CN104611291A - Culture medium capable of inducing embryonic stem cells into myocardial cells and application of culture medium - Google Patents
Culture medium capable of inducing embryonic stem cells into myocardial cells and application of culture medium Download PDFInfo
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Abstract
The invention provides a culture medium capable of inducing embryonic stem cells into myocardial cells. The culture medium is clear in chemical components, is a liquid culture medium with clear chemical components and comprises a basal culture medium RPMI-1640 and further comprises 1-3mg/L of L-carnitine, 0.5-2mg/L of ethanolamine, 10-20mg/L of putrescine, 0.01-0.02mg/L of selenite sodium, 0.5-2mg/L of linoleic acid, 3-6mg/L of transferrin, 50-200mg/L of vitamin C and/or vitamin C magnesium phosphate and 50-500upsilonM N-acetylcysteine. The invention also provides a preparation method and an application method of the culture medium. The culture medium is clear in chemical components, low in cost and very conductive to large-scale preparation and clinical application of myocardial cells.
Description
Technical field
The invention belongs to biological technical field, relating to a kind of substratum for by embryonic stem cell induction being myocardial cell.Particularly, the present invention relates to the substratum using chemical composition completely clear and definite, by human embryo stem cell for directional inductive formation myocardial cell efficiently; The invention still further relates to a kind of method and application thereof for by embryonic stem cell induction being myocardial cell.
Background technology
Research in recent years shows, heart disease is positioned at first of high fatal disease always, and in heart disease, myocardial infarction is again topmost Death causes.In recent years, along with improving constantly of people's living standard, particularly high hot high lipid food absorption and lack enough physical activitys, the sickness rate of myocardial infarction is in the trend risen year by year.Myocardial infarction mainly causes lumen of vessels narrow due to coronary atherosclerosis, and blood supply is not enough, thus causes myocardium cell necrosis.As everyone knows, the myocardial cell of myocardial cell, particularly grownup no longer has multiplication capacity.Therefore, after myocardial infarction, infarcted region forms scar tissue primarily of fibrocyte and matrix, causes left ventricular remodeling, and the later stage occurs in heart failure.
The method of traditional treatment myocardial infarction mainly contains following several: one, conservative medication; Two, medicine thromboembolism treatment; Three, Stent; Four, coronary bypass Coronary Artery Bypass.These treatment meanss can only mitigate the disease, improves the part quality of life of patient, can not prevent remodeling ventricle and heart failure subsequently.In addition, clinically, the heart disease patient for whole latter stage can also carry out heart transplantation, but heart donor source is very limited, there will be immunological rejection after heart transplantation simultaneously.Therefore, from the strict sense, healing that can not be real by these treatment meanss after myocardial infarction.
In recent years, along with going deep into of stem-cell research, many have generate myocardial cell potential multipotential stem cell by the seed cell as cell therapy, such as the stem cell of derived from bone marrow, hemopoietic stem cell, mescenchymal stem cell, Cardiac Stem Cells and endothelial precursor cell etc.But the efficiency and the curative effect that generate myocardial cell after these Transplanted cellss are in vivo controversial.Human embryonic stem cell (human embryonicstem cell, hESc) frontier of regenerative medicine has been started in foundation, compared with other therapeutic cells, embryonic stem cell can infinitely breed, can be divided into myocardial cell efficiently in vitro by self in vitro, and these features make it have broad application prospects at regenerative medicine and field of tissue engineering technology.
In order to advance the clinical application of embryonic stem cell series products, people are constantly attempting the culture systems improving and optimizating human embryo stem cell always.James Thomson laboratory is in the forefront of research always in this field.From the initial substratum containing 20% foetal calf serum (FBS), to the serum replacement (KOSR) finding alternative foetal calf serum, and comprise containing sero-abluminous mTSeR substratum.And optimize the best E8-vitronectin culture systems without feeder layer, non-animal derived, specific chemical components no more than its development in laboratory of improved culture medium.But unstable by embryonic stem cell Cardiomyocytes Induction of committed differentiation system at present, the otherness between as poor in differentiation gained cell uniformity, differentiation groups between efficiency and different clone is remarkable.Similar with the optimization of the culture systems of human embryo stem cell, differentiation culture system human embryo stem cell being divided into myocardial cell also experienced by composition component by the unknown to determining, by complexity to simple, by the future development of animal source to people source or specific chemical components.Early differentiation obtains myocardial cell mainly by splanchnic mesoderm like cell (the Mouse visceral endoderm-likecell with mouse, END-2) Dual culture, or obtained by the method for embryoid (Embryonic body, EB).But myocardial cell's efficiency that these methods obtain is low.Charles Murry development in laboratory monolayer cell differentiated system afterwards, by adding short Myocardium Differentiation factors A ctivin A and BMP4 in the basis differentiation liquid of tradition 1640 and B27 composition, this method can make the differentiation efficiency of cardiac muscle bring up to about 50%.Although serum-free additive eliminates serum composition, wherein still contain the serum albumin in a large amount of ox sources or people source.2014, the human serum albumin (rice-derived recombinant human albumin) that Joseph Wu seminar of Stanford university has only used three kinds of component: RPMI-1640, vitamins C phosphoric acid salt (L-Ascorbic acid 2-phosphate) and paddy rice to recombinate, adds synthetic small molecules CHIR99021 and Wnt-C59 on this basis again and just differentiation efficiency can be brought up to 80%.But the human serum albumin of paddy rice restructuring is required, and concentration is up to 0.5mg/mL, and the difference between serum albumin batch can cause the instability of differentiated system.
In sum, although groping of myocardium differentiation-inducing culture system has been carried out in a lot of laboratory, still exist significantly not enough.Human Cardiomyocytes has larger clinical application potential, and as cellular transplantation therapy myocardial infarction, broad-spectrum medicinal screening and the test of medicine cardiac muscle toxicity, and these all need a large amount of Human Cardiomyocytes.Therefore, the current needs of induction substratum also existed myocardium differentiation-inducing efficiency, non-animal derived composition, specific chemical components can be improved.
Summary of the invention
Therefore, for the deficiencies in the prior art, the present invention first to for by embryonic stem cell induction for the substratum of myocardial cell is studied, and be optimized to the state of specific chemical components, under the induction of the substratum of this optimization, acquisition myocardial cell that can be efficient, stable.
Object of the present invention is just to provide a kind of for being the substratum of myocardial cell by embryonic stem cell induction, and the specific chemical components of this substratum, thus lays a good foundation for the clinical application of myocardial cell.
On the one hand, the invention provides a kind of substratum for by embryonic stem cell induction being myocardial cell, described substratum is the liquid nutrient medium of specific chemical components, and described substratum comprises basic medium RPMI-1640;
Further, described substratum also comprises the N acetylcysteine of the L-BETAIN of 1 ~ 3mg/L, the thanomin of 0.5 ~ 2mg/L, the putrescine of 10 ~ 20mg/L, the Sodium Selenite of 0.01 ~ 0.02mg/L, the linolic acid of 0.5 ~ 2mg/L, the Transferrins,iron complexes of 3 ~ 6mg/L, the vitamins C of 50-200mg/L and/or Magnesium Ascorbyl Phosphate salt and 50-500 μM;
Preferably, described substratum also comprises the N acetylcysteine of the L-BETAIN of 2mg/L, the thanomin of 1mg/L, the putrescine of 16mg/L, the Sodium Selenite of 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L, the vitamins C of 100mg/L and/or Magnesium Ascorbyl Phosphate salt and 200 μMs;
Wherein, the osmotic pressure value of described basic medium is 295-305, is preferably 300;
Preferably, the sodium-chlor liquid storage of 5M is used to regulate the osmotic pressure value of described basic medium RPMI-1640;
Preferably, described substratum also comprises Regular Insulin;
Preferably, described Regular Insulin concentration is in the medium 2 ~ 6mg/L, is more preferably 4mg/L.
Preferably, described substratum also comprises induction small molecules;
Preferably, described induction small molecules is selective depressant CHIR99021, Wnt acceptor inhibitor IWR1 or the Wnt inhibitor wnt-c59 of liver starch synthesis kinases 3 β (GSK3 β) acceptor;
More preferably, described CHIR99021 concentration is in the medium 2-8 μM, also preferably, is 4 μMs; Further preferably, described IWR1 concentration is in the medium 2-8 μM, is also preferably 5 μMs; Again preferably, described wnt-c59 concentration is in the medium 1-5 μM, is also preferably 2 μMs.
Therefore, in a preferred embodiment, described substratum comprises the component shown in following table 1:
Table 1
Preferably, described substratum also comprises one or more in the component shown in following table 2;
Table 2
Preferably, described stem cell is clinical grade human embryonic stem cell (CTS-hES) or H7 human embryo stem cell; In a specific embodiment, described clinical grade human embryonic stem cell (CTS-hES) can purchased from Beijing stem cell bank.
On the other hand, present invention also offers the preparation method of substratum of the present invention, said method comprising the steps of:
1) according to the cumulative volume of substratum to be prepared, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) be dissolved in the deionized water of sterilizing by RPMI-1640 dry powder, stir, often liter adds 2g sodium bicarbonate, and regulates pH to be 7.2, then osmotic pressure value is adjusted to 295-305, frit, 4 DEG C of preservations;
Preferably, described osmotic pressure value is 300;
Preferably, 5M sodium-chlor liquid storage is used to regulate described osmotic pressure value;
Preferably, the frit of 0.22 μm is used;
Preferably, described pH value uses concentrated hydrochloric acid to regulate;
3) according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 1 ~ 3mg/L, 0.5 ~ 2mg/L, 10 ~ 20mg/L, 0.01 ~ 0.02mg/L, the linolic acid of 0.5 ~ 2mg/L, the Transferrins,iron complexes of 3 ~ 6mg/L prepare 50 × liquid storage;
Preferably, described 50 × the preparation method of liquid storage identical with the method for preparation 50 × B27 liquid storage;
More preferably, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 2mg/L, 1mg/L, 16mg/L, 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L prepare 50 × liquid storage;
4) according to the ratio of the N acetylcysteine of the vitamins C of 50-200mg/L and/or Magnesium Ascorbyl Phosphate salt, 50-500 μM prepare 1000 × liquid storage;
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in obtained basic medium, both can.
Preferably, described step 5) in also add mycillin and mix;
Wherein, the working concentration of described mycillin streptomycin is 100mg/l, and the working concentration of penicillin is 100,000U/L;
Preferably, during use, described substratum is heated to 37 DEG C.
Preferably, described step 3) be that method by comprising the following steps realizes:
According to the volume of liquid storage to be prepared, calculate the amount of required L-BETAIN, thanomin, putrescine, Sodium Selenite, linolic acid and Transferrins,iron complexes, be dissolved in the deionized water of sterilizing, stir, filter;
Preferably, the filter of described filtration use 0.22 μm;
Preferably, described stirring uses magnetic stirring apparatus;
Preferably, can by described liquid storage packing; More preferably, by described liquid storage as-20 DEG C or-80 DEG C of preservations.
Preferably, described step 4) be that method by comprising the following steps realizes:
According to the volume of liquid storage of preparation, calculate the amount of required vitamins C and/or Magnesium Ascorbyl Phosphate salt, N acetylcysteine, be dissolved in the deionized water of sterilizing, stir, filter;
Preferably, the filter of described filtration use 0.22 μm;
Preferably, described stirring uses magnetic stirring apparatus;
Preferably, can by described liquid storage packing; More preferably, by described liquid storage as-20 DEG C or-80 DEG C of preservations.
In a preferred embodiment, described substratum is prepared by method by comprising the following steps:
1) according to the cumulative volume of substratum to be prepared, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) RPMI-1640 dry powder is directly dissolved in the deionized water of sterilizing, stir, obtain basic medium, then often liter of basic medium adds 2g sodium bicarbonate, concentrated hydrochloric acid is used to adjust pH to be 7.2, with 5M sodium-chlor liquid storage, the osmotic pressure value of basic medium is adjusted to 300 again, with the frit of 0.22 μm, preserves the basic medium obtained for 4 degree;
3) according to the volume of 50 × liquid storage of preparation, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 2mg/L, 1mg/L, 16mg/L, 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
4) according to the volume of 1000 × liquid storage of preparation, according to the vitamins C of 100mg/L and/or Magnesium Ascorbyl Phosphate salt, the ratio of N acetylcysteine of 200 μMs, weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in the basic medium prepared, then mix with mycillin, be heated to 37 DEG C of uses,
Wherein, the working concentration of described mycillin streptomycin is 100mg/l, and the working concentration of penicillin is 100,000U/L;
Preferably, described method also comprises the step of adding Regular Insulin;
Preferably, the step of described interpolation Regular Insulin is that method by comprising the following steps realizes:
Be 3 by the pH regulator of described substratum, add the Regular Insulin of aequum, fully dissolve, until solution turned clear.
Preferably, described method also comprises the micromolecular step of interpolation induction;
Preferably, the described micromolecular step of induction of adding is that method by comprising the following steps realizes;
Induction small molecules is dissolved in the DMSO of trace, be then prepared as 1000 × liquid storage, described liquid storage is joined in basic medium according to required amount;
Preferably, the induction small molecules of aequum is dissolved in the DMSO of trace, is then dissolved in the deionized water of the sterilizing of aequum, stir, filter, thus obtained 1000 × liquid storage;
Wherein, the amount of the DMSO of use is no more than 0.5% of described liquid storage cumulative volume.
Again on the one hand, the present invention also provides a kind of method of being induced by embryonic stem cell as myocardial cell, and described method comprises the step using culture medium culturing cell of the present invention;
Preferably, described embryonic stem cell is human embryo stem cell; More preferably, be clinical grade human embryonic stem cell (CTS-hES) or H7 human embryo stem cell; In a specific embodiment, described clinical grade human embryonic stem cell (CTS-hES) can purchased from Beijing stem cell bank.
Preferably, said method comprising the steps of:
1) embryonic stem cell is used to prepare single cell suspension;
Preferably, the density of described single cell suspension is 1 ~ 1.5 × 10
5/ cm
2;
2) by step 1) in cell use E8 culture medium culturing 2-3 days, preferably, make cell grow to the saturation ratio of about 90%; Then use the culture medium culturing cell of interpolation of the present invention 2 ~ 8 μMs of CHIR99021, thus inducing cell starts differentiation;
Preferably, the usage quantity of described substratum is every 2-3 × 10
5unicellularly add 1mL substratum;
Preferably, in described substratum, the concentration of GSK3 beta inhibitor CHIR99021 is 4 μMs;
3) after 24 hours, siphon away old substratum, add new substratum, and cultured continuously 44-48 hour, liquid is not changed in centre;
Wherein, induction small molecules and Regular Insulin is not contained in described substratum;
4) after 44-48 hour, siphon away old substratum, then use the culture medium culturing cell containing Wnt inhibitor of the present invention, and do not change liquid in the middle of cultured continuously 44-48 hour;
5) after 44-48 hour, siphon away old substratum, then use culture medium culturing cell of the present invention, and cultured continuously did not change liquid in the middle of 72 hours;
Preferably, when using clinical grade human embryonic stem cell (CTS-hES) to carry out inducing cardiomyocytes, described substratum is the substratum containing Wnt inhibitor;
Preferably, when using H7 human embryo stem cell to carry out inducing cardiomyocytes, described substratum is not containing the substratum of Wnt inhibitor;
6) after 72 hours, siphon away old substratum, add the substratum containing Regular Insulin of the present invention, do not change liquid in the middle of cultured continuously 48-72 hour, thus obtain myocardial cell;
Preferably, when using clinical grade human embryonic stem cell (CTS-hES) to carry out inducing cardiomyocytes, within continuous 72 hours, liquid is not changed;
Preferably, when using H7 human embryo stem cell to carry out inducing cardiomyocytes, liquid was changed at the 48th hour.
Preferably, after obtaining myocardial cell, changed once the fresh substratum of the present invention containing Regular Insulin every 2-3 days;
Preferably, described method also comprises the step detecting myocardium induced efficiency;
Further preferably, the described induced efficiency detecting cardiac muscle starts carrying out for the 14th day of differentiation at cell;
Preferably, described detection uses flow cytometer to carry out;
Preferably, described detection uses the special marker cTNT of cardiac muscle to dye.
Preferably, described step 1) be that method by comprising the following steps realizes:
To cultivate the embryonic stem cell Stempro Accutase enzyme of 5-6 days, 37 DEG C of digestion 10min, make stem cell clone be digested to unicellular, wash once, 1000rpm with DMEMF12 substratum, centrifugal 5min; Siphon away supernatant, resuspended with the E8 substratum of interpolation 10 μMs of ROCK inhibitor Y27632, then with 1 ~ 1.5 × 10
5/ cm
2inoculum density inoculation.
Preferably, the myocardium induced efficiency of described detection is that method by comprising the following steps realizes:
1) myocardial cell will be obtained and digest 6-7min with 0.25%trypsin-EDTA 37 DEG C, and add 100%FBS and stop pancreatin, with rifle piping and druming to individual cells; The centrifugal 5min of 1000rpm, removes supernatant;
2) add 4mL buffer B/ to manage, wash once, the centrifugal 5min of 1000rpm, abandons buffer B;
Wherein, the component of described buffer B is 1 × PBS+0.5%BSA+0.05%NaN
3;
3) 4% paraformaldehyde, 4 DEG C of fixing 15min are used;
4) add buffer A 4mL/ to manage, 1000rpm centrifugal twice, each 5min;
Wherein, the component of buffer A is 1 × PBS+0.5%BSA+0.2% Saponin/TSM+0.05%NaN
3;
5) leave the liquid of about 50 μ l, the cTNT antibody that the buffer A adding same volume dilutes or isotype control Ab, hatch 40min for 4 DEG C, the final concentration of cTNT antibody is 2 μ g/mL;
6) add buffer A 4mL/ again to manage, 1000rpm centrifugal twice, each 5min;
7) dilute goat anti-mouse FITC or PE with buffer A (1:400), often pipe adds 100 μ l, dark, hatches 40min for 4 DEG C;
8) 4mL buffer B is added, 1000rpm centrifugal twice, each 5min;
9) remove supernatant, it is resuspended to add 200-400 μ l 4% paraformaldehyde;
10) re-suspension liquid is placed on flow cytometer detects, do negative control with isotype control Ab.
Again on the one hand, present invention also offers a kind of myocardial cell, and the purposes of described myocardial cell in drug screening and medicine detect the toxicity of cardiac muscle.
Preferably, the invention provides myocardial cell of the present invention for the identification of the purposes promoted in the growth of myocardial cell or the novel factor of induction, regeneration, existence etc. or the screening of the potential chemotherapeutics of tool.
Present invention also offers myocardial cell of the present invention for the preparation for the treatment of for the purposes in the medicine of the heart of heart disease state.
Compared with existing substratum, culture medium prescription of the present invention is simple, cost is lower, do not relate to any ethics problem, can improve induced efficiency, the cell of induction has more advantage, and the prospect of this substratum clinical application is also very good, no matter so be from fundamental research or marketing, the present invention has prospect widely.
Main prospect of the present invention and meaning comprise:
1) compared with traditional substratum, use substratum inducing cardiomyocytes of the present invention, though from differentiation efficiency, final yield of cardiomyocytes or batch and batch between stability, all increase significantly.
2) Human Cardiomyocytes that success obtains the toxicity detection to cardiac muscle can provide good research platform for drug screening and medicine.
3) first the clinical application of Human Cardiomyocytes must guarantee it is other seed stem cell of clinical grade.Multipotent stem cells related products could be applied to clinical like this, the animal derived materials in conventional husbandry layer differential method and nutrient solution all can limit its application.
Embryo stem cell for directional is induced efficiently as myocardial cell by the present invention under the completely clear and definite condition of chemical composition, and condition is relative maturity, so can promote clinical conversion in the future faster.
4) when there being the demand of a large amount of Human Cardiomyocytes, the present invention infinitely can expand in proportion according to the requirement of above-mentioned aequum, and obtains result without significant difference under acquired results and small scale systems.A large amount of myocardial cells can pass through cellular transplantation therapy myocardial infarction, observes the improvement effect of its heart function, can for providing reliable foundation by cellular transplantation therapy heart stalk patient in the future.
The present invention relates to a kind of substratum improving the specific chemical components of induction myocardial efficiency.Substratum of the present invention is applicable to clinical application in the future.In the present invention, each component of culture medium prescription is clear and definite, with low cost, is very beneficial for myocardial cell and prepares on a large scale and be applied to clinical.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 illustrates that the clinical grade human embryonic stem cell (CTS-hES) of Beijing stem cell bank foundation is at non-trophoblast, non-animal derived, after the E8-vitronectin culture of human embryonic stem cells cultivation of specific chemical components, use in traditional RPMI-1640+B27 (xeno-free) induction substratum B27 (XF) and obtain myocardial cell; Wherein:
A figure is the roughly flow scheme of differentiation;
B figure be clinical grade human embryonic stem cell (CTS-hES) at non-trophoblast, non-animal derived, the E8-vitronectin culture of human embryonic stem cells cultivation conditions of specific chemical components; Bars=200 μm;
C figure is that differentiation uses in traditional RPMI-1640+B27 (xeno-free) induction substratum and obtains myocardial cell, Bars=200 μm on the 14th day;
D figure is the ratio that differentiation obtains myocardial cell on the 14th day in traditional RPMI-1640+B27 (xeno-free) induction substratum, i.e. the differentiation efficiency (myocardium special marker cTNT positive cell accounts for the per-cent of total cell) of cardiac muscle;
E figure is the actual number that differentiation obtains myocardial cell on the 14th day in traditional RPMI-1640+B27 (xeno-free) induction substratum is 10
5/ cm
2.
Fig. 2 illustrates and is using the induction substratum (VN) that chemical composition of the present invention is completely clear and definite, by regulating the micromolecular best inductive dose of induction and induction time window, optimizes differentiation efficiency and the output of myocardial cell; Wherein:
A figure is that CHIR99021 (0-1 days), IWR1 (3-5 days) break up flow scheme;
B figure is under A figure differentiation scheme, breaks up the 14th day, the difference cardiac muscle differentiation efficiency under various dose CHIR99021 (0-1 days) induction, and the Myocardium Differentiation wherein under CHIR99021 4 μMs induction is most effective is 21.4%;
C figure is the differentiation efficiency T% of early stage mesoderm marker T after various dose CHIR99021 induces 1 day, and under wherein CHIR99021 is more than or equal to the dosage of 4 μMs, its T% no longer increases;
D figure is differentiation the 14th day, and the difference cardiac muscle differentiation efficiency under various dose CHIR99021 (0-1 days) induction, the Myocardium Differentiation wherein under CHIR99021 4 μMs induction is most effective.
E figure is that CHIR99021 (0-1 days), IWR1 (3-5 days, 5-8 days) break up flow scheme;
F figure is under E figure differentiation scheme, break up the 14th day, various dose CHIR99021 (0-1 days) and various dose IWR1 (3-5 days, 5-8 days) the lower myocardium differentiation efficiency of difference of induction, Myocardium Differentiation wherein under CHIR99021 4 μMs, IWR1 5 μMs induction is most effective is 55.3%;
G figure is under E figure differentiation scheme, break up the 14th day, various dose CHIR99021 (0-1 days) and various dose IWR1 (3-5 days, 5-8 days) the lower myocardium differentiation efficiency of difference of induction, Myocardium Differentiation wherein under CHIR99021 4 μMs, IWR1 5 μMs induction is most effective.
Fig. 3 illustrates under identical small molecules inductive condition, use substratum (VN) and traditional RPMI-1640+B27 (xeno-free) inducing culture B27 (XF) that chemical composition of the present invention is completely clear and definite, the differentiation efficiency and ultimate capacity of cardiac muscle show difference; Wherein
A figure is the differentiation-inducing flow scheme of optimum small molecules optimized;
B figure is two kinds of different inducing cultures, Myocardium Differentiation efficiency shows difference, the highest induced efficiency of induction substratum that wherein chemical composition of the present invention is completely clear and definite is 67.6%, and the highest induced efficiency of traditional RPMI-1640+B27 (xeno-free) inducing culture is 28.8%;
C figure is two kinds of different inducing cultures, and myocardium ultimate capacity shows difference, and wherein substratum production peak of the present invention is 2 × 10
5/ cm
2, and traditional RPMI-1640+B27 (xeno-free) inducing culture production peak is 10
5/ cm
2.
Fig. 4 illustrates and differentiation-inducingly in the induction substratum (VN) that chemical composition of the present invention is completely clear and definite obtains myocardial cell after percoll density gradient centrifugation, cardiac muscle can be further purified, and can expand differentiation-inducing cultivation on a large scale in proportion; Wherein:
A figure breaks up the myocardial cell that obtains after percoll density gradient centrifugation in 24 holes, can be divided into non-myocardial infarction layer and myocardial cell's layer, the purity of its cardiac myocyte layer cardiac muscle is 95.0%, and the purity of non-myocardial infarction layer cardiac muscle is 29.7%;
B figure breaks up the myocardial cell that obtains after percoll density gradient centrifugation in 24 holes, can be divided into the ultimate capacity of non-myocardial infarction layer and myocardial cell's layer Myocardial; The ultimate capacity of its cardiac myocyte layer cardiac muscle is greater than 2 × 10
5/ cm
2, the ultimate capacity of non-myocardial infarction layer cardiac muscle is then less than 0.5 × 10
5/ cm
2;
C figure be in T225 bottle extensive expand differentiation-inducing after, break up similar with 24 orifice plates after percoll density gradient centrifugation of the myocardial cell that obtains, 3 independently revision tests, the purity of its cardiac myocyte layer cardiac muscle is all more than 90%.
D figure is C Fig. 3 time independently revision test, and the myocardium purity of myocardial cell's layer is 92.8%+/-2.2%, and the ultimate capacity of cardiac muscle is (2.2+/-0.3) × 10
5/ cm
2.
Fig. 5 illustrates under identical small molecules inductive condition, is come differentiation efficiency and the ultimate capacity of optimization cardiac muscle by the concentration changing antioxidant in the completely clear and definite induction substratum of chemical composition of the present invention; Wherein:
A figure is fixing Antioxidant N-acetyl-cysteine (N-Ac) is 50 μMs, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), and the differentiation-inducing flow scheme of myocardial cell;
B figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the differentiation-inducing efficiency of cardiac muscle shows difference, then myocardial cell is can not get when wherein not adding V, and the concentration of V is 200 μ g/mL, the concentration of N-Ac is under the condition of 50 μMs (V200N50), and the differentiation efficiency of cardiac muscle is 81%;
C figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is 200 μ g/mL, the concentration of N-Ac is under 50 μMs of conditions, and the differentiation efficiency of cardiac muscle is the highest;
D figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the ultimate capacity of cardiac muscle shows difference, and wherein the concentration of V is 200 μ g/mL, the concentration of N-Ac is under 50 μMs of conditions, and the ultimate capacity of cardiac muscle is up to 3.3 × 10
5/ cm
2;
E figure is the concentration of fixing antioxidant vitamin C and/or Magnesium Ascorbyl Phosphate salt (V) is 200 μ g/mL, under the N-acetylcystein condition of different concns, and the differentiation-inducing flow scheme of cardiac muscle;
F figure is under the differentiation scheme of E figure, and under the N-acetylcystein of different concns, the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the differentiation efficiency of cardiac muscle is up to 72%;
G figure is under the differentiation scheme of E figure, and under different concns N-acetylcystein condition, the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the differentiation efficiency of cardiac muscle is the highest;
H figure is under the differentiation scheme of E figure, and ultimate capacity myocardium under different concns N-acetylcystein condition shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the ultimate capacity of cardiac muscle is up to 2.5 × 10
5/ cm
2.
Embodiment
Unless specifically stated otherwise, cell used herein, purchased from Institute of Zoology, Academia Sinica.
Unless specifically stated otherwise, reagent used in following examples is analytical pure level reagent, and commercially available acquisition.
the preparation of embodiment 1 substratum of the present invention
1. the component provided according to table 3 and proportioning prepare liquid nutrient medium of the present invention, and the step of described substratum is as follows:
1) according to the cumulative volume of preparation, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) RPMI-1640 powder is directly dissolved in the deionized water of sterilizing, stirs; Often liter is added 2g sodium bicarbonate, adjusts pH to be 7.2 with concentrated hydrochloric acid.With 5M sodium-chlor liquid storage, the osmotic pressure value of basic medium is heightened to 295 again, with the frit of 0.22 μm, 4 DEG C of preservations.
3) according to the volume of 50 × liquid storage of preparation, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 1mg/L, 0.5mg/L, 10mg/L, 0.01mg/L, the linolic acid of 0.5mg/L, the Transferrins,iron complexes of 3mg/L, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
4) according to the volume of 1000 × liquid storage of preparation, according to the vitamins C of 50mg/L and/or Magnesium Ascorbyl Phosphate salt, the ratio of N acetylcysteine of 50 μMs, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
Wherein, table 3 illustrates the quality of each component needed for preparation 50 × liquid storage and 1000 × liquid storage.
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in the basic medium prepared, then mix with commercial mycillin, be heated to 37 DEG C of uses.
Wherein, the working concentration of described mycillin streptomycin is 100mg/L, and the working concentration of penicillin is 100,000U/L.
The composition of table 3 liquid storage and ratio
Unless stated otherwise, in table 3, the concentration unit of each component is mg/l
2. prepare the substratum of the substratum of Regular Insulin, the substratum containing the CHIR99021 of 2 μMs and the wnt-c59 of IWR1 or 1 μM containing 2 μMs containing 2mg/L respectively.
According to the amount described in table 4, induction small molecules is dissolved in the DMSO of trace respectively, is then dissolved in the deionized water of sterilizing, stir, filter, be prepared as 1000 × liquid storage, during use, join in basic medium according to required amount;
Wherein, the amount of the DMSO of use is no more than 0.5% of described cumulative volume.
Table 4 induces micromolecular composition and ratio
the preparation of embodiment 2 substratum of the present invention
1. the component provided according to table 5 and proportioning prepare liquid nutrient medium of the present invention, and the step of described substratum is as follows:
1) according to the cumulative volume of preparation, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) RPMI-1640 powder is directly dissolved in the deionized water of sterilizing, stirs; Often liter is added 2g sodium bicarbonate, adjusts pH to be 7.2 with concentrated hydrochloric acid.With 5M sodium-chlor liquid storage, the osmotic pressure value of basic medium is heightened to 300 again, with the frit of 0.22 μm, 4 degree of preservations.
3) according to the volume of 50 × liquid storage of preparation, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 3mg/L, 2mg/L, 20mg/L, 0.02mg/L, the linolic acid of 2mg/L, the Transferrins,iron complexes of 6mg/L, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
4) according to the volume of 1000 × liquid storage of preparation, according to the vitamins C of 200mg/L and/or Magnesium Ascorbyl Phosphate salt, the ratio of N acetylcysteine of 500 μMs, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
Table 5 illustrates the quality of each component needed for preparation 50 × liquid storage and 1000 × liquid storage.
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in the basic medium prepared, then mix with mycillin, be heated to 37 DEG C of uses.
Wherein, the working concentration of described mycillin streptomycin is 100mg/l, and the working concentration of penicillin is 100,000U/L.
The composition of table 5 liquid storage and ratio
Unless stated otherwise, in table 5, the concentration unit of each component is mg/l
2. prepare the substratum of the substratum of Regular Insulin, the substratum containing the CHIR99021 of 8 μMs and the wnt-c59 of IWR1 or 5 μM containing 8 μMs containing 6mg/L respectively,
According to the amount described in table 6, induction small molecules is dissolved in the DMSO of trace respectively, is then dissolved in the deionized water of sterilizing, stir, filter, be prepared as 1000 × liquid storage, during use, join in basic medium according to required amount; Wherein, the amount of the DMSO of use is no more than 0.5% of described cumulative volume.
Table 6 induces micromolecular composition and ratio
the preparation of embodiment 3 substratum of the present invention
1. the component provided according to table 7 and proportioning prepare liquid nutrient medium of the present invention, and the step of described substratum is as follows:
1) according to the cumulative volume of preparation, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) RPMI-1640 powder is directly dissolved in the deionized water of sterilizing, stirs; Often liter is added 2g sodium bicarbonate, adjusts pH to be 7.2 with concentrated hydrochloric acid.With 5M sodium-chlor liquid storage, the osmotic pressure value of basic medium is heightened to 300 again, with the frit of 0.22 μm, 4 degree of preservations.
3) according to the volume of 50 × liquid storage of preparation, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 2mg/L, 1mg/L, 16mg/L, 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
4) according to the volume of 1000 × liquid storage of preparation, according to the vitamins C of 100mg/L and/or Magnesium Ascorbyl Phosphate salt, the ratio of N acetylcysteine of 250 μMs, to weigh described component, and be dissolved in the deionized water of sterilizing, magnetic stirrer is even, the frit packing of 0.22 μm ,-20 DEG C or-80 DEG C of preservations;
Table 3 illustrates the quality of each component needed for preparation 50 × liquid storage and 1000 × liquid storage.
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in the basic medium prepared, then mix with the commercial mycillin of 1%, be heated to 37 DEG C of uses.
Wherein, the working concentration of described mycillin streptomycin is 100mg/L, and the working concentration of penicillin is 100,000U/L.
The composition of table 7 liquid storage and ratio
Unless stated otherwise, in table 7, the concentration unit of each component is mg/l
2. prepare the substratum of the substratum of Regular Insulin, the substratum containing the CHIR99021 of 4 μMs and the wnt-c59 of IWR1 and 4 μM containing 5 μMs containing 4mg/L respectively,
According to the amount described in table 8, induction small molecules is dissolved in the DMSO of trace respectively, is then dissolved in the deionized water of sterilizing, stir, filter, be prepared as 1000 × liquid storage, during use, join in basic medium according to required amount; Wherein, the amount of the DMSO of use is no more than 0.5% of described cumulative volume.
Table 8 induces micromolecular composition and ratio
embodiment 4 uses the substratum of the embodiment of the present invention 3 by clinical grade human embryo stem cell
(CTS-hES) cardioblast is induced
Concrete implementation step:
1) will the clinical grade human embryo stem cell Stempro Acc μ tase enzyme 37 DEG C digestion about 10 minutes of 5-6 days be cultivated, and stem cell clone will be digested to unicellular, wash once with DMEMF12 substratum, 1000rpm, centrifugal 5min.
Siphon away supernatant, resuspended with the E8 substratum of interpolation 10 μMs of ROCK inhibitor Y27632, rolling counters forward.With 10
5-1.5 × 10
5/ cm
2inoculum density inoculation, for 24 orifice plates, in 24 orifice plates, the area in 1 hole is close to 2cm
2, 2-3 × 10 can be inoculated in a hole that is in 24 orifice plates
5/ cm
2human embryo stem cell, every hole 1mL E8 substratum.
2) by step 1) the middle cell use E8 culture medium culturing 2-3 days inoculated; Cell is made to grow to the saturation ratio of about 90%; Siphon away old E8 substratum, add the substratum of interpolation 4 μMs of GSK3 beta inhibitor CHIR99021 (activator of Wnt), every hole 1mL substratum, is designated as the 0th day.
For clinical grade human embryonic stem cell (CTS-hES), 4 μMs of CHIR99021 are best activities.
3) after 24 hours, siphon away old substratum, add the new substratum not containing CHIR99021, every hole 1mL substratum, is designated as day1.Cultured continuously did not change liquid in the middle of 48 hours.
4) after 48 hours, siphon away old substratum, add the substratum of interpolation 5 μMs of IWR1 or 2 μM Wnt-C59, every hole 1mL substratum, is designated as day3.Cultured continuously did not change liquid in the middle of 48 hours.In first day these 48 hours by the 3rd day, 3-4 hour can be shifted to an earlier date and change liquid, but liquid is changed in postponement.5) after 48 hours, siphon away old substratum, continue the substratum adding interpolation 5 μMs of IWR1 or 2 μM Wnt-C59, every hole 1mL substratum, is designated as the 5th day, and cultured continuously did not change liquid in the middle of 72 hours.
Day5 to day8 needs continuation interpolation Wnt inhibitor to promote Cardiomyocyte Differentiation in these 72 hours for clinical grade human embryonic stem cell (CTS-hES).
6) after 72 hours, siphon away old substratum, add the substratum adding 4mg/L Regular Insulin, every hole 1mL substratum, is designated as day8.Cultured continuously did not change liquid in the middle of 72 hours.
Within 8th day to the 11 day these 72 hours, liquid can not be changed for clinical grade human embryonic stem cell (CTS-hES).
7) myocardial cell generally starts to beat in the time of about day10, just has and beat when the 8th day the earliest, changes once the fresh substratum containing 4mg/L Regular Insulin every 2-3 days.Generally, after the 14th day, nearly all myocardial cell can beat.
At this moment just can dye with the marker cTNT that cardiac muscle is special, can cTNT% be detected by flow cytometer, be i.e. myocardium final induced efficiency.
Wherein, myocardium induced efficiency step is detected as follows:
1) myocardial cell will be obtained and digest 6-7min with 0.25%trypsin-EDTA 37 DEG C, and add 100%FBS and stop pancreatin, with rifle piping and druming to individual cells; The centrifugal 5min of 1000rpm, removes supernatant;
2) add 4mL buffer B/ to manage, wash once, the centrifugal 5min of 1000rpm, abandons buffer B;
Wherein, the component of described buffer B is 1 × PBS+0.5%BSA+0.05%NaN3;
3) 4% paraformaldehyde, 4 DEG C of fixing 15min are used;
4) add buffer A 4mL/ to manage, 1000rpm centrifugal twice, each 5min;
Wherein, the component of buffer A is 1 × PBS+0.5%BSA+0.2% Saponin/TSM+0.05%NaN
3;
5) leave the liquid of about 50 μ l, the cTNT antibody that the buffer A adding same volume dilutes or isotype control Ab, hatch 40min for 4 DEG C, the final concentration of cTNT antibody is 2 μ g/mL;
6) add buffer A 4mL/ again to manage, 1000rpm centrifugal twice, each 5min;
7) dilute goat anti-mouse FITC or PE with buffer A (1:400), often pipe adds 100 μ l, dark, hatches 40min for 4 DEG C;
8) 4mL buffer B is added, 1000rpm centrifugal twice, each 5min;
9) remove supernatant, it is resuspended to add 200-400 μ l 4% paraformaldehyde;
10) re-suspension liquid is placed on flow cytometer detects, do negative control with isotype control Ab.
embodiment 5 uses the substratum according to embodiment 3 preparation of the present invention and adds B27 substratum
the comparison of inducing cardiomyocytes
Use the substratum in embodiment 3 and the method inducing cardiomyocytes of RPMI-1640 substratum according to embodiment 4 adding B27 respectively; And carry out flow cytometer showed.
Wherein, B27 additive is a kind of conventional serum-free additive for culturing cell, and the B27 serum-free additive in the present embodiment is purchased from Life Technology company.
Result as Figure 1-3, wherein;
Fig. 1 illustrates that the clinical grade human embryonic stem cell (CTS-hES) of Beijing stem cell bank foundation is at non-trophoblast, non-animal derived, after the E8-vitronectin culture of human embryonic stem cells cultivation of specific chemical components, use in traditional RPMI-1640+B27 (xeno-free) induction substratum and obtain myocardial cell; Wherein:
A figure is the roughly flow scheme of differentiation;
B figure be clinical grade human embryonic stem cell (CTS-hES) at non-trophoblast, non-animal derived, the E8-vitronectin culture of human embryonic stem cells cultivation conditions of specific chemical components; Bars=200 μm;
C figure is that differentiation uses in traditional RPMI-1640+B27 (xeno-free) induction substratum and obtains myocardial cell, Bars=200 μm on the 14th day;
D figure is the ratio that differentiation obtains myocardial cell on the 14th day in traditional RPMI-1640+B27 (xeno-free) induction substratum, and namely the differentiation efficiency (myocardium special marker cTNT positive cell accounts for the per-cent of total cell) of cardiac muscle is 41.2%;
E figure is the actual number that differentiation obtains myocardial cell on the 14th day in traditional 1640+B27 (xeno-free) induction substratum is 10
5/ cm
2.
Fig. 2 illustrates and is using the induction substratum that chemical composition of the present invention is completely clear and definite, by regulating the micromolecular best inductive dose of induction and induction time window, optimizes differentiation efficiency and the output of myocardial cell; Wherein:
A figure is that CHIR99021 (0-1 days), IWR1 (3-5 days) break up flow scheme;
B figure is under A figure differentiation scheme, breaks up the 14th day, the difference cardiac muscle differentiation efficiency under various dose CHIR99021 (0-1 days) induction, and the Myocardium Differentiation wherein under CHIR99021 4 μMs induction is most effective is 21.4%;
C figure is the differentiation efficiency T% of early stage mesoderm marker T after various dose CHIR99021 induces 1 day, and under wherein CHIR99021 is more than or equal to the dosage of 4 μMs, its T% no longer increases;
D figure is differentiation the 14th day, and the difference cardiac muscle differentiation efficiency under various dose CHIR99021 (0-1 days) induction, the Myocardium Differentiation wherein under CHIR99021 4 μMs induction is most effective.
E figure is that CHIR99021 (0-1 days), IWR1 (3-5 days, 5-8 days) break up flow scheme;
F figure is under E figure differentiation scheme, break up the 14th day, various dose CHIR99021 (0-1 days) and various dose IWR1 (3-5 days, 5-8 days) the lower myocardium differentiation efficiency of difference of induction, Myocardium Differentiation wherein under CHIR99021 4 μMs, IWR1 5 μMs induction is most effective is 55.3%;
G figure is under E figure differentiation scheme, break up the 14th day, various dose CHIR99021 (0-1 days) and various dose IWR1 (3-5 days, 5-8 days) the lower myocardium differentiation efficiency of difference of induction, Myocardium Differentiation wherein under CHIR99021 4 μMs, IWR1 5 μMs induction is most effective.
Fig. 3 illustrates under identical small molecules inductive condition, use substratum and traditional RPMI-1640+B27 (xeno-free) inducing culture that chemical composition of the present invention is completely clear and definite, the differentiation efficiency and ultimate capacity of cardiac muscle show difference; Wherein
A figure is the differentiation-inducing flow scheme of optimum small molecules optimized;
B figure is two kinds of different inducing cultures, Myocardium Differentiation efficiency shows difference, the highest induced efficiency of induction substratum that wherein chemical composition of the present invention is completely clear and definite is 67.6%, and the highest induced efficiency of traditional 1640+B27 (xeno-free) inducing culture is 28.8%;
C figure is two kinds of different inducing cultures, and myocardium ultimate capacity shows difference, and wherein substratum production peak of the present invention is 2 × 10
5/ cm
2, and traditional RPMI-1640+B27 (xeno-free) inducing culture production peak is 10
5/ cm
2.
embodiment 6 uses substratum of the present invention to induce Human Cardiomyocytes on a large scale
Use the differentiation-inducing human embryo stem cell of substratum in embodiments of the invention 1 to be myocardial cell, method and step are shown in embodiment 4.
Wherein, described human embryo stem cell is the clinical grade human embryonic stem cell (CTS-hES) that Beijing stem cell bank is set up.
The results are shown in Figure 4, illustrate and differentiation-inducingly in the induction substratum that chemical composition of the present invention is completely clear and definite obtain myocardial cell after percoll density gradient centrifugation, cardiac muscle can be further purified, and can expand differentiation-inducing cultivation on a large scale in proportion; Wherein:
A figure breaks up the myocardial cell that obtains after percoll density gradient centrifugation in 24 holes, can be divided into non-myocardial infarction layer and myocardial cell's layer, the purity of its cardiac myocyte layer cardiac muscle is 95.0%, and the purity of non-myocardial infarction layer cardiac muscle is 29.7%;
B figure breaks up the myocardial cell that obtains after percoll density gradient centrifugation in 24 holes, can be divided into the ultimate capacity of non-myocardial infarction layer and myocardial cell's layer Myocardial; The ultimate capacity of its cardiac myocyte layer cardiac muscle is greater than 2 × 10
5/ cm
2, the ultimate capacity of non-myocardial infarction layer cardiac muscle is then less than 0.5 × 10
5/ cm
2;
C figure be in T225 bottle extensive expand differentiation-inducing after, break up similar with 24 orifice plates after percoll density gradient centrifugation of the myocardial cell that obtains, 3 independently revision tests, the purity of its cardiac myocyte layer cardiac muscle is all more than 90%.
D figure is C Fig. 3 time independently revision test, and the myocardium purity of myocardial cell's layer is 92.8%+/-2.2%, and the ultimate capacity of cardiac muscle is (2.2+/-0.3) × 10
5/ cm
2.
the inducing effect of embodiment 7 optimization substratum of the present invention
Use the differentiation-inducing human embryo stem cell of substratum in embodiments of the invention 3 to be myocardial cell, method and step are shown in embodiment 4.
The results are shown in Figure 5, illustrate under identical small molecules inductive condition, come differentiation efficiency and the ultimate capacity of optimization cardiac muscle by the concentration changing antioxidant in the completely clear and definite induction substratum of chemical composition of the present invention; Wherein:
A figure is fixing Antioxidant N-acetyl-cysteine (N-Ac) is 50 μMs, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), and the differentiation-inducing flow scheme of myocardial cell;
B figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the differentiation-inducing efficiency of cardiac muscle shows difference, then myocardial cell is can not get when wherein not adding V, and the concentration of V is 200 μ g/mL, the concentration of N-Ac is under the condition of 50 μMs (V200N50), and the differentiation efficiency of cardiac muscle is 81%;
C figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is 200 μ g/mL, the concentration of N-Ac is under 50 μMs of conditions, and the differentiation efficiency of cardiac muscle is the highest;
D figure is under A figure differentiation scheme, under the vitamins C of different concns and/or the condition of Magnesium Ascorbyl Phosphate salt (V), the ultimate capacity of cardiac muscle shows difference, and wherein the concentration of V is 200 μ g/mL, the concentration of N-Ac is under 50 μMs of conditions, and the ultimate capacity of cardiac muscle is up to 3.3 × 10
5/ cm
2;
E figure is the concentration of fixing antioxidant vitamin C and/or Magnesium Ascorbyl Phosphate salt (V) is 200 μ g/mL, under the N-acetylcystein condition of different concns, and the differentiation-inducing flow scheme of cardiac muscle;
F figure is under the differentiation scheme of E figure, and under the N-acetylcystein of different concns, the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the differentiation efficiency of cardiac muscle is up to 72%;
G figure is under the differentiation scheme of E figure, and under different concns N-acetylcystein condition, the differentiation-inducing efficiency of cardiac muscle shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the differentiation efficiency of cardiac muscle is the highest;
H figure is under the differentiation scheme of E figure, and ultimate capacity myocardium under different concns N-acetylcystein condition shows difference, and wherein the concentration of V is the concentration of 200 μ g/mL, N-Ac is under the condition of 100 μMs, and the ultimate capacity of cardiac muscle is up to 2.5 × 10
5/ cm
2.
Claims (10)
1. the substratum for by embryonic stem cell induction being myocardial cell, described substratum is the liquid nutrient medium of specific chemical components, and described substratum comprises basic medium RPMI-1640;
Further, described substratum also comprises the N acetylcysteine of the L-BETAIN of 1 ~ 3mg/L, the thanomin of 0.5 ~ 2mg/L, the putrescine of 10 ~ 20mg/L, the Sodium Selenite of 0.01 ~ 0.02mg/L, the linolic acid of 0.5 ~ 2mg/L, the Transferrins,iron complexes of 3 ~ 6mg/L, the vitamins C of 50-200mg/L and/or Magnesium Ascorbyl Phosphate salt and 50-500 μM;
Preferably, described substratum also comprises the N acetylcysteine of the L-BETAIN of 2mg/L, the thanomin of 1mg/L, the putrescine of 16mg/L, the Sodium Selenite of 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L, the vitamins C of 100mg/L and/or Magnesium Ascorbyl Phosphate salt and 200 μMs;
Wherein, the osmotic pressure value of described basic medium is 295-305, is preferably 300.
2. substratum according to claim 1, is characterized in that, described substratum also comprises Regular Insulin; Preferably, described Regular Insulin concentration is in the medium 2 ~ 6mg/L, is more preferably 4mg/L.
3. substratum according to claim 1, is characterized in that, described substratum also comprises induction small molecules; Preferably, described induction small molecules is CHIR99021, Wnt acceptor inhibitor IWR1 or Wnt inhibitor wnt-c59; More preferably, described CHIR99021 concentration is in the medium 2-8 μM, is also preferably 4 μMs; Further preferably, described IWR1 concentration is in the medium 2-8 μM, is also preferably 5 μMs; Again preferably, described wnt-c59 concentration is in the medium 1-5 μM, is also preferably 2 μMs.
4. the substratum according to any one of claims 1 to 3, is characterized in that, described substratum comprises the component shown in following table 1:
Table 1
Preferably, described substratum also comprises one or more in the component shown in following table 2;
Table 2
。
5. the substratum according to any one of Claims 1 to 4, is characterized in that, described embryonic stem cell is human embryo stem cell, is preferably clinical grade human embryonic stem cell or H7 human embryo stem cell.
6. the preparation method of the substratum according to any one of Claims 1 to 5, said method comprising the steps of:
1) according to the cumulative volume of substratum to be prepared, the amount of required basic medium RPMI-1640 dry powder is calculated;
2) be dissolved in the deionized water of sterilizing by RPMI-1640 dry powder, stir, often liter adds 2g sodium bicarbonate, and regulates pH to be 7.2, then osmotic pressure value is adjusted to 295-305, and preferably 300, frit, 4 DEG C of preservations;
Preferably, 5M sodium-chlor liquid storage is used to regulate described osmotic pressure value;
Preferably, the frit of 0.22 μm is used;
Preferably, described pH value uses concentrated hydrochloric acid to regulate;
3) according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 1 ~ 3mg/L, 0.5 ~ 2mg/L, 10 ~ 20mg/L, 0.01 ~ 0.02mg/L, the linolic acid of 0.5 ~ 2mg/L, the Transferrins,iron complexes of 3 ~ 6mg/L prepare 50 × liquid storage;
Preferably, described 50 × the preparation method of liquid storage identical with the method for preparation 50 × B27 liquid storage;
More preferably, according to the ratio of the Sodium Selenite of the putrescine of the thanomin of the L-BETAIN of 2mg/L, 1mg/L, 16mg/L, 0.016mg/L, the linolic acid of 1mg/L, the Transferrins,iron complexes of 5mg/L prepare 50 × liquid storage;
4) according to the ratio of the N acetylcysteine of the vitamins C of 50-200mg/L and/or Magnesium Ascorbyl Phosphate salt, 50-500 μM prepare 1000 × liquid storage;
5) by step 3) liquid storage prepared and step 4) liquid storage prepared joins step 2 according to required amount) in obtained basic medium;
Preferably, described step 5) in also comprise and add mycillin and the step mixed;
Wherein, the working concentration of described mycillin streptomycin is 100mg/l, and the working concentration of penicillin is 100,000U/L;
Preferably, during use, described substratum is heated to 37 DEG C;
Preferably, described step 3) be that method by comprising the following steps realizes:
According to the volume of the liquid storage of preparation, calculate the amount of required L-BETAIN, thanomin, putrescine, Sodium Selenite, linolic acid and Transferrins,iron complexes, be dissolved in the deionized water of sterilizing, stir, filter;
Preferably, the filter of described filtration use 0.22 μm;
Preferably, described stirring uses magnetic stirring apparatus;
Preferably, can by described liquid storage packing; More preferably, described liquid storage is placed in-20 DEG C or-80 DEG C of preservations;
Preferably, described step 4) be that method by comprising the following steps realizes:
According to the volume of liquid storage of preparation, calculate the amount of required vitamins C and/or Magnesium Ascorbyl Phosphate salt, N acetylcysteine, be dissolved in the deionized water of sterilizing, stir, filter;
Preferably, the filter of described filtration use 0.22 μm;
Preferably, described stirring uses magnetic stirring apparatus;
Preferably, can by described liquid storage packing; More preferably, described liquid storage is placed in-20 DEG C or-80 DEG C of preservations.
7. preparation method according to claim 6, is characterized in that, described method also comprises the step of adding Regular Insulin;
Preferably, the step of described interpolation Regular Insulin is that method by comprising the following steps realizes:
Be 3 by the pH regulator of described substratum, add the Regular Insulin of aequum, fully dissolve, until solution turned clear.
8. preparation method according to claim 6, is characterized in that, described method also comprises adds the micromolecular step of induction;
Preferably, the described micromolecular step of induction of adding is that method by comprising the following steps realizes;
Induction small molecules is dissolved in the DMSO of trace, be then prepared as 1000 × liquid storage, described liquid storage is joined in described substratum according to required amount;
Preferably, the induction small molecules of aequum is dissolved in the DMSO of trace, is then dissolved in the deionized water of the sterilizing of aequum, stir, filter, thus obtained 1000 × liquid storage;
Wherein, the amount of the DMSO of use is no more than 0.5% of described liquid storage cumulative volume.
9. be a myocardial cell's method by embryonic stem cell induction, described method comprises the step of the culture medium culturing cell used according to any one of Claims 1 to 5;
Preferably, described embryonic stem cell is human embryo stem cell; More preferably, be clinical grade human embryonic stem cell or H7 human embryo stem cell;
Preferably, said method comprising the steps of:
1) embryonic stem cell is used to prepare single cell suspension;
Preferably, the density of described single cell suspension is 1 ~ 1.5 × 10
5/ cm
2;
2) by step 1) in cell use E8 culture medium culturing 2-3 days, preferably, make cell grow to the saturation ratio of about 90%; Then use the culture medium culturing cell containing 2 ~ 8 μMs of CHIR99021 according to any one of Claims 1 to 5, thus inducing cell starts differentiation;
Preferably, the usage quantity of described substratum is every 2-3 × 10
5unicellularly add 1mL substratum;
Preferably, in described substratum, the concentration of GSK3 beta inhibitor CHIR99021 is 4 μMs;
3) after 24 hours, siphon away old substratum, add the new substratum according to any one of Claims 1 to 5, and cultured continuously 44-48 hour, liquid is not changed in centre;
Wherein, described substratum is not containing induction small molecules and Regular Insulin;
4) after 44-48 hour, siphon away old substratum, then use the culture medium culturing cell containing Wnt inhibitor according to any one of Claims 1 to 5, and do not change liquid in the middle of cultured continuously 44-48 hour;
5) after 44-48 hour, siphon away old substratum, then use the culture medium culturing cell according to any one of Claims 1 to 5, and cultured continuously does not change liquid in the middle of 72 hours;
Preferably, when using clinical grade human embryonic stem cell to carry out inducing cardiomyocytes, described substratum is the substratum containing Wnt inhibitor;
Preferably, when using H7 human embryo stem cell to carry out inducing cardiomyocytes, described substratum is not containing the substratum of Wnt inhibitor;
6) after 72 hours, siphon away old substratum, add the substratum containing 2 ~ 6mg/L Regular Insulin according to any one of Claims 1 to 5, do not change liquid in the middle of cultured continuously 48-72 hour, thus obtain myocardial cell;
Preferably, when using clinical grade human embryonic stem cell to carry out inducing cardiomyocytes, within continuous 72 hours, liquid is not changed;
Preferably, when using H7 human embryo stem cell to carry out inducing cardiomyocytes, liquid was changed at the 48th hour;
Preferably, after obtaining myocardial cell, changed once the fresh substratum containing Regular Insulin according to any one of Claims 1 to 5 every 2-3 days;
Preferably, described step 1) be that method by comprising the following steps realizes:
To cultivate the embryonic stem cell Stempro Accutase enzyme of 5-6 days, 37 DEG C of digestion 10min, make stem cell clone be digested to unicellular, wash once with DMEMF12 substratum, the centrifugal 5min of 1000rpm; Siphon away supernatant, resuspended with the E8 substratum of interpolation 10 μMs of ROCK inhibitor Y27632, then with 1 ~ 1.5 × 10
5/ cm
2inoculum density inoculation.
10. method according to claim 9, is characterized in that, described method also comprises the step detecting myocardium induced efficiency;
Preferably, described induced efficiency the carrying out for the 14th day in cytodifferentiation detecting cardiac muscle;
Preferably, described detection uses flow cytometer to carry out;
Preferably, described detection uses the special marker cTNT of cardiac muscle to dye.
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Cited By (4)
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| CN105838676A (en) * | 2016-05-06 | 2016-08-10 | 中国科学院动物研究所 | Culture solution for retinal pigment epitheliums and preparation method and application thereof |
| CN105950548A (en) * | 2016-05-26 | 2016-09-21 | 南京医科大学附属南京儿童医院 | Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells |
| CN107858331A (en) * | 2017-11-02 | 2018-03-30 | 北京全式金生物技术有限公司 | A kind of method for inducing people's multipotent stem cells to be divided into spinal motor nerve precursor |
| CN114438030A (en) * | 2020-11-05 | 2022-05-06 | 中国科学院动物研究所 | Culture medium for inducing embryonic stem cells into retinal pigment epithelial cells and application |
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| WO2013056072A1 (en) * | 2011-10-13 | 2013-04-18 | Wisconsin Alumni Research Foundation | Generation of cardiomyocytes from human pluripotent stem cells |
| EP2808383A1 (en) * | 2012-01-27 | 2014-12-03 | Kyoto University | Method for inducing differentiation of pluripotent stem cell into cardiac muscle |
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| EP2808383A1 (en) * | 2012-01-27 | 2014-12-03 | Kyoto University | Method for inducing differentiation of pluripotent stem cell into cardiac muscle |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838676A (en) * | 2016-05-06 | 2016-08-10 | 中国科学院动物研究所 | Culture solution for retinal pigment epitheliums and preparation method and application thereof |
| CN105838676B (en) * | 2016-05-06 | 2020-06-05 | 中国科学院动物研究所 | Culture solution for retinal pigment epithelial cells and preparation method and application thereof |
| CN105950548A (en) * | 2016-05-26 | 2016-09-21 | 南京医科大学附属南京儿童医院 | Method for efficiently obtaining beating functional cardiomyocytes from mesenchymal stem cells |
| CN107858331A (en) * | 2017-11-02 | 2018-03-30 | 北京全式金生物技术有限公司 | A kind of method for inducing people's multipotent stem cells to be divided into spinal motor nerve precursor |
| CN114438030A (en) * | 2020-11-05 | 2022-05-06 | 中国科学院动物研究所 | Culture medium for inducing embryonic stem cells into retinal pigment epithelial cells and application |
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| CN104611291B (en) | 2020-04-28 |
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