately resulting in the excessive generation of mitochondrial ROS (reactive oxygen species). Excessive accumulation of mitochondrial ROS not only further exacerbates cardiolipin damage, creating a vicious cycle, but also damages cellular DNA, proteins, and lipids, leading to decreased cell viability, increased apoptosis rate, and seriously affecting cell culture efficiency, cell quality, and the reliability of subsequent experimental or production results.

[0004] Currently, the mitochondrial function protection components used in cell culture have significant defects: (1) Single antioxidants such as glutathione, taurine, and MitoTEMPO can only remove mitochondrial ROS, but cannot stabilize mitochondrial cardiolipin or maintain mitochondrial morphology; (2) Membrane potential maintainers such as coenzyme Q10 have poor solubility and no protective effect on mitochondrial cardiolipin; (3) Peptide mitochondrial cardiolipin protectants such as SPN10 and SS-31 peptide have controversial sequences and are easily degraded by enzymes, and their effect on maintaining mitochondrial morphology is limited when used alone. Existing technologies simply superimpose multiple single functional components, which easily leads to problems such as component interference, cytotoxicity, and inability to simultaneously achieve mitochondrial cardiolipin stabilization, morphology maintenance, and membrane potential protection, making it difficult to meet the comprehensive needs of in vitro cell culture. Summary of the Invention

[0005] The purpose of this invention is to provide a mitochondrial function-optimizing additive for cell culture, its preparation method, and its application, to solve the technical problems in existing cell culture methods, such as mitochondrial susceptibility to damage, limited protective agent function, poor synergistic effect, and inability to simultaneously address mitochondrial cardiolipin stability, morphology maintenance, membrane potential preservation, and mitochondrial ROS clearance. To achieve the above objective, this invention adopts the following technical solution: In a first aspect, this application provides a mitochondrial function optimization additive for cell culture, the additive comprising a core active ingredient, auxiliary ingredients, and a carrier, wherein the core active ingredient comprises D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid.

[0006] Secondly, this application provides a method for preparing a mitochondrial function-optimizing additive for cell culture, the method comprising: The core active ingredient and auxiliary ingredient from the first aspect are respectively added to the carrier to form the core active solution and the auxiliary solution; The core active solution was slowly added to the auxiliary solution, stirred evenly at 4°C, and then filtered through a 0.22 μm filter membrane for sterilization to obtain a mitochondrial function optimization additive for cell culture.

[0007] Thirdly, this application provides an application of a mitochondrial function optimization additive for cell culture, namely, for stabilizing mitochondrial cardiolipin, maintaining mitochondrial morphology and membrane potential, and reducing mitochondrial ROS generation in cell culture.

[0008] Fourthly, this application provides a method for using a mitochondrial function optimization additive for cell culture, comprising: mixing the mitochondrial function optimization additive for cell culture from the first aspect with the cell culture system at a volume ratio of 1:(100-500), and then culturing at 37°C and 5% CO2.

[0009] The present invention has the following beneficial effects: (1) Precise protection of mitochondrial cardiolipin: D-Arg-Cha-Lys-Phe-NH2 is a mitochondrial-targeting tetrapeptide containing the non-natural amino acid Cha. It specifically binds to mitochondrial cardiolipin through electrostatic and hydrophobic interactions, preventing oxidative degradation and eversion, maintaining the inner membrane structure and cristae morphology, and is resistant to enzymatic degradation and has strong binding force.

[0010] (2) Highly efficient and stable membrane potential: ubiquinone-10 phosphate participates in electron transfer, inhibits the opening of permeability transition pores, reduces proton leakage, stabilizes membrane potential, and works synergistically with peptides to enhance mitochondrial structure and function.

[0011] (3) Synergistic antioxidant effect: Reduced glutathione directly removes mitochondrial ROS, α-lipoic acid regenerates GSH and prolongs the duration of action, forming a closed-loop antioxidant system.

[0012] (4) Multi-target synergy: The combination of four core components achieves triple protection of stabilizing mitochondrial cardiolipin, maintaining morphology and membrane potential, and reducing mitochondrial ROS, breaking through the limitations of single function or simple superposition.

[0013] (5) Significant effects: mitochondrial cardiolipin content increased by more than 50%, mitochondrial abnormality rate ≤12%, membrane potential positive cells ≥86%, mitochondrial ROS decreased by more than 40%, and cell proliferation rate increased by more than 25%, which is superior to existing single or compound components.

[0014] (6) Safe and easy to use: low concentration, no cytotoxicity, suitable for eukaryotic cells such as stem cells, primary cells, tumor cells, and immune cells; simple process, low cost, suitable for mass production and promotion. Attached Figure Description

[0015] Figure 1 A comparison chart of mitochondrial cardiolipin content in each group provided in the embodiments of this application; Figure 2 TEM comparison images of various groups of mitochondria provided in the embodiments of this application; Figure 3 Comparison diagrams of mitochondrial abnormalities provided in the embodiments of this application; Figure 4 This is a comparison chart of the proportion of mitochondrial membrane potential positive cells in each group provided in the embodiments of this application; Figure 5 Comparison of mitochondrial ROS fluorescence intensity for each group provided in the embodiments of this application; Figure 6 This is a comparison chart of the relative values ​​of mitochondrial cell proliferation rates for each group provided in the embodiments of this application. Detailed Implementation

[0016] In a first aspect, this application provides a mitochondrial function optimization additive for cell culture, the additive comprising a core active ingredient, an auxiliary ingredient, and a carrier, wherein the core active ingredient comprises D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid.

[0017] Specifically, the D-Arg-Cha-Lys-Phe-NH2 peptide is a novel mitochondrial-targeting cardiolipin-protecting tetrapeptide with a molecular weight of approximately 601.78 Da and a purity ≥98%, and it incorporates cyclohexylalanine (Cha). Cyclohexylalanine (Cha) is a non-natural alicyclic amino acid with a hydrophobic alicyclic structure. When the D-Arg-Cha-Lys-Phe-NH2 peptide is added during cell culture, it specifically targets the inner mitochondrial membrane. The N-terminal D-Arg group forms an electrostatic interaction with the phosphate group of cardiolipin, while the hydrophobic alicyclic structure of cyclohexylalanine (Cha) mimics the hydrophobic effects of natural aromatic amino acids, specifically binding to the hydrophobic pockets of mitochondrial cardiolipin to form a strong hydrophobic effect. Thus, by synergistically combining electrostatic and strong hydrophobic interactions, cardiolipin is effectively prevented from being oxidized and degraded by ROS, inhibiting its extravasation into the mitochondrial outer membrane, maintaining the normal content and structural integrity of cardiolipin, precisely protecting the morphology and spatial arrangement of mitochondrial cristae, and thereby maintaining the normal morphology of mitochondria.

[0018] The introduction of non-natural cyclohexylalanine (Cha) in this application enables the D-Arg-Cha-Lys-Phe-NH2 polypeptide to possess excellent anti-enzymatic stability and mitochondrial membrane binding affinity. It also enhances lipid solubility, membrane binding affinity, and anti-enzymatic stability, laying a core foundation for mitochondrial morphological stability.

[0019] The D-Arg-Cha-Lys-Phe-NH2 peptide in this application was purchased from GL Biochem, Ltd., catalog number GLB-1340093, specification 5mg / vial, purity ≥98%, synthesis cycle 14 days, and is provided in lyophilized powder form for easy direct dissolution to prepare the core active solution.

[0020] Ubiquinone-10 phosphate is a phosphate ester prepared from ubiquinone-10 with a purity ≥95%. After entering the mitochondria, ubiquinone-10 phosphate participates in the electron shuttle process from complex I / II to complex III in the electron transport chain, enhancing electron transport efficiency, inhibiting the opening of the mitochondrial permeability transition pore, and reducing proton leakage from the mitochondrial matrix. This stabilizes the mitochondrial membrane potential and prevents mitochondrial dysfunction caused by membrane depolarization. Simultaneously, ubiquinone-10 phosphate can synergistically interact with D-Arg-Cha-Lys-Phe-NH2 peptide to further enhance the integrity of mitochondrial structure and function.

[0021] In this application, the method for preparing ubiquinone-10 phosphate includes: S01: Under nitrogen protection and anhydrous and oxygen-free conditions, 1.0 g and 1.16 mmol of ubiquinone-10 were added to a dry 50 mL three-necked flask, followed by 20 mL of anhydrous dichloromethane. The flask was placed in an ice bath and cooled to 0 °C, with stirring to completely dissolve ubiquinone-10. Then, 1.5 g and 2.32 mmol of tetrabenzyl pyrophosphate, 0.37 g and 1.16 mmol of tetrabutylammonium bromide, and 0.35 mL and 2.5 mmol of triethylamine were added to the three-necked flask. The mixture was stirred thoroughly and reacted at 0 °C for 4 h. After stirring, the ice bath was removed, and the temperature was raised to 23-27 °C, with stirring continued for 12 h. During the reaction, the reaction progress was monitored by thin-layer chromatography (TLC) using a dichloromethane:methanol mixture (95:5 v / v). The reaction was considered complete when TLC showed that ubiquinone-10 had essentially disappeared and the target product spot appeared, yielding the reaction solution. Among them, ubiquinone-10 has the CAS number 303-98-0, a purity of ≥98%, and was purchased from MCE with the catalog number HY-N0111.

[0022] S02: Add saturated sodium chloride solution to the reaction solution, mix and separate into layers, add anhydrous sodium sulfate to the organic phase, let stand to dry, filter, concentrate under reduced pressure, and obtain an oily crude product.

[0023] Add 20 mL of saturated sodium chloride solution to the reaction mixture, shake thoroughly to mix, and allow to stand for separation. Collect the organic phase. Extract the aqueous phase twice with 10 mL of anhydrous dichloromethane. Combine all organic phases and add anhydrous sodium sulfate. Allow to stand and dry for 2 hours. The amount of anhydrous sodium sulfate added is 1 / 10 of the volume of the organic phase. After standing, filter to remove the anhydrous sodium sulfate, obtaining the filtrate. Place the filtrate in a rotary evaporator and concentrate under reduced pressure at 40 °C and 0.08 MPa to remove the solvent, yielding a pale yellow oily crude product.

[0024] S03: Dissolve the oily crude product in anhydrous methanol, add Pd / C catalyst, and stir the reaction at 23-27℃ for 6 hours under hydrogen atmosphere. After the reaction is completed, filter, wash, and concentrate under reduced pressure to obtain solid crude product.

[0025] The oily crude product was added to 20 mL of anhydrous methanol and stirred to dissolve. Then, 0.1 g of Pd / C catalyst was added, and the mixture was stirred and reacted for 6 h at a hydrogen pressure of 0.1 MPa and a temperature of 23-27 °C to carry out the benzyl protecting group removal reaction. After the reaction was completed, the Pd / C catalyst was removed by vacuum filtration, and the Pd / C catalyst was washed twice with 5 mL of anhydrous methanol. The washing liquid and filtrate were combined, and the methanol was removed by concentration under reduced pressure to obtain a pale yellow solid crude product.

[0026] S04: The crude solid product is dissolved in TFA aqueous solution, purified by high performance liquid chromatography, and the target eluent with a retention time of 28-30 min is collected and freeze-dried to obtain ubiquinone-10 phosphate.

[0027] The pale yellow crude solid was dissolved in 5 mL of 0.1% TFA (trifluoroacetic acid) aqueous solution and purified by preparative high performance liquid chromatography (HPLC). The target eluent with a retention time of 28-30 min was collected and placed in a freeze dryer and freeze-dried at -50℃ and 0.01 MPa for 12 h to obtain a pale yellow powder product, which is ubiquinone-10 phosphate. In this application, the preparative high-performance liquid chromatography (HPLC) conditions are as follows: the chromatographic column is a C18 column, 250 mm × 10 mm, 5 μm; mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% TFA acetonitrile solution; the elution gradient is as follows: 0-5 min, the volume fraction of mobile phase B remains at 20%; 5-35 min, the volume fraction of mobile phase B linearly increases from 20% to 80%; 35-40 min, the volume fraction of mobile phase B remains at 80%; 40-45 min, the volume fraction of mobile phase B linearly decreases from 80% to 20%; the flow rate is 3 mL / min; the detection wavelength is 275 nm; and the column temperature is 30 °C.

[0028] In this application, mass spectrometry (MS) was used to detect the molecular weight of the product. The calculated ESI-MS m / z [MH]⁻ value was 863.5, consistent with the calculated value, confirming that the prepared product was the target product, ubiquinone-10 phosphate. Furthermore, analytical high-performance liquid chromatography (HPLC) was used to detect the purity of the product. The chromatographic conditions were the same as those for preparative HPLC, except that the flow rate was adjusted to 1 mL / min. The purity of ubiquinone-10 phosphate was found to be ≥95%, making it suitable for direct use in the formulation of the core active ingredient of this invention.

[0029] Reduced glutathione (GSH) can directly scavenge mitochondrial reactive oxygen species (ROS), while alpha-lipoic acid (ALA) can regenerate GSH, prolonging its antioxidant activity. Thus, GSH and ALA form a synergistic antioxidant system. Furthermore, GSH and ALA can inhibit NADPH oxidase activity, reducing enzymatic ROS production, and are effective even at low concentrations, avoiding the interference of high-concentration antioxidants on the cell culture environment.

[0030] Therefore, this application achieves stable cardiolipin, maintains mitochondrial morphology and membrane potential, and reduces ROS through the synergistic effect of the core active ingredients, thus solving the problems of existing additives having single function, insufficient synergistic effect, and technical controversy.

[0031] In this application, the additive comprises, by weight, 0.001-0.05 parts of core active ingredient, 0.5-2 parts of auxiliary ingredient, and 97.95-99.499 parts of carrier, and the mass ratio of D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid is 1:(3-5):(8-12):(2-4).

[0032] Preferably, the additive comprises, by weight, 0.01-0.03 parts of core active ingredient, 0.8-1.5 parts of auxiliary ingredient, and 98.47-99.19 parts of carrier; wherein, the mass ratio of D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid is 1:4:10:3. At this ratio, the optimal synergistic effect of stabilizing cardiolipin, maintaining membrane potential, and reducing ROS can be achieved, and the effects of each component promote each other without antagonistic effects.

[0033] In this application, the auxiliary components include mannitol and vitamin E succinate in a mass ratio of (2-3):1. Mannitol, as a lyophilization protectant and osmotic pressure regulator, can prevent the core active ingredient from denaturing during storage and use, while maintaining the osmotic pressure stability of the cell culture system. Vitamin E succinate, as a lipid-soluble antioxidant, can protect membrane lipids from ROS oxidation, reduce ROS damage to mitochondria, reduce ROS oxidative attack on cardiolipin, and help maintain the integrity of the mitochondrial membrane. Together with the core active ingredient, it can enhance the mitochondrial protective effect, forming a virtuous cycle of ROS scavenging, cardiolipin protection, and membrane potential stabilization.

[0034] In this application, the carrier includes sterile physiological saline or serum-free cell culture basal medium to ensure compatibility between the additives and the cell culture system without additional toxic interference. The serum-free cell culture basal medium in this application is selected from serum-free DMEM (Dulbecco's Modified Eagle Medium), serum-free RPMI-1640 (RoswellPark Memorial Institute-1640) medium, or serum-free MEM (Minimum Essential Medium) medium.

[0035] Secondly, this application provides a method for preparing a mitochondrial function-optimizing additive for cell culture, the method comprising: S01: The core active ingredient and auxiliary ingredient from the first aspect are added to the carrier to form the core active solution and the auxiliary solution, respectively.

[0036] D-Arg-Cha-Lys-Phe-NH2 peptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid were added to the carrier in a specific ratio and stirred at 4°C and 150-200 rpm for 30-60 min until completely dissolved to obtain the core active solution. Mannitol and vitamin E succinate were added to the carrier, and the mixture was heated in a water bath at 60-70°C with stirring for 20-30 min. After cooling to room temperature, the auxiliary solution was obtained.

[0037] S02: Slowly add the core active solution to the auxiliary solution, stir evenly at 4°C for 1-2 hours, and then filter through a 0.22μm filter membrane for sterilization to obtain the mitochondrial function optimization additive for cell culture. This additive is aliquoted into sterile centrifuge tubes and stored frozen at -20°C. Before use, thaw to room temperature and gently invert to mix.

[0038] Thirdly, this application provides an application of a mitochondrial function optimization additive for cell culture, namely, its application in stabilizing mitochondrial cardiolipin, maintaining mitochondrial morphology and membrane potential, and reducing mitochondrial ROS generation.

[0039] Fourthly, this application provides a method for using a mitochondrial function optimization additive for cell culture, comprising: mixing the mitochondrial function optimization additive for cell culture from the first aspect with the cell culture system, and then culturing at 37°C and 5% CO2.

[0040] The cells in the cell culture system of this application include stem cells, primary cells, tumor cell lines, or immune cells. Preferably, the volume ratio of the additive to the cell culture system is 1:(200-300).

[0041] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0042] Example 1 This application provides a ubiquinone-10 phosphate ester, the preparation method of which includes: 101: Under nitrogen protection and anhydrous and oxygen-free conditions, 1.0 g and 1.16 mmol of ubiquinone-10 were added to a dry 50 mL three-necked flask, followed by 20 mL of anhydrous dichloromethane. The flask was placed in an ice bath and cooled to 0 °C, with stirring to completely dissolve ubiquinone-10. Then, 1.5 g and 2.32 mmol of tetrabenzyl pyrophosphate, 0.37 g and 1.16 mmol of tetrabutylammonium bromide, and 0.35 mL and 2.5 mmol of triethylamine were added to the three-necked flask. The mixture was stirred thoroughly and reacted at 0 °C for 4 h. After stirring, the ice bath was removed, and the temperature was raised to 23-27 °C, with stirring continued for 12 h. During the reaction, the reaction progress was monitored by thin-layer chromatography (TLC) using a dichloromethane:methanol mixture (95:5 v / v). The reaction was considered complete when TLC showed that ubiquinone-10 had essentially disappeared and the target product spot appeared, yielding the reaction solution. Among them, ubiquinone-10 has the CAS number 303-98-0, a purity of ≥98%, and was purchased from MCE with the catalog number HY-N0111.

[0043] S102: Add 20 mL of saturated sodium chloride solution to the reaction solution, shake thoroughly to mix, and allow to stand for separation. Collect the organic phase. Extract the aqueous phase twice with 10 mL of anhydrous dichloromethane. Combine all organic phases and add anhydrous sodium sulfate. Allow to stand and dry for 2 hours. The amount of anhydrous sodium sulfate added is 1 / 10 of the volume of the organic phase. After standing, filter to remove the anhydrous sodium sulfate, obtaining the filtrate. Place the filtrate in a rotary evaporator and concentrate under reduced pressure at 40 °C and 0.08 MPa to remove the solvent, obtaining a pale yellow oily crude product.

[0044] S103: The oily crude product was added to 20 mL of anhydrous methanol and stirred to dissolve. Then, 0.1 g of Pd / C catalyst was added, and the mixture was stirred and reacted for 6 h at a hydrogen pressure of 0.1 MPa and a temperature of 23-27 °C to carry out the benzyl protecting group removal reaction. After the reaction was completed, the Pd / C catalyst was removed by vacuum filtration, and the Pd / C catalyst was washed twice with 5 mL of anhydrous methanol. The washing liquid and filtrate were combined, and the methanol was removed by concentration under reduced pressure to obtain a pale yellow solid crude product.

[0045] S104: The pale yellow crude solid was dissolved in 5 mL of 0.1% TFA (Trifluoroacetic acid) aqueous solution and purified by preparative high performance liquid chromatography. The target eluent with a retention time of 28-30 min was collected and placed in a freeze dryer and freeze-dried at -50℃ and 0.01 MPa for 12 h to obtain a pale yellow powder product, which is ubiquinone-10 phosphate. In this application, the preparative high-performance liquid chromatography (HPLC) conditions are as follows: the chromatographic column is a C18 column, 250 mm × 10 mm, 5 μm; mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% TFA acetonitrile solution; the elution gradient is as follows: 0-5 min, the volume fraction of mobile phase B is maintained at 20%; 5-35 min, the volume fraction of mobile phase B linearly increases from 20% to 80%; 35-40 min, the volume fraction of mobile phase B is maintained at 80%; 40-45 min, the volume fraction of mobile phase B linearly decreases from 80% to 20%; the flow rate is 3 mL / min; the detection wavelength is 275 nm; and the column temperature is 30 °C.

[0046] Example 2 This application provides a mitochondrial function optimization additive for cell culture. The additive comprises, by weight, 0.01 parts of a core active ingredient, 0.8 parts of auxiliary ingredients, and 99.19 parts of sterile physiological saline. The core active ingredient includes D-Arg-Cha-Lys-Phe-NH2 polypeptide in a mass ratio of 1:4:10:3, ubiquinone-10 phosphate from Example 1, reduced glutathione, and α-lipoic acid; the auxiliary ingredients include mannitol and vitamin E succinate in a mass ratio of 2:1.

[0047] The method for preparing the additive provided in this application includes: S201: D-Arg-Cha-Lys-Phe-NH2 peptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid were added to sterile physiological saline in the above proportions. The mixture was stirred at 4°C and 180 rpm for 45 min until completely dissolved to obtain the core active solution. Mannitol and vitamin E succinate were added to sterile physiological saline, heated in a 65°C water bath and stirred for 25 min. After cooling to room temperature, the auxiliary solution was obtained.

[0048] S202: The core active solution is slowly added to the auxiliary solution, stirred evenly at 4°C for 1.5 hours, and then filtered through a 0.22μm filter membrane for sterilization to obtain the additive.

[0049] Example 3 This application provides a mitochondrial function optimization additive for cell culture. The additive comprises, by weight, 0.03 parts of a core active ingredient, 1.5 parts of auxiliary ingredients, and 98.47 parts of serum-free DMEM culture medium. The core active ingredient includes D-Arg-Cha-Lys-Phe-NH2 polypeptide in a mass ratio of 1:5:10:3, ubiquinone-10 phosphate from Example 1, reduced glutathione, and α-lipoic acid; the auxiliary ingredients include mannitol and vitamin E succinate in a mass ratio of 2:1.

[0050] The preparation method of the additives provided in this application embodiment is the same as that in Embodiment 2.

[0051] Example 4 This application provides a mitochondrial function optimization additive for cell culture. The additive comprises, by weight, 0.005 parts of a core active ingredient, 0.5 parts of auxiliary ingredients, and 99.495 parts of serum-free RPMI-1640 culture medium. The core active ingredient includes D-Arg-Cha-Lys-Phe-NH2 polypeptide in a mass ratio of 1:3:8:2, ubiquinone-10 phosphate from Example 1, reduced glutathione, and α-lipoic acid; the auxiliary ingredients include mannitol and vitamin E succinate in a mass ratio of 2:1.

[0052] The preparation method of the additives provided in this application embodiment is the same as that in Embodiment 2.

[0053] To verify that the additives provided in this application can stabilize mitochondrial cardiolipin, mitochondrial morphology, maintain mitochondrial morphology and membrane potential, and reduce mitochondrial ROS generation, human umbilical cord mesenchymal stem cells were used as experimental subjects in this application. Five groups of experiments were set up, with three replicate wells in each group. After culturing for 72 h, indicators such as mitochondrial cardiolipin content, mitochondrial membrane potential, mitochondrial ROS level, and cell viability were detected. Among them, Group 1: no mitochondrial protective additives were added; Group 2: only D-Arg-Cha-Lys-Phe-NH2 polypeptide was added as a mitochondrial protective agent, and the concentration was consistent with the concentration of D-Arg-Cha-Lys-Phe-NH2 polypeptide, the core active ingredient in Example 2; Group 3: a compound additive group composed of coenzyme Q10 and glutathione was added, and the concentration was consistent with the concentration of the corresponding component in Example 2; Group 4: the additive prepared in Example 2 of this invention was added, and added to the cell culture system at a volume ratio of 1:200; Group 5: the additive prepared in Example 3 of this invention was added, and added to the cell culture system at a volume ratio of 1:200. 1. Detection of mitochondrial cardiolipin content (1) Sample preparation: Cells from three replicate wells in each group were digested with trypsin and centrifuged at 1000 rpm for 5 min at 4 °C. The cell pellet was collected. The cell pellet was washed twice with pre-chilled PBS buffer, centrifuged under the same conditions each time, and the supernatant was discarded. 500 μL of a 2:1 chloroform-methanol mixture was added to the pellet, vortexed for 10 min, and then incubated on ice for 30 min to fully extract lipids. After incubation, 125 μL of pre-chilled physiological saline was added, vortexed for 5 min, and then centrifuged at 12000 rpm at 4 °C for 15 min. The lower organic phase was collected, dried under nitrogen, dissolved in 100 μL of methanol, and filtered through a 0.22 μm organic filter membrane to obtain the sample to be tested.

[0054] (2) Detection by HPLC Chromatographic conditions: A C18 column (4.6 mm × 250 mm, 5 μm) was used; the mobile phase was methanol-phosphate buffer (85:15 v / v), and the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 205 nm; and the injection volume was 20 μL. Detection and calculation: Cardiac phospholipid standards of different concentrations were prepared and detected under the above chromatographic conditions to plot a standard curve; the test sample was injected into the HPLC instrument, and the corresponding peak area was detected. The content of central phospholipids in the sample was calculated based on the standard curve. Finally, using a blank control group as a reference, the relative values ​​of cardiac phospholipid content in each group were calculated to obtain the results. Figure 1 .

[0055] From the appendix Figure 1 As can be seen, the mitochondrial cardiolipin content in group 1 was 1.00±0.05, which served as the baseline. Compared with group 1, the relative content of mitochondrial cardiolipin in the other groups increased to varying degrees: the relative content of cardiolipin in group 2 was 1.28±0.06, an increase of 28% compared to group 1, indicating that the single D-Arg-Cha-Lys-Phe-NH2 polypeptide can stabilize mitochondrial cardiolipin to a certain extent and reduce its oxidative degradation; the relative content of cardiolipin in group 3 was 1.12±0.05, an increase of 12% compared to group 1, with a weaker increase than group 2, indicating that the existing compound additive has limited stabilizing effect on cardiolipin; the relative content of cardiolipin in group 4 was 1.52±0.07, an increase of 52% compared to group 1, significantly higher than groups 2 and 3, demonstrating a significant enhancement of the cardiolipin protective effect under synergistic effect; the relative content of cardiolipin in group 5 was 1.55±0.08, an increase of 55% compared to group 1, with no significant difference from group 4, further confirming the highly efficient stabilizing effect of the additive provided in this application on cardiolipin.

[0056] 2. Mitochondrial morphology detection (1) Cell fixation: Cells from 3 replicate wells in each group were digested with trypsin and the cell pellet was collected. The pellet was then fixed at 4°C for 2 h with pre-cooled 2.5% glutaraldehyde fixative. After fixation, the cells were washed 3 times with PBS buffer for 10 min each time and the supernatant was discarded. Then, 1% osmium tetroxide fixative was added and the cells were fixed at 4°C for 1 h. The cells were then washed 3 times with PBS buffer for 10 min each time.

[0057] (2) Dehydration and embedding: The cells were dehydrated sequentially with 50%, 70%, 80%, 90%, and 100% ethanol, for 15 min at each concentration; then, acetone was used to replace the ethanol twice, for 15 min each time; the dehydrated cell pellet was mixed with the embedding agent at a 1:1 ratio and incubated at 37°C for 2 h, then pure embedding agent was added and polymerized at 60°C for 24 h to prepare the embedding block. The embedding agent was prepared by mixing epoxy resin 812 embedding agent and acetone at a volume ratio of 1:1, and the pure embedding agent was epoxy resin 812 embedding agent.

[0058] (3) Sectioning and staining: Cut the embedded block into ultrathin sections of 50-70 nm and attach them to a copper grid; stain with uranium acetate for 15 min and lead citrate for 5 min, and let them air dry for later use.

[0059] (4) TEM detection: The mitochondrial morphology of each group of cells was observed using a transmission electron microscope at a magnification of 10000×. Ten fields of view were randomly selected from each sample, and 50 mitochondria were counted in each field of view. The number of swollen (increased mitochondrial volume, sparse cristae) and fragmented (mitochondria broken into multiple small fragments) mitochondria were counted, and the proportion of abnormal mitochondria was calculated to obtain the mitochondrial morphology. Figure 2 , 3 The percentage of abnormal mitochondria is calculated as the number of abnormal mitochondria / total number of mitochondria × 100%.

[0060] From the appendix Figure 2 , 3As can be seen, the mitochondrial abnormality rate in group 1 was 38.6±4.2%, and under electron microscopy, a large number of mitochondria showed obvious swelling, cristae breakage, and fragmentation, with severe damage to structural integrity; the mitochondrial abnormality rate in group 2 was 27.5±3.5%, a decrease of 28.8% compared to group 1, and under electron microscopy, the degree of mitochondrial swelling and fragmentation was reduced, and structural integrity was improved to some extent; the mitochondrial abnormality rate in group 3 was 29.8±3.7%, a decrease of 22.8% compared to group 1, but under electron microscopy, a large number of mitochondria were still swollen, and the improvement effect was weaker than that in group 2; the mitochondrial abnormality rate in group 4 was 12.3±2.1%, a decrease of 68.1% compared to group 1, significantly lower than groups 2 and 3, and under electron microscopy, the mitochondrial morphology was close to the normal physiological state, with only a small amount of slight swelling and no obvious fragmentation; the mitochondrial abnormality rate in group 5 was 11.8±2.0%, a decrease of 69.4% compared to group 1, with no significant difference from group 4, and under electron microscopy, the mitochondrial morphology was regular and the cristae structure was clear, which indicates that the additive provided in this application has a highly effective protective effect on mitochondrial morphology.

[0061] 3. Mitochondrial membrane potential detection The JC-1 staining method was used, and the membrane potential stability was represented by the proportion of membrane potential-positive cells by flow cytometry. The specific steps are as follows: (1) Preparation of staining solution: Dilute JC-1 staining solution to a final concentration of 5 μmol / L with serum-free culture medium, mix thoroughly, and store in the dark for later use.

[0062] (2) Cell staining: Cells from 3 replicate wells in each group were digested with trypsin, and the cell pellet was collected. The pellet was washed twice with PBS buffer and the cell concentration was adjusted to 1×10⁻⁶. 6 Add an equal volume of diluted JC-1 staining solution and incubate at 37°C in a 5% CO2 incubator for 20 minutes in the dark. (3) Washing and resuspension: After incubation, centrifuge at 1000 r / min and 4℃ for 5 min and discard the supernatant; wash the cell pellet twice with pre-cooled JC-1 washing solution, with the same centrifugation conditions each time; add 500 μL PBS buffer, gently pipette to resuspend the cells and prepare a single-cell suspension.

[0063] (4) Flow cytometry detection and statistics: Flow cytometry was used for detection. The excitation wavelength was 488 nm, and the emission wavelengths were 530 nm and 590 nm. The 488 nm light emitted green fluorescence, used to detect cells with decreased membrane potential; the 530 nm light emitted red fluorescence, used to detect cells with normal membrane potential. 10,000 cells were analyzed for each sample. Flow cytometry analysis software was used to distinguish between membrane potential-positive cells (red fluorescence positive) and membrane potential-negative cells (green fluorescence positive). The proportion of membrane potential-positive cells was calculated to obtain the results. Figure 4 .

[0064] From the appendix Figure 4 As can be seen, the proportion of cells positive for three-dimensional membrane potential in group 1 was 52.3±3.8%, indicating a high degree of mitochondrial membrane potential depolarization under conventional culture. The proportion of cells positive for membrane potential in group 2 was 60.1±4.1%, an increase of 14.9% compared to group 1, but the increase was limited, indicating that the single D-Arg-Cha-Lys-Phe-NH2 polypeptide had a weak effect on maintaining membrane potential. The proportion of cells positive for membrane potential in group 3 was 65.4±3.9%, an increase of 25.0% compared to group 1, superior to group 2, demonstrating the protective effect of coenzyme Q10+glutathione on membrane potential. The proportion of cells positive for membrane potential in group 4 was 86.7±4.5%, an increase of 65.8% compared to group 1, significantly higher than groups 2 and 3, indicating that the additive provided in this application can efficiently maintain mitochondrial membrane potential stability; the proportion of cells positive for membrane potential in group 5 was 88.2±4.3%, an increase of 68.6% compared to group 1, with no significant difference from group 4, further confirming the efficient maintenance effect of the additive provided in this application on membrane potential.

[0065] 4. Mitochondrial ROS level detection The DCFH-DA staining method was used, and the ROS content was detected by flow cytometry, with fluorescence intensity representing the ROS content. The specific steps are as follows: (1) Preparation of staining solution: Dilute the DCFH-DA staining solution to a final concentration of 10 μmol / L with serum-free culture medium, mix thoroughly, and store in the dark for later use.

[0066] (2) Cell staining: Cells from 3 replicate wells in each group were digested with trypsin, and the cell pellet was collected. The pellet was washed twice with PBS buffer and the cell concentration was adjusted to 1×10⁻⁶. 6 Add the diluted DCFH-DA staining solution to the sample and incubate at 37°C in a 5% CO2 incubator for 30 minutes in the dark, gently shaking the container every 10 minutes to ensure uniform staining.

[0067] (3) Washing and resuspension: After incubation, centrifuge at 1000 r / min and 4℃ for 5 min and discard the supernatant; wash the cell pellet three times with pre-cooled PBS buffer, centrifuging under the same conditions each time to remove staining solution that did not enter the cells; add 500 μL PBS buffer and gently pipette to resuspend the cells to make a single-cell suspension.

[0068] (4) Flow cytometry detection and statistics: Flow cytometry was used for detection, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. 10,000 cells were analyzed for each sample. The average fluorescence intensity of each group was calculated using flow cytometry software. Using the fluorescence intensity of group 1 as a reference, the relative values ​​of ROS fluorescence intensity for each group were calculated to obtain the results. Figure 5 .

[0069] From the appendix Figure 5As can be seen, the ROS fluorescence intensity of group 1 was 1.00±0.07, which served as the baseline level; the ROS fluorescence intensity of group 2 was 0.85±0.06, a decrease of 15% compared to group 1, indicating that the single D-Arg-Cha-Lys-Phe-NH2 polypeptide could only partially clear mitochondrial ROS; the ROS fluorescence intensity of group 3 was 0.72±0.05, a decrease of 28% compared to group 1, which was better than group 2, demonstrating the antioxidant effect of coenzyme Q10+glutathione; the ROS fluorescence intensity of group 4 was 0.58±0.04, a decrease of 42% compared to group 1, which was significantly lower than groups 2 and 3, indicating that the additive provided in this application can efficiently clear mitochondrial ROS; the ROS fluorescence intensity of group 5 was 0.56±0.04, a decrease of 44% compared to group 1, with no significant difference from group 4, further verifying the highly efficient antioxidant effect of the additive provided in this application.

[0070] 5. Cell viability detection Cell proliferation rate was detected using the CCK-8 assay, and the relative value with group 1 was calculated. The specific steps are as follows: (1) Inoculation and culture: Cells in each group were inoculated at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 1 cell per well in 96-well plates, with 3 replicate wells in each group. Group 1 was set up as a blank control well (only culture medium was added, without cells). The cells were incubated at 37°C and 5% CO2 for 72 hours, consistent with the culture conditions of the experimental groups.

[0071] (2) Staining and incubation: After the culture is completed, add 10 μL of CCK-8 reagent to each well, gently shake the 96-well plate to ensure that the reagent and culture medium are fully mixed; incubate at 37℃ and 5% CO2 in the dark for 2 h.

[0072] (3) Absorbance detection: The absorbance (OD) value of each well at a wavelength of 450 nm was detected using an ELISA reader. The blank control well was first zeroed, and then the OD values ​​of each group of sample wells were detected sequentially. The data were recorded, and the relative values ​​of cell proliferation rates for each group were calculated to obtain the absorbance. Figure 6 The cell proliferation rate (%) was calculated as follows: (OD value of experimental group - OD value of blank control well) / (OD value of blank control group - OD value of blank control well) × 100%. The relative value of cell proliferation rate for each group was calculated with the proliferation rate of the blank control group being 100%.

[0073] From the appendix Figure 6As can be seen, the cell proliferation rate of group 1 was 100.0±2.5%, which served as the baseline level; the cell proliferation rate of group 2 was 105.3±2.8%, an increase of 5.3% compared to group 1, with a limited increase; the cell proliferation rate of group 3 was 108.6±3.0%, an increase of 8.6% compared to group 1, with no significant improvement; the cell proliferation rate of group 4 was 125.8±3.2%, an increase of 25.8% compared to group 1, significantly higher than groups 2 and 3, indicating that the additive provided in this application can significantly improve cell activity and proliferation capacity; the cell proliferation rate of group 5 was 132.4±2.4%, an increase of 32.4% compared to group 1, with no significant difference from group 4, further confirming the promoting effect of the additive provided in this application on cell proliferation.

[0074] In summary, compared to Group 1, while the use of a single component in Group 2 increased cardiolipin content and reduced the proportion of abnormal mitochondria, its effects on membrane potential maintenance and ROS scavenging were limited. Compared to Group 1, while the use of coenzyme Q10 + glutathione in Group 3 reduced ROS and maintained membrane potential, its stabilizing effect on cardiolipin was weak, and the increase in cell proliferation rate was not significant. Compared to Groups 1-3, the additives prepared in Examples 2 and 3 of this application can increase mitochondrial cardiolipin content by more than 50%, reduce the proportion of abnormal mitochondria to below 12%, increase the proportion of membrane potential-positive cells to more than 86%, reduce mitochondrial ROS levels by more than 40%, and increase cell proliferation rate by more than 25%.

[0075] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mitochondrial function-optimizing additive for cell culture, characterized in that, It includes a core active ingredient, auxiliary ingredients, and a carrier, wherein the core active ingredient includes D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid.

2. The mitochondrial function-optimizing additive for cell culture according to claim 1, characterized in that, The mass ratio of the D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione and α-lipoic acid is 1:(3-5):(8-12):(2-4).

3. The mitochondrial function-optimizing additive for cell culture according to claim 1, characterized in that, The auxiliary components include mannitol and vitamin E succinate in a mass ratio of (2-3):1; the carrier includes sterile physiological saline or serum-free cell culture basal medium.

4. The mitochondrial function-optimizing additive for cell culture according to claim 3, characterized in that, The serum-free cell culture basal medium includes serum-free DMEM medium, serum-free RPMI-1640 medium, or serum-free MEM medium.

5. The mitochondrial function-optimizing additive for cell culture according to claim 1, characterized in that, Based on weight parts, it includes 0.001-0.05 parts of core active ingredient, 0.5-2 parts of auxiliary ingredients, and 97.95-99.499 parts of carrier.

6. The mitochondrial function-optimizing additive for cell culture according to claim 1, characterized in that, The product comprises, by weight, 0.01-0.03 parts of core active ingredient, 0.8-1.5 parts of auxiliary ingredient, and 98.47-99.19 parts of carrier; the mass ratio of D-Arg-Cha-Lys-Phe-NH2 polypeptide, ubiquinone-10 phosphate, reduced glutathione, and α-lipoic acid is 1:4:10:

3.

7. The mitochondrial function-optimizing additive for cell culture according to claim 1, characterized in that, The preparation method of the ubiquinone-10 phosphate includes: Ubiquinone-10 was dissolved in anhydrous dichloromethane under nitrogen protection, 0°C, and anhydrous and oxygen-free conditions. Tetrabenzyl pyrophosphate, tetrabutylammonium bromide and triethylamine were added and the mixture was stirred for 4 h. The temperature was then raised to 23-27°C and the reaction was carried out for 12 h to obtain the reaction solution. Saturated sodium chloride solution was added to the reaction solution and mixed and separated into layers. Anhydrous sodium sulfate was added to the organic phase, and the mixture was allowed to stand, dried, filtered, and concentrated under reduced pressure to obtain an oily crude product. The oily crude product was dissolved in anhydrous methanol, a Pd / C catalyst was added, and the mixture was stirred and reacted at 23-27°C for 6 hours under a hydrogen atmosphere. After the reaction was completed, the product was filtered, washed, and concentrated under reduced pressure to obtain a solid crude product. The crude solid product was dissolved in TFA aqueous solution, purified by high performance liquid chromatography, and the target eluent with a retention time of 28-30 min was collected and freeze-dried to obtain ubiquinone-10 phosphate.

8. A method for preparing the mitochondrial function-optimizing additive for cell culture according to any one of claims 1-7, characterized in that, include: The core active ingredient and auxiliary ingredient described in any one of claims 1-7 are respectively added to the carrier to form a core active solution and an auxiliary solution; The core active solution was slowly added to the auxiliary solution, stirred evenly at 4°C, and then filtered through a 0.22 μm filter membrane for sterilization to obtain a mitochondrial function optimization additive for cell culture.

9. The application of the mitochondrial function-optimizing additive for cell culture according to any one of claims 1-7 in stabilizing mitochondrial cardiolipin, maintaining mitochondrial morphology and membrane potential, and reducing mitochondrial ROS generation.

10. A method of using the mitochondrial function-optimizing additive for cell culture according to any one of claims 1-7, comprising: The mitochondrial function optimization additive for cell culture according to any one of claims 1-7 and the cell culture system are mixed at a volume ratio of 1:(100-500) and cultured at 37°C and 5% CO2.

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