CN108373992A - A kind of composition and its application for embryonic stem cell culture - Google Patents
A kind of composition and its application for embryonic stem cell culture Download PDFInfo
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- CN108373992A CN108373992A CN201711451313.9A CN201711451313A CN108373992A CN 108373992 A CN108373992 A CN 108373992A CN 201711451313 A CN201711451313 A CN 201711451313A CN 108373992 A CN108373992 A CN 108373992A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The present invention provides a kind of composition for embryonic stem cell culture, can ensure that embryonic stem cell is chronically at undifferentiated state and the ability with infinite multiplication.It includes LIF ELISA, transforminggrowthfactor-β1, epithelical cell growth factor, basic fibroblast growth factor, Rho kinase inhibitors Y27632, mek inhibitor and bone morphogenetic protein inhibitor Noggin.The present invention also provides the cell culture fluid comprising the composition and its applications.
Description
Technical field
The present invention relates to cytology fields, and in particular, to a kind of composition for embryonic stem cell culture and its answers
With.
Background technology
HESC's is successfully separated the impressive progress that culture is current life science, it is from mankind's morning
It is being detached in the inner cell mass of phase preimplantation embryo, can keep undifferentiated in vitro and constantly be proliferated under felicity condition one
Kind stem cell.Theoretically, it has the potential for being divided into all cell tissues of body, is referred to as " seed cell ".People can be with
It induces differentiation to generate a large amount of different types of cells embryo stem cell for directional using different condition of culture and is used as cell shifting
Plant, the tissue substitute even cell donor of organ cloning, for treat in the future many refractory disease of the mankind provide it is unlimited thin
Born of the same parents source.Therefore it has a extensive future in the application of the clinical medicine in future, becomes another research hotspot in 21 century life science.
In addition, human embryonic cells also has other many purposes, for example, its carrier as gene therapy, by homologous heavy
Group mode carries out gene knockout or modifier is imported into stem cell by transgenosis, can reach gene therapy purpose:It can
The Normal human cells for providing any organization type provide a large amount of samples for developing new drug, and can be used for screening drug, identification
Gene of the target gene site, screening of new drug effect for regeneration gene therapy.The embryonic stem cell of mammal is then
Important tool in biomedical research and model.Efficient embryonic stem cell culture systems will promote the regeneration based on stem cell
Biomedical research and clinical treatment.
The culture of embryonic stem cell needs to pay attention to following two points：One, ensure that embryonic stem cell is in undifferentiated state:Two,
Ensure the ability that embryonic stem cell has infinite multiplication.However, existing culture medium can't reach ideal effect.
Invention content
The technical problem to be solved in the present invention is to provide a kind of compositions for embryonic stem cell culture, can ensure that embryo
Tire stem cell is chronically at undifferentiated state and the ability with infinite multiplication.
Another technical problems to be solved of the invention are to provide one kind comprising the composition and can be done suitable for embryo
The cell culture fluid of cell culture.
In order to solve the above technical problems, a kind of composition for embryonic stem cell culture of the present invention, including leukaemia
Inhibiting factor, transforminggrowthfactor-β1, epithelical cell growth factor, basic fibroblast growth factor, Rho kinase inhibitors
Y27632, mek inhibitor and bone morphogenetic protein inhibitor Noggin, the content of each composition is as follows in the composition：
Further, the basic culture solution includes
Wherein nonessential amino acid is by the alanine of isoconcentration, arginine, asparatate, cystine, proline and junket
Propylhomoserin forms.
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
Further, the content of each composition is as follows in the composition：
The experiment proved that human embryo stem cell is including LIF ELISA, transforminggrowthfactor-β1, epidermal cell
Egg occurs for growth factor, basic fibroblast growth factor, Rho kinase inhibitors Y27632, mek inhibitor and Bones morphology
Grow and significantly improve in the cell culture fluid of white inhibitor Noggin, versatility expression enhances, and can test bar in vitro
It is divided into different germinal layers under part, expresses specific molecular marker, the specific gene of the germinal layer, and can be formed after blastaea injection
Chimeric animal：I.e. embryonic stem cell is at undifferentiated state and the ability with infinite multiplication in the cultivating system.
Specific implementation mode
Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under
State the technology disclosed in example represent inventor discovery can be used for implement the present invention technology, therefore can be considered as implementation this
The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification
Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention particular embodiment many equivalent technologies.These will equally be comprised in claims.
Embodiment 1
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 2
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 3
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 4
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 5
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 6
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 7
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 8
Composition provided in this embodiment for embryonic stem cell culture includes：
Embodiment 9
New zealand rabbit (being purchased from Nanjing Nanjing kind warren) is handled using superfecundation, i.e., injects total amount 2.4mg ovum for three days on end
Bubble irritates plain (FSH, Foll tropin, Bioniche, Canada), then inject 200IU human chorionic gonadotrophins (hCG,
Chorulon, Intervet, USA).It mates with buck by female rabbit of super row, ovum is taken after 18 hours, Embryo Culture is 2.5%
In the B2 embryo mediums (Laboratories CCD, France) of FBS, 38.5 degree, 3 days are cultivated under conditions of 5%CO2 extremely
Blastaea.Blastaea is respectively put into FL cultivating systems and control group and is cultivated, the culture medium for studying the present invention is dry to rabbit embryo thin
Born of the same parents obtain the effect and influence with maintenance.The results show that in the medium of the present invention, apparent rabbit is presented in the growth of cell
The queueing discipline of embryonic stem cell characteristic, cell is neat, and cell contact is exquisite, and cell growth aggregation embodies clone cell
Growth pattern.In the control group of base culture base, cell clone growth is disintegrated, and cell is divided into rapidly fibroblast
Type.
Rabbit embryonic stem cell is fixed on 4% poly first and wakes up (Sigma, USA), is rinsed with PBS, then in alkaline phosphatase
In (Alkaline phosphatase, AP) dyeing liquor, cell is observed under inverted microscope, and the cell that red staining is presented is
Has the cell of stem cell potential.After cell is fixed with 4% poly first is awake, through 0.2% TritonX-100,0.1%Tween oozes
The cell membrane of saturating stem cell, with the anti-human Oct4 first antibodies staining cell of mouse, then the anti-mouse IgG secondary antibodies of donkey with FITC labels
Processing checks the staining conditions of cell under fluorescence microscope.Oct4 is the albumen of stem cell-specific marker, is present in cell
In core, it is the cell for having stem cell potential to show that the cell of fluorescence can be assumed that.We carry out rabbit embryo with embryo respectively
The acquisition of stem cell is tested, every group of 12 embryos, in the experimental group using the culture solution of embodiment 1-8, what acquisition can pass on
4-10 cell line, the cell line for embodying stem cell properties (AP alkaline phosphatases and Oct4) account for 40% or more；It is cultivated on basis
In liquid, rabbit embryonic stem cell line is not induced, and concrete outcome is as shown in table 1.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of composition for embryonic stem cell culture, which is characterized in that including LIF ELISA, conversion growth because
Sub- β 1, epithelical cell growth factor, basic fibroblast growth factor, Rho kinase inhibitors Y27632, mek inhibitor and
Bone morphogenetic protein inhibitor Noggin, the content of each composition is as follows in the composition：
2. composition according to claim 1, it is characterised in that：The basic culture solution includes
3. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
297.5 μ g/L of bone morphogenetic protein inhibitor Noggin.
4. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
5. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
6. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
7. composition according to claim 1 or 2, which is characterized in that each composition in the composition
8. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
9. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
10. composition according to claim 1 or 2, which is characterized in that the content of each composition is as follows in the composition：
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711451313.9A CN108373992A (en) | 2017-12-27 | 2017-12-27 | A kind of composition and its application for embryonic stem cell culture |
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| CN201711451313.9A CN108373992A (en) | 2017-12-27 | 2017-12-27 | A kind of composition and its application for embryonic stem cell culture |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774803A (en) * | 2014-01-10 | 2015-07-15 | 北京爱思益普生物科技股份有限公司 | Novel embryonic stem cell culturing system and embryonic stem cell line establishment method |
| CN105062958A (en) * | 2015-08-26 | 2015-11-18 | 兰诺生物技术无锡有限公司 | Composition for embryonic stem cell culture and application thereof |
| CN106754657A (en) * | 2017-03-28 | 2017-05-31 | 北京赛斯达生物技术有限公司 | A kind of serum free medium of monkey embryonic stem cell |
-
2017
- 2017-12-27 CN CN201711451313.9A patent/CN108373992A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774803A (en) * | 2014-01-10 | 2015-07-15 | 北京爱思益普生物科技股份有限公司 | Novel embryonic stem cell culturing system and embryonic stem cell line establishment method |
| CN105062958A (en) * | 2015-08-26 | 2015-11-18 | 兰诺生物技术无锡有限公司 | Composition for embryonic stem cell culture and application thereof |
| CN106754657A (en) * | 2017-03-28 | 2017-05-31 | 北京赛斯达生物技术有限公司 | A kind of serum free medium of monkey embryonic stem cell |
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