Myocardial cell in vitro maturation culture solution additive and application thereof

Abstract

The application provides a myocardial cell in-vitro maturation culture solution additive and application thereof, and belongs to the technical field of biology. The application provides an innovative chemical induction scheme aiming at the key problem of low maturity of human induced pluripotent stem cell derived myocardial cells (hiPSC-CMs) in in-vitro culture. The scheme combines the addition of benfotiamine and Mdivi-1 in the myocardial cell maturation culture solution, utilizes the synergistic effect of the two on optimizing cell metabolism and promoting mitochondrial function, and significantly drives the metabolic mode of myocardial cells from glycolysis to oxidative phosphorylation. The experimental results show that, compared with the use of any one of the drugs alone, the combined use can more significantly improve the oxygen consumption rate and ATP production of myocardial cells, effectively improves the maturation rate and functional quality of the cells. The application is simple to operate, provides an efficient method for obtaining a myocardial cell model closer to the adult state, and has important application value in heart disease modeling and drug screening.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to an additive for in vitro maturation culture medium of cardiomyocytes and its application. Background Technology

[0002] Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for disease modeling, drug screening, and regenerative medicine. However, under conventional in vitro culture conditions, they often maintain an immature fetal-like phenotype, exhibiting a metabolic pattern dominated by glycolysis, incomplete mitochondrial function, and immature electrophysiological characteristics, which severely limits their application value. Therefore, developing methods to effectively promote the structural, functional, and metabolic maturation of hiPSC-CMs is crucial.

[0003] Currently, strategies to improve cardiomyocyte maturation include physical stimulation (such as electrical stimulation and mechanical stretching), three-dimensional culture, and chemical regulation of the culture medium (such as adding thyroid hormones and fatty acids). However, these methods are either complex to operate or have limited effectiveness, and there is still no simple, efficient, and universal chemical additive solution. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies of the prior art and provide an additive for in vitro maturation culture medium of cardiomyocytes and its application. The additive of this invention can improve the in vitro maturation rate of cardiomyocytes.

[0005] To achieve the objectives of this invention, the technical solution is as follows: One objective of this invention is to provide an additive for in vitro maturation culture medium of cardiomyocytes, the additive comprising phenylphosphine and Mdivi-1, the structural formula of which is shown below: .

[0006] Preferably, in some embodiments, the concentration of phenylphosphine combined with Mdivi-1 in the in vitro maturation culture medium additive for cardiomyocytes is from 12.5 μM to 100 μM / mL. Therefore, the cardiomyocytes cultured with the additive containing phenylphosphine combined with Mdivi-1 within this concentration range have a higher maturation rate and better quality.

[0007] More preferably, in some embodiments, the concentration of phenylphosphine combined with Mdivi-1 in the in vitro maturation culture medium additive for cardiomyocytes is 50 μM / mL. Therefore, cardiomyocytes cultured in vitro with phenylphosphine combined with Mdivi-1 at a concentration of 50 μM / mL exhibit the highest maturation rate and the best quality.

[0008] The second objective of this invention is to provide an in vitro maturation culture medium for cardiomyocytes, wherein the in vitro maturation culture medium for cardiomyocytes contains phenylphosphine and Mdivi-1.

[0009] In some embodiments, the in vitro maturation culture medium for cardiomyocytes comprises: cardiomyocyte basal culture medium, 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum (FBS), and phenylphosphine combined with Mdivi-1. Therefore, this culture medium can significantly improve the in vitro maturation rate and quality of cardiomyocytes.

[0010] In some embodiments, the in vitro maturation culture medium for cardiomyocytes comprises: cardiomyocyte basal culture medium, 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum (FBS), and 12.5 μM to 100 μM / mL phenylphosphine combined with Mdivi-1. Therefore, this culture medium can significantly improve the in vitro maturation rate and quality of cardiomyocytes.

[0011] In some embodiments, the in vitro maturation culture medium for cardiomyocytes comprises: cardiomyocyte basal culture medium, 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum (FBS), and 50 μM / mL phenylphosphine combined with Mdivi-1. Therefore, this culture medium is optimally effective in improving the in vitro maturation rate and quality of cardiomyocytes.

[0012] In one specific embodiment, the basic culture medium for cardiomyocytes is RPMI 1640 Medium (purchased from STEMCELL, catalog number 36750).

[0013] A third objective of this invention is to provide the application of the aforementioned cardiomyocyte in vitro maturation culture medium additive in improving the in vitro cardiomyocyte maturation rate.

[0014] The fourth objective of this invention is to provide the application of the aforementioned cardiomyocyte in vitro maturation culture medium in improving the in vitro cardiomyocyte maturation rate.

[0015] Preferably, the combined concentration of phenylphosphine and Mdivi-1 in the additive is from 12.5 μM to 100 μM / mL. More preferably, the combined concentration is 50 μM / mL.

[0016] The present invention has the following beneficial effects: 1. Synergistic Effect: This invention is the first to combine fenfosinate and Mdivi-1 in the in vitro maturation culture of cardiomyocytes. Experiments have shown that the combined use of the two drugs produces a synergistic effect superior to using either drug alone in promoting the metabolic maturation of cardiomyocytes (e.g., increasing cellular oxygen consumption rate (OCR) and ATP production) (see Example 1). Figure 3-4 ).

[0017] 2. Significant effect: Maturation culture medium containing fenpropathrin and Mdivi-1 (especially at a concentration of 50 μM / mL) can significantly promote the transformation of hiPSC-CMs from glycolytic metabolism to oxidative phosphorylation metabolism, effectively improving cell maturation rate and functional quality.

[0018] 3. Simple operation and wide applicability: This invention can achieve efficient maturation induction by simply adding culture medium, without the need for complex equipment, and is easy to standardize. It has broad application prospects in the fields of drug cardiotoxicity screening, construction of heart disease models and regenerative medicine research. Attached Figure Description

[0019] Figure 1 Effects of different concentrations of fenprophane on the viability of hiPSC-CMs (Schematic diagram of CCK8 assay results).

[0020] Figure 2 Effects of different concentrations of Mdivi-1 on the viability of hiPSC-CMs (Schematic diagram of CCK8 assay results).

[0021] Figure 3 Schematic diagram of the effects of different treatment groups on myocardial oxygen consumption rate (OCR) levels. Among them, the OCR value of the 50 μM / mL fenfotiam combined with Mdivi-1 group was significantly higher than that of the control group, the fenfotiam monotherapy group (50 μM / mL), and the Mdivi-1 monotherapy group (50 μM / mL).

[0022] Figure 4 Schematic diagram showing the effect of different treatment groups on ATP production in cardiomyocytes. Among them, the ATP production of the 50 μM / mL phosphatidylcholine combined with Mdivi-1 group was significantly higher than that of the other groups. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0024] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0026] To address the technical problem of this invention, the overall concept of this invention is as follows: The combination of fenpropathrin and Mdivi-1 can significantly promote the metabolic maturation of cardiomyocytes, shifting them from a metabolic mode dominated by glycolysis to one dominated by mitochondrial oxidative phosphorylation, thereby greatly improving the maturity rate and quality of cardiomyocytes.

[0027] The combination of phenylphosphine and Mdivi-1 is mainly used to optimize the in vitro culture system of cardiomyocytes. As an additive to the cardiomyocyte maturation culture medium, it can significantly improve the cardiomyocyte oxygen consumption rate (OCR) and ATP production, and significantly improve the in vitro cardiomyocyte maturation rate. This can greatly improve the maturation rate and quality of cardiomyocytes, and has broad application prospects in the fields of cardiac disease modeling, drug safety screening and regenerative medicine. It can significantly improve the reliability and success rate of related research and applications.

[0028] The present application will now be described in detail with reference to embodiments and experimental data.

[0029] Example 1: Effects of phenylphosphine and Mdivi-1 on the viability of hiPSC-CMs and screening of safe concentrations I. Experimental Objective To evaluate the effects of different concentrations of fenprophalezamide and Mdivi-1 monotherapy (purchased from MedChemExpress Monmouth Junction, NJ, USA, HY-15886) on the viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and to determine the safe and effective concentration range for subsequent combined experiments.

[0030] II. Experimental Methods Add 1 mL of ready-to-use matrix gel (e.g., ABS9410) to each well of a 6-well plate containing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and 0.5 mL to each well of a 12-well plate. Gently agitate to ensure the matrix gel completely covers the bottom of the plate. Incubate at 37°C for 1-2 hours. Before the experiment, remove the plate and allow it to equilibrate at room temperature for 20 minutes in a clean bench. If not used immediately, seal with Parafilm and store at 2-8°C for up to one week. After cell resuscitation, rapidly thaw the cryovials at 37°C for 1-2 minutes; add 2-3 mL of preheated culture medium and centrifuge at 1000 rpm for 5 minutes; seeding density: 300-500 cell colonies / well (6-well plate); detect the effects of phenylphosphine (...) using the CCK8 assay. Figure 1 ) 12.5μm, 25μm, 50μm, 75μm, 100μm, 150μm; given Mdivi-1 ( Figure 2 Cell viability after treatment at 12.5μm, 25μm, 50μm, 75μm, 100μm, and 150μm.

[0031] III. Experimental Results like Figure 1 The results showed that within the tested concentration range, fenprophos did not exhibit significant cytotoxicity to hiPSC-CMs, and cell viability remained at a high level, providing a safety basis for selecting 50 μM / mL as its optimal experimental concentration.

[0032] like Figure 2 The results showed that within the tested concentration range, Mdivi-1 did not exhibit significant cytotoxicity to hiPSC-CMs, and cell viability remained at a high level, providing a safety basis for subsequently selecting 50 μM / mL as its optimal experimental concentration.

[0033] Example 2: Synergistic effect of phosphatidylcholine combined with Mdivi-1 on myocardial oxygen consumption rate (OCR) I. Experimental Objective To verify the effect of combined application of phenylphosphine and Mdivi-1 on the key indicator of metabolic maturation of hiPSC-CMs—cellular oxygen consumption rate (OCR), and to assess whether there is a synergistic effect.

[0034] II. Experimental Methods Add 1 mL of ready-to-use matrix gel (e.g., ABS9410) to each well of a 6-well plate containing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and 0.5 mL to each well of a 12-well plate. Gently agitate to ensure the matrix gel completely covers the bottom of the plate. Incubate at 37°C for 1-2 hours. Before the experiment, remove the plate and allow it to equilibrate at room temperature for 20 minutes in a clean bench. If not used immediately, seal with Parafilm and store at 2-8°C for up to one week. After cell resuscitation, rapidly thaw the cryovials at 37°C for 1-2 minutes; add 2-3 mL of preheated culture medium and centrifuge at 1000 rpm for 5 minutes; seeding density: 300-500 cell colonies / well (six-well plate); the mature culture medium is: modified to mature cardiomyocyte culture containing cardiomyocyte RPMI 1640 basal culture medium, 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum, and thiamine.

[0035] Mature cardiomyocyte culture was divided into 4 groups: ① Control group (mature culture medium without the addition of fenprophal and Mdivi-1), ② fenprophal optimal concentration group (50 μM / mL), ③ Mdivi-1 optimal concentration group (50 μM / mL), and ④ fenprophal and Mdivi-1 combined treatment as follows: 12.5 μM / mL group (mature culture medium with 10 μM / mL fenprophal and Mdivi-1), 25 μM / mL group (mature culture medium with 25 μM / mL fenprophal and Mdivi-1), 50 μM / mL group (mature culture medium with 50 μM / mL fenprophal and Mdivi-1), and 100 μM / mL group (mature culture medium with 100 μM / mL fenprophal and Mdivi-1).

[0036] Intracellular ROS levels in cardiomyocytes were detected using a ROS detection kit (Sigma-Aldrich). Cardiomyocytes were cultured in RPMI 1640 medium containing 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum, phenylphosphine, and Mdivi-1. Fluorescence signals were detected and photographed using a laser confocal microscope, and the relative ratios of fluorescence intensities were analyzed using ImageJ software. All five groups of cardiomyocytes underwent the same procedure, including incubation, washing, and imaging. After 24 h of maturation culture, the cardiomyocyte oxygen consumption rate (OCR) was measured using appropriate methods.

[0037] III. Experimental Results like Figure 3 The results showed that after 24 hours of maturation culture, the OCR levels of both the fenfotiam monotherapy group (50 μM / mL) and the Mdivi-1 monotherapy group (50 μM / mL) were improved compared with the control group. However, the OCR level of the 50 μM / mL fenfotiam combined with Mdivi-1 group was significantly improved (P<0.05), and its effect was better than either monotherapy group, indicating that the two have a synergistic effect in improving the oxidative metabolism capacity of cardiomyocytes.

[0038] Example 3: Synergistic effect of phosphatidylcholine combined with Mdivi-1 on ATP production in cardiomyocytes I. Experimental Objective The study verified the effect of combined application of phenylphosphine and Mdivi-1 on ATP production, a key energy metabolism indicator in hiPSC-CMs, and further confirmed its synergistic promoting effect on cardiomyocyte metabolic maturation.

[0039] II. Experimental Methods Intracellular ATP levels in cardiomyocytes were detected using an ATP assay kit (Abcam, ab83355). Cardiomyocytes were cultured in RPMI 1640 medium containing 1 μg / mL glutathione, 1 μg / mL para-aminobenzoic acid (PABA), 0.2 μg / mL biotin, 0.005 μg / mL vitamin B12, 35 μg / mL inositol, 3 μg / mL choline chloride, 2 mM glutamine, 1 mM sodium pyruvate, 5 μg / mL phenol red, 10% fetal bovine serum, and fenfosinate combined with Mdivi-1. One × 10⁶ cells were collected from each well of a 96-well plate, homogenized, lysed, and the supernatant was collected for ATP measurement.

[0040] Add 50 μL of the reaction mixture to an equal volume of sample. Incubate the plate at room temperature for 30 minutes. After incubation, measure the absorbance at 570 nm using a spectrophotometer. Protein concentration was determined using a BCA kit. ATP levels were expressed as the relative ratio of fluorescence intensity in analyzed cardiomyocytes in picomoles per microgram of protein (pmol / μg protein). All five cardiomyocyte assays followed the same procedure, including incubation, washing, and assay. Cardiomyocytes were cultured for 24 hours after maturation.

[0041] III. Experimental Results like Figure 4 The results showed that after 24 hours of maturation culture, ATP production increased in both the fenfotiam and Mdivi-1 single-drug groups compared to the control group. The ATP production increase was most significant in the 50 μM / mL fenfotiam combined with Mdivi-1 group (P<0.05), with a significantly higher enhancement effect than the two single-drug groups. This result is consistent with the OCR detection results, jointly demonstrating that the combined application of fenfotiam and Mdivi-1 can synergistically enhance the mitochondrial oxidative phosphorylation function of hiPSC-CMs, thereby effectively promoting their metabolic maturation.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An additive for in vitro maturation culture medium of cardiomyocytes, characterized in that, The additive includes phenylphosphine and Mdivi-1, the structural formula of which is shown below: 。 2. The cardiomyocyte in vitro maturation culture medium additive according to claim 1, characterized in that, The concentration of phenylphosphine and Mdivi-1 added is 12.5 μM-100 μM / mL.

3. An in vitro maturation culture medium for cardiomyocytes, characterized in that, The in vitro maturation culture medium for cardiomyocytes contains the additives described in any one of claims 1-2.

4. The in vitro maturation culture medium for cardiomyocytes according to claim 3, characterized in that, The in vitro maturation culture medium for cardiomyocytes also includes a basic culture medium for cardiomyocytes, glutathione, para-aminobenzoic acid, biotin, vitamin B12, inositol, choline chloride, glutamine, sodium pyruvate, phenol red, and fetal bovine serum.

5. The in vitro maturation culture medium for cardiomyocytes according to claim 4, characterized in that, The addition ratio of each component is as follows: the final concentration of phenylphosphine and Mdivi-1 is 12.5 μM-100 μM / mL.

6. The use of the cardiomyocyte in vitro maturation culture medium additive according to claim 1 in promoting cardiomyocyte in vitro maturation.

7. The use of the cardiomyocyte in vitro maturation culture medium according to claim 3 in promoting cardiomyocyte in vitro maturation.

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