Novel culture method for in vitro differentiation from human embryonic stem cells to functional myocardial cells

Abstract

The invention discloses a novel culture method for in vitro differentiation from human embryonic stem cells to functional myocardial cells. The novel culture method is different from a conventional typical hanging-drop culture method. According to the novel culture method, an embryoid body is formed by adopting a simplified suspension differentiation culture method, serum-free culture liquid is adopted for improvement, and various inducing and nutrition-enriching substances are added into the culture liquid to increase the differentiation rate of the myocardial cells and prolong the survival time of the myocardial cells; a method for differentiation from embryonic stem cells to myocardial cells is further effectively updated and perfected, the proportion of the myocardial cells differentiated by the embryoid body is greatly increased, and a large quantity of myocardial cell sources with powerful functions can be provided for stem cell clinical transplantation treatment technology, drug screening and the like; the method is simple and feasible to carry out, the culture time is shortened, the functions for in vitro differentiation from the embryonic stem cells to the myocardial cells are improved and include pulsation time as well as autorhythmicity and rhythmicity of myocardial cell beating, and the method is good in repeatability.

Description

technical field

[0001] The invention relates to an effective culture method for human embryonic stem cells differentiated into functional cardiomyocytes in vitro, and belongs to the field of stem cell biotechnology. Background technique

[0002] Heart disease is the number one killer of human health. According to the statistics of the World Heart Federation, for every three deaths in the world, the cause of death of one person is related to cardiovascular diseases. Now it has reached the "blowout period" of cardiovascular diseases, especially in the age group of 35-50 years old. Such as ischemic heart disease, myocardial infarction, heart failure and cardiomyopathy have become common clinical diseases today. The number of stem cells in human myocardial tissue is limited, and their differentiation ability is poor. After myocardial cell necrosis or apoptosis, the myocardium is difficult to repair, and then the number of myocardial cells decreases, ventricular remodeling, arrh...

Examples

specific Embodiment 1

[0031] Specific embodiment 1: the formation of embryoid body

[0032] 1. Prepare a gelatin-coated culture dish, the steps include: prepare a 0.1% gelatin solution, spread the gelatin solution on the bottom of a sterile tissue culture dish and incubate for at least 1 hour, then suck off the excess gelatin solution, and then dry it for later use. That is, the gelatin-coated culture dish is prepared.

[0033] 2. Prepare serum-free embryoid body formation medium: 80% DMEM medium for basal medium; add 20% serum substitute, 1mmol / L essential amino acid, 2mmol / LL-glutamine, 0.1mmol / L β-mercaptoethanol , 50IU / mL penicillin, 10μg / mL streptomycin.

[0034] 3. Formation of embryoid bodies: at a concentration of 2×10 5 Individuals / ml inoculated human embryonic stem cells into gelatin-coated culture dishes, adding culture medium and pipetting every day to avoid cell adhesion. CO 2 and 95% saturated humidity. On the 3rd day, embryoid bodies can be formed and suspended in the culture me...

specific Embodiment 2

[0035] Specific embodiment 2: cardiomyocyte differentiation culture

[0036] 1. Preparation of cardiomyocyte differentiation medium: 80% DMEM medium for basal medium, 20% serum substitute, and added inducing substances, respectively: L-glutamine 2mmol / L, L-sodium pyruvate 0.182mmol / L L. Non-essential amino acid 1mmol / L, β-mercaptoethanol 0.1mmol / L, transferrin 5.6mg / L, sodium selenite 20mg / L and p38-MAPK inhibitor 5μmol / L.

[0037] 2. Transfer the cultured embryoid bodies to a 24-well culture plate, place 1 embryoid body in each well, the medium used is the cardiomyocyte differentiation medium, and add nutrient-enhancing substances during the cardiomyocyte differentiation culture process, and at the same time It includes vitamin C, protein hydrolyzate and bovine serum albumin, and its concentrations are vitamin C 0.1mmol / L, protein hydrolyzate 50μg / ml and bovine serum albumin 50μg / ml. Culture conditions are 37°C, 5% CO 2 and 95% saturated humidity. The differentiation mediu...

specific Embodiment 3

[0038] Specific embodiment 3: the differentiation rate experiment of human embryonic stem cells to cardiomyocytes

[0039] 1. Experimental steps and methods for differentiating embryoid bodies formed under different conditions into cardiomyocytes: transfer the cultured embryoid bodies to a 96-well culture plate, inoculate one embryoid body in each well, and do 3 experiments in both the experimental group and the control group. plate parallel experiments. Then place at 37°C, 95% saturated humidity and 5% CO 2 Static culture under the conditions; the medium was changed once a day. Among them, the experimental group adopts the embryoid body formed under the condition of suspension culture, and adds nutrition-enhancing substances, and the control group adopts the embryoid body formed under the traditional inverted suspension culture condition, without adding nutrition-enhancing substances; There are spontaneously beating cell clumps, and the presence of rhythmically beating cell...

technical field

[0001] The invention relates to an effective culture method for human embryonic stem cells differentiated into functional cardiomyocytes in vitro, and belongs to the field of stem cell biotechnology. Background technique

[0002] Heart disease is the number one killer of human health. According to the statistics of the World Heart Federation, for every three deaths in the world, the cause of death of one person is related to cardiovascular diseases. Now it has reached the "blowout period" of cardiovascular diseases, especially in the age group of 35-50 years old. Such as ischemic heart disease, myocardial infarction, heart failure and cardiomyopathy have become common clinical diseases today. The number of stem cells in human myocardial tissue is limited, and their differentiation ability is poor. After myocardial cell necrosis or apoptosis, the myocardium is difficult to repair, and then the number of myocardial cells decreases, ventricular remodeling, arrh...

Examples

specific Embodiment 1

[0031] Specific embodiment 1: the formation of embryoid body

[0032] 1. Prepare a gelatin-coated culture dish, the steps include: prepare a 0.1% gelatin solution, spread the gelatin solution on the bottom of a sterile tissue culture dish and incubate for at least 1 hour, then suck off the excess gelatin solution, and then dry it for later use. That is, the gelatin-coated culture dish is prepared.

[0033] 2. Prepare serum-free embryoid body formation medium: 80% DMEM medium for basal medium; add 20% serum substitute, 1mmol / L essential amino acid, 2mmol / LL-glutamine, 0.1mmol / L β-mercaptoethanol , 50IU / mL penicillin, 10μg / mL streptomycin.

[0034] 3. Formation of embryoid bodies: at a concentration of 2×10 5 Individuals / ml inoculated human embryonic stem cells into gelatin-coated culture dishes, adding culture medium and pipetting every day to avoid cell adhesion. CO 2 and 95% saturated humidity. On the 3rd day, embryoid bodies can be formed and suspended in the culture me...

specific Embodiment 2

[0035] Specific embodiment 2: cardiomyocyte differentiation culture

[0036] 1. Preparation of cardiomyocyte differentiation medium: 80% DMEM medium for basal medium, 20% serum substitute, and added inducing substances, respectively: L-glutamine 2mmol / L, L-sodium pyruvate 0.182mmol / L L. Non-essential amino acid 1mmol / L, β-mercaptoethanol 0.1mmol / L, transferrin 5.6mg / L, sodium selenite 20mg / L and p38-MAPK inhibitor 5μmol / L.

[0037] 2. Transfer the cultured embryoid bodies to a 24-well culture plate, place 1 embryoid body in each well, the medium used is the cardiomyocyte differentiation medium, and add nutrient-enhancing substances during the cardiomyocyte differentiation culture process, and at the same time It includes vitamin C, protein hydrolyzate and bovine serum albumin, and its concentrations are vitamin C 0.1mmol / L, protein hydrolyzate 50μg / ml and bovine serum albumin 50μg / ml. Culture conditions are 37°C, 5% CO 2 and 95% saturated humidity. The differentiation mediu...

specific Embodiment 3

[0038] Specific embodiment 3: the differentiation rate experiment of human embryonic stem cells to cardiomyocytes

[0039] 1. Experimental steps and methods for differentiating embryoid bodies formed under different conditions into cardiomyocytes: transfer the cultured embryoid bodies to a 96-well culture plate, inoculate one embryoid body in each well, and do 3 experiments in both the experimental group and the control group. plate parallel experiments. Then place at 37°C, 95% saturated humidity and 5% CO 2 Static culture under the conditions; the medium was changed once a day. Among them, the experimental group adopts the embryoid body formed under the condition of suspension culture, and adds nutrition-enhancing substances, and the control group adopts the embryoid body formed under the traditional inverted suspension culture condition, without adding nutrition-enhancing substances; There are spontaneously beating cell clumps, and the presence of rhythmically beating cell...

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