CN111621470A - Efficient low-toxicity myocardial purification culture medium and method - Google Patents
Efficient low-toxicity myocardial purification culture medium and method Download PDFInfo
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- CN111621470A CN111621470A CN202010509784.6A CN202010509784A CN111621470A CN 111621470 A CN111621470 A CN 111621470A CN 202010509784 A CN202010509784 A CN 202010509784A CN 111621470 A CN111621470 A CN 111621470A
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Abstract
The invention discloses a high-efficiency low-toxicity myocardial purification culture medium and a method. The culture medium of some examples of the invention can be purified to obtain iPS-CM unexpectedly faster, and the components of the culture medium have lower toxicity to myocardial cells, so that the survival rate of the myocardial cells can be better maintained.
Description
Technical Field
The invention relates to a biological culture medium and a cell culture method, in particular to a high-efficiency low-toxicity myocardial purification culture medium and a method.
Background
Cardiovascular disease is a major health problem and leading cause of death in today's society. Nearly 2 thousand 6 million people worldwide are suffering from heart failure with a survival rate of less than 53% within 5 years. With the development of iPS technology and the establishment of a cardiomyocyte differentiation and purification method, people can simulate the cardiomyocytes of the heart phenotype of a patient in vitro. These cardiomyocytes (iPS-CM) differentiated from iPS can mimic the donor's cardiac phenotype and are not subject to ethical debate, allowing us to study the pathogenesis of cardiovascular diseases (e.g., long QT interval syndrome, arrhythmia, hypertrophic cardiomyopathy, dilated cardiomyopathy, mitochondrial cardiomyopathy, etc.), cardiotoxicity tests for evaluating drugs (e.g., aldosterone antagonists, angiotensin converting enzyme inhibitors, angiotensin ii receptor blockers, beta receptor blockers, angiotensin enkephalinase receptor inhibitors, etc.), and seed cells for myocardial tissue engineering.
Generally, the viability of the cells decreased with the increase of the culture time, and the induced iPS-CM had many non-cardiomyocytes and was difficult to use without purification. The conventional method for purifying the myocardial cells mainly uses lactate to carry out metabolic selection, but the method needs long time, generally needs 6-10 days, and is difficult to obtain the myocardial cells with high purity in a short time (see Xu C. Differentiation and expression of myocardial cells from human pluralityot cells [ J ]. Journal of molecular and cellular science 2012, 52(6): 1203-1212), and the used basal medium is a sugar-free DMEM medium. The inventors have also tried purification using lipids (lipids used in cardiomyocyte purification cultures generally include one or more unsaturated fatty acids, saturated fatty acids, and certain emulsifiers added to make them uniformly dispersible in the Culture medium) instead of lactate, but lipids are toxic to cells, and although the purification rate is fast, the cell viability is poor (see: Lin B, Lin X, Stachel M, et al. Culture in glucose-depleted medium with fatty acids and 3, 3', 5-triiodol-thonine surfactants purification and purification of human pluripotent stem cell-derived cardiac cells [ J ]. department of kinetics, 2017, 8: 253.).
How to obtain the iPS-CM by rapid purification under the condition of keeping the survival rate of the myocardial cells is a technical problem to be solved.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provide a high-efficiency and low-toxicity myocardial purification culture medium and a method.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
an additive for an iPS-CM purification medium, the additive consisting of lactate, lipid and L-, carnitine, the amounts of each component added to the iPS-CM purification medium being: lactate with the concentration of 2-4 mM; 0.1-0.5 v/v% of lipid; l-carnitine, 2-10 μ M.
In some examples, L-carnitine is added in an amount of 4 to 10. mu.M.
In some examples, the lipid is added in an amount of 0.2-0.4 v/v%.
In some examples, the additive further comprises a pH stabilizer.
In some examples, the pH stabilizer is selected from at least one of HEPES and MOPS.
In some examples, the lactate salt is selected from sodium lactate, potassium lactate, calcium lactate, magnesium lactate, zinc lactate, ferrous lactate.
In some examples, the lipid contains at least one fatty acid of arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, octadecenoic acid.
In some examples, the commercial product of the lipid is selected from: item numbers L9655, L0288 manufactured by Sigma; the products of HyClone are LS1000 and HyClone LS 250.
In a second aspect of the present invention, there is provided:
an iPS-CM purification medium, comprising a basic medium, wherein the basic medium is added with: lactate with the concentration of 2-4 mM; 0.1-0.5 v/v% of lipid; l-carnitine, 2-10 μ M.
In some examples, the L-carnitine is added in an amount of 4-10 μ M in the basal medium.
In some examples, the base medium comprises lipids in an amount of 0.2-0.4 v/v%.
In some examples, the basal medium is further supplemented with a pH stabilizer.
In some examples, the pH stabilizer is selected from HEPES, MOPS, MHM.
In some examples, the lactate salt is selected from sodium lactate, potassium lactate, calcium lactate, magnesium lactate, zinc lactate, ferrous lactate.
In some examples, the lipid contains at least one fatty acid of arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, octadecenoic acid.
In some examples, the commercial product of the lipid is selected from: item numbers L9655, L0288 manufactured by Sigma; the products of HyClone are LS1000 and HyClone LS 250.
In some examples, the basal medium is selected from the group consisting of sugarless DMEM medium, sugarless 1640 medium, sugarless DMEM/F12.
In some examples, the composition of the iPS-CM purification medium is:
sodium lactate: 3 mM
Lipid: 0.25v/v%, Cat number Sigma, L0288
L-carnitine: 5 μ M
HEPES：1 v/v%
Sugar-free DMEM medium, and the balance.
In a third aspect of the present invention, there is provided:
a method for culturing iPS-CM by purification, comprising adding iPS-CM obtained by iPS differentiation into the iPS-CM purified culture medium according to the second aspect of the present invention for metabolic purification culture.
The invention has the beneficial effects that:
the culture medium of some examples of the invention can be purified to obtain iPS-CM unexpectedly faster, and the components of the culture medium have lower toxicity to myocardial cells, so that the survival rate of the myocardial cells can be better maintained.
According to the culture method of some examples of the invention, the iPS-CM can be purified more quickly, the toxicity to cells is lower, and the survival rate of the cardiac muscle cells can be better maintained.
Drawings
FIG. 1 is a graph of the effect of different media on cardiomyocyte viability;
FIG. 2 shows the results of purity measurements of cardiomyocytes after purification in different media;
FIG. 3 shows the results of flow assays of iPS-CM purified media of examples 2 to 4;
FIG. 4 shows the results of the determination of the viability of the iPS-CM purified medium of examples 2 to 4.
Detailed Description
Unless otherwise specified, the abbreviations in the present invention have the following meanings:
HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
MOPS: 3-morpholinepropanesulfonic acid
Unexplained abbreviations have the meaning commonly indicated in the art.
Lactate is not particularly required, and lactate that is less toxic to myocardial cells is preferred. Practical lactate salts include, but are not limited to, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, zinc lactate, ferrous lactate.
The lipid of the present invention refers to a lipid used for the purification and culture of cardiac muscle, which is a mixture of one or more of unsaturated fatty acids and saturated fatty acids after emulsification, as exemplified by lipid L9655 manufactured by Sigma, which contains 2. mu.g/ml arachidonic acid, and linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid, each at a concentration of 10. mu.g/ml, 0.22 mg/ml cholesterol, 2.2 mg/ml Tween 80, 70. mu.g/ml tocopherol acetate, and 100 mg/ml Pluronic F-68, and the solvent is water for cell culture.
The basic culture medium is a culture medium without extra additives for purifying the cardiac muscle, and includes but is not limited to a culture medium used for purifying the cardiac muscle, such as a sugar-free DMEM culture medium, a sugar-free 1640 culture medium, a sugar-free DMEM/F12 and the like.
The pH stabilizer can increase the buffer capacity of the culture medium, can better stabilize the pH of the culture medium, and can be selected as required to be suitable for cell culture, including but not limited to HEPES and MOPS. The pH stabilizer can be used alone or in combination according to needs, and the dosage of the pH stabilizer can be adjusted correspondingly according to the buffering effect. Of course, the pH of the culture medium during the purification culture can be controlled by other known means, and the addition of the pH stabilizer can be omitted.
The technical scheme of the invention is further explained by combining the examples and experiments.
For convenience of comparison, the following examples or comparative examples used the following starting materials, unless otherwise specified:
lactate: sigma, L4263 sodium lactate
Lipid: sigma, L0288
L-carnitine: sigma, C0158
HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid): GIBCO, 15630080
Sugar-free DMEM: GIBCO, 11966025
Example 1
The iPS-CM purification medium comprises the following components:
example 2
The iPS-CM purification medium comprises the following components:
example 3
The iPS-CM purification medium comprises the following components:
example 4
The iPS-CM purification medium comprises the following components:
example 5
The iPS-CM purification medium comprises the following components:
comparative example 1
The iPS-CM purification medium comprises the following components:
comparative example 2
The iPS-CM purification medium comprises the following components:
comparative example 3
The iPS-CM purification medium comprises the following components:
test of Effect of different media on myocardial cell viability
Culturing the myocardial cells: cardiomyocytes ((Cor.4U, Axon BioSystems, cat.no. Ax-B-HC 02-1M) were inoculated in RPMI1640 medium with 3% serum replacement (Gibco, 10828-2And (3) standing and culturing for 24 hours, respectively replacing the three culture mediums in the example 1 after the cells adhere to the wall and grow well, replacing the culture mediums once every 2 days, continuously culturing for 8 days, and sampling every day to detect the cell survival rate.
And (3) detecting the survival rate: cardiomyocytes were digested into single cells with 0.05% trypsin, treated with AO/PI dye as described, and cell viability calculated using the COUNTSTAR FL assay, the results of which are shown in FIG. 1.
As can be seen from FIG. 1, the cell viability rate decreased with the increase of the culture time, wherein the cell viability rate decreased slowly in example 1 and comparative example 1, and the difference between the two was not significant; the cell viability rate of the comparative example 2 is reduced rapidly, the cell viability rate is obviously different from that of the example 1 and the comparative example 1, and p is less than 0.05; comparative example 3 the cell viability rate decreased slightly lower than that of comparative example 2, but the cell viability rate was also significantly different from that of example 1 and comparative example 1, with p < 0.05.
Culture medium for testing purification efficiency of differentiated hiPS-CM
The culture of (2): human induced pluripotent stem cells DYR0100(ATCC) were inoculated on a plate coated with Matrigel matrix (corning, 354277), followed by culture in Stemflex medium (Gibco, A3349401). The StemFlex medium was changed every two days. hipscs were passaged every 3 days, or when cell cultures reached 80-90% confluence. During the passage, the cells were washed 1 time with 1 XDPBS (Gibco, 14040133) and then treated in 0.5 mM EDTA (Invitrogen, 15575020) diluted with 1 XDPBS (Gibco, 14190144) for 10 min at room temperature. The passage ratio is 1: 3-1: 6.
differentiation of (2): hipscs were treated with small molecule CHIR99021(Tocris, 4423, final concentration 10 mM) in RPMI-BSA medium [ RPMI1640 medium (HyClone, SH30027.01) +213 μ g/mLAA2P (l-ascorbic acid 2-magnesium phosphate) (Sigma, a8960) and 0.1% bovine serum albumin (Sigma, a1470) ] for 24 h, followed by RPMI-BSA incubation. On day 4 of differentiation, cells were treated with small molecule IWP2(Tocris, 3533, final concentration 5. mu.M) in RPMI-BSA medium. After 48h, the medium was replaced with RPMI-BSA. In subsequent experiments, cardiomyocytes were cultured in RPMI1640 medium plus 3% serum replacement (Gibco, 10828-028).
And (3) purifying the cells: iPS-CM was purified using a metabolic selection method. And (3) purifying the differentiated cells by using different culture media, changing the cells once every 2 days, continuously culturing for 8 days, and sampling every day to detect the purity of the myocardial cells.
And (3) detecting the purity of the cells: after 15 days of differentiation of the cells were digested into individual cells with 0.05% trypsin and labeled with anti-Cardiac Troponin T antibody, Cardiac Troponin T was a marker of cardiomyocytes, and the purity of cardiomyocytes was confirmed by flow cytometry to calculate the proportion of cells containing Cardiac Troponin T. Analysis was performed using a FACSAria ™ II flow cytometer (BD). The results are shown in FIG. 2.
As can be seen from fig. 2, the cardiomyocyte occupancy increased with time, the acceleration of the cardiomyocyte occupancy was faster in example 1, comparative example 2, and comparative example 3, the acceleration of the comparative example 1 was slower, the cardiomyocyte occupancy reached 90% or more in example 1, comparative example 2, and comparative example 3 by day 4, and the cardiomyocyte occupancy was greater than 95% in example 1, comparative example 2, and comparative example 3 by day 6.
In conclusion:
1) the comparative example 1 and the example 1 can better maintain the survival rate of the cells;
2) comparative example 2 and example 1 enable more efficient purification of cardiomyocytes.
3) While example 1 enables efficient purification of cardiomyocytes while maintaining cell viability, sufficiently pure cardiomyocytes were obtained on day 4 or day 5, while significantly higher viability was maintained.
Example 2-4 experiments on cardiomyocyte purification
The iPS-CM purified medium of examples 2 to 4 was used to purify cardiomyocytes differentiated from hipscs for 20 days, and the culture was changed every 2 days, and the cells were harvested by day 6 and subjected to flow assay (results are shown in FIG. 3) and viability assay (results are shown in FIG. 4), respectively, as described in the "medium-to-differentiated hiPS-CM purification efficiency test".
As can be seen from FIGS. 3 and 4, the percentage of cardiomyocytes reached 95% or more and the viability remained 80% or more by the time of purification on day 6, further demonstrating that the ratio in the range of the present invention can efficiently purify cardiomyocytes while maintaining viability.
Claims (10)
1. An additive for an iPS-CM purification medium, the additive consisting of lactate, lipid and L-, carnitine, the amounts of each component added to the iPS-CM purification medium being: lactate with the concentration of 2-4 mM; 0.1-0.5 v/v% of lipid; 2-10 mu M of L-carnitine;
preferably, the addition amount of the L-carnitine is 4-10 mu M;
preferably, the addition amount of the lipid is 0.2-0.4 v/v%.
2. Additive according to claim 1, characterized in that: also comprises a pH stabilizing agent.
3. Additive according to claim 2, characterized in that: the pH stabilizer is at least one selected from HEPES and MOPS.
4. An additive according to any one of claims 1 to 3, wherein: the lactate is selected from sodium lactate, potassium lactate, calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.
5. An additive according to any one of claims 1 to 3, wherein: the lipid contains at least one fatty acid selected from arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, and octadecenoic acid.
6. An additive according to any one of claims 1 to 3, wherein: the commercial products of the lipids are selected from: item numbers L9655, L0288 manufactured by Sigma; the products of HyClone are LS1000 and HyClone LS 250.
7. An iPS-CM purification culture medium, which comprises a basic culture medium and is characterized in that: the basal medium is also added with the additive of claims 1-6.
8. The iPS-CM purification media of claim 7, wherein: the basic culture medium is selected from a sugar-free DMEM culture medium, a sugar-free 1640 culture medium and sugar-free DMEM/F12.
9. The iPS-CM purification media of claim 7, wherein: the composition is as follows:
sodium lactate: 3 mM
Lipid: 0.25v/v%, Cat number Sigma, L0288
L-carnitine: 5 μ M
HEPES：1 v/v%
Sugar-free DMEM medium, and the balance.
10. An iPS-CM purification culture method comprises the steps of adding iPS-CM obtained by iPS differentiation into an iPS-CM purification culture medium for metabolic purification culture, and is characterized in that: the iPS-CM purification medium is as defined in any one of claims 7 to 9.
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| CN117866884A (en) * | 2024-03-13 | 2024-04-12 | 中国医学科学院北京协和医院 | Construction method of glycogen accumulating disease IIIa type myocardial cell model |
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