Culture medium and culture method for promoting stem cells to be differentiated into myocardial cells

Abstract

The invention discloses a culture medium for promoting stem cells to differentiate into myocardial cells. The culture medium comprises the following components: a basic culture solution, growth factors, extracellular matrix components, inorganic salt, vitamin C, vitamin E, an antioxidant, glucose, sodium pyruvate, amino acid and water. The invention also provides a culture method for promoting stem cells to be differentiated into myocardial cells by using the culture medium. According to the culture medium provided by the invention, by optimizing the components and the concentration, the differentiation efficiency of stem cells to myocardial cells can be remarkably improved, and meanwhile, good cell activity and functions are kept.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium and a culture method for promoting stem cells to differentiate into cardiomyocytes. The culture medium can effectively induce stem cells to differentiate into cardiomyocytes, providing a new technical means for the treatment of heart diseases and regenerative medicine research. Background Art

[0002] Stem cells have the potential for self-renewal and multidirectional differentiation and are an important tool for tissue engineering and regenerative medicine research. Cardiomyocyte damage or dysfunction is the main cause of heart disease. Therefore, the development of a culture medium that can effectively promote the differentiation of stem cells into cardiomyocytes has important clinical significance. However, existing culture media have problems such as low efficiency and poor stability in promoting the differentiation of stem cells into cardiomyocytes, and they are in urgent need of improvement. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a culture medium and a culture method that can effectively promote the differentiation of stem cells into cardiomyocytes. The culture medium can significantly improve the differentiation efficiency of stem cells into cardiomyocytes by optimizing the components and concentrations, while maintaining good cell activity and function.

[0004] The culture medium of the present invention comprises the following components:

[0005] Basic culture medium: DMEM / F12 mixed solution, which provides essential nutrients and growth factors for stem cells while maintaining appropriate pH and osmotic pressure.

[0006] Growth factors: recombinant human insulin-like growth factor-1 (rhIGF-1) and recombinant human fibroblast growth factor-2 (rhFGF-2), these two growth factors can promote the proliferation and differentiation of stem cells.

[0007] Extracellular matrix components: recombinant human laminin (rhLaminin) and recombinant human fibronectin (rhFibronectin), simulate the environment of the extracellular matrix, which helps the adhesion and directional differentiation of stem cells.

[0008] Inorganic salts: potassium chloride, magnesium sulfate, calcium chloride. Inorganic salts maintain the ionic balance of the culture medium and are essential for the physiological functions of cells.

[0009] Vitamin C, Vitamin E: Antioxidant vitamins can protect cells from damage caused by oxidative stress.

[0010] Antioxidants: N-acetylcysteine ​​(NAC), NAC is a potent antioxidant that scavenges free radicals and reduces cell damage.

[0011] Glucose and sodium pyruvate: These two energy substances can provide the necessary energy for cell growth and metabolism.

[0012] Amino acids: arginine, glutamine and cysteine, amino acids are essential precursors for cells to synthesize proteins and other biological macromolecules.

[0013] Component concentration:

[0014] DMEM / F12 mixed solution: The content is 90-95% of the total volume of the culture medium.

[0015] Recombinant human insulin-like growth factor-1 (rhIGF-1): 10-50 ng / mL, preferably 30 ng / mL.

[0016] Recombinant human fibroblast growth factor-2 (rhFGF-2): 5-20 ng / mL, preferably 10 ng / mL.

[0017] Recombinant human laminin (rhLaminin): 1-10 μg / mL, preferably 5 μg / mL.

[0018] Recombinant human fibronectin (rhFibronectin): 5-20 μg / mL, preferably 10 μg / mL.

[0019] Vitamin C: 50-150 μM, preferably 100 μM.

[0020] Vitamin E: 10-50 μM, preferably 30 μM.

[0021] Glucose: 1-4.5 g / L, preferably 3.5 g / L.

[0022] Sodium pyruvate: 1-5 mM, preferably 3 mM.

[0023] N-acetylcysteine ​​(NAC): 1-10 mM, preferably 5 mM.

[0024] Arginine: 0.1-1 mM, preferably 0.5 mM.

[0025] Glutamine: 0.1-3 mM, preferably 2 mM.

[0026] Cysteine: 0.05-0.5 mM, preferably 0.25 mM.

[0027] Cultivation method:

[0028] S1: Inoculate stem cells (embryonic stem cells or induced pluripotent stem cells) into the culture medium at a density of 10^4 to 10^6 cells per square centimeter.

[0029] S2: Stem cells are cultured in an incubator at 37°C and 5% CO2. The humidity and gas concentration in the incubator are crucial to the growth and differentiation of cells.

[0030] S3: During the culture process, the culture medium is replaced every 24 hours to ensure that the cells obtain sufficient nutrients and growth factors while removing metabolic waste.

[0031] S4: After 7-14 days of culture, the differentiation of cardiomyocytes can be evaluated by immunofluorescence staining or electrophysiological testing. Immunofluorescence staining can detect cardiomyocyte-specific markers, such as cardiac troponin T (cTnT) and cardiac troponin I (cTnI), while electrophysiological testing can evaluate the electrophysiological properties of cardiomyocytes, such as the morphology and duration of action potentials.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) Improving the efficiency of stem cell differentiation into cardiomyocytes: Through a carefully designed culture medium formula, including specific growth factors, extracellular matrix components, antioxidants and hormones, the culture medium can significantly improve the efficiency and quality of stem cell differentiation into cardiomyocytes.

[0034] (2) Enhance cell function: Vitamin C, vitamin E, and antioxidants such as N-acetylcysteine ​​(NAC) in the culture medium help protect cells from damage by oxidative stress while promoting cell metabolism and functional maturation.

[0035] (3) Precise control of cell differentiation process: By regulating the concentration of growth factors and hormones, such as recombinant human insulin-like growth factor-1 (rhIGF-1) and recombinant human fibroblast growth factor-2 (rhFGF-2), the differentiation process of stem cells into cardiomyocytes can be precisely controlled.

[0036] (4) Simplified operation procedures: The culture method includes simple operation steps, such as regular replacement of culture medium and cultivation under standard conditions, making the method easy to implement and promote in the laboratory.

[0037] In summary, the present invention provides a culture medium and method for differentiating stem cells into cardiomyocytes that is efficient, controllable and easy to operate, which has important scientific value and broad application prospects. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] 1. Prepare the culture medium:

[0041] According to the following formula, accurately weigh each component:

[0042] Recipe 1:

[0043] DMEM / F12 mixed solution: The content is 90% of the total volume of the culture medium.

[0044] Recombinant human insulin-like growth factor-1 (rhIGF-1): 10 ng / mL.

[0045] Recombinant human fibroblast growth factor-2 (rhFGF-2): 5ng / mL.

[0046] Recombinant human laminin (rhLaminin): 1 μg / mL.

[0047] Recombinant human fibronectin (rhFibronectin): 5 μg / mL.

[0048] Vitamin C: 50 μM.

[0049] Vitamin E: 10 μM.

[0050] Glucose: 1g / L.

[0051] Sodium pyruvate: 13 mM.

[0052] N-Acetylcysteine ​​(NAC): 1 mM.

[0053] Arginine: 0.1 mM.

[0054] Glutamine: 0.1 mM.

[0055] Cysteine: 0.05 mM.

[0056] Recipe 2:

[0057] DMEM / F12 mixed solution: 95%.

[0058] Recombinant human insulin-like growth factor-1 (rhIGF-1): 50 ng / mL. Recombinant human fibroblast growth factor-2 (rhFGF-2): 20 ng / mL. Recombinant human laminin (rhLaminin): 10 μg / mL.

[0059] Recombinant human fibronectin (rhFibronectin): 20 μg / mL.

[0060] Vitamin C: 150 μM.

[0061] Vitamin E: 50 μM.

[0062] Glucose: 4.5g / L.

[0063] Sodium pyruvate: 5 mM.

[0064] N-Acetylcysteine ​​(NAC): 10 mM.

[0065] Arginine: 1 mM.

[0066] Glutamine: 3 mM.

[0067] Cysteine: 0.5 mM.

[0068] Recipe 3:

[0069] DMEM / F12 mixed solution: content is 92% of the total volume of the culture medium. Recombinant human insulin-like growth factor-1 (rhIGF-1): 30 ng / mL. Recombinant human fibroblast growth factor-2 (rhFGF-2): 10 ng / mL. Recombinant human laminin (rhLaminin): 5 μg / mL.

[0070] Recombinant human fibronectin (rhFibronectin): 10 μg / mL.

[0071] Vitamin C: 100 μM.

[0072] Vitamin E: 30 μM.

[0073] Glucose: 3.5g / L.

[0074] Sodium pyruvate: 3 mM.

[0075] N-Acetylcysteine ​​(NAC): 5 mM.

[0076] Arginine: 0.5 mM.

[0077] Glutamine: 2 mM.

[0078] Cysteine: 0.25 mM.

[0079] Dissolve the above formula components separately in the DMEM / F12 mixed solution and ensure that they are fully mixed.

[0080] The culture medium was sterilized by filtration using a 0.22 μm filter membrane, and then dispensed into sterile culture bottles for later use.

[0081] 2. Stem cell vaccination:

[0082] Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) in a good growth state were seeded in a 6-well plate containing the above culture medium at a density of 60,000 per well.

[0083] 3. Culture conditions:

[0084] The seeded stem cells were placed at 37°C and 5% CO 2 Cultured in a constant temperature incubator.

[0085] During the culture process, freshly prepared culture medium is replaced every 24 hours to maintain a stable culture environment and sufficient nutrients.

[0086] 4. Differentiation assessment:

[0087] After 7 and 14 days of culture, the cell morphology was observed under a microscope, and myocardial-specific markers, such as cardiac troponin T (cTnT), cardiac troponin I (cTnI), and α-sarcomeric actinin, were detected by immunofluorescence staining and flow cytometry to evaluate the differentiation of cardiomyocytes.

[0088] The electrophysiological properties of differentiated cardiomyocytes were evaluated by patch clamp technique.

[0089] 5. Data recording and analysis:

[0090] The time of each culture medium change, cell morphology changes, expression levels of differentiation markers, and electrophysiological characteristics were recorded.

[0091] Data were analyzed using statistical software to determine optimal parameters for media formulation and culture conditions.

[0092] During the above operation, all steps should be carried out under sterile conditions to avoid contamination; the preparation and replacement of culture medium should be carried out strictly according to the prescribed concentration and time.

[0093] Cell morphology observation:

[0094] After 7 days of culture, it was observed under a microscope that the cell morphology gradually changed from round or polygonal to long spindle, which is a typical morphological feature of cardiomyocytes.

[0095] After 14 days of culture, it was observed that the cells were arranged more orderly, forming a structure similar to myocardial tissue, with obvious muscle fiber arrangement between cells.

[0096] Immunofluorescence staining results:

[0097] Immunofluorescence staining was performed using the myocardial specific markers cardiac troponin T (cTnT) and α-sarcomericactinin.

[0098] The results showed that after 7 days of culture, about 30% of the cells expressed cTnT, and after 14 days of culture, this proportion increased to 60%.

[0099] The expression of α-sarcomeric actinin also showed a similar trend, with about 25% of cells expressing it after 7 days and increasing to 55% after 14 days.

[0100] Flow cytometry detection:

[0101] Cell surface and internal marker expression were analyzed using flow cytometry.

[0102] The results showed that the proportion of positive cells for cardiomyocyte-specific markers increased with the increase of culture time, which was consistent with the results of immunofluorescence staining.

[0103] Electrophysiological testing:

[0104] The electrophysiological properties of cardiomyocytes were recorded using the patch clamp technique.

[0105] The results showed that after 14 days of culture, about 40% of the cells exhibited spontaneous action potentials.

[0106] The action potentials recorded had typical rising and falling phases, indicating that the cells had the electrophysiological characteristics of cardiomyocytes.

[0107] Statistical analysis:

[0108] Statistical software was used to analyze the above data and calculate the mean, standard deviation and confidence interval.

[0109] The results showed that the differentiation efficiency of cardiomyocytes increased significantly with the increase of culture time (p<0.05).

[0110] These data results show that by using this patented culture medium and culture method, stem cells can effectively differentiate into cells with cardiomyocyte characteristics, including morphology, molecular marker expression, and electrophysiological properties. These results provide strong support for subsequent regenerative medicine research and clinical applications.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The components and preparation methods involved in the present invention can be adjusted accordingly according to the implementation method, but shall not exceed the protection scope of the invention.

Claims

1. A culture medium for promoting differentiation of stem cells into cardiomyocytes, characterized in that: The culture medium comprises the following components: basic culture fluid, growth factors, extracellular matrix components, inorganic salts, vitamin C, vitamin E, antioxidants, glucose, sodium pyruvate, amino acids and water.

2. The culture medium according to claim 1, characterized in that The basic culture fluid is a DMEM / F12 mixed solution, and its content is 90-95% of the total volume of the culture medium; the growth factors are recombinant human insulin-like growth factor-1 (rhIGF-1) and recombinant human fibroblast growth factor-2 (rhFGF-2), and the inorganic salts are potassium chloride, magnesium sulfate, and calcium chloride.

3. The culture medium according to claim 1, characterized in that The extracellular matrix components are recombinant human laminin (rhLaminin) and recombinant human fibronectin (rhFibronectin); the antioxidant is N-acetylcysteine ​​(NAC); and the amino acids are arginine, glutamine and cysteine.

4. The culture medium according to claim 2, characterized in that The concentration of the recombinant human insulin-like growth factor-1 (rhIGF-1) is 10-50 ng / mL, and the concentration of the recombinant human fibroblast growth factor-2 (rhFGF-2) is 5-20 ng / mL.

5. The culture medium according to claim 3, characterized in that The concentrations of the recombinant human laminin (rhLaminin) and the recombinant human fibronectin (rhFibronectin) are 1-10 μg / mL and 5-20 μg / mL respectively.

6. The culture medium according to claim 1, characterized in that The content of vitamin C is 50-150 μM, the content of vitamin E is 10-50 μM, the content of glucose is 1-4.5 g / L, and the content of sodium pyruvate is 1-5 mM.

7. The culture medium according to claim 3, characterized in that The concentration of N-acetylcysteine ​​(NAC) is 1-10 mM, the content of arginine is 0.1-1 mM, the content of glutamine is 0.1-3 mM, and the content of cysteine ​​is 0.05-0.5 mM.

8. A method for promoting stem cells to differentiate into cardiomyocytes using the culture medium according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1, inoculating stem cells in the culture medium; S2, culturing stem cells in an incubator at 37°C and 5% CO2; S3, during the culture process, the culture medium was replaced every 24 h; S4, after 7-14 days of culture, observe and evaluate the differentiation of cardiomyocytes.

9. The culture method according to claim 8, characterized in that: The stem cells are embryonic stem cells or induced pluripotent stem cells.

10. The culture method according to claim 8, characterized in that: During the culture process, the differentiation of cardiomyocytes is evaluated by immunofluorescence staining or electrophysiological detection.