Serum-free culture medium for promoting directional amplification of muscle stem cells and preparation method of serum-free culture medium

Abstract

The invention provides a serum-free medium for promoting directional amplification of muscle stem cells and a preparation method, and relates to the technical field of cell culture. All the components in the serum-free culture medium form a synergistic effect through a specific synergistic ratio, proliferation of the muscle stem cells is directionally promoted through multidimensional synergism of cell signal regulation, epigenetic modification, metabolism support, cytoskeleton stability and the like, meanwhile, abnormal differentiation of the muscle stem cells is inhibited, and the characteristics of the stem cells are maintained. The serum-free culture medium adopts a serum-free system, so that the problems of complex serum components, large batch difference, possible carrying of pathogens or heterologous proteins and the like in the traditional serum-containing culture medium are solved, the safety and stability of muscle stem cell culture are improved, and meanwhile, the culture conditions are conveniently and accurately regulated and controlled. All the components in the serum-free culture medium act according to a specific proportion and sequence to form a complete network of proliferation driving, stemness maintenance, metabolism support and negative regulation removal, and directional efficient amplification of muscle stem cells can be achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and relates to a serum-free culture medium for promoting directional expansion of muscle stem cells and a preparation method. BACKGROUND

[0002] Muscle stem cells are mononuclear somatic stem cells under the basement membrane of skeletal muscle, which maintain self-renewal ability in a resting state, are activated to differentiate into muscle cells and then fuse to form muscle fibers when skeletal muscle is damaged, and have great application potential in the fields of muscle damage repair, regenerative medicine and biopharmaceuticals.

[0003] The culture of muscle stem cells usually uses a culture medium containing serum. However, the serum composition is complex, and there are large batch differences, and there is a risk of contamination by pathogens such as viruses and mycoplasma; at the same time, the serum contains unknown components, which is not conducive to the accurate regulation of the cell culture microenvironment, and also limits the directional expansion of muscle stem cells and subsequent clinical application and research. Therefore, it is of great significance to develop a serum-free culture medium with clear composition, stable performance and effective promotion of directional expansion of muscle stem cells.

[0004] At present, when the existing serum-free culture medium is used to promote the directional expansion of muscle stem cells, there are problems such as low cell expansion efficiency, unstable functional characteristics of the cells after expansion, and the expansion effect is not ideal. SUMMARY

[0005] The purpose of the present application is to provide a serum-free culture medium for promoting directional expansion of muscle stem cells and a preparation method, so as to solve the problem that the existing serum-free culture medium is difficult to achieve efficient directional expansion of muscle stem cells.

[0006] To achieve the above-mentioned purpose, the following technical solutions are adopted in the present application: The present application provides a serum-free culture medium for promoting directional expansion of muscle stem cells, which takes a basic culture medium as a substrate and includes, according to the final concentration, PEG-PDLLA-IGF-1 conjugate 20-80 ng / ml, PEG-PDLLA-bFGF conjugate 0.05-0.5% w / v, seleno-cysteine 3-5 nM, alpha-ketobutyric acid 1-1.5 mM, BIX01294 0.5-2 μM, Entinostat 0.3-0.8 μM, Fc-TβRIII fusion protein 80-120 ng / mL, Mstn-NP 1-3 μg / ml, Jasplakinolide 0.1-0.3 nM, and Icariin 5-10 μM.

[0007] In the present application, the basic culture medium is DMEM / F12 culture medium, wherein the mass ratio of DMEM culture medium to F12 culture medium is 1:1. The muscle stem cells are human muscle stem cells.

[0008] PEG-PDLLA-IGF-1 conjugate is prepared by covalently connecting IGF-1 (Chinese name: insulin-like growth factor 1) in the hydrophobic core of PEG-PDLLA amphiphilic block copolymer micelles. PEG-PDLLA-IGF-1 conjugate can prolong the plasma half-life from less than 10 min to 3-7 days, and continuously activate IGF-1 R / PI3K / Akt axis in serum-free culture system, maintain Pax7 + Self-renewal state and inhibit MyoD premature up-regulation. The PEG (polyethylene glycol) chain in PEG-PDLLA-IGF-1 conjugate can reduce cross-binding with insulin receptors and avoid the risk of hypoglycemia; the degradability of PDLLA (poly-d,l-lactic acid) block can realize diffusion-degradation release and form a stable local IGF-1 concentration gradient.

[0009] In the embodiments of the present application, the preparation method of PEG-PDLLA-IGF-1 conjugate comprises: S01: Under nitrogen protection, 1 mmol of polyethylene glycol and 5 mmol of polylactic acid monomer were dissolved in 10 ml of dichloromethane, 0.006 mmol of stannous octoate was added, and the reaction was stirred at 60°C for 24 h to obtain reaction liquid a.

[0010] S02: After the reaction was completed, the reaction liquid was dropped into excess anhydrous ethanol for precipitation, centrifuged at a speed of 3000 rpm for 10 min, and then freeze-dried and vacuum-dried to obtain PEG-PDLLA copolymer.

[0011] S03: 1g of PEG-PDLLA copolymer, 0.2g of succinimidyl propionate, 0.15g of N,N'-dicyclohexyl carbodiimide, and 0.08g of N-hydroxysuccinimide were dissolved in 20ml of dichloromethane, stirred at room temperature and in the dark for 12h, the filtrate after removing the by-product dicyclohexyl urea was dropped into excess anhydrous ethanol for precipitation, centrifuged, freeze-dried and vacuum-dried to obtain PEG-PDLLA-SPA.

[0012] S04: 1 mg IGF-1 was dissolved in 1 ml PBS (phosphate buffered saline) to form an IGF-1 / PBS solution. 80 mg PEG-PDLLA-SPA was dissolved in 5 ml PBS to form a PEG-PDLLA-SPA / PBS solution. Under the condition of magnetic stirring at pH 7.2-7.4, the IGF-1 / PBS solution was slowly added to the carrier PEG-PDLLA-SPA / PBS solution, and reacted for 4 h at 25°C in the dark to obtain reaction liquid b. The excess of the carrier PEG-PDLLA-SPA / PBS solution can improve the coupling rate of IGF-1, and the neutral environment at pH 7.2-7.4 can avoid destroying the activity of IGF-1.

[0013] S05: The reaction liquid b was loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and dialysis was performed at 4°C for 48 h using PBS at pH 7.4 as the dialysate, and the PBS was replaced every 6 h to facilitate the removal of free IGF-1 and unreacted small molecules by dialysis. After lyophilization, the PEG-PDLLA-IGF-1 conjugate was obtained as a white powder.

[0014] The PEG-PDLLA-bFGF conjugate encapsulates bFGF in a similar core-shell structure, and constructs a nano-micelle with a particle size of 50-200 nm using the hydrophobic core of PDLLA and the hydrophilic shell of PEG. The PEG-PDLLA-bFGF conjugate releases in a zero-order manner within 7 days, continuously activates the FGFR1-ERK pathway, promotes cell proliferation, and prevents protease degradation.

[0015] The synergistic effect of the PEG-PDLLA-IGF-1 conjugate and the PEG-PDLLA-bFGF conjugate can provide a "long-acting + targeted" dual growth factor signal under serum-free conditions, significantly improving the expansion efficiency and phenotype stability of muscle stem cells.

[0016] In the present application, the preparation method of the PEG-PDLLA-bFGF conjugate comprises: S01: 1 mmol of polyethylene glycol and 5 mmol of polylactic acid monomer were dissolved in 10 ml of dichloromethane under nitrogen protection, 0.006 mmol of stannous octoate was added, and the reaction was stirred at 60°C for 24 h to obtain reaction liquid a.

[0017] S02: After the reaction was completed, the reaction liquid was dropped into excess anhydrous ethanol for precipitation, centrifuged at a speed of 3000 rpm for 10 min, and then freeze-dried to obtain the PEG-PDLLA copolymer.

[0018] S03: 1 g of PEG-PDLLA copolymer, 0.2 g of succinimidyl propionate, 0.15 g of N,N'-dicyclohexyl carbodiimide, and 0.08 g of N-hydroxysuccinimide were dissolved in 20 ml of dichloromethane, stirred at room temperature for 12 h in the dark, and then precipitated by adding the filtrate after removing the by-product dicyclohexyl urea into excess anhydrous ethanol. After centrifugation, freezing, and vacuum drying, PEG-PDLLA-SPA was obtained.

[0019] S04: 1 mg of bFGF was dissolved in 1 ml of PBS to form a bFGF / PBS solution. 100 mg of PEG-PDLLA-SPA was dissolved in 5 ml of PBS to form a PEG-PDLLA-SPA / PBS solution. Under magnetic stirring and nitrogen filling, the bFGF / PBS solution was slowly added to the carrier PEG-PDLLA-SPA / PBS solution, and reacted for 6 h at 20°C in the dark to obtain reaction liquid b. The stability of bFGF is poor, and the reaction at a low temperature of 20°C can reduce the loss of activity and improve the activity of the PEG-PDLLA-bFGF conjugate. In addition, the nitrogen environment can avoid the oxidation of bFGF, further reducing the loss of activity of bFGF.

[0020] S05: Reaction liquid b was loaded into a dialysis bag with a molecular weight cut-off of 15 kDa, and PBS with a final concentration of 0.1% BSA (bovine albumin) was used as the dialysis liquid. The reaction liquid b was dialyzed at 4°C for 72 h, and the PBS was replaced every 6 h to facilitate the removal of free bFGF and unreacted small molecules by dialysis. After lyophilization, white powder PEG-PDLLA-bFGF conjugate was obtained. Among them, BSA can protect the activity of bFGF.

[0021] Selenocysteine, as a key antioxidant, can protect cells from oxidative stress damage and improve cell viability and functional stability. After the synergistic use of selenocysteine and α-ketobutyric acid, α-ketobutyric acid, as an important intermediate of cell metabolism, can promote energy metabolism and amino acid synthesis, support cell metabolic balance and growth.

[0022] BIX01294 is an inhibitor of histone methyltransferase G9a, which promotes muscle stem cell fate remodeling by regulating epigenetic modification. Entinostat is a histone deacetylase inhibitor, which can synergistically regulate chromatin structure and gene expression when compounded with BIX01294 at a molar ratio of 3:1, promoting the activation of cell differentiation potential.

[0023] The Fc-TβRIII fusion protein can reduce the inhibition of cell proliferation and differentiation by binding and neutralizing TGF-β signal, and help the expansion and function maintenance of muscle stem cells. Mstn-NP, as a myostatin-related peptide, can regulate the growth inhibition mechanism of muscle cells. After Mstn-NP is compounded with Icaritin at a molar ratio of 4:1, Icaritin has the effects of anti-inflammatory, anti-oxidation and promoting the proliferation and differentiation of skeletal muscle cells, and further promotes the growth and repair of muscle stem cells. Jasplakinolide is a natural cyclic peptide extracted from marine sponges, which has the effects of inducing actin polymerization and stabilizing actin filaments, can stabilize actin in the cytoskeleton, regulate cell morphology and migration ability, and is beneficial to the maintenance of cell function and the formation of tissue structure.

[0024] In the present application, the directional expansion of muscle stem cells is effectively promoted by reasonably combining various growth factors, nutrients and signal pathway regulators, the cell proliferation speed is improved, the cell stemness and directional differentiation ability are maintained, the components are clear, the quality is stable, the defects of traditional serum-containing culture medium are overcome, and the present application has a broad application prospect in the field of muscle stem cell-related research and application.

[0025] More preferably, the serum-free culture medium in the present application comprises, by final concentration: PEG-PDLLA-IGF-1 conjugate 50 ng / ml, PEG-PDLLA-bFGF conjugate 0.1% w / v, seleno-cysteine 5 nM, α-ketobutyric acid 1 mM, BIX01294 1 μM, Entinostat 0.5 μM, Fc-TβRIII fusion protein 100 ng / mL, Mstn-NP 2 μg / ml, Jasplakinolide 0.2 nM, and Icaritin 6 μM.

[0026] The present application also provides a preparation method of the serum-free culture medium for promoting the directional expansion of muscle stem cells, which comprises: S01: Dissolve DMEM / F12 dry powder in water for injection to obtain a DMEM / F12 culture medium. While stirring, sequentially add PEG-PDLLA-IGF-1 conjugate, PEG-PDLLA-bFGF conjugate, seleno-cysteine, α-ketobutyric acid, BIX01294, Entinostat, Fc-TβRIII fusion protein, Mstn-NP, Jasplakinolide and Icaritin into the DMEM / F12 culture medium, respectively, to dissolve and mix each component to obtain a mixture. The addition interval of PEG-PDLLA-IGF-1 conjugate and PEG-PDLLA-bFGF conjugate is ≥5 min.

[0027] More preferably, the mass ratio of the PEG-PDLLA-IGF-1 conjugate and the PEG-PDLLA-bFGF conjugate is 1:1000. The synergistic molar concentration ratio of selenocysteine and alpha-ketobutyric acid is 1:75, the synergistic molar concentration ratio of BIX01294 Entinostat is 3:1, and the synergistic molar concentration ratio of icariin and Mstn-NP is 4:1.

[0028] S02: After adjusting the pH value of the mixture to 7.2-7.4 by using 1M HCl or 1M NaOH, sterilization is removed by using a sterile filter membrane with a pore size of 0.22 μm, and the filtrate is the serum-free medium.

[0029] The present application has the following beneficial effects: (1) The conjugate design enhances the efficacy of growth factors: the conjugate structure of PEG-PDLLA and IGF-1, bFGF can reduce the risk of bFGF oxidation through the carrier effect of PEG-PDLLA, avoid the inactivation of IGF-1 under acidic conditions, achieve the purpose of protecting the activity of growth factors, and then prolong the half-life of growth factors, reduce the degradation of free growth factors, and thus enhance the efficiency of promoting muscle stem cell proliferation.

[0030] (2) Multi-component synergistic effect, directional promotion of expansion: each component forms a synergistic effect through specific synergistic ratio, multi-dimensional synergies from cell signal regulation, epigenetic modification, metabolic support, and cytoskeleton stabilization, directional promotion of muscle stem cell expansion, while inhibiting abnormal differentiation and maintaining stem cell characteristics.

[0031] (3) Combination of natural ingredients and synthetic ingredients, considering efficacy and safety: natural ingredient icariin and synthetic ingredient Mstn-NP synergistically act in a specific ratio to enhance the expansion effect while possibly reducing the potential toxicity of chemical synthetic substances and improving the biocompatibility of the culture medium.

[0032] (4) Serum-free culture, avoiding the drawbacks of serum: using a serum-free system avoids the problems of complex serum components, large batch differences, possible pathogen or xenogeneic protein carrying in traditional serum-containing culture medium, improves the safety and stability of muscle stem cell culture, and facilitates precise regulation of culture conditions.

[0033] (5) Adapt to the needs of clinical transformation: the serum-free system reduces the introduction of xenogeneic proteins, reducing the risk of immunogenicity; the controllable ingredients are free of potential contaminants, making the cultured muscle stem cells more suitable for the safety requirements of clinical applications, providing high-quality cell sources for cell therapy fields such as muscle injury repair and myopathy treatment.

[0034] (6) The components are clear and controllable, and the culture repeatability is improved: the types, concentrations and ratios of each component in the culture medium are clearly defined, and the uniformity of the components is ensured through specific addition sequence, pH adjustment and sterile filtration process. The operation is controllable, which greatly improves the repeatability of muscle stem cell culture experiments, facilitates standardized research and application, provides a feasible technical support for the large-scale expansion of muscle stem cells, and meets the demand for a large number of high-quality muscle stem cells in scientific research and clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A comparison chart of the proliferation effects of muscle stem cells cultured by the A, B and C groups of culture media in the embodiments of the present application; Figure 2 A comparison chart of the expression effects of muscle stem cell marker PAX7 cultured by the A, B and C groups of culture media in the embodiments of the present application; Figure 3 A comparison chart of the expression results of muscle stem cell myosin heavy chain cultured by the A, B and C groups of culture media in the embodiments of the present application. DETAILED DESCRIPTION

[0036] The technical solutions of the present application will be further explained and described below through specific embodiments.

[0037] Embodiment 1 The embodiments of the present application provide a serum-free culture medium for promoting directional expansion of muscle stem cells. The culture medium takes DMEM / F12 medium as a substrate and includes, according to the final concentration: PEG-PDLLA-IGF-1 conjugate 50 ng / ml, PEG-PDLLA-bFGF conjugate 0.1% w / v, seleno-cysteine 5 nM, alpha-ketobutyric acid 1 mM, BIX01294 1 μM, Entinostat 0.5 μM, Fc-TβRIII fusion protein 100 ng / mL, Mstn-NP 2 μg / ml, Jasplakinolide 0.2 nM, Icariin 6 μM.

[0038] The present application also provides a preparation method of the serum-free culture medium for promoting directional expansion of muscle stem cells. The method comprises: S101: Dissolve DMEM / F12 dry powder in water for injection to obtain DMEM / F12 culture medium. Add PEG-PDLLA-IGF-1 conjugate, PEG-PDLLA-bFGF conjugate, seleno-cysteine, a-ketobutyric acid, BIX01294, Entinostat, Fc-TβRIII fusion protein, Mstn-NP, jasplakinolide and icariin to the DMEM / F12 culture medium in sequence under stirring, dissolve and mix each component to obtain a mixture. The interval time of adding PEG-PDLLA-IGF-1 conjugate and PEG-PDLLA-bFGF conjugate is 5 min.

[0039] The preparation method of the PEG-PDLLA-IGF-1 conjugate comprises: S1011: Under nitrogen protection, dissolve 1 mmol polyethylene glycol and 5 mmol polylactic acid monomer in 10 ml dichloromethane, then add 0.006 mmol stannous octoate, and stir at 60°C for 24 h to obtain reaction liquid a.

[0040] S1012: After the reaction is completed, drop the reaction liquid into excessive anhydrous ethanol for precipitation, centrifuge at a rotation speed of 3000 rpm for 10 min, freeze, and vacuum dry to obtain the PEG-PDLLA copolymer.

[0041] S1013: Dissolve 1 g PEG-PDLLA copolymer, 0.2 g succinimidyl propionate, 0.15 g N,N'-dicyclohexyl carbodiimide and 0.08 g N-hydroxysuccinimide in 20 ml dichloromethane, stir at room temperature and avoid light for 12 h, drop the filtrate after removing the by-product dicyclohexyl urea by filtration into excessive anhydrous ethanol for precipitation, centrifuge, freeze and vacuum dry to obtain the PEG-PDLLA-SPA.

[0042] S1014: Dissolve 1 mg IGF-1 in 1 ml PBS (phosphate buffered saline) to form an IGF-1 / PBS solution. Dissolve 80 mg PEG-PDLLA-SPA in 5 ml PBS to form a PEG-PDLLA-SPA / PBS solution. Under the condition of magnetic stirring at a pH value of 7.2-7.4, slowly add the IGF-1 / PBS solution to the carrier PEG-PDLLA-SPA / PBS solution, react at 25°C and avoid light for 4 h to obtain reaction liquid b. The excessive carrier PEG-PDLLA-SPA / PBS solution can improve the conjugation rate of IGF-1, and the neutral environment with a pH value of 7.2-7.4 can avoid destroying the activity of IGF-1.

[0043] S1015: The reaction liquid b is loaded into a dialysis bag with a molecular weight cut-off of 10 kDa, and PBS with a pH value of 7.4 is used as the dialysis liquid. The reaction liquid b is dialyzed at 4°C for 48 h, and the PBS is replaced every 6 h to remove free IGF-1 and unreacted small molecules by dialysis. After lyophilization, the PEG-PDLLA-IGF-1 conjugate in the form of white powder is obtained.

[0044] The preparation method of the PEG-PDLLA-bFGF conjugate includes: S1021: Under the protection of nitrogen, 1 mmol of polyethylene glycol and 5 mmol of polylactic acid monomer are dissolved in 10 ml of dichloromethane, and then 0.006 mmol of stannous octoate is added. The reaction is stirred at 60°C for 24 h to obtain reaction liquid a.

[0045] S1022: After the reaction is completed, the reaction liquid is dropped into excess anhydrous ethanol for precipitation. Centrifugation is performed at a rotation speed of 3000 rpm for 10 min, and then the sample is frozen, vacuum dried, and PEG-PDLLA copolymer is obtained.

[0046] S1023: 1 g of PEG-PDLLA copolymer, 0.2 g of succinimidyl propionate, 0.15 g of N,N'-dicyclohexyl carbodiimide, and 0.08 g of N-hydroxysuccinimide are dissolved in 20 ml of dichloromethane. The solution is stirred at room temperature in the dark for 12 h. After filtering to remove the byproduct dicyclohexyl urea, the filtrate is dropped into excess anhydrous ethanol for precipitation. Centrifugation, freezing, and vacuum drying are performed to obtain PEG-PDLLA-SPA.

[0047] S1024: 1 mg of bFGF is dissolved in 1 ml of PBS to form a bFGF / PBS solution. 100 mg of PEG-PDLLA-SPA is dissolved in 5 ml of PBS to form a PEG-PDLLA-SPA / PBS solution. Under the conditions of magnetic stirring and nitrogen filling, the bFGF / PBS solution is slowly added to the carrier PEG-PDLLA-SPA / PBS solution. The reaction is carried out at 20°C in the dark for 6 h to obtain reaction liquid b. The stability of bFGF is poor, and the reaction at a low temperature of 20°C can reduce the loss of activity and improve the activity of the PEG-PDLLA-bFGF conjugate. In addition, the nitrogen environment can avoid the oxidation of bFGF, further reducing the loss of activity of bFGF.

[0048] S1025: The reaction solution b is loaded into a dialysis bag with a molecular weight cut-off of 15 kDa, and PBS with a final concentration of 0.1% BSA (bovine albumin) is used as the dialysis solution. The reaction solution b is dialyzed at 4°C for 72 hours, and the PBS is replaced every 6 hours to remove free bFGF and unreacted small molecules by dialysis. After lyophilization, the PEG-PDLLA-bFGF conjugate is obtained as a white powder. The BSA can protect the activity of bFGF.

[0049] S102: After adjusting the pH value of the mixture to 7.3 with 1M HCl, sterilization is performed by filtering with a sterile filter membrane with a pore size of 0.22μm, and the filtrate is the serum-free culture medium.

[0050] Example 2 The serum-free culture medium for promoting directional expansion of muscle stem cells provided by the embodiments of the present application is based on DMEM / F12 medium, and includes PEG-PDLLA-IGF-1 conjugate 30ng / ml, PEG-PDLLA-bFGF conjugate 0.5% w / v, seleno-cysteine 3nM, α-ketobutyric acid 1.5mM, BIX01294 1.5μM, Entinostat 0.6μM, Fc-TβRIII fusion protein 80ng / mL, Mstn-NP 3μg / ml, Jasplakinolide 0.1nM, and Icariin 8μM.

[0051] The embodiments of the present application also provide a preparation method of the serum-free culture medium for promoting directional expansion of muscle stem cells, which is the same as that in Example 1.

[0052] Example 3 The serum-free culture medium for promoting directional expansion of muscle stem cells provided by the embodiments of the present application is based on DMEM / F12 medium, and includes PEG-PDLLA-IGF-1 conjugate 30ng / ml, PEG-PDLLA-bFGF conjugate 0.5% w / v, seleno-cysteine 3nM, α-ketobutyric acid 1.5mM, BIX01294 1.5μM, Entinostat 0.6μM, Fc-TβRIII fusion protein 80ng / mL, Mstn-NP 3μg / ml, Jasplakinolide 0.1nM, and Icariin 8μM.

[0053] The embodiments of the present application also provide a preparation method of the serum-free culture medium for promoting directional expansion of muscle stem cells, which is the same as that in Example 1.

[0054] Example 4 The embodiment of the application provides a serum-free culture medium for promoting directional expansion of muscle stem cells, which takes DMEM / F12 culture medium as a matrix and comprises the following components in a final concentration: PEG-PDLLA-IGF-1 conjugate 50 ng / ml, PEG-PDLLA-bFGF conjugate 0.05% w / v, seleno-cysteine 3.5 nM, alpha-ketobutyric acid 1.1 mM, BIX01294 2 muM, Entinostat 0.5 muM, Fc-TbetaRIII fusion protein 90 ng / mL, Mstn-NP 2 mu g / ml, Jasplakinolide 0.15 nM and Icariin 5 muM.

[0055] The embodiment of the application also provides a preparation method of the serum-free culture medium for promoting directional expansion of muscle stem cells, which is the same as the method in the embodiment 1.

[0056] Example 5 The embodiment of the application provides a serum-free culture medium for promoting directional expansion of muscle stem cells, which takes DMEM / F12 culture medium as a matrix and comprises the following components in a final concentration: PEG-PDLLA-IGF-1 conjugate 20 ng / ml, PEG-PDLLA-bFGF conjugate 0.5% w / v, seleno-cysteine 3 nM, alpha-ketobutyric acid 1.5 mM, BIX01294 0.5 muM, Entinostat 0.8 muM, Fc-TbetaRIII fusion protein 80 ng / mL, Mstn-NP 3 mu g / ml, Jasplakinolide 0.1 nM and Icariin 10 muM.

[0057] The embodiment of the application also provides a preparation method of the serum-free culture medium for promoting directional expansion of muscle stem cells, which is the same as the method in the embodiment 1.

[0058] Comparative Example 1 The comparative example of the application provides a serum-free culture medium for promoting directional expansion of muscle stem cells, which takes DMEM / F12 culture medium as a matrix and comprises the following components in a final concentration: FBS 10%, and PS (Phosphatidylserine) 1%.

[0059] The preparation method of the serum-free culture medium comprises the following steps: D101: DMEM / F12 dry powder is dissolved in water for injection to obtain DMEM / F12 culture medium. FBS 10% and PS 1% are sequentially added into the DMEM / F12 culture medium, and the mixture is uniformly stirred to obtain a mixture.

[0060] D102: After the pH value of the mixture is adjusted to 7.3 by using 1M HCl, the mixture is filtered and sterilized by using a sterile filter membrane with a pore size of 0.22 mu m, and the filtrate is the serum-free culture medium.

[0061] Comparative Example 2 The commercially available serum-free medium STEMPRO® was used as Comparative Example 2.

[0062] The serum-free medium in Example 1, the conventional serum-containing medium, and the commercially available serum-free medium in Comparative Example 2 were used to culture muscle stem cells respectively, and the cultured muscle stem cells were subjected to cell proliferation ability detection, stem cell property detection, and differentiation ability detection. The following will be described in detail respectively.

[0063] Muscle stem cell culture: The primary muscle stem cells were inoculated in a culture dish at a density of 1×10^4 / cm 2 , and the prepared serum-free medium was added. The culture was placed in a 37°C, 5% CO2 incubator for culture. The culture medium was replaced every 2-3 days, and the cell growth was observed and counted.

[0064] 1. CCK8 detection of the proliferation ability of muscle stem cells The muscle stem cells cultured to P3 were suspended in the culture medium to prepare a cell suspension. The cell suspension was inoculated in a 96-well plate at a density of 2×10 4 / cm, and 100 μL of the cell suspension was inoculated in each well. The inoculated culture plate was placed in an incubator for pre-culture at 37°C, 5% CO2 for 4 h. The pre-cultured cell suspension was divided into A, B, and C groups, with 3 repeated wells in each group.

[0065] After the pre-culture was completed, the culture medium was discarded. The serum-free medium in Example 1 was added to the cell suspension in group A, an equal volume of the conventional serum-containing medium was added to the cell suspension in group B, and an equal volume of the commercially available serum-free medium in Comparative Example 2 was added to the cell suspension in group C. The conventional serum-containing medium was VivaCel from Shanghai Dartxill Biotech Co., Ltd., with batch number 2452576. The cell suspensions in groups A, B, and C were all placed in an incubator for culture at 37°C, 5% CO2 for 48 h. After the culture was completed, 10 ul of CCK-8 reagent was added to each well, and the culture was continued for 2 h. The absorbance value at 450 nm was detected by using an enzyme label instrument to determine the cell proliferation rate, and the results are shown in Table 1. Figure 1 .

[0066] The results in Table 1 show that the serum-free medium in Example 1 has the best effect on the proliferation of muscle stem cells, and the commercially available serum-free medium in Comparative Example 2 has the worst effect on the proliferation of muscle stem cells. Figure 1It can be seen that, during the culture of muscle stem cells, the OD value of the cells in group A is above 0.75, the OD value of the cells in group B is about 0.55, and the OD value of the cells in group C is about 0.52. This shows that the serum-free culture medium prepared in Example 1 of the present application can effectively promote the proliferation of muscle stem cells and keep the cells in a high activity. This shows that the addition of the PEG-PDLLA-IGF-1 conjugate and the PEG-PDLLA-bFGF conjugate in the serum-free culture medium simultaneously activates the IGF-1 / PI3K / Akt and bFGF / ERK pathways, effectively promoting the proliferation and survival of muscle stem cells.

[0067] 2. Immunofluorescence detection of the expression of muscle stem cell stemness marker PAX7 The muscle stem cells in groups A, B and C cultured with different culture media were taken, trypsinized, and adjusted to a cell concentration of 1 x 10 5 / ml, and then added dropwise to a 6-well plate for adhesion to a glass slide and cultured for 24 h. After the culture, the cell culture plate was taken out, the culture solution was aspirated, and the plate was washed with PBS for 3 times. Then, 4% paraformaldehyde was added for room temperature fixation for 20 min. After the fixation, the plate was washed with PBS for 3 times, and then 0.1% Triton X-100 was added for membrane permeation for 10 min. The plate was washed with PBS for 3 times, and then 5% BSA was added for blocking for 30 min. After the blocking, the primary antibody Pax7 was added at a volume ratio of 1:500, and the plate was incubated at 4°C overnight. After the incubation, the plate was washed with PBS for 3 times, and then the secondary antibody Alexa Fluor 488 was added at a volume ratio of 1:10000, and the plate was incubated at 37°C for 1 h. After the incubation, the plate was washed with PBS for 3 times, and then DAPI (4', 6-diamidino-2-phenylindole) was added for nuclear staining for 15 min. The plate was washed with PBS for 3 times, and then photographed by a fluorescence microscope to obtain the attached TM fluorescence images. Figure 2 .

[0068] The attached Figure 2It can be seen that the PAX7 expression of the muscle stem cells in group A is strong, and the fluorescence intensity is about 90%; the PAX7 expression of the muscle stem cells in group B is weakened, and is about 58%; and the PAX7 expression of the muscle stem cells in group C is weakened, and is about 70%. This shows that the serum-free culture medium prepared in Example 1 of the present application can effectively enhance the expression of the PAX7 marker. This shows that BIX01294 in the serum-free culture medium provided in Example 1 of the present application can reduce the histone H3K9 dimethylation in the PAX7 gene promoter region, and play an inhibitory modification role; at the same time, it can also remove the silencing of the PAX7 gene, and directly promote the transcription and expression thereof; the addition of Entinostat can increase the histone acetylation level in the PAX7 gene promoter region by inhibiting class I histone deacetylase HDAC1 / 3, and play an activating modification role, thereby enhancing the binding ability of the transcription factor and the promoter. As a result, the synergistic effect of BIX01294 and Entinostat can effectively maintain the undifferentiated state of the muscle stem cells, inhibit the premature differentiation thereof, and thereby enhance the stemness expression thereof.

[0069] 3. Detection of myogenic gene expression by qRT-PCR The muscle stem cells in groups A, B and C cultured by using different culture media were taken, and the RNAs of the muscle stem cells in the three groups were extracted. After reverse transcription was performed by using a 20 μL reverse transcription system, qPCR amplification reaction was performed for detection, and the results shown in FIG. 6 were obtained. Figure 3 .

[0070] The process of extracting the RNA is as follows: 1 mL of TRIzol reagent was added to each well TM The muscle stem cells were lysed by using the reagent, and chloroform was added for layering treatment, and the upper water phase was taken. Isopropanol was added to the upper water phase to precipitate the RNA, and the RNA precipitate was washed with 75% ethanol, and the supernatant was discarded. After the ethanol was evaporated, the RNA was dissolved in DEPC (diethyl pyrocarbonate, diethyl pyrocarbonate) water. The purity and concentration of the RNA were determined by using a Nanodrop TM The A260 / A280 ratio was required to be between 1.8 and 2.0, and the concentration was required to be ≥200 ng / μL.

[0071] The reverse transcription system: the RNA was pre-denatured at 65°C for 5 min, and then immediately placed on ice; the components and contents of the reverse transcription system were as follows: 4 μL of 4×DN Master Mix, 0.5 pg-0.5 ug of RNA, the total volume was 16 μL, the rest was supplemented with RNase-free water, and the RNA was added according to the concentration of different samples; each component was mixed, and the reaction was performed at 37°C for 5 min; 4 μL of 5×RT Master Mix II was added and mixed, and the final volume was 20 μL.

[0072] Reverse transcription procedure: 37℃ 15min→ 98℃ 5min→ 4℃ 5min.

[0073] The myogenesis marker primer sequences for qPCR amplification are shown in Table 1: Table 1: Myogenesis marker primer sequences for qPCR amplification qPCR amplification system: 10 μL PCR amplification premix solution SYBR Green Master, 2 μL Mix 10 μL cDNA template, 0.8 μL 10 μM forward primer F, 0.8 μL 10 μM reverse primer R, 6.4 μL RNase-free water.

[0074] qPCR amplification procedure: Pre-denaturation: 95℃ for 30 seconds; PCR cycle: 95℃ denaturation for 5 seconds, then annealing for 30 seconds according to the annealing temperature of each primer pair, and cycling for 40 times; Melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, 95℃ for 15 seconds.

[0075] The results are shown in Figure 2. Figure 3 It can be seen that the RNA relative expression amounts of the myogenesis genes MyhC, Myogenin and Myod of the muscle stem cells cultured in group A are about 1, 1.4 and 1.8 respectively; the RNA relative expression amounts of the myogenesis genes MyhC, Myogenin and Myod of the muscle stem cells cultured in group B are about 0.5, 0.3 and 1.1 respectively; and the RNA relative expression amounts of the myogenesis genes MyhC, Myogenin and Myod of the muscle stem cells cultured in group C are about 0.6, 0.8 and 1.3 respectively. This shows that the serum-free culture medium provided in Example 1 of the present application can more effectively promote the expression of muscle stem cell myogenesis-related genes and maintain the myogenic potential of muscle stem cells. This shows that the serum-free culture medium provided in Example 1 of the present application has more significant advantages in promoting the expression of muscle stem cell myogenesis genes. Its TβRIII is a transforming growth factor β family receptor, and the fusion protein can competitively bind to TGF-β superfamily members that inhibit myogenesis, such as myostatin, to relieve its inhibition on myogenesis and promote muscle stem cell proliferation. At the same time, the myostatin nanobody Mstn-NP can specifically neutralize myostatin and block its signal of inhibiting muscle cell proliferation, enhance the myogenic effect, and effectively promote the expression of MyhC, Myogenin and Myod.

[0076] In summary, in the serum-free culture medium for promoting directional expansion of muscle stem cells provided by the embodiments of the present application, the PEG-PDLLA-IGF-1 conjugate and the PEG-PDLLA-bFGF conjugate are modified by polymers to prolong the half-life, continuously activate the PI3K / Akt and MAPK pathways to promote proliferation, and support the cell microenvironment through signal regulation and extracellular matrix synthesis, respectively; the selenocysteine enhances the antioxidant capacity, and the alpha-ketobutyric acid provides energy metabolism substrates, which together maintain cell homeostasis; BIX01294 and Entinostat synergistically activate PAX7 and other stemness genes through demethylation and acetylation epigenetic modification, and inhibit abnormal differentiation; the Fc-TβRIII fusion protein and the Mstn-NP block the negative regulation signals of TGF-β and myostatin, respectively, and relieve the inhibition of myogenesis; the jasplakinolide stabilizes the cytoskeleton, enhances adhesion and morphology maintenance; the icariin synergistically promotes the balance of proliferation and differentiation through natural activity, and through the above multi-dimensional synergistic effects, the survival, proliferation and stemness maintenance of muscle stem cells are precisely regulated. At the same time, the components in the serum-free culture medium act in a specific ratio and order to form a complete network of "proliferation drive-stemness maintenance-metabolic support-negative regulation relief", which can realize the directional and efficient expansion of muscle stem cells.

[0077] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A serum-free culture medium for promoting directed expansion of muscle stem cells, characterized in that: Using basal culture medium as the matrix, the final concentrations include: PEG-PDLLA-IGF-1 conjugate 20-80 ng / ml, PEG-PDLLA-bFGF conjugate 0.05-0.5% w / v, selenocysteine ​​3-5 nM, α-ketobutyrate 1-1.5 mM, BIX01294 0.5-2 μM, Entinostat 0.3-0.8 μM, Fc-TβRIII fusion protein 80-120 ng / mL, Mstn-NP 1-3 μg / ml, Jasplakinolide 0.1-0.3 nM, and icariin 5-10 μM.

2. The serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 1, characterized in that The final concentrations included: PEG-PDLLA-IGF-1 conjugate 50 ng / ml, PEG-PDLLA-bFGF conjugate 0.1% w / v, selenocysteine ​​5 nM, α-ketobutyrate 1 mM, BIX01294 1 μM, Entinostat 0.5 μM, Fc-TβRIII fusion protein 100 ng / mL, Mstn-NP 2 μg / ml, Jasplakinolide 0.2 nM, and icariin 6 μM.

3. The serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 1, characterized in that The preparation method of the PEG-PDLLA-IGF-1 conjugate comprises: Under nitrogen protection, polyethylene glycol and polylactic acid monomers were dissolved in dichloromethane, and stannous octoate was added. The mixture was stirred and reacted at 60°C for 24 hours to obtain reaction solution a. The reaction solution was added dropwise to excess anhydrous ethanol for precipitation, and the PEG-PDLLA copolymer was obtained by centrifugation, freezing, and drying; The PEG-PDLLA copolymer, succinimidyl propionate, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide were dissolved in dichloromethane, stirred at room temperature in the dark for 12 hours, and the filtered filtrate was dropped into excess anhydrous ethanol for precipitation, centrifuged, frozen, and dried to obtain PEG-PDLLA-SPA; The PEG-PDLLA-SPA was dissolved in PBS, and IGF-1 was slowly added under magnetic stirring at a pH of 7.2-7.4, and the mixture was reacted at 25° C. in the dark for 4 hours to obtain a reaction solution b; The reaction solution b was dialyzed in PBS at pH 7.4 and 4° C. for 48 h using a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysate was lyophilized to obtain the PEG-PDLLA-IGF-1 conjugate.

4. The serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 1, characterized in that The preparation method of the PEG-PDLLA-bFGF conjugate comprises: Under nitrogen protection, polyethylene glycol and polylactic acid monomers were dissolved in dichloromethane, and stannous octoate was added. The mixture was stirred and reacted at 60°C for 24 hours to obtain reaction solution a. The reaction solution was added dropwise to excess anhydrous ethanol for precipitation, and the PEG-PDLLA copolymer was obtained by centrifugation, freezing, and drying; The PEG-PDLLA copolymer, succinimidyl propionate, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide were dissolved in dichloromethane, stirred at room temperature in the dark for 12 hours, and the filtered filtrate was dropped into excess anhydrous ethanol for precipitation, centrifuged, frozen, and dried to obtain PEG-PDLLA-SPA; The PEG-PDLLA-SPA was dissolved in PBS, bFGF was slowly added under a nitrogen atmosphere and magnetic stirring at a pH of 7.2-7.4, and the mixture was reacted at 20° C. in the dark for 6 h to obtain a reaction solution b; The reaction solution b was dialyzed in PBS containing 0.1% BSA at 4° C. for 72 h using a dialysis bag with a molecular weight cutoff of 15 kDa. The dialyzate was lyophilized to obtain the PEG-PDLLA-bFGF conjugate.

5. The serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 1, characterized in that The basic culture medium is DMEM / F12 culture medium.

6. A method for preparing a serum-free culture medium for promoting directed expansion of muscle stem cells, characterized in that: include: sequentially adding PEG-PDLLA-IGF-1 conjugate, PEG-PDLLA-bFGF conjugate, selenocysteine, α-ketobutyric acid, BIX01294, Entinostat, Fc-TβRIII fusion protein, Mstn-NP, Jasplakinolide, and icariin to the basal culture medium, dissolving the mixture, and mixing the mixture to obtain a mixture; After adjusting the pH value of the mixture to 7.2-7.4, the mixture was filtered through a sterile filter membrane with a pore size of 0.22 μm to obtain a serum-free culture medium.

7. The method for preparing a serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 6, wherein: The mass ratio of the PEG-PDLLA-IGF-1 conjugate to the PEG-PDLLA-bFGF conjugate is 1:1000, and the addition interval is ≥5 min.

8. The method for preparing a serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 6, wherein: The synergistic molar concentration ratio of the selenocysteine ​​and the α-ketobutyrate is 1:

75.

9. The method for preparing a serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 6, wherein: The synergistic molar concentration ratio of the BIX01294 and the Entinostat is 3:

1.

10. The method for preparing a serum-free culture medium for promoting directed expansion of muscle stem cells according to claim 6, wherein: The synergistic molar concentration ratio of the icariin and the Mstn-NP is 4:1.

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