CN113061573B - Method for promoting myocardial cell maturation by intermittent starvation - Google Patents
Method for promoting myocardial cell maturation by intermittent starvation Download PDFInfo
- Publication number
- CN113061573B CN113061573B CN202110559884.4A CN202110559884A CN113061573B CN 113061573 B CN113061573 B CN 113061573B CN 202110559884 A CN202110559884 A CN 202110559884A CN 113061573 B CN113061573 B CN 113061573B
- Authority
- CN
- China
- Prior art keywords
- intermittent
- myocardial
- starvation
- cells
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Cardiology (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for promoting myocardial cell maturation by intermittent starvation. The invention uses balanced salt solution to conduct intermittent starvation treatment for 1-5 hours/day and lasts for 5-15 days, and the medium is changed into the culture medium of the myocardial cells after the treatment is finished, so that the detection of related maturity indexes is improved. The intermittent starvation treatment of the myocardial cells by the balanced salt solution can induce the morphological structure, metabolism and electrophysiological maturation of the myocardial cells, and has simple operation and low cost. Therefore, the method is an innovative way for inducing myocardial maturation, is beneficial to solving the problem of low myocardial cell maturation degree, and can promote the clinical application of the pluripotent stem cells in the cardiovascular diseases.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for promoting maturation of myocardial cells by intermittent starvation.
Background
Cardiovascular disease has become the first killer of human health, and the most common type of disease is myocardial infarction, which is characterized primarily by myocardial necrosis caused by acute, persistent ischemic hypoxia in the coronary arteries. Because of the limited ability of adult cardiomyocytes to regenerate, primary human cardiomyocytes are difficult to obtain and maintain in vitro, and heart disease research and treatment faces bottlenecks. Human Pluripotent Stem Cells (PSCs) mainly include Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (ipscs), which can be induced to differentiate into cardiomyocytes efficiently in vitro and are simple to operate. Has great potential in the aspects of modeling heart diseases, discovering drugs, treating cells and the like. However, due to the different culture environments in vitro and the limitations of culture techniques, the physiological state of PSC-derived cardiomyocytes (PSC-CM) cannot mature as that of adult cardiomyocytes, but is closer to that of fetal cardiomyocytes. The immature nature of PSC-CM hampers the development and therapeutic efficacy of cardiovascular disease. Therefore, increasing the maturity of PSC-CM is an urgent problem to be solved.
The main characteristics of cardiomyocyte maturation include mature hypertrophy, organized regular myofibril arrangement, increased mitochondrial DNA copy number, increased oxidative phosphorylation capacity, and enhanced electrophysiological and calcium physiological capacities. Currently, methods for increasing PSC-CM maturation include long-term culture methods, tissue engineering, drug stimulation, and bioelectric stimulation.
(1) Culturing the cardiomyocytes for a long period of time. The PSC-CM was continuously cultured in vitro for 60 days to 1 year, and the cardiomyocytes were shown to be in a mature myocardial state, such as: myocardial cell elongation, cell size increase, regular arrangement of muscle segments, etc. However, long-term culture is time-consuming, the maturity degree is still low, and the stability is poor.
(2) And (5) tissue engineering culture. With the improvement of tissue engineering in PSC-CM culture schemes, novel materials such as matrigel or hydrogel are used as media to form various types of myocardial/cardiac tissue engineering, such as: the heart patch, 3D heart organs and the like can obviously promote the maturation of the myocardial cell structure. However, the special materials are difficult to obtain, the operation technology is difficult, and the influence on the physiological state of cells is large.
(3) Electrical stimulation is used to promote the maturation of cardiomyocytes. Most commonly, cell alignment and sarcomere structure are enhanced by electric field stimulation. For example, electrical stimulation of PSC-CM at frequencies close to physiological frequencies, with increasing stimulation intensity from 1 hz to 6 hz, has been found to improve various aspects of cardiomyocyte maturation over time, including enhancement of cardiomyocyte alignment, sarcomere structure, calcium handling capacity and action potential capacity. The limitations are that it requires high equipment requirements, is not suitable for large-scale culture processes, requires considerable time for stimulation and data analysis, and has high downstream analysis difficulties.
Therefore, the methods have higher requirements on time, cost and technology and are difficult to popularize. Therefore, there is a pressing need for more efficient methods to drive the maturation of PSC-CMs in a sustainable time and cost.
Disclosure of Invention
In order to solve the technical problems, the invention simulates the process of myocardial maturation in a physiological state and establishes a method for improving the maturity of the PSC-CM by intermittently hungry balanced salt solution. The method is simple to operate, low in cost, short in maturation period and capable of comprehensively promoting myocardial maturation.
The first purpose of the invention is to provide a method for promoting the maturation of myocardial cells by intermittent starvation, which comprises the following steps:
s1, obtaining purified myocardial cells, and cleaning to remove surface culture solution;
s2, adding balanced salt solution to perform hunger treatment for 1-5 hours, and replacing the myocardial cell culture medium to culture for 19-23 hours;
s3, according to the processing method of the steps S1-S2, intermittent hunger is performed after continuous processing for 5-15 days;
and S4, continuously culturing after intermittent hunger to obtain mature myocardial cells.
Further, the cardiomyocytes are pluripotent stem cell differentiated cardiomyocytes or isolated primary cardiomyocytes or commercialized cardiomyocyte cell lines.
Further, commercially available cardiomyocyte cell lines include the HL-1 cardiomyocyte cell line or the H9C2 cardiomyocyte cell line.
Further, in step S1, washing is performed using a buffer.
Further, the buffer solution is Dulbecco's Phosphate Buffer Solution (DPBS) and D-Hanks balanced salt solution.
Further, in the step S2, the balanced salt solution is Earle 'S balanced salt solution, dulbecco' S phosphate buffer solution or D-Hanks balanced salt solution.
Further, the cardiomyocyte culture medium is CDM3 medium (RPIM-1640 basic medium plus ascorbic acid, fetal bovine serum albumin and penicillin streptomycin solution) or commercial cardiomyocyte culture medium.
Further, the commercialized cardiomyocyte culture medium is DMEM/F12 medium, CMM cardiomyocyte culture medium or CGM cardiomyocyte growth medium.
Further, in step S4, the culture is continued for 3 to 10 days after intermittent starvation.
Further, pluripotent Stem Cells (PSCs) include Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (ipscs).
The embryonic stem cell is an original pluripotent stem cell derived from a blastocyst inner cell mass and obtained by in vitro separation and culture, has the potential of differentiating various cells in an organism and has the characteristics of unlimited proliferation and self-renewal. To eliminate ethical concerns, the present invention uses human embryonic stem cells that are ethically approved, including commercially available human embryonic stem cells (e.g., H1, H7, H9, ES3, etc.).
The induced pluripotent stem cell refers to a stem cell with differentiation potential obtained by introducing a specific transcription factor into terminally differentiated somatic cells through a reprogramming technology. Induced pluripotent stem cells are similar to pluripotent stem cells in morphology, gene and protein expression, and differentiation capacity. Commercially available induced pluripotent stem cells, or induced pluripotent stem cells obtained by reprogramming somatic cells are used in the present invention.
In particular, the human pluripotent stem cells used in the present invention, whether human embryonic stem cells or human induced pluripotent stem cells, are not capable of developing into whole individuals and are all pluripotent stem cells that have been established through ethical examination.
By means of the scheme, the invention at least has the following advantages:
in the process of researching the maturation of the differentiated cardiac muscle cells of the commercialized human embryonic stem cells, the invention can simulate the physiological state of the cardiac muscle of a human at birth and promote the maturation of the cardiac muscle cells by carrying out the hunger treatment of balanced salt solution at the post-purification stage of the cardiac muscle cells. The method has the advantages of short experimental period, low cost and easy operation, and can promote the myocardial maturation from the morphological structure and promote the myocardial to reach a more mature state from the aspects of metabolism and electrophysiology. Therefore, the method is an innovative method for promoting myocardial maturation, solves the problem of low myocardial cell maturation degree, and can promote the clinical application of the pluripotent stem cells in cardiovascular diseases.
Meanwhile, the maturity of the PSC-CM is low at present, so that the heart transplantation of the immature myocardial cells easily causes arrhythmia and can not be well integrated into the heart, thereby greatly reducing the treatment effect. The invention utilizes an intermittent starvation method to promote the maturation of the myocardial cells, is expected to solve the problem, and utilizes that the functions of mitochondria of the myocardial cells after starvation treatment are close to those of adult myocardial cells, so that the oxidative phosphorylation capacity is enhanced. Under the stimulation of hunger in a relatively harsh environment, the myocardial cells may have a greater resistance to the harsh environment than those of other mature-mode-derived myocardial cells. Therefore, after the implant is transplanted into a heart with myocardial infarction, the survival of myocardial cells can be more favorably realized, so that the effect is realized, and the structural function of the heart is more effectively remodeled and repaired.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is an immunofluorescence photograph (63-fold oil lens) of embryonic stem cell-derived cardiomyocytes in the control group and intermittent starvation-treated group without starvation treatment according to the present invention, showing mitochondrial distribution and sarcomere arrangement, and statistics on cell area and sarcomere length.
FIG. 2 shows quantitative comparison of the expression of maturation-associated genes in cells of a control group and intermittently starved cells after treatment of embryonic stem cell-derived cardiomyocytes by intermittent starvation according to the present invention.
Fig. 3 is a graph showing comparison of oxidative phosphorylation energy metabolism in embryonic stem cell-derived cardiomyocytes in the control group and the intermittent starvation-treated group, which were not subjected to starvation treatment, according to the present invention.
FIG. 4 is a graph showing a comparison of electrophysiological correlation indices in embryonic stem cell-derived cardiomyocytes in the control group without starvation treatment and the intermittent starvation treatment group in accordance with the present invention.
FIG. 5 is an immunofluorescence photograph of cardiomyocytes in suckling mice in the control group and intermittent starvation treatment group without starvation treatment, and statistics and comparison of cell area and sarcomere length.
FIG. 6 shows quantitative comparison of the expression of mature genes after intermittent starvation treatment for 10 days.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1: intermittent starvation treatment of human embryonic stem cell-derived cardiomyocytes with balanced salt solution
Specific examples of the method employed in the present invention are as follows:
after the commercial human embryonic stem cells are induced to differentiate and purified myocardial cells are obtained, the cells are washed three times by DPBS, cell surface culture solution is washed away, then balanced salt solution (such as 1 xEarle's balanced solution) is added for 2 hours of starvation, after the starvation time is over, the culture medium of the myocardial cells is changed back, and the starvation treatment is continuously carried out for 10 days for 2 hours/day; after 10 consecutive days of treatment, the cells were cultured in a cardiomyocyte medium, and the maturation index of the cardiomyocytes was examined on day 35.
(1) Detection of myocardial structure. According to the invention, immunofluorescence staining and confocal microscope observation are combined, the arrangement (alpha-actin staining, green) and mitochondrial distribution (MitoTracker staining, red) of the sarcomere in the myocardial cells are evaluated, and maturation indexes such as cell area and sarcomere length are compared, so that the cell morphology after intermittent hunger is closer to the mature myocardial morphology, the cell area is increased, and the sarcomere length is increased. Indicating that EBSS starvation promotes structural maturation of cardiomyocytes (fig. 1);
(2) detecting the expression of the myocardial maturation gene. Quantitatively analyzing the expression of structural genes such as MYH6 and MYH7 and metabolism related gene PGC 1-alpha by adopting a real-time fluorescent quantitative PCR method, and showing that intermittent hunger remarkably improves the expression of maturation related genes in the cardiac muscle cells (figure 2);
(3) detection of myocardial cell energy metabolism. And detecting the change of oxidative phosphorylation by adopting an energy metabolism analyzer, and performing data analysis by combining the result of cell protein quantification. It was found that compared with the cardiomyocytes in the control group, the cardiomyocytes in the starvation treatment group were significantly increased in both Basal respiration (Basal respiration) and ATP production (ATP production) or maximum respiration (maximum respiration) and reserve (spark respiration), indicating that the mitochondrial oxidative phosphorylation of the cardiomyocytes was enhanced after starvation treatment (fig. 3);
(4) detection of myocardial electrophysiology. And recording the action potential of the myocardial cells by using a patch clamp instrument, and collecting and analyzing data. As a result, the duration of 90% repolarization of the action potential (APD 90) of the cardiomyocytes in the starved group was significantly increased compared to the control group, and the duration of 50% repolarization of the action potential (APD 50) was also higher than that in the control group, which indicates that the EBSS starvation treatment can promote the electrophysiological maturation of the cardiomyocytes (fig. 4).
The detection results show that the intermittent starvation of the myocardial cells through the balanced salt solution can effectively promote the maturation of the myocardial cells, and a large number of functional mature cardiac muscles can be provided for clinical treatment of myocardial infarction and heart disease modeling.
Example 2: intermittent starvation treatment of neonatal suckling mouse cardiac muscle cells with balanced salt solution
The invention uses balanced salt solution to intermittently starve and treat myocardial cells of the suckling mice to promote the maturation of the myocardial cells. Such as: after extracting myocardial cells of suckling mice, cleaning with DPBS, washing off culture solution on the cell surface, then adding balanced salt solution (such as DPBS balanced solution) for starvation for 1 hour, after the starvation time is over, changing back to the myocardial cell culture medium, and continuously starving for 5 days for 1 hour/day; after 5 consecutive days of treatment, the culture was continued using a cardiomyocyte culture medium, and the maturation indicator of the cardiomyocytes was examined on day 10.
(1) Detection of myocardial structures. The invention utilizes a confocal microscope and stains myocardial sarcomere through immunofluorescence staining, wherein the length and the stability of the sarcomere are observed by alpha-actin (alpha-actin) and troponin (CTNT), and the sarcomere is found to be uniformly distributed in cells, and the cell morphology after intermittent hunger is closer to the mature myocardial morphology. This may indicate that DPBS starvation promotes structural maturation of cardiomyocytes (fig. 5);
(2) detecting myocardial maturation gene expression, structural changes and metabolic changes. The expression of the structural and metabolic genes ANP, MYH6, SERCA2A, i.e. the changes in the expression of these genes, were quantitatively analyzed, e.g. using real-time fluorescent quantitative PCR (fig. 6).
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A method for promoting the maturation of cardiac muscle cells by intermittent starvation is characterized by comprising the following steps:
s1, obtaining purified myocardial cells, cleaning and removing surface culture solution;
s2, adding balanced salt solution to perform starvation treatment for 1~5 hours, and changing back to the myocardial cell culture medium to culture for 19-23 hours;
s3, according to the processing method of the steps S1-S2, intermittent hunger is performed after continuous processing for 5-15 days;
s4, continuously culturing after intermittent hunger to obtain mature myocardial cells;
the cardiac muscle cell is differentiated cardiac muscle cell of pluripotent stem cell.
2. The method according to claim 1, wherein in the step S1, the washing is performed using a buffer.
3. The method of claim 2, wherein the buffer is Dulbecco's phosphate buffer or D-Hanks balanced salt solution.
4. The method according to claim 1, wherein in the S2 step, the balanced salt solution is Earle 'S balanced salt solution, dulbecco' S phosphate buffer solution or D-Hanks balanced salt solution.
5. The method according to claim 1, wherein the cardiomyocyte culture medium is a CDM3 medium, DMEM/F12 medium, CMM cardiomyocyte culture medium or CGM cardiomyocyte growth medium.
6. The method of claim 5, wherein the CDM3 medium is prepared by adding ascorbic acid, fetal bovine serum albumin and penicillin streptomycin solution to RPIM-1640 basic medium.
7. The method according to claim 1, wherein in step S4, the cultivation is continued for 3 to 10 days after intermittent hunger.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110559884.4A CN113061573B (en) | 2021-05-21 | 2021-05-21 | Method for promoting myocardial cell maturation by intermittent starvation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110559884.4A CN113061573B (en) | 2021-05-21 | 2021-05-21 | Method for promoting myocardial cell maturation by intermittent starvation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113061573A CN113061573A (en) | 2021-07-02 |
| CN113061573B true CN113061573B (en) | 2023-03-24 |
Family
ID=76568710
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110559884.4A Active CN113061573B (en) | 2021-05-21 | 2021-05-21 | Method for promoting myocardial cell maturation by intermittent starvation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113061573B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060125A (en) * | 2018-02-07 | 2018-05-22 | 苏州大学 | Promote the method that pluripotent stem cell differentiation is cardiac muscle cell's maturation |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1693451B1 (en) * | 2003-11-21 | 2011-11-09 | Daiichi Sankyo Company, Limited | Method of growing myocardial cells |
| US20210002615A1 (en) * | 2018-03-16 | 2021-01-07 | The Board Of Trustees Of The Leland Stanford Junior University | Reagents and methods with wnt agonists and bioactive lipids for generating and expanding cardiomyocytes |
| KR102224273B1 (en) * | 2019-10-10 | 2021-03-08 | 고려대학교 산학협력단 | Stem Cell-derived Mature Cardiomyocytes and Cardiovascular Disease Model Using the Same |
| CN111187750B (en) * | 2020-01-17 | 2022-08-16 | 复旦大学附属中山医院 | Method for inducing pluripotent stem cells to differentiate into myocardial cells by biogenic amine and application |
-
2021
- 2021-05-21 CN CN202110559884.4A patent/CN113061573B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060125A (en) * | 2018-02-07 | 2018-05-22 | 苏州大学 | Promote the method that pluripotent stem cell differentiation is cardiac muscle cell's maturation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113061573A (en) | 2021-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hawrot et al. | [53] Long-term culture of dissociated sympathetic neurons | |
| Judson et al. | Isolation, culture, and differentiation of fibro/adipogenic progenitors (FAPs) from skeletal muscle | |
| CN1860222A (en) | Stem cells for clinical and commercial uses | |
| CN101765657A (en) | Method for isolating and culturing adult stem cells derived from human amniotic epithelium | |
| CN104762257B (en) | A kind of method preparing mescenchymal stem cell from umbilical cord | |
| CN106085952A (en) | A kind of placental villi plate mescenchymal stem cell and extracting method thereof | |
| CN106265740B (en) | Umbilical cord mesenchymal stem cells combine application of the astragalus polyose in treatment hyperglycaemia and medicine for treating diabetic nephropathy is prepared | |
| CN109251889A (en) | A kind of transplanting preparation system of dental pulp mescenchymal stem cell microballoon | |
| TW201510223A (en) | Method for separating living cell and constructing cell bank by means of tissue homogenate method | |
| CN106282100A (en) | A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle | |
| CN106350480B (en) | A method of the purifying tissue-derived sarcoblast of ox fetal skeletal muscle | |
| CN104745526B (en) | A kind of zebra fish primary embryonic cells vitro differentiation is the new method of cardiac muscle cell | |
| CN106119191A (en) | A kind of placental villi plate mescenchymal stem cell and clinicization preparation method | |
| CN106399248B (en) | Method for inducing transdifferentiation of fibroblasts into nerve cells | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN113061573B (en) | Method for promoting myocardial cell maturation by intermittent starvation | |
| CN109234230A (en) | A kind of primary separation method of skin mesenchymal stem cells | |
| CN111575227B (en) | Method for establishing human-derived diabetic cardiomyopathy model | |
| CN107557332A (en) | CD29+People's Mesenchymal Stem Cells from Umbilical Cord and its purposes in skeletal muscle atrophy medicine under preparing treatment high glucose and high fat environment | |
| CN106244522A (en) | A kind of stem cell cultivating system and cultural method thereof | |
| CN105624115B (en) | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into nerve-like cells and induction method thereof | |
| CN113755553A (en) | Myocardial cell culture medium and method and application thereof for screening drug cardiotoxicity | |
| CN116486918A (en) | Stem cell quality evaluation method | |
| CN114480268B (en) | Preparation method of human umbilical cord mesenchymal stem cells | |
| CN117821376A (en) | Primary culture method of cynomolgus monkey ligament fibroblasts |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |