CN103031272A - Culture medium for rat embryonic stem cells - Google Patents
Culture medium for rat embryonic stem cells Download PDFInfo
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Abstract
The invention discloses a culture medium for rat embryonic stem cells. The culture medium comprises basal culture media and additives, wherein the additives comprise differentiation inhibitors and Pluripotin; and moreover, the differentiation inhibitors comprise GSK (Glycogen Synthase Kinase) inhibitors, FGFR (Fibroblast Growth Factor Receptor) inhibitors and MAPK (Mitogen-activated Protein Kinases) inhibitors. The invention also discloses a culture medium kit of the rat embryonic stem cells, a method for culturing the rat embryonic stem cells and applications of the culture medium and the kit, wherein the culture medium kit comprises the basal culture media, the differentiation inhibitors and the Pluripotin. With the adoption of the differentiation inhibitors and the Pluripotin, the culture medium for the rat embryonic stem cells can inhibit the differentiation of the rat embryonic stem cells and can further guarantee the stability of karyotype for reproductive potential.
Description
Technical field
The present invention relates to field of cell culture.More specifically, the present invention relates to substratum and the corresponding cultural method cultivated for Embryonic Stem Cell.
Background technology
The mankind, mouse, rat and many vegeto-animal gene sequencing are finished, and indicate that life science has begun to stride forward the genome times afterwards comprehensively, i.e. the epoch of gene function analysis.Present gene function analysis method has: gene knockout, antisense technology, dominant, gene induced overexpression, RNA interference and information biology etc., the method of standard the most take the gene knockout method as gene function analysis wherein, the gene knockout analysis has become the gold standard of gene function analysis.
Model animals (comprising gene knockout rat and mouse) will be brought into play the vital role that can not be substituted in functional genomics research.Abroad, mouse, rat, fruit bat, nematode and Xenopus laevis etc. are indispensable model animalss in the biological study always, and the research that utilizes these model animalss to do has directly expedited the emergence of the multinomial Nobel prize.The genetic modification mouse that utilization knocks out the technology development can be used for Research foundation biology problem, as human diseases model, drug screening and treatment plan evaluation, has extremely important value aspect fundamental research and the applied research.Regrettably, compare with functional genomicses such as information biology, biochip, proteomics, the research of model animals association area is not subject to enough approvals and attention at home, relevant fundamental research is also quite weak, is just becoming to hinder domestic functional genomics to the more profound bottleneck that further develops.
The most normal animal model that uses of gene Knockout is mouse at present, mainly comprises complete gene knockout and condition type gene knockout.The key of homologous recombination gene knockout is the embryonic stem cell that obtains to have reproductive function.
1981, British scientist Martin Ai Wensi successfully extracted the embryonic stem cell of mouse, can allow scientist carry out genetic manipulation outside Mice Body, determined the effect of specific gene in growth, physiology and pathologic process.After this scientist changes the research rat over to, because compare and mouse, the anatomy of rat and physiologic character and the mankind are more approaching, but uses with it the rat not breakthrough in more than 20 year because the extraction of mouse embryo stem cell and cultural method can't overlap.
Want to obtain rat gene and knock out model, key wherein just is to set up the embryonic stem cell strain of the rat of can reproduction going down to posterity.In this area, although successfully from rat embryo, extract stem cell, and by suppressing the specific gene path of Embryonic Stem Cell differentiation, suppress the Embryonic Stem Cell differentiation, thereby remain on original embryo stage.Yet the caryogram of its large murine stem cell is still very unstable behind medicine and gene targeting, must again screen and just can obtain the stable rat stem cell line that is used for injection.
Therefore, a kind of novel technology of still needing can be broken up by the establishment Embryonic Stem Cell, the embryonic stem cell strain of the rat that the reproduction of foundation energy is gone down to posterity, and make the large murine stem cell of cultivation after practicing shooting, stable caryogram be arranged.
Summary of the invention
The object of the present invention is to provide a kind of substratum and corresponding cultural method for the Embryonic Stem Cell cultivation.
A first aspect of the present invention, a kind of substratum for Embryonic Stem Cell is provided, described substratum comprises basic medium and additive, wherein additive comprises differentiation inhibitors and Pluripotin, and described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
In another preference, described basic medium comprises: DMEM substratum, DMEM/F12 substratum, neurocyte basic medium or its combination.
In another preference, described basic medium be DMEM/F12 substratum and neurocyte basic medium by volume 0.5～1.5: 0.5～2 mixed preparing form.
In another preference, described additive also comprises:
0.1～10mmol/L beta-mercaptoethanol;
0.1～5mmol/L L-glutaminate.
In another preference, described additive also comprises: Thiazovivin, and concentration is 0.1～10 μ mol/L, and is better, and concentration is 0.5～5 μ mol/L, and more preferably, concentration is 0.8～2 μ mol/L.
In another preference, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L;
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
Preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 1～10 μ mol/L; The concentration of described FGFR inhibitor is 0.5～5 μ mol/L; The concentration of described MAPK inhibitor is 0.5～5 μ mol/L; The concentration of described Pluripotin is 0.5～5 μ mol/L.
More preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 2～5 μ mol/L; The concentration of described FGFR inhibitor is 0.8～2 μ mol/L; The concentration of described MAPK inhibitor is 0.8～2 μ mol/L; The concentration of described Pluripotin is 0.8～2 μ mol/L.
In another preference, described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
A second aspect of the present invention provides a kind of substratum test kit for Embryonic Stem Cell, comprises following component:
(a) basic medium;
(b) differentiation inhibitors;
(c)Pluripotin；
Wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
In another preference, described test kit comprises following component:
(a) basic medium of interpolation differentiation inhibitors;
(b)Pluripotin；
Wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
In another preference, described basic medium comprises: DMEM substratum, DMEM/F12 substratum, neurocyte basic medium or its combination.
In another preference, described basic medium be DMEM/F12 substratum and neurocyte basic medium by volume 0.5～1.5: 0.5～2 mixed preparing form.
In another preference, described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
In another preference, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L,
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
Preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 1～10 μ mol/L; The concentration of described FGFR inhibitor is 0.5～5 μ mol/L; The concentration of described MAPK inhibitor is 0.5～5 μ mol/L; The concentration of described Pluripotin is 0.5～5 μ mol/L Pluripotin.
More preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 2～5 μ mol/L; The concentration of described FGFR inhibitor is 0.8～2 μ mol/L; The concentration of described MAPK inhibitor is 0.8～2 μ mol/L; The concentration of described Pluripotin is 0.8～2 μ mol/L Pluripotin.
A third aspect of the present invention provides a kind of method of cultivating large murine stem cell, comprising:
In the presence of differentiation inhibitors and Pluripotin, cultivate large murine stem cell, wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
In another preference, described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
In another preference, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L,
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
In another preference, described GSK inhibitor is CHIR99021; Described FGFR inhibitor is PD0325901 or SU5402; Described MAPK inhibitor is PD0325901 or PD184352.
A fourth aspect of the present invention provides a kind of Embryonic Stem Cell, adopts the described method of the third aspect to obtain.
A fifth aspect of the present invention provides the application of the described substratum of first aspect or the described test kit of second aspect:
(a) as the substratum of cultivating Embryonic Stem Cell;
(b) be used for promotion and/or raising Embryonic Stem Cell clone formation; Or
(c) building for Embryonic Stem Cell is cultivation.
A sixth aspect of the present invention provides the purposes of a kind of composition or mixture, and described composition or mixture are differentiation inhibitors and Pluripotin.Purposes is:
(a) for the preparation of the substratum of cultivating Embryonic Stem Cell; Or
(b) as the additive of the substratum of Embryonic Stem Cell.
The present invention adopts differentiation inhibitors and Pluripotin, can suppress the Embryonic Stem Cell differentiation and guarantee that caryogram is stable, has reproductive potential.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Fig. 1 is the method synoptic diagram of the signal path of blocking-up Embryonic Stem Cell differentiation.
Fig. 2 is the picture that blastaea forms to embryonic stem cell, (A) and (B) be 4,5 days be the form of blastaea, the form when (C) planting first day on the feeder layer for blastaea, form when (D) planting on the feeder layer the 4th day for blastaea has the clone to form.
Fig. 3 is planted in behind the rat substratum clone's form of the 2nd day, wherein, (C) and (D) all illustrates and has good clone and form, and cloning cluster (E) is not completed into, and (F) is dead cell mass.
Embodiment
The inventor is through extensive and deep research, the unexpected discovery: the Pluripotin of differentiation inhibitors and promotion self adds in the basic medium, signalling channel that can not only the establishment differentiation of stem cells, make Embryonic Stem Cell be in my update mode always, and can effectively keep its caryogram stable.
Embryonic stem cell (Embryonic Stem cell, ES cell)
The ES cell is to come from the totipotent clone of a kind of tool that body early embryo inner cell mass (Inner cell mass, ICM) clones through the vitro inhibition differentiation culture.
Because in embryo and body development process, differentiation and division growth generally carry out simultaneously, and building of ES cell is, requires fast infinite multiplication of ES cell, is again not break up attitude.Inhibited differentiation and amplification are conflicts, must screen suitable substratum and raising and build and be tied to form power.
Separation and Culture ES cell, most important precondition are the differentiation that will suppress to greatest extent cell.Adopt the earliest and be the method for the most generally using at present, be to prepare feeder layer cells with 12 days to 14 days mice embryonics, and make inoblast lose mitogen activation with ametycin (Mitomycin C, MMC) processing.It is generally acknowledged that feeder layer cells produces leukemia growth inhibiting factor (LIF), mainly contains the consolidated form that secretor type and extracellular matrix link.
The requirement of the ES Growth of Cells of different plant species is also inconsistent.Mouse embryo stem cell needs trophocyte and leukemia growth inhibiting factor (LIF) at present, adopt the embryo fibroblast substratum to add the training method of LIF, use bovine serum, can play the effect of better anti-differentiation and the distortion of anti-caryogram to mouse embryo stem cell; Human embryo stem cell needs trophocyte and leukemia growth inhibiting factor (LIF), substitutes bovine serum with plasma substitute (KSR), also need keep its propagation with 4ng/ml Prostatropin (bFGF).
But adopt the embryo fibroblast substratum to add the training method of LIF, rat and people's embryonic stem cell is not also had the ability of enough Inhibited differentiations, simultaneously because the effect of MMC, thereby and cause the growth of the dead ES of interference of some feeder layer cells cells.
Differentiation inhibitors
How to keep its long-term external self in the process that embryonic stem cell is set up is a core topic always, even in vivo, the inner cell mass that is equivalent to embryonic stem cell in blastaea also can through after the of short duration growth to lower differentiation.
As shown in Figure 1, the differentiation inhibitors of the present invention that can be used for can be GSK inhibitor, FGFR inhibitor, MAPK inhibitor, suppresses GSK3, MAPK and the FGFR three barss passage that goes down to posterity, and guarantees that large murine stem cell is basicly stable.
In a preference, the differentiation inhibitors of the present invention that is used for is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
Wherein, CHIR99021 is the GSK inhibitor; PD0325901, SU5402 are the FGFR inhibitor; PD0325901, PD184352 are the MAPK inhibitor.
The differentiation inhibitors that the present invention adopts is commercialization, can buy from companies such as Axon, such as small molecules CHIR99021 (Axon Medchem BV, cat.no.Axon 1386); Small molecules PD0325901 (Axon Medchem BV, cat.no.Axon 1408).
Pluripotin
Pluripotin is that the hope such as Sheng Ding can be found a kind of composition that can replace feeder cell and cofactor, thousands of kinds of synthesized micromolecules in the Scripps Research storehouse are carried out high flux screening, filter out at first one group can Promote cell's growth pyrimidine, then research group has developed the analogue of this group pyrimidine, and prove that these analogues only contain unique a kind of identical component, with this composition called after " pluripotin ".
Pluripotin can assist alone the mouse embryo stem cell self in the standard base substratum, keep totipotency.
Pluripotin is commercialization, can buy, such as Stemolecule
TMSC1 (Pluripotin) (Stemgent, cat.no.04-0011).
Substratum (or nutrient solution)
The DMEM that high concentration glucose is often arranged in the conventional ES cell culture fluid, L-glutaminate, non-essential amino acid, beta-mercaptoethanol and foetal calf serum, concentration and quality thereof that wherein it should be noted that foetal calf serum are the important factors that affects the ES Growth of Cells, the foetal calf serum of excessive concentrations also is unfavorable for the growth of ES cell, generally adds about 10% calf serum or about 10% foetal calf serum at substratum.
In order to make large murine stem cell basicly stable, prevent vitro differentiation and keep caryogram stable, the substratum of the Embryonic Stem Cell that the present invention adopts, comprise basic medium and additive, wherein additive comprises differentiation inhibitors and Pluripotin, and described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
Preferably, described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
Further, described additive also contains:
0.1～10mmol/L beta-mercaptoethanol;
0.1～5mmol/L L-glutaminate.
Described basic medium comprises: DMEM substratum, DMEM/F12 substratum, neurocyte basic medium or its combination.
In another preference, described basic medium is by DMEM/F12 substratum and neurocyte basic medium by volume (0.5～1.5): (0.5～2) mixed preparing forms.
DMEM/F12 is commercial basic medium commonly used in the cell cultures, and this substratum is comprised of many kinds of substances such as glucose, amino acid, VITAMIN, inorganic salt.Can buy acquisition from Sigma, Gibco company etc., such as the D6421 of Sigma company.
Further, also can add the N2 additive in the described DMEM/F12 substratum, by with the DMEM/F12 culture volume than (0.8～1.5): 100 add.The N2 additive is nerve growth additive (N2Supplement), is commercial additive, can buy from Gibco, Invitrogen company etc. to obtain.
Neurocyte basic medium (Neurobasal medium) also is commercial substratum, be the substratum that is suitable for its growth that aims at the external Training Design of neurocyte, this substratum is comprised of many kinds of substances such as glucose, amino acid, VITAMIN, inorganic salt, neurone additives (such as NGS).Can buy acquisition from Sigma, Invitrogen company etc., such as the 21103-049 of Invitrogen.
Further, also can add neuronal cell cultures recruitment factor B27 (B27-supplement is such as the 17504-044 of Invitrogen) in the neurocyte basic medium, with the volume ratio of neurocyte basic medium be (1～5): 100.
Substratum provided by the invention, described substratum have 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L,
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
Preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 1～10 μ mol/L; The concentration of described FGFR inhibitor is 0.5～5 μ mol/L; The concentration of described MAPK inhibitor is 0.5～5 μ mol/L; The concentration of described Pluripotin is 0.5～5 μ mol/L.
More preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 2～5 μ mol/L; The concentration of described FGFR inhibitor is 0.8～2 μ mol/L; The concentration of described MAPK inhibitor is 0.8～2 μ mol/L; The concentration of described Pluripotin is 0.8～2 μ mol/L.
Substratum provided by the invention, described GSK inhibitor is CHIR99021; Described FGFR inhibitor is PD0325901 or SU5402; Described MAPK inhibitor is PD0325901 or PD184352.
The substratum test kit
Test kit for Embryonic Stem Cell provided by the invention comprises following component:
(a) basic medium;
(b) differentiation inhibitors;
(c)Pluripotin；
Wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
In another preference, described test kit comprises following component:
(a) basic medium of interpolation differentiation inhibitors;
(b)Pluripotin；
Wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
Described basic medium comprises: DMEM substratum, DMEM/F12 substratum, neurocyte basic medium or its combination.
In another preference, described basic medium be DMEM/F12 substratum and neurocyte basic medium by volume 0.5～1.5: 0.5～2 mixed preparing form.
Preferably, described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
In another preference, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L,
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
Preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 1～10 μ mol/L; The concentration of described FGFR inhibitor is 0.5～5 μ mol/L; The concentration of described MAPK inhibitor is 0.5～5 μ mol/L; The concentration of described Pluripotin is 0.5～5 μ mol/L.
More preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 2～5 μ mol/L; The concentration of described FGFR inhibitor is 0.8～2 μ mol/L; The concentration of described MAPK inhibitor is 0.8～2 μ mol/L; The concentration of described Pluripotin is 0.8～2 μ mol/L.
Substratum (nutrient solution) or substratum test kit purposes
Substratum provided by the invention (or nutrient solution) or substratum test kit purposes are used for:
(a) as the substratum of cultivating Embryonic Stem Cell;
(b) be used for promotion and/or raising Embryonic Stem Cell clone formation; Or
(c) building for Embryonic Stem Cell is cultivation.
Substratum of the present invention or substratum test kit are used for the Embryonic Stem Cell clone and build is cultivation, the establishment vitro differentiation, and can keep caryogram stable.
Cultural method
The method of the large murine stem cell of cultivation provided by the invention comprises:
In the presence of differentiation inhibitors and Pluripotin, cultivate large murine stem cell, wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
Described differentiation inhibitors is CHIR99021 and PD0325901.
In another preference, described differentiation inhibitors is CHIR99021, SU5402 and PD184352.
In another preference, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L,
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
Preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 1～10 μ mol/L; The concentration of described FGFR inhibitor is 0.5～5 μ mol/L; The concentration of described MAPK inhibitor is 0.5～5 μ mol/L; The concentration of described Pluripotin is 0.5～5 μ mol/L.
More preferably, by the cumulative volume of the substratum of Embryonic Stem Cell, the concentration of described GSK inhibitor is 2～5 μ mol/L; The concentration of described FGFR inhibitor is 0.8～2 μ mol/L; The concentration of described MAPK inhibitor is 0.8～2 μ mol/L; The concentration of described Pluripotin is 0.8～2 μ mol/L.
Beneficial effect of the present invention is: provide a kind of and cultivate rat and reach the intimate all-round stem cell media of other animal, compare with traditional substratum, the Embryonic Stem Cell positive colony rate that cultivation obtains is higher, add CHIR99021, PD0325901, Pluripotin (SC1) in the substratum, reinforcement guarantees its dryness growth and does not break up; And the stem cell caryogram is more stable.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and usefulness, each feature that discloses in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
The substratum preparation
In 100ml N2B27 mixed culture medium, add successively 10mM CHIR99021,10mMPD0325901 and 10mM Pluripotin (SC1), so that the final concentration of CHIR99021 is 3 μ mol/L; The final concentration of PD0325901 is 1 μ mol/L; The final concentration of Pluripotin (SC1) is that 1 μ mol/L obtains the preferred substratum of the present invention, can preserve 1 month in 4 ℃.
Wherein, the N2B27 mixed culture medium is prepared according to the following formulation:
DMEN/F12-N12 substratum 100ml;
Neurocyte basic medium/B27 substratum 100ml;
0.1M beta-mercaptoethanol 200 μ l.
The DMEN/F12-N12 substratum is prepared according to the following formulation:
DMEN/F12 substratum 100ml;
N2 additive 1ml.
Neurocyte basic medium/B27 substratum is prepared according to the following formulation:
Neurocyte basic medium 100ml;
Neuronal cell cultures recruitment factor B27 2ml;
200mM L-glutaminate 0.5ml.
Embodiment 2
The substratum preparation
In 100ml DMEM substratum, add successively 10mM CHIR99021,10mMPD0325901, so that the final concentration of CHIR99021 is 3 μ M; The final concentration of PD0325901 is 1 μ M, obtains the substratum of large murine stem cell, can preserve 1 month in 4 ℃.
Wherein, the N2B27 mixed culture medium is prepared according to the following formulation:
DMEN/F12-N12 substratum 100ml;
Neurocyte basic medium/B27 substratum 100ml;
0.1M beta-mercaptoethanol 200 μ l.
The DMEN/F12-N12 substratum is prepared according to the following formulation:
DMEN/F12 substratum 100ml;
N2 additive 1ml.
Neurocyte basic medium/B27 substratum is prepared according to the following formulation:
Neurocyte basic medium 100ml;
Neuronal cell cultures recruitment factor B27 2ml;
200mM L-glutaminate 0.5ml.
Embodiment 3
Former primary cultures of rat embryonic stem cell
Inoblast with MMC processes makes it lose mitogen activation, plants in 96 orifice plates, makes feeder layer.
Use the as stated above feeder layer of preparation in the 1-3.Absorb the supernatant liquor of 96 orifice plates, add the substratum of embodiment 1 preparation, every hole 150 μ l.
Draw the 4.5th day white rat blastaea with the mouth keyholed back plate, its form is shown in Fig. 2 (A) and Fig. 2 (B), and every hole adds one piece of blastaea, places 37 ℃, 5%CO
2And 5%O
2In the incubator.The form of plantation first day is shown in Fig. 2 (C).Cultivated altogether 3 days, during need not change liquid.
Cultivation to the 4 days, shown in Fig. 2 (D), blastaea is fixed on the feeder layer, forms larger cell mass.Choose the middle body of cell mass with mechanical means, be transferred in the other hole that contains feeder layer, be designated as first-generation Embryonic Stem Cell.
Go down to posterity and analyze
Depending on the size of first-generation Embryonic Stem Cell cloning cluster, in 1-3 days, go down to posterity.Embryonic Stem Cell after going down to posterity is cultivated in the substratum of embodiment 1 preparation, as shown in Figure 3, although had part not be completed into the clone in the 2nd day or die, shown in Fig. 3 (E) and Fig. 3 (F), but the cloning cluster that major part can form, shown in Fig. 3 (C) and Fig. 3 (D), positive colony rate 60-90%.And after testing, be Embryonic Stem Cell more than 98%, show the differentiation of the large murine stem cell of substratum establishment of embodiment 1 preparation.
Concrete steps are:
The sucking-off supernatant adds the substratum that 150 μ l embodiment 1 prepare, and with the volley of rifle fire (12 road) machinery piping and druming about 10 times, goes down to posterity with 1: 3.
Choose at random 6～10 strains the 5th generation Embryonic Stem Cell, carry out according to a conventional method G and carry out karyotyping for dyeing, the result shows that caryogram is all stable normal.Major part is carried out through the normal stem cell strain of karyotyping result frozen, all the other further subcultures.Propagating method can adopt the piping and druming of above-mentioned volley of rifle fire machinery, also can adopt neutral enzymatic to digest had digestive transfer culture, further adds small molecules Stemolecule in the nutrient solution
TMThiazovivin (Stemgent, cat.no.04-0017) promotes growth.
Choose at random 6～10 strains the tenth generation Embryonic Stem Cell and carry out karyotyping, the result shows that caryogram is stable.Stay 3～4 strain Embryonic Stem Cells for detection of reproductive potential, normal each the stem cell strain of all the other lease making karyotyping results is carried out frozen.
Embodiment 5
The reproductive potential test
3～4 strain Embryonic Stem Cells that embodiment 4 stays are for detection of reproductive potential.
Experimental procedure is as follows:
Prepare the blastaea (3～4 strain Embryonic Stem Cells that embodiment 3 stays are from white rat) of black rat;
Large murine stem cell (15-30 the cell) microinjection of just going down to posterity one day is entered the rat segmentation cavity;
The above-mentioned rat blastaea of having injected Embryonic Stem Cell is injected the rat uterus angle of false pregnancy under surgical condition, wait for term birth;
The rat of assorted hair is chosen, after the sexual maturity with the rat mating of black, the filial generation that produces white.
Illustrate that 3～4 strain Embryonic Stem Cells that embodiment 4 stays are the embryonic stem cell with reproductive potential.The embryonic stem cell that shows culture medium culturing of the present invention can go down to posterity in reproduction, can be used in the rat that obtains gene knockout.
Embodiment 6
Practice shooting and test
Make up with the marker gene green fluorescence protein gene and contain the p53 targeting vector of homology arm (method is referring to document " Production of p53 gene knockout rats by homologous recombination in embryonic stem cells ", Nature.2010 September 9 with ordinary method; 467 (7312): 211-213).Described targeting vector changed in the intestinal bacteria increase, through plasmid extraction, ultra-high speed centrifugal purification or Promrga plasmid extraction test kit purifying.Adopt the specific site enzyme to be cut into strand.
Be ready to 2-3 diameter 10cm and contain the plate of mouse embryo fibroblasts MEF feeder cell, be used for electrotransfection in 2-4 days; Prepare the large murine stem cell in the tenth to 12 generation, it is electrotransfection that the second day that goes down to posterity is practiced shooting; Stem cell behind the electrotransfection changes in the plate that contains MEF.
Second day begins drug screening (G418 or Ganciclor); Stopped drug screening on the 3rd day, the propagation of cloning, selected clone carries out PCR and Southern hybridization after the week.
The checking caryogram is stable: pass 5-10 and carry out according to a conventional method G after generation for dyeing (human stem cell G is with dyeing process).
Microscopically is observed the karyomit(e) number and is contrasted with the large mouse chromosome of standard, determines the stability of the large murine stem cell of electrotransfection.
The result shows, large murine stem cell more than 80% keeps 42 karyomit(e)s behind electrotransfection, the large murine stem cell of electrotransfection keeps stable, and the effect of this expression basal culture medium is better than existing substratum (20%～30% large murine stem cell keeps 42 karyomit(e)s behind electrotransfection).
Embodiment 7
Stem cell is cultivated and analyzes
Adopt the experimental procedure of embodiment 3 to prepare former culture embryonic stem cell, different is the nutrient solution that adopts embodiment 2 preparations.
The method of employing embodiment 4 goes down to posterity and carries out karyotyping, the difference nutrient solution of the rear nutrient solution that uses as embodiment 2 preparations that be to go down to posterity, add in addition 10mM Pluripotin (SC1), so that the final concentration of Pluripotin (SC1) is 1 μ M.
Each is detected for large murine stem cell, is Embryonic Stem Cell more than 96%, and differentiation is by establishment, and the karyotyping result shows that caryogram is stable normal.
Adopt the method test reproductive potential of embodiment 5, the filial generation that produces white shows with the embryonic stem cell of the culture medium culturing of embodiment 2 preparations, can reproduction go down to posterity, and can be used in the rat that obtains gene knockout.
Adopt the test of practicing shooting of the method for embodiment 6, the result shows that the large murine stem cell 80% or more keeps 42 karyomit(e)s behind electrotransfection, and the murine stem cell caryogram is stablized greatly after target practice.
Embodiment 8
Stem cell is cultivated and analyzes
Adopt the preparation steps preparation nutrient solution of embodiment 1, difference is:
The final concentration of CHIR99021 is 1 μ mol/L;
The final concentration of PD0325901 is 0.5 μ mol/L;
The concentration of Pluripotin is 0.5 μ mol/L.
Adopt the experimental procedure of embodiment 3 to prepare former culture embryonic stem cell, difference is to adopt the nutrient solution of present embodiment preparation; The method of employing embodiment 4 goes down to posterity and carries out karyotyping, and difference is to adopt the nutrient solution of present embodiment preparation.
Each is detected for large murine stem cell, is Embryonic Stem Cell more than 95%, and differentiation is by establishment, and the karyotyping result shows that caryogram is stable normal.
Adopt the method test reproductive potential of embodiment 5, the filial generation that produces white shows with the embryonic stem cell of the culture medium culturing of present embodiment preparation, can reproduction go down to posterity, and can be used in the rat that obtains gene knockout.
Adopt the test of practicing shooting of the method for embodiment 6, the result shows that the large murine stem cell 75% or more keeps 42 karyomit(e)s behind electrotransfection, and the murine stem cell caryogram is stablized greatly after target practice.
Embodiment 9
Stem cell is cultivated and analyzes
Adopt the preparation steps preparation nutrient solution of embodiment 1, difference is:
The final concentration of CHIR99021 is 10 μ mol/L
The final concentration of PD0325901 is 5 μ mol/L
The concentration of Pluripotin is 5 μ mol/L
Adopt the experimental procedure of embodiment 3 to prepare former culture embryonic stem cell, difference is to adopt the nutrient solution of present embodiment preparation; The method of employing embodiment 4 goes down to posterity and carries out karyotyping, and difference is to adopt the nutrient solution of present embodiment preparation.
Each is detected for large murine stem cell, is Embryonic Stem Cell more than 99%, and differentiation is by establishment, and the karyotyping result shows that caryogram is stable normal.
Adopt the method test reproductive potential of embodiment 5, the filial generation that produces white shows with the embryonic stem cell of the culture medium culturing of present embodiment preparation, can reproduction go down to posterity, and can be used in the rat that obtains gene knockout.
Adopt the test of practicing shooting of the method for embodiment 6, the result shows that the large murine stem cell 80% or more keeps 42 karyomit(e)s behind electrotransfection, and the murine stem cell caryogram is stablized greatly after target practice.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. substratum that is used for Embryonic Stem Cell, it is characterized in that, described substratum comprises basic medium and additive, and wherein additive comprises differentiation inhibitors and Pluripotin, and described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
2. substratum as claimed in claim 1 is characterized in that, described basic medium comprises: DMEM substratum, DMEM/F12 substratum, neurocyte basic medium or its combination.
3. substratum as claimed in claim 1 is characterized in that, described substratum has 1-4 the feature that is selected from lower group:
(a) concentration of described GSK inhibitor is 0.5～20 μ mol/L;
(b) concentration of described FGFR inhibitor is 0.2～10 μ mol/L;
(c) concentration of described MAPK inhibitor is 0.2～10 μ mol/L;
(d) concentration of described Pluripotin is 0.1～10 μ mol/L;
Wherein, each concentration is by the cumulative volume of the substratum of Embryonic Stem Cell.
4. substratum as claimed in claim 1 is characterized in that, described differentiation inhibitors is CHIR99021 and PD0325901.
5. substratum test kit that is used for Embryonic Stem Cell is characterized in that described test kit comprises following component:
(a) basic medium;
(b) differentiation inhibitors;
(c)Pluripotin；
Wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
6. test kit as claimed in claim 5 is characterized in that, described differentiation inhibitors is CHIR99021 and PD0325901.
7. method of cultivating large murine stem cell is characterized in that described method comprises:
In the presence of differentiation inhibitors and Pluripotin, cultivate large murine stem cell, wherein, described differentiation inhibitors comprises GSK inhibitor, FGFR inhibitor and MAPK inhibitor.
8. an Embryonic Stem Cell is characterized in that, adopts method claimed in claim 7 to obtain.
9. the application of substratum as claimed in claim 1 or test kit claimed in claim 5 is characterized in that, (a) as the substratum of cultivating Embryonic Stem Cell; (b) be used for promotion and/or raising Embryonic Stem Cell clone formation; Or (c) to be used for that Embryonic Stem Cell builds be cultivation.
10. the purposes of a composition or mixture, described composition or mixture are differentiation inhibitors and Pluripotin, it is characterized in that, (a) for the preparation of the substratum of cultivating Embryonic Stem Cell; Or (b) as the additive of the substratum of Embryonic Stem Cell.
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| CN105483080A (en) * | 2015-12-15 | 2016-04-13 | 同济大学 | Efficient culture medium for rat embryonic stem cells |
| CN112877279A (en) * | 2019-12-11 | 2021-06-01 | 上海厚超生物科技股份有限公司 | Culture solution and culture method for rat embryonic stem cells and light source controllable cell culture box |
| CN112877279B (en) * | 2019-12-11 | 2023-03-28 | 上海厚超生物科技股份有限公司 | Light source controllable cell culture box for rat embryonic stem cells |
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