CN108456654A - Serum free culture system for promoting source of human stem cell cardiac muscle cell maturation - Google Patents
Serum free culture system for promoting source of human stem cell cardiac muscle cell maturation Download PDFInfo
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- CN108456654A CN108456654A CN201810154071.5A CN201810154071A CN108456654A CN 108456654 A CN108456654 A CN 108456654A CN 201810154071 A CN201810154071 A CN 201810154071A CN 108456654 A CN108456654 A CN 108456654A
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- 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
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Abstract
The present invention relates to the serum free culture systems for promoting source of human stem cell cardiac muscle cell maturation.Specifically, the present invention provides a kind of culture medium promoting cardiac muscle cell's maturation, the culture medium contains serum-free differentiation media and promotes the additive of cardiac muscle cell's maturation, wherein, the additive for promoting cardiac muscle cell's maturation contains steroids, lipid, amino acid derivativges, nonprotein amino acid and biostearin compound.The present invention improves the immature state of cardiac muscle cell, it is made to have more ripe phenotype in terms of size and form, molecular composition, metabolism and physiological function by the way that a series of additives for promoting cardiac muscle cell's maturation are added in serum-free differentiation media.The cardiac muscle cell being prepared in the system has normal function, can be preferably applied for the drug screening and detection of cardiotoxicity of medicine, disease modeling and stem cell regenerating medical domain.
Description
Technical field
The invention belongs to stem cell biologies and technical field of cell culture, it particularly relates to for promoting stem cell
The serum free culture system of source cardiac muscle cell maturation.
Background technology
Angiocardiopathy seriously threatens the life and health of the mankind, according to World Health Organization, accounts for global dead total
Several 30% or more.Myocardial infarction is a kind of common damage to cardiac tissue, and adult is after occurring myocardial infarction, the heart of itself
Myocyte does not have power of regeneration substantially.The exploitation of drug is merely able to delay the life of sufferer, clinical heart to a certain extent
The problems such as transplanting is again compatible there is organ donor finite sum semelincident immunization.
The listing of drug needs to spend a large amount of manpower financial capacity.In twenty or thirty year in past, there is the drug of one third to remove
The main reason for city is with cardiac toxic.Preclinical detection model differs greatly with human body, cannot predict heart well
Toxic reaction thus causes some drugs and is finally recalled by market, can not be listed.
Multipotential stem cell includes embryonic stem cell and induced multi-potent stem cell, they have unlimited proliferative capacity and differentiation
Versatility, all types of body cells can be differentiated to form, open the new page of Homo sapiens reproduction's medicine.Multipotential stem cell source
Cardiac muscle cell is in research heart development process, drug screening and the detection of cardiotoxicity of medicine, disease modeling and clinical application etc.
Aspect also brings completely new hope.
Contain fetal calf serum ingredient in the culture medium of multipotential stem cell culture and Myocardium Differentiation mostly, the ingredient of serum is not
Know and complicated, since the serum product of different manufacturers is batch widely different, causes the repeatability of experiment extremely low.In addition, blood
Pathogen and other virulence factors may be carried by checking up and returning, and greatly reduced cardiac muscle cell and controlled in cardiotoxicity of medicine detection or cell
The application value of treat etc. and safety.Therefore matter of utmost importance is to establish a serum-free Myocardium Differentiation culture medium.Although mesh
Had been directed to the content of serum free medium in preceding published patent, but there are still differentiate cardiac muscle cell not at
Ripe problem.
The identified analysis of cardiac muscle cell in multipotential stem cell source, no matter form, gene and protein marker expression,
And in the characteristic of action potential and ion channel, there is prodigious differences with the cardiac muscle cell in body maturation.Based on upper
Problem is stated, the present invention proposes in serum-free Myocardium Differentiation culture medium, and a series of additives for promoting cardiac muscle cell's maturation are added,
Make cardiac muscle cell that there is more ripe phenotype in terms of size and form, molecular composition, metabolism and physiological function, it can be preferably
Applied to the drug screening and detection of cardiotoxicity of medicine, disease modeling and stem cell regenerating medical domain.
Invention content
The purpose of the present invention is on the basis of serum-free differentiation media, add a series of substance stem cell is promoted to come
The maturation of source cardiac muscle cell makes cardiac muscle cell have more ripe phenotype, is preferably applied for drug screening and medicine heart
Detection, disease modeling and the stem cell regenerating medical domain of toxicity.
Therefore, first aspect present invention provide it is a kind of promote cardiac muscle cell's maturation culture medium, the culture medium containing whether there is or not
Serum differentiation culture medium and the additive for promoting cardiac muscle cell's maturation.
It is described to promote the additive of cardiac muscle cell's maturation for steroids, lipid, amino in one or more embodiments
Acid derivative, nonprotein amino acid and biostearin compound.
In one or more embodiments, the steroids compound is selected from trilute and adrenalone
In one kind or all two kinds.
In one or more embodiments, trilute in the culture medium for promoting cardiac muscle cell's maturation
A concentration of 2 μ g/L to 200 μ g/L, preferably 2 μ g/L to 100 μ g/L, more preferable 10 μ g/L to 50 μ g/L.
In one or more embodiments, trilute in the culture medium for promoting cardiac muscle cell's maturation
A concentration of 20 ± 5 μ g/L.
In one or more embodiments, adrenalone is a concentration of in the culture medium for promoting cardiac muscle cell's maturation
10nM to 1000nM, preferably 10nM to 500nM, more preferably 50nM to 200nM.
In one or more embodiments, adrenalone is a concentration of in the culture medium for promoting cardiac muscle cell's maturation
100±20nM。
In one or more embodiments, the lipoid substance includes that the oleic acid of 0.03mM to 3mM, 0.03mM are arrived
The palmitic acid of 3mM, 5 μ g/L to the lipoic acid of 500 μ g/L, the leukotrienes of 0.1mg/L to 10mg/L, 0.1mg/L to 10mg/L
One or more of the anabolic steroids of linoleic acid and 0.2mg/L to 20mg/L.
In one or more embodiments, the lipoid substance includes that the oleic acid of 0.03mM to 3mM, 0.03mM are arrived
The palmitic acid of 3mM, 5 μ g/L to the lipoic acid of 500 μ g/L, the leukotrienes of 0.1mg/L to 10mg/L, 0.1mg/L to 10mg/L
The anabolic steroids of linoleic acid and 0.2mg/L to 20mg/L.
In one or more embodiments, a concentration of 0.1-1.0mM of the oleic acid, preferably 0.1-0.5mM.
In one or more embodiments, a concentration of 0.1-1.0mM of the palmitic acid, preferably 0.1-0.5mM.
In one or more embodiments, a concentration of 5 μ g/L to 200 μ g/L of the lipoic acid, preferably 10 μ g/L are arrived
100 μ g/L, more preferable 30 μ g/L to 80 μ g/L.
In one or more embodiments, linolenic a concentration of 0.1mg/L to the 5mg/L, preferably 0.5mg/L
To 3mg/L, more preferably 0.8mg/L to 1.2mg/L.
In one or more embodiments, linoleic a concentration of 0.1mg/L to the 5mg/L, preferably 0.5mg/L
To 3mg/L, more preferably 0.8mg/L to 1.2mg/L.
In one or more embodiments, a concentration of 0.2mg/L to the 10mg/L of the anabolic steroids, preferably
0.5mg/L to 5mg/L, more preferably 1mg/L to 5mg/L, more preferably 1mg/L to 3mg/L.
In one or more embodiments, the amino acid derivativges are creatine, and a concentration of 0.5mM to 50mM is excellent
It is selected as 1mM to 20mM, more preferably 1mM to 10mM, more preferably 3mM to 8mM.
In one or more embodiments, the nonprotein amino acid is taurine, and a concentration of 0.5mM is arrived
50mM, preferably 1mM to 20mM, more preferably 1mM to 10mM, more preferably 3mM to 8mM.
In one or more embodiments, the biostearin compound is carnitine, and a concentration of 0.2mg/L is arrived
20mg/L, preferably 1mg/L to 10mg/L, more preferably 1mg/L to 5mg/L, more preferably 1mg/L to 3mg/L.
It is described that the culture medium of cardiac muscle cell's maturation is promoted to contain following promotion cardiac muscles carefully in one or more embodiments
The additive of born of the same parents' maturation：
(1) one kind in trilute and adrenalone or all two kinds of steroids compound,
In, when containing sometimes, a concentration of 10 μ g/L to 50 μ g/L of trilute, preferably 20 ± 5 μ g/L；When containing sometimes,
A concentration of 50nM to the 200nM of adrenalone, preferably 100 ± 20nM；
(2) it is selected from the fat of one or more of oleic acid, palmitic acid, lipoic acid, leukotrienes, linoleic acid and anabolic steroids
Class compound, wherein when containing sometimes, a concentration of 0.1-0.5mM of oleic acid, a concentration of 0.1-0.5mM of palmitic acid, lipoic acid
To 80 μ g/L, linolenic a concentration of 0.8mg/L to 1.2mg/L, linoleic concentration 0.8mg/L are arrived a concentration of 30 μ g/L
1.2mg/L, a concentration of 1mg/L to the 3mg/L of anabolic steroids；Preferably, the lipoid substance is by the oleic acid, palm
Acid, lipoic acid, leukotrienes, linoleic acid and anabolic steroids composition；
(3) creatine, a concentration of 3mM to 8mM；
(4) taurine, a concentration of 3mM to 8mM；With
(5) carnitine, a concentration of 1mg/L to 3mg/L.
The present invention also provides a kind of kit, the kit contain serum-free differentiation media and promote cardiac muscle cell at
Ripe additive.
It is described to promote the additive of cardiac muscle cell's maturation for steroids, lipid, amino in one or more embodiments
Acid derivative, nonprotein amino acid and biostearin compound.
In one or more embodiments, the steroids compound is selected from trilute and adrenalone
In one kind or all two kinds.
In one or more embodiments, the amount of trilute is sufficient to make final with obtained in kit
To promotion cardiac muscle cell's maturation culture medium in its a concentration of 2 μ g/L to 200 μ g/L, preferably 2 μ g/L are more excellent to 100 μ g/L
Select 10 μ g/L to 50 μ g/L, more preferably 20 ± 5 μ g/L.
In one or more embodiments, the amount of adrenalone is sufficient to make the promotion finally prepared and obtained in kit
Its a concentration of 10nM to 500nM, more preferably 50nM to 200nM in the culture medium of cardiac muscle cell's maturation, more preferably 100 ±
20nM。
In one or more embodiments, the amount of each lipoid substance described in kit makes what finally preparation obtained
The oleic acid containing 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM, 5 μ g/L in the culture medium of cardiac muscle cell's maturation are promoted to arrive
Lipoic acid, the leukotrienes of 0.1mg/L to 10mg/L, the linoleic acid of 0.1mg/L to 10mg/L and the 0.2mg/L of 500 μ g/L is arrived
It is one or more in the anabolic steroids of 20mg/L.
In one or more embodiments, the amount of each lipoid substance described in kit makes what finally preparation obtained
The oleic acid containing 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM, 5 μ g/L in the culture medium of cardiac muscle cell's maturation are promoted to arrive
Lipoic acid, the leukotrienes of 0.1mg/L to 10mg/L, the linoleic acid of 0.1mg/L to 10mg/L and the 0.2mg/L of 500 μ g/L is arrived
The anabolic steroids of 20mg/L.
In one or more embodiments, the amino acid derivativges are creatine, and the amount of creatine makes most in kit
Its a concentration of 0.5mM to 50mM, preferably 1mM to 20mM in the culture medium of obtained promotion cardiac muscle cell's maturation are prepared eventually, more
Preferably 1mM to 10mM, more preferably 3mM to 8mM.
In one or more embodiments, the nonprotein amino acid is taurine, the amount of taurine in kit
So that its a concentration of 0.5mM to 50mM in the final culture medium for preparing obtained promotion cardiac muscle cell's maturation, preferably 1mM are arrived
20mM, more preferably 1mM to 10mM, more preferably 3mM to 8mM.
In one or more embodiments, the biostearin compound is carnitine, the amount of carnitine in kit
So that its a concentration of 0.2mg/L to 20mg/L in the final culture medium for preparing obtained promotion cardiac muscle cell's maturation, preferably
1mg/L to 10mg/L, more preferably 1mg/L to 5mg/L, more preferably 1mg/L to 3mg/L.
In one or more embodiments, the steroids, lipid, amino acid derivativges, nonprotein amino acid with
And biostearin compound respective independent packaging in kit.
In one or more embodiments, the steroids, lipid, amino acid derivativges, nonprotein amino acid with
And two or more in biostearin compound exists as a mixture in kit.
In one or more embodiments, steroids, lipid, amino acid derivativges, nonprotein amino in mixture
Acid and the respective content of biostearin compound should make when the culture for using the mixture to prepare promotion cardiac muscle cell's maturation
Concentration of each ingredient in final culture medium is respectively when base：The oleic acid of 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM,
5 μ g/L to the lipoic acid of 500 μ g/L, the leukotrienes of 0.1mg/L to 10mg/L, the linoleic acid of 0.1mg/L to 10mg/L and
0.2mg/L to 20mg/L, the preferably respectively palmitic acid of the oleic acid of 0.1-0.5mM, 0.1-0.5mM, 30 μ g/L are to 80 μ g/L's
Lipoic acid, the leukotrienes of 0.8mg/L to 1.2mg/L, the linoleic acid and 1mg/L to 3mg/L of 0.8mg/L to 1.2mg/L synthesize class
Sterol.
In one or more embodiments, the kit contains the culture as described herein for promoting cardiac muscle cell's maturation
Base.
The present invention also provides a kind of additive for promoting cardiac muscle cell's maturation, the additive contains steroids, fat
Class, amino acid derivativges, nonprotein amino acid and biostearin compound, or by steroids, lipid, amino acid derivativges,
Nonprotein amino acid and biostearin compound composition.
The present invention also provides described for promoting the additive of cardiac muscle cell's maturation preparing promotion cardiac muscle cell's maturation
Application in culture medium.
The present invention also provides the methods for the cardiac muscle cell's maturation for promoting differentiation, and the method includes using rush as described herein
Into cardiac muscle cell's maturation medium culture broken up by stem cell made of cardiac muscle cell the step of.
Description of the drawings
Fig. 1 is the table of four ion channel genes (HCN4, GJA1, KCNJ2 and SCN5A) relative to early stage cardiac muscle cell
Up to situation.
Specific implementation mode
The present invention adds a series of substance to promote source of human stem cell cardiac muscle thin on the basis of serum-free differentiation media
The maturation of born of the same parents makes cardiac muscle cell have more ripe phenotype, is preferably applied for drug screening and the inspection of cardiotoxicity of medicine
It surveys, disease models and stem cell regenerating medical domain.
It is multipotential stem cell to be suitable for the invention stem cell, can be established human embryo stem cell, or at
Somatic stem cell such as epidermal stem cells, can also be induced multi-potent stem cell, as obtained through external reprogramming by people's foreskin cells
Stem cell.
It can be that the stem cell that is used for well known in the art breaks up to be suitable for the invention serum-free differentiation media, especially
Stem cell breaks up the various culture mediums of cardioblast.This kind of culture medium be typically basal medium well known in the art such as
One or more suitable additives are added on the basis of 1640 culture mediums of RPMI, DMEM, DMEM/F12, IMDM and BME, are used
Stem cell is cultivated, is broken up cardioblast.This kind of additive can promote stem cell differentiation deliberately for well known in the art
The additive of myocyte.
Commercially available serum-free differentiation media and well known differentiation method can be used that stem cell is broken up cardioblast.
For example, can refer to the differentiation that the method described in Lian et al.2013, Nature Protocol carries out stem cell.
For a period of time rear (such as after 14 days) with serum-free differentiation media culture stem cell, use can be added into culture
Original training is replaced in the additive for promoting cardiac muscle cell's maturation, or with the culture medium as described herein for promoting cardiac muscle cell's maturation
Base is supported, is cultivated, the maturation of cardiac muscle cell can be promoted.
Herein, described to promote the additive of cardiac muscle cell's maturation for steroids, lipid, amino acid derivativges, non-protein
Matter amino acid and biostearin compound.The one kind of steroids compound in trilute and adrenalone
Or all two kinds.Lipoid substance can be selected from one in oleic acid, palmitic acid, lipoic acid, leukotrienes, linoleic acid and anabolic steroids
Kind is several, preferably all uses.Amino acid derivativges are preferably creatine.Nonprotein amino acid is preferably taurine.Class
Element-vitamine compound is preferably carnitine.
In general, when containing sometimes, a concentration of the 2 of trilute in the culture medium for promoting cardiac muscle cell's maturation
μ g/L to 200 μ g/L, preferably 2 μ g/L are to 100 μ g/L, more preferable 10 μ g/L to 50 μ g/L, more preferably 20 ± 5 μ g/L；When containing
Sometimes, a concentration of 10nM to the 1000nM of adrenalone, preferably 10nM to 500nM, more preferably 50nM to 200nM are more excellent
It is selected as 100 ± 20nM.
Lipoid substance generally includes the oleic acid of 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM, 5 μ g/L to 500 μ g/
The lipoic acid of L, the leukotrienes of 0.1mg/L to 10mg/L, the linoleic acid of 0.1mg/L to 10mg/L and 0.2mg/L to 20mg/L
One or more of anabolic steroids.Preferably, the lipoid substance includes that the oleic acid of 0.03mM to 3mM, 0.03mM are arrived
The palmitic acid of 3mM, 5 μ g/L to the lipoic acid of 500 μ g/L, the leukotrienes of 0.1mg/L to 10mg/L, 0.1mg/L to 10mg/L
The anabolic steroids of linoleic acid and 0.2mg/L to 20mg/L.In certain embodiments, when containing sometimes, the concentration of oleic acid is preferred
For 0.1-1.0mM, more preferably 0.1-0.5mM；The concentration of palmitic acid is preferably 0.1-1.0mM, more preferably 0.1-0.5mM；
The concentration of lipoic acid is preferably 5 μ g/L to 200 μ g/L, more preferably 10 μ g/L to 100 μ g/L, more preferably 30 μ g/L to 80 μ
g/L；Linolenic a concentration of 0.1mg/L to 5mg/L, preferably 0.5mg/L to 3mg/L, more preferably 0.8mg/L to 1.2mg/
L；Linoleic a concentration of 0.1mg/L to 5mg/L, preferably 0.5mg/L to 3mg/L, more preferably 0.8mg/L to 1.2mg/L；
A concentration of 0.2mg/L to the 10mg/L of anabolic steroids, preferably 0.5mg/L to 5mg/L, more preferably 1mg/L to 5mg/L,
More preferably 1mg/L to 3mg/L.
In certain embodiments, a concentration of 0.5mM to the 50mM of creatine, preferably 1mM to 20mM, more preferably 1mM
To 10mM, more preferably 3mM to 8mM.
In certain embodiments, a concentration of 0.5mM to the 50mM of taurine, preferably 1mM to 20mM, more preferably
1mM to 10mM, more preferably 3mM to 8mM.
In certain embodiments, a concentration of 0.2mg/L to the 20mg/L of carnitine, preferably 1mg/L to 10mg/L, more
Preferably 1mg/L to 5mg/L, more preferably 1mg/L to 3mg/L.
The medium culture stem cell point of promotion cardiac muscle cell's maturation of the present invention can be used under conventional condition of culture
Change the cardiac muscle cell formed, a subculture was replaced per 2-3 days.
Culture medium using the present invention can improve the immature state of cardiac muscle cell, make its size and form, molecular composition,
There is more ripe phenotype in terms of metabolism and physiological function.
The present invention also provides a kind of kit, which contains serum-free differentiation media and for promoting cardiac muscle cell
Ripe additive.The additive for promoting cardiac muscle cell's maturation is steroids, lipid, amino acid derivativges, non-egg
White matter amino acid and biostearin compound.Serum-free differentiation media can be well known in the art for stem cell point
Change, especially the various culture mediums of stem cell differentiation cardioblast.This kind of culture medium is typically on basis well known in the art
It adds and one or more suitably adds on the basis of 1640 culture medium of culture medium such as RPMI, DMEM, DMEM/F12, IMDM and BME
Add agent, for cultivating stem cell, is broken up cardioblast.It is suitable to be used to promote adding for stem cell differentiation cardioblast
It is well known in the art to add agent, for example, seralbumin, insulin, transferrins etc..
In certain embodiments, which contains the culture medium as described herein for promoting cardiac muscle cell's maturation.Alternatively,
Serum-free differentiation media described in kit and the additive respectively independent packaging for promoting cardiac muscle cell's maturation, respectively
Additive also can independent packaging, or in the form of two or more mixture provide.When independent packaging, hormone
Class, lipid, amino acid derivativges, nonprotein amino acid and biostearin compound amount should be sufficient to make and matched using them
Their own concentration promotes cardiac muscle cell defined by the present invention in the culture medium for the promotion cardiac muscle cell's maturation being made
In ripe culture medium within the concentration range of each additive.For example, the amount of trilute is enough to make in kit
Its a concentration of 2 μ g/L to 200 μ g/L in the culture medium for finally preparing obtained promotion cardiac muscle cell's maturation is obtained, preferably 2 μ g/L are arrived
100 μ g/L, more preferable 10 μ g/L are to 50 μ g/L, more preferably 20 ± 5 μ g/L；The amount of adrenalone is sufficient to make final with obtained
To promotion cardiac muscle cell's maturation culture medium in its a concentration of 10nM to 500nM, more preferably 50nM to 200nM, more preferably
For 100 ± 20nM；The amount of each lipoid substance finally to prepare to be contained in the culture medium of obtained promotion cardiac muscle cell's maturation
Lipoic acid, 0.1mg/L to the 10mg/L of the oleic acid of 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM, 5 μ g/L to 500 μ g/L
Leukotrienes, one or more in the linoleic acid of 0.1mg/L to 10mg/L and the anabolic steroids of 0.2mg/L to 20mg/L；
The amount of creatine makes its a concentration of 0.5mM to 50mM in the culture medium for promoting cardiac muscle cell's maturation that finally preparation obtains, preferably
For 1mM to 20mM, more preferably 1mM to 10mM, more preferably 3mM to 8mM；The amount of taurine finally to prepare obtained rush
Into its a concentration of 0.5mM to 50mM, preferably 1mM to 20mM in the culture medium of cardiac muscle cell's maturation, more preferably 1mM is arrived
10mM, more preferably 3mM to 8mM；The amount of carnitine finally to prepare in the obtained culture medium for promoting cardiac muscle cell's maturation
Its a concentration of 0.2mg/L to 20mg/L, preferably 1mg/L to 10mg/L, more preferably 1mg/L to 5mg/L, more preferably 1mg/
L to 3mg/L.
Containing steroids, lipid, amino acid derivativges, nonprotein amino acid and biostearin compound, or by hormone
The mixture that class, lipid, amino acid derivativges, nonprotein amino acid and biostearin compound form is also in the guarantor of the present invention
Within the scope of shield.The content of each ingredient should to add them to prepare in basal medium and obtain herein in these mixtures
After the culture medium of the differentiation serum free medium or promotion cardiac muscle cell's maturation, the concentration of each ingredient is at this in culture medium
It invents within each embodiment limited range.For example, the amount of trilute is sufficient to make finally in mixture
Prepare its a concentration of 2 μ g/L to 200 μ g/L, preferably 2 μ g/L to 100 μ g/ in the culture medium of obtained promotion cardiac muscle cell's maturation
L, more preferable 10 μ g/L are to 50 μ g/L, more preferably 20 ± 5 μ g/L；The amount of adrenalone is sufficient to make the rush finally prepared and obtained
Into its a concentration of 10nM to 500nM, more preferably 50nM to 200nM in the culture medium of cardiac muscle cell's maturation, more preferably 100 ±
20nM；The amount of each lipoid substance, which finally to prepare, to be arrived in the culture medium of obtained promotion cardiac muscle cell's maturation containing 0.03mM
The oleic acid of 3mM, the palmitic acid of 0.03mM to 3mM, the lipoic acid of 5 μ g/L to 500 μ g/L, 0.1mg/L to 10mg/L leukotrienes,
It is one or more in the linoleic acid of 0.1mg/L to 10mg/L and the anabolic steroids of 0.2mg/L to 20mg/L；The amount of creatine makes
Its a concentration of 0.5mM to 50mM in the culture medium for finally preparing obtained promotion cardiac muscle cell's maturation is obtained, preferably 1mM is arrived
20mM, more preferably 1mM to 10mM, more preferably 3mM to 8mM；The amount of taurine finally to prepare obtained promotion cardiac muscle
Its in the culture medium of cell maturation a concentration of 0.5mM to 50mM, preferably 1mM to 20mM, more preferably 1mM to 10mM, it is more excellent
It is selected as 3mM to 8mM；The amount of carnitine makes its in the culture medium for promoting cardiac muscle cell's maturation that finally preparation obtains a concentration of
0.2mg/L to 20mg/L, preferably 1mg/L to 10mg/L, more preferably 1mg/L to 5mg/L, more preferably 1mg/L to 3mg/
L。
It should be understood that the concentration range of each ingredient given herein refers to that it is being trained when containing the ingredient in culture medium
Support the ultimate density range in base.
The present invention also provides described for promoting the additive of cardiac muscle cell's maturation preparing promotion cardiac muscle cell's maturation
Application in culture medium.The present invention also provides the methods for the cardiac muscle cell's maturation for promoting differentiation, and the method includes using herein
Made of the medium culture of promotion cardiac muscle cell's maturation is broken up by stem cell the step of cardiac muscle cell.
Further, the present invention also provides the methods for preparing cardiac muscle cell, and the method includes using the rush of the present invention
Into cardiac muscle cell's maturation medium culture broken up by stem cell made of cardiac muscle cell the step of.
As it was noted above, in addition to culture medium, it is ability to cultivate stem cell and other conditions such as temperature of cardiac muscle cell etc.
The condition of domain routine.
Moreover, it will be understood that each additive addressed herein, such as steroids, lipid, amino acid derivativges, non-protein
Matter amino acid and biostearin compound are the products of this field routine, can be obtained by commercially available approach；Each basal medium
Also it can obtain from commercially available approach, or voluntarily be prepared according to formula well known in the art.
Serum free culture system for promoting source of human stem cell cardiac muscle cell maturation is proposed using the present invention, is had following
Advantage：
(1) serum composition is not contained in Myocardium Differentiation culture medium, reduces the batch sex differernce of differentiation, is improved myocardium thin
The safety of born of the same parents and application value.
(2) a series of promotion cell maturation additives are added in serum-free differentiation media, improve cardiac muscle cell
Immature state, make its in terms of size and form, molecular composition, metabolism and physiological function have more ripe phenotype.
(3) cardiac muscle cell prepared has normal function, can be preferably applied for drug screening and cardiotoxicity of medicine
Detection, disease modeling and stem cell regenerating medical domain.
Further detailed description is carried out to the present invention with reference to embodiment, but they are not to the further of the present invention
Limitation.
Electronic Speculum imaging analysis can be used to observe the muscle fibril and sarcomere structural of cardiac muscle cell, the form for evaluating cell is special
The information for appearance of seeking peace；It is ripe related to cardiac muscle cell using Real-time quantitative PCR and the detection of immunostained for analysis method
Marker gene or albumen expression；(TMRM) staining analysis of tetramethylrhodamine ethyl ester can be used to obtain about external
The mitochondria activity of Culture Center myocyte is horizontal；Action potential and the ion that patch clamp technique announcement cardiac muscle cell can be used are logical
The functional character of the expression in road.These technologies and implement the reagent that the reagent and equipment needed for these technologies are all commonly used in the art
And equipment.
Embodiment 1：Stem cell myocardium differentiation is induced in serum free medium
The induced multi-potent stem cell that the present embodiment uses is that people's foreskin cells are obtained through external reprogramming, and text has been delivered in reference
Differentiation method (Lian et al.2013, Nature Protocol) in offering is broken up.It can be induced at the 7th day or so
The cardiac muscle cell now independently to beat continued culture by 14 days.
Embodiment 2：Promote the maturation of cardiac muscle cell
Following component is added in embodiment 1 in culture to the culture of the 14th day cardiac muscle cell, continues to cultivate：
Steroids：The adrenalone of the trilute and 100nM of 20 μ g/L；
Lipid：The Asia of the oleic acid of 0.3mM, the palmitic acid of 0.3mM, the lipoic acid of 50 μ g/L, the leukotrienes of 1mg/L, 1mg/L
The anabolic steroids of oleic acid and 2mg/L；
Amino acid derivativges：The creatine of 5mM；
Nonprotein amino acid：The taurine of 5mM；With
Biostearin compound：The carnitine of 2mg/L.
A concentration of final concentration of above-mentioned each ingredient.
A subculture was replaced per 2-3 days.
Using Real-time quantitative PCR and the detection of immunostained for analysis method and the ripe relevant mark base of cardiac muscle cell
The expression of cause or albumen.
The results are shown in Figure 1.The results show that prolonging with the incubation time for using serum free culture system of the invention
It is long, it is had differences really in the expression of important ion channel gene with end-stage cardiomyocytes in early days.
Embodiment 3：Promote the maturation of cardiac muscle cell
Following component is added in embodiment 1 in culture to the culture of the 14th day cardiac muscle cell, continues to cultivate：
Steroids：The adrenalone of the trilute and 100nM of 20 μ g/L；
Lipid：The Asia of the oleic acid of 0.3mM, the palmitic acid of 0.3mM, the lipoic acid of 50 μ g/L, the leukotrienes of 1mg/L, 1mg/L
The anabolic steroids of oleic acid or 2mg/L；
Amino acid derivativges：The creatine of 5mM；
Nonprotein amino acid：The taurine of 5mM；With
Biostearin compound：The carnitine of 2mg/L.
A concentration of final concentration of above-mentioned each ingredient.
A subculture was replaced per 2-3 days.
Using Real-time quantitative PCR and the detection of immunostained for analysis method and the ripe relevant mark base of cardiac muscle cell
The expression of cause or albumen.As a result show early stage with end-stage cardiomyocytes important ion channel gene HCN4, GJA1,
It is had differences in the expression of KCNJ2 and SCN5A.
Embodiment 4：Promote the maturation of cardiac muscle cell
Following component is added in embodiment 1 in culture to the culture of the 14th day cardiac muscle cell, continues to cultivate：
Steroids：The adrenalone of the trilute and 100nM of 20 μ g/L；
Lipid：The oleic acid of 0.3mM, the lipoic acid of 50 μ g/L, the leukotrienes of 1mg/L and the anabolic steroids of 2mg/L；
Amino acid derivativges：The creatine of 5mM；
Nonprotein amino acid：The taurine of 5mM；With
Biostearin compound：The carnitine of 2mg/L.
A concentration of final concentration of above-mentioned each ingredient.
A subculture was replaced per 2-3 days.
Using Real-time quantitative PCR and the detection of immunostained for analysis method and the ripe relevant mark base of cardiac muscle cell
The expression of cause or albumen.As a result show early stage with end-stage cardiomyocytes important ion channel gene HCN4, GJA1,
It is had differences in the expression of KCNJ2 and SCN5A.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by present embodiment
Limitation, the change made in the case of other any essence and principle without departing from the present invention replacement, are combined, simplification and modification,
Equivalence replacement mode is should be, is included within the scope of the present invention.
Claims (10)
1. a kind of culture medium promoting cardiac muscle cell's maturation, which is characterized in that the culture medium contains serum-free differentiation media
With promote cardiac muscle cell's maturation additive, wherein it is described promotion cardiac muscle cell's maturation additive contain steroids, lipid,
Amino acid derivativges, nonprotein amino acid and biostearin compound.
2. culture medium as described in claim 1, which is characterized in that the steroids compound is selected from trilute
With two kinds of one kind or whole in adrenalone；
Preferably, it is described promote cardiac muscle cell's maturation culture medium in trilute a concentration of 2 μ g/L to 200 μ
G/L, preferably 2 μ g/L are to 100 μ g/L, more preferable 10 μ g/L to 50 μ g/L, more preferable 20 ± 5 μ g/L；
Preferably, it is described promote cardiac muscle cell's maturation culture medium in adrenalone a concentration of 10nM to 1000nM, preferably
10nM to 500nM, more preferable 50nM to 200nM, more preferable 100 ± 20nM.
3. culture medium as claimed in claim 1 or 2, which is characterized in that the lipoid substance includes the oil of 0.03mM to 3mM
Acid, the palmitic acid of 0.03mM to 3mM, 5 μ g/L to the lipoic acid of 500 μ g/L, leukotrienes, the 0.1mg/L of 0.1mg/L to 10mg/L
To the linoleic acid of 10mg/L and one or more of the anabolic steroids of 0.2mg/L to 20mg/L；
Preferably, the lipoid substance includes the oleic acid of 0.03mM to 3mM, the palmitic acid of 0.03mM to 3mM, 5 μ g/L to 500
The linoleic acid and 0.2mg/L to 20mg/L of the lipoic acid of μ g/L, the leukotrienes of 0.1mg/L to 10mg/L, 0.1mg/L to 10mg/L
Anabolic steroids.
4. culture medium as claimed in claim 3, which is characterized in that
A concentration of 0.1-1.0mM of the oleic acid, preferably 0.1-0.5mM；
A concentration of 0.1-1.0mM of the palmitic acid, preferably 0.1-0.5mM；
A concentration of 5 μ g/L to 200 μ g/L of the lipoic acid, preferably 10 μ g/L are to 100 μ g/L, more preferable 30 μ g/L to 80 μ g/
L；
Linolenic a concentration of 0.1mg/L to the 5mg/L, preferably 0.5mg/L to 3mg/L, more preferable 0.8mg/L to 1.2mg/
L；
Linoleic a concentration of 0.1mg/L to the 5mg/L, preferably 0.5mg/L to 3mg/L, more preferable 0.8mg/L to 1.2mg/
L；With
A concentration of 0.2mg/L to the 10mg/L of the anabolic steroids, preferably 0.5mg/L to 5mg/L, more preferable 1mg/L are arrived
5mg/L, more preferable 1mg/L to 3mg/L.
5. culture medium as claimed in claim 1 or 2, which is characterized in that
The amino acid derivativges be creatine, a concentration of 0.5mM to 50mM, preferably 1mM to 20mM, more preferable 1mM to 10mM,
More preferable 3mM to 8mM；
The nonprotein amino acid is taurine, and a concentration of 0.5mM to 50mM, preferably 1mM to 20mM, more preferable 1mM arrives
10mM, more preferable 3mM to 8mM；And/or
The biostearin compound is carnitine, and a concentration of 0.2mg/L to 20mg/L, preferably 1mg/L to 10mg/L are more excellent
Select 1mg/L to 5mg/L, more preferable 1mg/L to 3mg/L.
6. culture medium as described in claim 1, which is characterized in that the culture medium contains following promotion cardiac muscle cells maturations
Additive：
(1) one kind in trilute and adrenalone or all two kinds of steroids compound, wherein when
Containing sometimes, a concentration of 10 μ g/L to 50 μ g/L of trilute, preferably 20 ± 5 μ g/L；When containing sometimes, adrenal gland
A concentration of 50nM to the 200nM of ketone, preferably 100 ± 20nM；
(2) it is selected from the lipid of one or more of oleic acid, palmitic acid, lipoic acid, leukotrienes, linoleic acid and anabolic steroids
Close object, wherein when containing sometimes, a concentration of 0.1-0.5mM of oleic acid, a concentration of 0.1-0.5mM of palmitic acid, the concentration of lipoic acid
For 30 μ g/L to 80 μ g/L, linolenic a concentration of 0.8mg/L to 1.2mg/L, linoleic concentration 0.8mg/L to 1.2mg/L,
A concentration of 1mg/L to the 3mg/L of anabolic steroids；Preferably, the lipoid substance by the oleic acid, palmitic acid, lipoic acid,
Leukotrienes, linoleic acid and anabolic steroids composition；
(3) creatine, a concentration of 3mM to 8mM；
(4) taurine, a concentration of 3mM to 8mM；With
(5) carnitine, a concentration of 1mg/L to 3mg/L.
7. a kind of kit, the kit contains serum-free differentiation media and promotes the additive of cardiac muscle cell's maturation,
In, the additive for promoting cardiac muscle cell's maturation is steroids, lipid, amino acid derivativges, nonprotein amino acid
And biostearin compound；
Preferably, the kit contains the culture medium described in any one of claim 1-6；Or
Preferably, the additive of serum-free differentiation media described in the kit and promotion cardiac muscle cell's maturation independently wraps
Dress, wherein described to promote the additive of cardiac muscle cell's maturation as described in any one of claim 2-6.
8. a kind of for promoting the additive of cardiac muscle cell's maturation, the additive contains steroids, lipid, amino acid derived
Object, nonprotein amino acid and biostearin compound, or by steroids, lipid, amino acid derivativges, nonprotein amino acid
It is formed with biostearin compound；Wherein, the steroids compound is in trilute and adrenalone
It is a kind of or two kinds whole；The lipoid substance includes oleic acid, palmitic acid, lipoic acid, leukotrienes, linoleic acid and anabolic steroids
One or more of；The amino acid derivativges are creatine；The nonprotein amino acid is taurine；The biostearin
Compound is carnitine；
Preferably, steroids described in additive, lipid, amino acid derivativges, nonprotein amino acid and biostearin chemical combination
The content of object matches so that the additive is added in the culture medium prepared and obtained afterwards in serum-free differentiation media：Triiodo first
A concentration of 2 μ g/L to 200 μ g/L of shape gland original ammonia acid, a concentration of 10nM to the 1000nM of adrenalone, oleic acid it is a concentration of
A concentration of 5 μ g/L to 500 μ g/L, linolenic dense of 0.03mM to 3mM, a concentration of 0.03mM to the 3mM of palmitic acid, lipoic acid
Degree is 0.1mg/L to 10mg/L, a concentration of 0.2mg/L of linoleic a concentration of 0.1mg/L to 10mg/L, anabolic steroids is arrived
20mg/L, a concentration of 0.5mM to the 50mM of creatine, a concentration of 0.5mM to the 50mM of taurine and carnitine it is a concentration of
0.2mg/L to 20mg/L.
9. application of the additive according to any one of claims 8 in preparing the culture medium for promoting cardiac muscle cell's maturation.
10. a kind of method for the cardiac muscle cell's maturation for promoting to break up, the method includes using any one of claim 1-6
Made of the medium culture of promotion cardiac muscle cell's maturation is broken up by stem cell the step of cardiac muscle cell.
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