CN109722411A - A kind of application method for the small molecule promoting embryonic stem cell self-renewing state - Google Patents
A kind of application method for the small molecule promoting embryonic stem cell self-renewing state Download PDFInfo
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Abstract
The invention discloses a kind of application methods of small molecule for promoting embryonic stem cell self-renewing state, observation and detection of passage and culture and the present invention of the embryonic stem cell including people and mouse under this small molecule state of the invention to cell self-renewal state.The present invention solves mouse and human embryo stem cell state under current traditional culture conditions by adding CID755673 small molecule and easily breaks up, passes on the problems such as complicated for operation, condition of culture is at high cost and is unfavorable for follow-up mechanism research, have many advantages, such as effect stability, economical and efficient, it is easy to operate, convenient for research, it also gives a clue simultaneously to improve and establishing the condition of culture of the multipotential stem cell of other types and species, there is positive impetus for the smooth development of the following stem cell basis and application study.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of small molecules for promoting embryonic stem cell self-renewing state
Application method.
Background technique
Stem cell is one kind in fetus, embryo or adult and under certain condition with unlimited self-renewing and increasing
Grow a kind of cell of differentiation capability.Embryonic stem cell can be induced to differentiate into various tissues under certain condition, this characteristic makes it
There is tempting application prospect in basic research, transplantation treatment and gene therapy, embryonic stem cell is in vitro under suitable condition,
Can be in infinite multiplication under undifferentiated state, this provides nexhaustible cell for the research and application of embryonic stem cell progress
Source.Both key properties of embryonic stem cell have become research gene function, screening drug and manufacture diseased n animal mould
One of powerful tool of type has huge application prospect in regenerative medicine field.Study at present most commonly used dry thin
Born of the same parents are mouse embryo stem cell (mo μ se Embryonic Stem Cells, mESCs), and mouse embryo stem cell derives from implantation
The inner cell mass of preceding embryo (pregnancy the 3.5th day) has the versatility of development, this be also first plant be successfully established in vitro it is dry
Cell line.Later, embryonic stem cell (human Embryonic Stem Cells, hESCs) system of people is also successfully constructed,
HESCs is to inhibit culture by vitro differentiation by the body early embryo inner cell mass or hPGCs of people, and the pluripotency separated is thin
Born of the same parents have extremely wide application prospect.Although mouse and people ESCs possess some common features, but the biological characteristics of the two
Property but has many differences.CID755673 small molecule maintains the self-renewing state of people and mouse embryo stem cell, also avoids
People and mouse occur differentiation state during the cultivation process and provide a new approaches.Itself leads also for entire stem cell
The expansion of depth and broadness is probed into domain, thus has important scientific research value and academic significance, and related ends facilitate following right
In the security application of stem cell.
The maintenance of the embryonic stem cell of people and mouse needs specific nutriment and cell factor, if without these cells because
Sub- embryonic stem cell will appear differentiating phenomenon, it has been found that CID755673 chemical small molecule can be added without some cells
Promote the self-renewing of embryonic stem cell under conditions of the factor, and this can be used as following condition of culture of embryonic stem cell,
The type for enriching embryonic stem cell condition of culture optimizes the condition of culture of embryonic stem cell.
Although the CID755673 condition of culture after optimization can maintain the self-renewing state of embryonic stem cell, due to
Related cell factor type is excessive, is unfavorable for the subsequent deep spy for intracellular associated signal paths and molecular mechanism
Study carefully, and toxigenic capacity is high.The present invention on this basis, by successively decrease one by one from the trial to different permutation and combination, further change
Into the self-renewing condition with optimization people and mouse, two kinds of small molecules, which are used only, in the condition after optimization can maintain mouse embryonic stem
The self-renewing state of cell, small molecule are the self-renewing that can promote the embryo of people, reach identical as former condition of culture
Culture effect, have many advantages, such as it is time saving, laborsaving, economical, be conducive to follow-up study.
Summary of the invention
The present invention mentions to solve to occur easily breaking up in embryonic stem cell incubation, pass on the problems such as inconvenient for operation
For a kind of application method of small molecule for promoting embryonic stem cell self-renewing state.
The present invention is achieved by the following technical solutions:
A kind of application method for the small molecule promoting embryonic stem cell self-renewing state, in DMEM culture medium, N2B27+KSR
In culture medium, CID755673 small molecule compound is added to maintain the self-renewing state of the embryonic stem cell of mouse and people, packet
The embryonic stem cell of passage and culture and mouse of the embryonic stem cell of people under the conditions of CID755673 is included in CID755673 item
Passage and culture under part.
Passage and culture of the embryonic stem cell of the people under the conditions of CID755673, comprising the following steps:
(1) 6 porocyte culture plates are taken, 2 ml DMEM+10 μ l Matrigel wrapper sheets of every hole are placed in 37 DEG C, 5% CO2
In the cell incubator of concentration, it is coated with 4 h；
(2) embryonic stem cell for growing to the people of 70-80% density is taken, culture solution is abandoned, is washed cell 1 time with PBS, removes cell
Culture solution remained on surface；
(3) 1 ml CTK is added and digests embryonic stem cell, after 6-8min, cell edges float, and abandon CTK, add 2 ml containing 10%
The regular growth culture solution of FBS, is blown and beaten with pipettor, and cell is blown and beaten from culture plate, be then transferred to 15 ml without
In bacterium centrifuge tube；
(4) 1200 rpm suck supernatant after being centrifuged 4 min；
(5) plus cell is resuspended in N2B27 of 2 ml containing 10%KSR；
(6) cell in suspension is counted using blood counting chamber under the microscope, and calculates the density of cell with this；
(7) culture dish being coated with is taken, wrapper sheet solution is abandoned, PBS is washed one time, and 2 ml N2B27+ are added into the every hole of culture plate
KSR culture medium；
(8) 5 × 10 are added into culture medium4A cell, planche cross shape are shaken, and cell is uniformly distributed；
(9) the CID755673 small molecule of 10 μm of ol is added, planche cross shape is shaken, it is uniformly mixed；
(10) Tissue Culture Dish is placed in 37 DEG C, 5% CO2Culture in the cell incubator of concentration.
CTK in the step 3 contains 1% Collagenase IV, 0.25% Trypsin, 20% KSR, 1mM
CaCl2PBS.
The every 5ml of N2B27 in the step 5 contains 1.875 ml DMEM/F12,0.25 ml N2,1.875 ml
Neurobasal medium, 0.5 ml B27,2 mM L-gl μ tamine, 0.1 mM β-mercaptoethanol.
The long-term self-renewing shape that small molecule CRT0066101 can maintain the embryonic stem cell of people is added in the application
State.
Passage and culture of the embryonic stem cell of the mouse under the conditions of CID755673, comprising the following steps:
(1) 6 porocyte culture plates are taken, 2 ml Gelatin wrapper sheets of every hole are placed in 37 DEG C, 5% CO2The cell of concentration
In incubator, it is coated with 1 h；
(2) the stable cell line of stand density 55-65% is taken, culture solution is abandoned, PBS buffer solution is carefully washed 1 time, and net residual is abandoned
Cleaning solution；
(3) 1 ml pancreatin digestive juice vitellophag is added immediately, cell colony edge floats after several minutes, uses micropipettor
It carefully dispels and is transferred in the 15 ml centrifuge tubes containing 2 ml DMEM serum free culture system liquid, continue careful piping and druming and mix, terminate
Digestion；
(4) the mixing suspension in centrifuge tube is centrifuged about 3 min in 1000 rpm, abandons supernatant and the washing of 1 ml PBS buffer solution is added
And it is centrifuged again；
(5) it abandons supernatant and appropriate DMEM serum free culture system liquid resuspension cell is added, and carry out cell count；
(6) it takes appropriate cell suspension in 15 ml centrifuge tubes, appropriate DMEM blood serum medium is added to 2 ml of total volume, is added 2
μm ol CID755673 small molecule, careful piping and druming mix；
(7) Tissue Culture Dish being coated with is taken, gelatin is abandoned, the suspension of 2 ml of upper step is transferred to culture dish, horizontal cross ten
The sliding of word method, makes cell and small molecule be evenly distributed；
(8) Tissue Culture Dish is placed in 37 DEG C, 5% CO2Culture in the cell incubator of concentration, observes cellular morphology and life daily
Long situation, and culture solution is changed every other day.
The embryonic stem cell of the mouse be added in the passage and culture under the conditions of CID755673 CID755673 and
Two kinds of small molecules of PD0325901 are able to maintain that the embryonic stem cell of mouse maintains long-term self-renewing state.
The invention has the advantages that
(1) present invention is small using CID755673 is added in the DMEM culture medium of 10% FBS content, the culture medium of N2B27+KSR
The method of molecule promotes the self-renewing of the embryonic stem cell of mouse and people, under such condition of culture, cell growth status
It well, being capable of the of short duration self-renewing for maintaining people and mouse embryo stem cell；
(2) CID755673 and two kinds of small molecules of PD0325901, human embryo stem cell are added in the culture of mouse embryo stem cell
CRT0066101 small molecule is added in culture, cell can be made to maintain optimal growth conditions, promote embryonic stem cell for a long time
Self-renewing, simplify operating procedure and process, conducive to the development of basic research；
(3) and for existing people and Mouse Embryonic Stem Cell Culture condition, the present invention has the advantages that more economically efficient, changes
It has been apt to the differentiating phenomenon occurred during people and Mouse Embryonic Stem Cell Culture, toxigenic capacity and time can be saved significantly on；Meanwhile
The addition type for simplifying cell factor, optimizes cultivating system, is conducive to subsequent to intracellular associated signal paths and molecule tune
The expansion of control mechanism is deeply probed into；
(4) condition of culture that the present invention is found out can be the current people of improvement and mouse embryo stem cell and other types
The condition of culture of multipotent stem cells is given a clue.
Detailed description of the invention
Aspect graph and embryo of the P5 that Fig. 1 is cultivated under the conditions of showing three kinds for mouse embryo stem cell under phase contrast microscope
The result of tire stem cell AP dyeing.
Aspect graph and embryo of the P0 embryonic stem cell that Fig. 2 is cultivated under the conditions of showing three kinds under phase contrast microscope are dry thin
The result of born of the same parents AP dyeing.
The P3 that Fig. 3 is cultivated under the conditions of showing three kinds carries out the immune of self-renewing marker protein for mouse embryo stem cell
Fluorescent staining detection.
Specific embodiment
Below in conjunction with specific example, technical scheme is described further:
The embryonic stem cell strain HES2 of people used in experiment, the embryonic stem cell strain 46C of mouse is by University of Southern California, the U.S.
Professor Ying Qilong provides.
Embodiment 1
Passage and culture of the embryonic stem cell of people under the conditions of CID755673
(1) 6 porocyte culture plates are taken, 2 ml DMEM+10 μ l Matrigel wrapper sheets of every hole are placed in 37 DEG C, 5%
CO2In the cell incubator of concentration, it is coated with 4h；
(2) embryonic stem cell for growing to the people of 70-80% density is taken, culture solution is abandoned, is washed cell 1 time with PBS, it is thin to remove
The remaining culture solution of cellular surface；
(3) 1 ml CTK is added and contains (+20% KSR+1mM CaCl of 1%Collagenase IV+0.25% Trypsin2PBS)
Embryonic stem cell is digested, 7 min or so, cell edges float, and abandon CTK, and add 2 ml to contain the regular growth culture solution of 10% FBS,
It is blown and beaten with pipettor, cell is blown and beaten from culture plate, be then transferred in 15 ml sterile centrifugation tubes；
(4) 1200 rpm suck supernatant after being centrifuged 4 min；
(5) plus N2B27(5 ml N2B27 of 2 ml containing 10%KSR is containing 1.875 ml DMEM/F12,0.25 ml N2, and 1.875
Ml Ne μ robasal medium, 0.5 ml B27,2 mM L-glutamine, 0.1 mM β-mercaptoethanol) weight
Outstanding cell；
(6) cell in suspension is counted using blood counting chamber under the microscope, and calculates the density of cell with this；
(7) culture dish being coated with is taken, wrapper sheet solution is abandoned, PBS is washed one time, and 2 ml N2B27+ are added into the every hole of culture plate
KSR culture medium；
(8) 5 × 10 are added into culture medium4A cell, planche cross shape are shaken, and cell is uniformly distributed；
(9) CID755673 of 10 μm of ol is added, planche cross shape is shaken, and is uniformly mixed it, while two groups of culture items are arranged
For part as control, one group is not added the factor, one group of CID755673 for only adding 10 μm of ol；
(10) Tissue Culture Dish is placed in 37 DEG C, cultivated in the cell incubator of 5% CO2 concentration.
Embodiment 2
Passage and culture of the embryonic stem cell of mouse under the conditions of CID755673
(1) 6 porocyte culture plates are taken, 2 ml Gelatin wrapper sheets of every hole are placed in 37 DEG C, 5% CO2The cell of concentration
In incubator, it is coated with 1 h；
(2) the stable cell line of stand density about 60% is taken, culture solution is abandoned, PBS buffer solution is carefully washed 1 time, is abandoned net residual and is washed
Wash liquid；
(3) 1 ml pancreatin digestive juice (Trypsin-EDTA) vitellophag is added immediately, cell colony edge is floating after several minutes
It rises, is carefully dispelled and be transferred in the 15ml centrifuge tube containing 2 ml DMEM serum free culture system liquid with micropipettor, continue small
Heart piping and druming mixes, and terminates digestion；
(4) the mixing suspension in centrifuge tube is centrifuged about 3 min in 1000 rpm, abandons supernatant and the washing of 1 ml PBS buffer solution is added
And it is centrifuged again；
(5) it abandons supernatant and appropriate DMEM serum free culture system liquid resuspension cell is added, and carry out cell count；
(6) appropriate cell suspension is taken in 15 ml centrifuge tubes, and appropriate DMEM blood serum medium is added to 2 ml of total volume, in proportion
The CID755673 of 10 μm of ol is added, careful piping and druming mixes；
(7) Tissue Culture Dish being coated with is taken, gelatin is abandoned, the suspension of 2 ml of upper step is transferred to culture dish, horizontal cross ten
The sliding of word method, makes cell and small molecule be evenly distributed；
(8) Tissue Culture Dish is placed in 37 DEG C, 5% CO2Culture in the cell incubator of concentration, observes cellular morphology and life daily
Long situation, and culture solution is changed every other day.
Embodiment 3
Use the self-renewing Indexs measure after CID755673 small molecule
(1) morphologic observation
Using Leica DMi8 inverted microscope respectively to do not add any small molecule (as a control group), only add
CID755673 small molecule, different cultivated days the embryonic stem cell of mouse observed, cell is in compared with the control group for discovery
Reveal the close single layer configuration of accumulation, colony sharpness of border meets the morphological feature of self-renewing, however, it was found that being only added
The self-renewing that CID755673 can only maintain mouse embryo stem cell of short duration, thus again discovery be added CID755673 and
Two kinds of small molecules of PD0325901 are able to maintain that the embryonic stem cell of mouse maintains long-term self-renewing state.
Using Leica DMi8 inverted microscope respectively to do not add any small molecule (as a control group), only add
CID755673 small molecule, different cultivated days the embryonic stem cell of people observe, cell is presented compared with the control group for discovery
Close single layer configuration, and colony sharpness of border are accumulated out, meets the morphological feature of self-renewing, and discovery is only added
The self-renewing state that CID755673 is only capable of maintaining the embryonic stem cell of people of short duration filters out small molecule by deeper probing into
CRT0066101 can maintain the long-term self-renewing state of the embryonic stem cell of people.
After cell is repeatedly passed on, the culture form of different algebra cells is observed, the feature of self-renewing is kept.
(2) AP dyes (alkaline phosphatase dyeing) detection cell either with or without differentiation
Under different condition of culture subculture 5 instead of after, carry out AP dyeing.Coloring agent is configured first, by a 8523R glue
Capsule, which dissolves in the water of 48ml, to be placed on 4 degree and is kept in dark place, and takes appropriate, it is sufficiently mixed into SLBJ6060V in the ratio of 25:1
Coloring agent.The stem cell rested abandons training liquid, and PBS is added to wash one time；The fixed cell 2 of 4% paraformaldehyde (PFA) of 1 ml is added to divide
Clock；PFA is abandoned, PBS is added to wash twice, PFA residual is washed off；Each culture hole adds the coloring agent of 2 ml；It is protected from light standing 40 minutes；It abandons
Dyeing liquor is washed twice with PBS；Add to be placed on to observe under Leica DMi8 inverted microscope after appropriate PBS and take pictures.The coloring shape of observation
State, to judge the self-renewing state of cell.
The P5 that cultivates is dry for aspect graph of the mouse embryo stem cell under phase contrast microscope and embryo under the conditions of Fig. 1 is three kinds
It is that cell AP dyes (alkaline phosphatase dyeing) as a result, the wherein aspect graph under top half phase contrast microscope, upper left side is does not add
Add the mouse embryo stem cell cultivated under the conditions of any factor, the state in differentiation；Middle upside is addition CID755673 condition
The cell of lower culture meets the feature of short-term self-renewing state；Upper right side is addition CID755673 and PD0325901 culture
Cell, meet the feature of long-term self-renewing state.
Lower half portion is then AP dyeing as a result, lower left side is not add the mouse embryonic stem cultivated under the conditions of any factor
Cell, middle downside are the embryonic stem cell for adding the mouse cultivated under the conditions of CID755673, and lower right side is addition CID755673
With the embryonic stem cell of the mouse of PD0325901 culture, undifferentiated cell can be colored, and the cell of differentiation will not be colored.
Aspect graph and embryonic stem cell AP of the P0 embryonic stem cell that Fig. 2 is cultivated under the conditions of being three kinds under phase contrast microscope
Dye (alkaline phosphatase dyeing) as a result, the wherein aspect graph under top half phase contrast microscope, upper left side are any not add
The embryonic stem cell of the people cultivated under the conditions of the factor, the state in differentiation；It is cultivated under the conditions of being addition CID755673 middle upside
People embryonic stem cell, meet the feature of short-term self-renewing state；Upper right side is the people for adding CRT0066101 culture
Embryonic stem cell meets the feature of long-term self-renewing state.
And lower half portion is then AP dyeing as a result, lower left side is not add the embryo of the people cultivated under the conditions of any factor
Stem cell, middle downside are the embryonic stem cell for adding the people cultivated under the conditions of CID755673, and lower right side is addition CRT0066101
Embryonic stem cell under cultivation conditions, undifferentiated cell can be colored, and the cell of differentiation will not be colored.
(3) expression of immunofluorescence dyeing detection self-renewing marker protein Oct4
By human embryo stem cell and mouse embryo stem cell under the conditions of adding CID755673 small molecule after 5 generation of secondary culture, training is abandoned
Nutrient solution is rinsed cell 1 time with PBS, and the PFA(paraformaldehyde of 4% concentration is added), room temperature fixes 20 min；
PFA is abandoned, is rinsed cell 1 time with PBS, confining liquid is then added and (contains 5% concentration bovine serum albumin(BSA), 0.2% concentration
The PBS of Triton X-100), cell is placed in 37 DEG C of incubators, is incubated for 1 h；
Confining liquid is abandoned, is rinsed cell 1 time, is then separately added into containing Oct4(1:200, Santa Cr μ z, μ SA with PBS) primary antibody
Dilution (contain 5% concentration bovine serum albumin(BSA), 0.2% concentration Triton X-100, the PBS of 1% concentration N3Na), 4 DEG C of mistakes
Night；
Next day abandons primary antibody；It is rinsed cell 3 times with PBS, every 5 min is washed once, is then separately added into and is carried 488 fluorophors
Secondary antibody (1:1000, the green skies) and Hoechst (1:5000, Invitrogen) dyestuff dilution (contain 5% concentration ox
Sero-abluminous PBS), culture plate is encased with masking foil, 37 DEG C are protected from light 1 h of incubation；
Secondary antibody diluent is abandoned, is rinsed cell 3 times with PBS, every 3min is washed once, is then inverted fluorescence in Leica DMi3000B type
Microscopically observation is simultaneously taken pictures；
Fig. 3 is the detection for carrying out self-renewing marker protein for mouse embryo stem cell to the P3 cultivated under the conditions of three kinds, used
Detection method be immunofluorescence dyeing.In fluorescence microscopy microscopic observation, green fluorescence is presented in Oct4 albumen；Hoechst dye
Material is used to show the position of nucleus, and blue-fluorescence is presented under fluorescence microscope；Merge is then that Oct4 albumen presentation green is glimmering
The figure of light is merged with the figure of the position of Hoechst dyestuff display nucleus.
Immunofluorescence results are shown, add the embryonic stem cell of the mouse of exogeneous small molecule CID755673 and the embryo of people
Tire stem cell height expresses Oct4, illustrates that cell is in the state of self-renewing, and the cell differentiation what is all not added.
Claims (7)
1. a kind of application method for the small molecule for promoting embryonic stem cell self-renewing state, which is characterized in that cultivated in DMEM
Base, N2B27+KSR culture medium in, CID755673 small molecule compound is added to maintain the embryonic stem cell of mouse and people
The embryo of self-renewing state, passage and culture and mouse of the embryonic stem cell including people under the conditions of CID755673 is dry thin
Passage and culture of the born of the same parents under the conditions of CID755673.
2. application method according to claim 1, which is characterized in that the embryonic stem cell of the people is in CID755673
Under the conditions of passage and culture, comprising the following steps:
(1) 6 porocyte culture plates are taken, 2 ml DMEM+10 μ l Matrigel wrapper sheets of every hole are placed in 37 DEG C, 5% CO2
In the cell incubator of concentration, it is coated with 4 h；
(2) embryonic stem cell for growing to the people of 70-80% density is taken, culture solution is abandoned, is washed cell 1 time with PBS, removes cell
Culture solution remained on surface；
(3) 1 ml CTK is added and digests embryonic stem cell, after 6-8min, cell edges float, and abandon CTK, add 2 ml containing 10%
The regular growth culture solution of FBS, is blown and beaten with pipettor, and cell is blown and beaten from culture plate, be then transferred to 15 ml without
In bacterium centrifuge tube；
(4) 1200 rpm suck supernatant after being centrifuged 4 min；
(5) plus cell is resuspended in N2B27 of 2 ml containing 10%KSR；
(6) cell in suspension is counted using blood counting chamber under the microscope, and calculates the density of cell with this；
(7) culture dish being coated with is taken, wrapper sheet solution is abandoned, PBS is washed one time, and 2 ml N2B27+ are added into the every hole of culture plate
KSR culture medium；
(8) 5 × 10 are added into culture medium4A cell, planche cross shape are shaken, and cell is uniformly distributed；
(9) the CID755673 small molecule of 10 μm of ol is added, planche cross shape is shaken, it is uniformly mixed；
(10) Tissue Culture Dish is placed in 37 DEG C, 5% CO2Culture in the cell incubator of concentration.
3. application method according to claim 2, which is characterized in that the CTK in the step 3 contains 1%
Collagenase IV、0.25% Trypsin 、20% KSR、1mM CaCl2PBS.
4. application method according to claim 2, which is characterized in that the every 5ml of N2B27 in the step 5 contains 1.875
Ml DMEM/F12,0.25 ml N2,1.875 ml Neurobasal medium, 0.5 ml B27,2 mM L-gl μ
Tamine, 0.1 mM β-mercaptoethanol.
5. application method according to claim 2, which is characterized in that small molecule CRT0066101, which is added, can maintain people's
The long-term self-renewing state of embryonic stem cell.
6. application method according to claim 1, which is characterized in that the embryonic stem cell of the mouse exists
Passage and culture under the conditions of CID755673, comprising the following steps:
(1) 6 porocyte culture plates are taken, 2 ml Gelatin wrapper sheets of every hole are placed in 37 DEG C, 5% CO2The cell of concentration is trained
It supports in case, is coated with 1 h；
(2) the stable cell line of stand density 55-65% is taken, culture solution is abandoned, PBS buffer solution is carefully washed 1 time, and net residual is abandoned
Cleaning solution；
(3) 1 ml pancreatin digestive juice vitellophag is added immediately, cell colony edge floats after several minutes, uses micropipettor
It carefully dispels and is transferred in the 15 ml centrifuge tubes containing 2 ml DMEM serum free culture system liquid, continue careful piping and druming and mix, terminate
Digestion；
(4) the mixing suspension in centrifuge tube is centrifuged about 3 min in 1000 rpm, abandons supernatant and the washing of 1 ml PBS buffer solution is added
And it is centrifuged again；
(5) it abandons supernatant and appropriate DMEM serum free culture system liquid resuspension cell is added, and carry out cell count；
(6) it takes appropriate cell suspension in 15 ml centrifuge tubes, appropriate DMEM blood serum medium is added to 2 ml of total volume, is added 2
μm ol CID755673 small molecule, careful piping and druming mix；
(7) Tissue Culture Dish being coated with is taken, gelatin is abandoned, the suspension of 2 ml of upper step is transferred to culture dish, horizontal cross ten
The sliding of word method, makes cell and small molecule be evenly distributed；
(8) Tissue Culture Dish is placed in 37 DEG C, 5% CO2Culture in the cell incubator of concentration, observes cellular morphology and life daily
Long situation, and culture solution is changed every other day.
7. application method according to claim 6, which is characterized in that CID755673 and two kinds small point of PD0325901 is added
Son is able to maintain that the embryonic stem cell of mouse maintains long-term self-renewing state.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724664A (en) * | 2019-11-07 | 2020-01-24 | 安徽大学 | Culture method for maintaining self-renewal of mouse embryonic stem cells |
| CN115029302A (en) * | 2022-06-10 | 2022-09-09 | 安徽大学 | Novel method for in vitro induction and establishment of pluripotent stem cells |
| CN115029302B (en) * | 2022-06-10 | 2023-07-11 | 安徽大学 | Culture method for maintaining mouse embryo stem cell stem property |
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