CN109609446A - It is a kind of for being separately cultured the culture solution and method of rabbit embryonic stem cell - Google Patents
It is a kind of for being separately cultured the culture solution and method of rabbit embryonic stem cell Download PDFInfo
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- CN109609446A CN109609446A CN201811354111.7A CN201811354111A CN109609446A CN 109609446 A CN109609446 A CN 109609446A CN 201811354111 A CN201811354111 A CN 201811354111A CN 109609446 A CN109609446 A CN 109609446A
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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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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Abstract
The invention discloses a kind of for being separately cultured the culture solution and method of rabbit embryonic stem cell.The culture solution includes a kind of for being separately cultured the composition of rabbit embryonic stem cell, and the composition is made of bFGF, LIF, CHIR99021 and XAV-939.BFGF, LIF, CHIR99021, XAV939 are added in culture solution can maintain the versatility of rbESCs, the rbESCs form turned out is similar with mouse embryo stem cell clone, in " island " shape convex growth, three-dimensional sense is strong, and there is the original state versatility in part, it remains to form monoclonal after pancreatin had digestive transfer culture.In addition, the rbESCs turned out can express higher levels of stem cell versatility related gene, and with the feature differentiation capability of Na ve state and Primed state stem cell, and can carry out long-term in vitro secondary culture in the culture solution, it can be passaged to for 14 generations.
Description
Technical field
The present invention relates to cell engineering fields, and in particular, to a kind of for being separately cultured rabbit embryonic stem cell
Culture solution and method.
Background technique
Rabbit is a kind of common laboratory animal, is easy raising, cheap, the breeding cycle is short, and litter size is more, already
For biomedical research.Rabbit volume ratio mouse is big, convenient experimental operation, so that operation is easier, more effectively obtains full-page proof
This.In biological medicine, rabbit is widely used in biomedical acute experiment, endocrine experiment, metabolism experiment, medicine
Neo-Confucianism experiment etc..In human diseases using upper, rabbit is in upper opposite rodent of evolving closer to the mankind.Human diseases exists
The cause of disease ratio shown on rabbit is more like on mouse, and transgene rabbit is that heredity and the excellent of acquired human diseases move again
Object model, thus rabbit be largely used as treatment human diseases experimental model: as diabetes, hypertrophic cardiomyopathy, hypertension,
Atherosclerosis, hyperlipidemia, Lipoprotein Disorders, retinal degeneration, injury of lungs, inflammation disease etc..Rabbit another
Advantage is classical as one laboratory species in terms of reproductive technology.It ovulates in hypervelocity, in vitro fertilization, sperm injection, essence
Liquid freezen protective, ovum and Embryo freezing preservation, pronuclear microinjection, nuclear transfer clone etc., rabbit suffers from as experiment
The significant superiority of animal.Therefore, from the rabbit embryonic stem cell of rabbit blastaea ICM (Rabbit Embryonic Stem
Cells, rbESCs) research be also taken seriously, if can obtain rbESCs for human diseases research, production transgenosis
Rabbit, test stem cell therapy, gene targeting research, will all have great importance.However, with regard to current progress,
There is still a need for further investigations for be separately cultured system of the people to rbESCs.
Currently, commonly the method for separation ICM has Mechanical Method, immunosurgery method and micro- partition method etc..WANG et al. benefit
With the embryo of lonely female activation, the rbESCs of energy Long Term Passages is turned out, immunosurgery method separation effect is found when separating ICM
Fruit is bad.Japanese Scientists Honda then utilizes micromanipulation instrument technology, provides preferable method for the separation of ICM.In difference
In the stem-cell research of species, mouse embryo stem cell (Embryonic Stem Cells, mESCs) research is more.MESCs is only
Needing to add LIF can pass on steadily in the long term, keep having edge bright, relief clone.In contrast, rbESCs, people
It is more difficult that embryonic stem cell (human Embryonic Stem Cells, hESCs), which is cultivated, built,.RbESCs, hESCs's
Culture medium often adds bFGF, and rbESCs, the hESCs for just having larger probability to be cloned, and turned out show flat cloning,
It is not amenable to pancreatin digestion, needs carefully to pass on.Though there are multiple laboratories to be separately cultured out rbESCs and successfully built to be now,
But this is also the success of the sub-fraction obtained after a large number of experiments, and different experiments room operation technique, condition of culture difference
Larger, there is also differences by the rbESCs obtained out.Even if using identical culture medium, identical cultivation temperature, but operation technique
It can not be controlled with embryo's individual difference.Most rbESCs cannot stablize passage, gradually break up during the cultivation process, only
Only a few rbESCs can long-term cultivation, some laboratory report rbESCs can pass for tens generations, and some laboratories report
RbESCs can only pass several generations.
In research reported at present, about the ingredient of rbESCs cultivating system, there are still larger disputes.Pierre
Osteil et al. discovery serum substitute (Knockout Serum Replacement, KOSR) is for the culture of ESCs must not
It can lack, the culture medium that fetal calf serum (Fetal bovine serum, FBS) is only supplemented without KOSR cannot all generate
rbESCs.AP Catunda et al. thinks have LIF that can preferably prevent from breaking up, and occurs when rabbit embryonic develops into blastaea first
LIF, LIFR, gp130 expression when secondly cultivating rbESC, remove LIF and occur cardiac-like muscle cell later.Honda thinks
Acitvin/Nodal-SMAD2/3 signal path is vital to the maintenance of rbESCs stemness, and LIF is not essential,
Presence or absence does not affect the expression of the multipotencys gene such as OCT4, NANOG of rbESCs, does not influence colony morphology, the AP of rbESCs
Dyeing.The STAT3 protein level of phosphorylation does not also change with LIF concentration in rbESCs, and also there is the STAT3 of phosphorylation in when no LIF
Albumen.Intawicha P etc. then think add LIF be it is necessary, LIF can improve primary colonies quantity, pass for a long time after removing LIF
In generation, will lead to cell differentiation.Intawicha P it is subsequent studies have shown that in culture medium add suitable concentration bFGF, can improve
The activity of ERK1/2 and AKT in rbESCs promotes the maintenance and self-renewing of its versatility.In culture medium simultaneously add LIF with
BFGF can be such that the STAT3/AKT/ERK level of rbESCs is substantially improved, be more advantageous to the maintenance of rbESCs versatility.Even if
Under condition of culture without feeder layer, LIF and bFGF also contribute to the maintenance of rbESCs colony morphology.Xue F is by not homologous feeding
The LIF combination collocation culture rbESCs of layer and separate sources is supported, after the discovery homologous LIF of rabbit is used together with the homologous feeder layer of rabbit,
Establish rbESC cell line.Pierre Osteil et al. think the conduction of LIF/JAK signal in rbESCs self more
It is newly unnecessary, but add the rbESCs clone that LIF is turned out there is certain chimeric ability, is chimera embryo, it can be more
It effectively is colonized in ectoderm, is hadThe characteristics of state ESCs.Pierre Osteil et al. also provides a comparison of addition growth factor
The culture medium of LIF, bFGF, and do not add the culture medium of growth factor and respectively turn out rbESCs system efficiency.The result shows that adding
Add the culture medium establishment efficiency highest of bFGF.
To sum up, bFGF and LIF has use in rabbit embryonic stem cell culture at present, but the embryonic stem cell obtained is more
Energy property reduces, and cannot maintain the distinctive morphological feature of its stem cell and biological characteristics for a long time.Therefore, it is necessary to study a kind of new
Rabbit embryonic stem cell cultivating system and method, to realize the external Long Term Passages culture of rbESCs, and maintain its morphology special
It seeks peace biological characteristics.
Summary of the invention
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, provide a kind of dry for being separately cultured rabbit embryonic
The composition of cell, in the rbESCs that bFGF, LIF, CHIR99021 and XAV-939 in the composition can be such that culture obtains
The expression of stem cell versatility related gene is higher, and withThe feature of state and Primed state stem cell is realized
External Long Term Passages culture, and there is differentiation capability.
Another object of the present invention is to provide a kind of to be used to be separately cultured rabbit embryonic stem cell comprising above-mentioned composition
Culture solution.
Another object of the present invention is to provide a kind of methods for being separately cultured rabbit embryonic stem cell.
To achieve the goals above, the present invention is achieved by following scheme:
It is a kind of for being separately cultured the composition of rabbit embryonic stem cell, the composition is by Basic Fibroblast Growth Factor
BFGF, LIF ELISA LIF, GSK3 inhibitor C HIR99021 and β-catenin specific inhibitor XAV-939 composition.
Wherein, small molecule compound CHIR99021 (GSK3 inhibitor), XAV-939 (β-catenin specific inhibitor)
It is the related inhibitors on Wnt/ β-catenin access.CHIR99021 inhibits the accumulation of GSK3 promotion β-catenin,
CHIR99021 is culture mouse embryo stem cell, maintains the crucial additive of its original state multipotency state.The effect of XAV939 is
Stablize the Axin in β-catenin protein degradation compound, Axin can inhibit β-catenin to enter nucleus, inhibit β-
The expression of catenin induction differentiation gene.Therefore, in bFGF (Basic Fibroblast Growth Factor), LIF (LIF ELISA)
On the basis of add CHIR99021, XAV939, be conducive to maintain rbESCs versatility, make it maintain stem cell peculiar for a long time
Morphological feature and biological characteristics.
Preferably, the final concentration ratio of bFGF, LIF, CHIR99021, XAV-939 are (4~6ng/mL) in the composition:
(9~11ng/mL): (2.5~3.5 μM): (1.5~2.5 μM).
The present invention be also claimed it is a kind of for being separately cultured the culture solution of rabbit embryonic stem cell, every 50mL culture solution by with
The following group is grouped as: 37mL KnockOut DMEM culture solution, 7.5mL serum substitute, 2.5mL fetal calf serum, 0.5mL blueness strepto-
Element, 0.5mL nonessential amino acid, 0.5mL glutamic acid, 0.5mL L-Glutamine, 0.5mL beta -mercaptoethanol, 0.5mL nucleosides,
The bFGF of final concentration of 4~6ng/mL, the LIF of final concentration of 9~11ng/mL, final concentration of 2.5~3.5 μM
CHIR99021, final concentration of 1.5~2.5 μM of XAV-939.
It is turned out using culture solution of the present invention compared to KBL group (being not added with CHIR99021 and XAV939)
RbESCs has the advantage that (1) form is similar with mouse embryo stem cell clone, is in " island " shape convex growth, and three-dimensional sense is strong；
(2) there is the original state versatility in part, remain to form monoclonal after pancreatin had digestive transfer culture；(3) the rbESCs energy turned out
Express higher levels of stem cell versatility related gene；(4) withState and the differentiation of the feature of Primed state stem cell
Ability can be divided into the cardiac muscle cell of bounce；(5) long-term in vitro secondary culture can be carried out in the culture solution, can be passaged to 14
Generation.
Preferably, in the culture solution final concentration of bFGF, LIF, CHIR99021, XAV-939 be respectively 4ng/mL,
10ng/mL、3μM、2μM。
A kind of method for being separately cultured rabbit embryonic stem cell is also claimed in the present invention, includes the following steps: using machinery
Partition method handles rabbit blastaea, and the isolated entire ICM of each blastaea is inoculated into the feeder layer containing above-mentioned culture solution, in 37
DEG C, 5%CO2, cultivate under conditions of relative humidity 95%, originally culture 4.31~5.63 days；ReLeSR digestion method is used later
Clone's passage is carried out to get rabbit embryonic stem cell, gained rabbit embryonic stem cell in vitro culture can be passaged to for 14 generations.
The present invention from the processing method of blastaea, the generation time of clone, the propagating method of clone, cultivating system etc. into
Go optimization:
(1) the blastaea processing method after optimizing is mechanical phonograph recorder separation, handles blastaea, inner cell mass using mechanical phonograph recorder separation
(Inner Cell Mass, ICM) adherence rate and cloning efficiency respectively reach 87.93% and 70.69%, are significantly higher than
Micro- partition method and full embryo culture method.
(2) clone's generation time after optimizing carries out for 4.31 days to 5.63 days preferably after ICM inoculation, is because of 84.2%
ICM cell in the 4-5 days formation primary colonies being inoculated on feeder layer, the average time that primary colonies are formed is 4.31
It；92.1% ICM cell starts differentiating phenomenon occur the 5th~6 day be inoculated on feeder layer, average time 5.63
It.
(3) clone's propagating method after optimizing is ReLeSR digestion method, and ReLeSR digestion method optionally makes undifferentiated
Clone cell be detached from culture dish, be digested to small cell cluster of uniform size, the clone's quantity formed after passage is more, and energy
Evenly laid out growth, passage effect are better than Mechanical Method and trypsin digestion.
Preferably, the step of mechanical treatment process be first with chain protease digest blastaea, remove embryo mucoprotein and
Oolemma；ICM is separated with trophocyte again, then ICM suction is transferred in feeder layer.
Preferably, the processing time of the ReLeSR digestion method is 4~6min.
As a preferred embodiment, the step of ReLeSR digestion method are as follows: discard rabbit embryonic stem cell culture
Liquid is washed 2 times with PBS, and appropriate ReLeSR is added and carries out digestion 1min；Extra digestive juice is siphoned away, a small amount of liquid is stayed not have cell,
It is put into 3~5min of hatching in incubator；New 0.5mL rabbit embryonic stem cell culture solution is added and terminates digestion, liquid is transferred to
EP pipe；Add the PBS of 0.5mL to clean digestion hole, PBS is also transferred to EP pipe；It is centrifuged 1000rpm 3min, supernatant is discarded, stays
Bottom cell precipitation；Appropriate rabbit embryonic stem cell culture solution is added, cell precipitation is resuspended in piping and druming, by 1~2 × 105/ mL cell
Liquid is transferred in 24 orifice plates of new feeder layer or 12 orifice plates by quantity, is rocked mixing, is put into incubator and cultivates, carry out down
Had digestive transfer culture.
As another preferred embodiment, the step of the ReLeSR digestion method are as follows: first picking shape under the microscope
State is preferably cloned, and is transferred in 96 orifice plates containing 100 μ L ReLeSR liquid, is placed and is digested 3~5min in incubator；Digestion
It is gently that clone's piping and druming is loose after complete, liquid is transferred to EP pipe, supplements 900 μ L rabbit embryonic stem cell culture solutions；Centrifugation
1000rpm 3min, discards supernatant, portion's cell precipitation of keeping on file；Appropriate rabbit embryonic stem cell culture solution is added, piping and druming makes cell
Precipitating is resuspended, by 1~2 × 105Liquid is transferred in 24 orifice plates of new feeder layer or 12 orifice plates by/mL cell quantity, is rocked
It mixes, is put into incubator and cultivates, carry out had digestive transfer culture next time.
As a kind of selectable specific embodiment, the method for being separately cultured rabbit embryonic stem cell includes following tool
Body step: being denoted as 0 day with female rabbit mating time, is taken female rabbit of mating 2 days to carry out living body and is rushed embryo operation, the embryo gone out is in vitro
Culture.When mating 3 days, the fibroblast feeder layer of defrosting ICR tire rat tissue production.It is in mating the 4th day, feeder layer is thin
Born of the same parents' culture medium changes the CXBL stem cell media (specific formula in following examples 1) of preparation into, and takes and be developed to blastaea rank
The embryo of section digests mucoprotein, the oolemma of blastaea in chain protease drop.After mucoprotein, oolemma digestion, blastaea is moved
To in the glass dish containing CXBL stem cell media.Under Stereo microscope, two 1mL syringes, needle point phase interworking are utilized
It closes, the trophocyte around blastaea inner cell mass is removed, is inoculated on feeder layer after isolating the inner cell mass of blastaea.It is interior
After cell mass is inoculated in feeder layer, average 4.31d forms clone, grows to certain amount, cell in clone cell and does not divide largely
Before change, with ReLeSR reagent 4~6min of vitellophag, cell is collected after culture medium is added, centrifugation carries out cell passage, and cell passes
Continue to cultivate to new containing in feeder layer CXBL stem cell media.
Compared with prior art, the invention has the following advantages:
Provided culture solution through the invention, the rbESCs form turned out in conjunction with isolated culture method and Development of Mouse Embryos
Tire stem cell clone is similar, is in " island " shape convex growth, and three-dimensional sense is strong, and has the original state versatility in part, disappears by pancreatin
It remains to form monoclonal after changing passage.In addition, the rbESCs turned out can express higher levels of stem cell versatility dependency basis
Cause, and withThe feature differentiation capability of state and Primed state stem cell, and long-term in vitro can be carried out in the culture solution
Secondary culture can be passaged to for 14 generations.
Detailed description of the invention
Fig. 1 is photo of the blastaea of rabbit under inverted microscope, and amplification factor 10 × 20, scale bar is 100 μm.
Fig. 2 is primary colonies forming process and differentiation situation；Wherein, A, B, C, D, E, F are respectively after ICM is cultivated
D2, D3, D4, D5, D6, D6, arrow direction region indicate to clone herein crowded；Occurs the clone broken up centered on G, arrow is directed toward
Region indicates clone's differentiation herein.
Fig. 3 is that ReLeSR digestion method passes on rbESCs clone；Wherein, A: the cell just digested by ReLeSR；B:ReLeSR
Remaining cell after had digestive transfer culture；C, the clone's colony formed after the passage of D:ReLeSR digestion method.
Fig. 4 is the primary colonies of mTesR culture medium culture；Wherein, A: without feeder layer；B: there is feeder layer；Divide from left to right
Not Wei ICM culture D1, D2, D4, D5.
Fig. 5 is the colony morphology of KB, KBL, CXBL culture medium culture；Wherein, the clone of A:KB culture medium culture；B:KBL
The clone of culture medium culture；The clone of C:CXBL culture medium culture.
Fig. 6 is the clone of different cultivating systems；Wherein, the generation clone of A:KB culture medium culture to 12；The training of B:KBL culture medium
It supports to 12 generation clone；The culture of C:CXBL culture medium to 14 generations clone.
Fig. 7 is the clone that grows after the rbESCs pancreatin had digestive transfer culture in CXBL cultivating system；Wherein, A: D1 after passage；
B: D3 after passage.
Fig. 8 is the multipotency expression conditions of rbESCs under different condition of culture.
Fig. 9 is that rbESCs clones AP Activity determination result.
Figure 10 is rbESCs multipotency gene RT-PCR testing result；Wherein, swimming lane 1:rbESCs；Swimming lane 2:REF；Swimming lane 3:
BLACK。
Figure 11 is the identified by immunofluorescence result of rbESCs versatility gene；Wherein, respectively fluorescence, core from left to right
Dye, ordinary light.
Figure 12 is the differentiation qualification result of rbESCs；Wherein, A: epithelioid cell；B, E: neural-like cells；C: at fiber
Like cell；D: the cardiac-like muscle cell of bounce.
Figure 13 is the embryoid body that rbESCs is formed；Wherein, A: the embryoid body of suspension；B: adherent embryoid body；From left to right,
Epithelioid cell, fibroblast-like cells, neural-like cells have been grown around embryoid body respectively.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The preparation of 1 rbESCs cultivating system of embodiment
50mL culture medium KB includes following each component:
Culture medium KBL: LIF (final concentration 10ng/mL) is added on the basis of KB culture medium.
Culture medium C XBL: on the basis of KBL culture medium (eventually plus CHIR99021 (3 μM of final concentration) and XAV-939
2 μM of concentration).
The preparation of 2 feeder layer of embodiment
ICR mouse natural mating is stayed overnight, and second day is shown in that vaginal plug is denoted as 0.5 day morning.It is real by post-coitum 13 days pregnant mouse
Apply cervical dislocation execution.Pregnant mouse position is ajusted, makes abdomen upward, abdomen sprays alcohol disinfecting.Abdominal cavity is cut off by U-shaped, is taken out
Uterus.Suitable alcohols are sprayed to uterus, embathe uterus 3 with PBS (containing 3% mycillin) solution of suitable antibiotic containing high power
~5 times, remove bloodstain.Uterus is cut off in super-clean bench, and tire mouse and the fetal membrane, amniotic fluid, part placenta that are wrapped in it are shelled together
Uterus out after impregnating 20~30s in 70% alcohol, is embathed 3~5 times in PBS (containing 1% mycillin).Fetal membrane is cut off, it will
Tire mouse is taken out.Head, tail, four limbs, the internal organ of tire mouse are carefully removed with eye scissors, ophthalmic tweezers, remaining tissue is embathed 3 times with PBS
It is transferred to the EP pipe of new 1.5mL afterwards.Tissue is shredded into rotten shape as far as possible with eye scissors, 3 times of tissue block volume or so of addition
Ordinary culture medium is blown and beaten into tissue mass suspension.It draws appropriate suspension and is added drop-wise to 60mm culture dish ware bottom, it will by liquid-transfering gun pipette tips
Suspension is uniformly spreadable, and tissue block is made to be uniformly distributed in ware bottom.Culture dish side is put, excessive liquid is siphoned away, places next ware weight
Multiple aforesaid operations.Culture dish is in 37 DEG C, 5%CO2Incubator in be inverted culture 2~4h (keeping tissue block adherent more firm).It is inverted
Period checks that the liquid on primary structure block, overdrying are then suitably added every half an hour.After inversion, every ware is carefully added
The ordinary culture medium of 1mL is just setting culture.It changes within second day liquid, adds 2mL ordinary culture medium, third day is shown in slightly more at fiber finer
Born of the same parents in tissue block after climbing out of, then adds culture medium to 4mL.
It is about passed on according to 1:3 after cell covers with.Take part cell cryopreservation when P2, remaining cell culture to P3 or
When P4, mitomycin C is added by culture volume and handles 3h, PBS, which is frozen or passed on after cleaning 5 times, makees feeder layer.It needs
When using feeder layer, the previous day preparation is proposed.Appropriate Matrigel covering culture dish ware bottom is added, incubator is placed and is incubated for 30min,
The processed l cell of mitomycin C is thawed or passed on, by 4 × 105/cm2The density of left and right is inoculated into
(extra Matrigel is first siphoned away when inoculation) on the processed ware of Matrigel, change MEF culture solution accordingly within second day
Embryonic stem cell medium (mTeSR culture medium).
The female superfecundation of rabbit of embodiment 3 and the acquisition of embryo
(1) superfecundation of female rabbit: afternoon the red female rabbit of a little micro mist of 4:00 picking private parts, femoribus internus intramuscular injection
Female rabbit private parts, breeding when becoming scarlet or bright red are checked after PMSG 35IU, 48h.Female rabbit is put into buck cage and is handed over naturally
Match, occur after buck mounting movement that significant twitch falls down to the ground be breed it is successfully primary.Female rabbit is brought out, hip are gently thrown,
Buck sperm is set to further flow into private parts.In female rabbit femoribus internus intramuscular injection LH 35IU, post-coitum is denoted as 0 day, to prevent mother
Rabbit breeding is once fertilized unsuccessful, and female rabbit is allowed to continue to mate with buck, breed successfully three times after put back to former cage again.
(2) production of culture plate: female rabbit mating makes culture plate one day after.By the disposable Tissue Culture Dish of diameter 35mm
External ware bottom be equally divided into 4 sectors with marking pen, the successively number of label 1 to 4 on each sector.Each sector production 3
35 μ L embryo culture medium droplets are dripped, adds 2.5mL paraffin oil to cover droplet, is put into 38 DEG C, 5%CO2It is balanced in incubator.
(3) it the acquisition of embryo: takes post-coitum 2 days female rabbits to carry out living body and rushes embryo operation.From female rabbit auricular vein when operation
Injecting anesthetic medicine shaves the hair in abdominal cavity after efficacy stability, net hair portion successively applies Iodophor, alcohol carries out disinfection.Against mother rabbit
Ovary approximate location cuts open skin layer, abdominal cavity layer according to ventrimeson using scalpel, and the edge of a knife is about 5cm.After abdominal cavity is opened, look for
To uterus, fallopian tubal, ovary.When rushing embryo, a people polishes the steel of syringe needle from the uterus position insertion apart from palace pipe junction 1cm
Needle, syringe needle pass through palace pipe jointing part insertion portion fallopian tubal, the plastic straw alignment fimbria ovarica portion insertion of another people's scalp acupuncture
And it fixes.The CCM of 10mL preheating is injected at steel needle end, and the other end of tubule collects the liquid gone out, with picking up under stereomicroscope
Ovum needle chooses the embryo in liquid.Embryo washes 3 times in the medium, removes impurity, embryo is finally moved on to the droplet of culture plate
Middle culture.
Female rabbit post-coitum 48h, intracorporal embryo have been developed to 16 cell stages, and the embryo of 16 cell stages of acquisition is existed
It is cultivated in culture plate, cultivates embryonic development two days later into blastaea.As shown in Figure 1, the ICM of rabbit blastaea is obvious, with other animals
Unlike embryo, oolemma outer layer is accompanied by a thick layer mucoprotein.
Separation, the culture of 4 rbESCs of embodiment
The third day of female rabbit post-coitum gets out feeder cells, and all feeder layers are all seeded on 24 orifice plates.Female rabbit
4th day separation blastaea of post-coitum, feeder layer culture medium is replaced as corresponding rbESCs culture solution by 3h before separating.
(1) micro- partition method: first under stereomicroscope, blastaea is transferred in the droplet of chain protease with ovum needle is picked up, gently
20s digestion mucoprotein is beaten in featheriness.The blastaea for removing mucoprotein (is covered with paraffin with the droplet that ovum needle is transferred in culture dish is picked up
Oil), place the operation of micromanipulation platform.Fixed pin fixes blastaea, and injection needle is inserted into blastaea, the cell of ICM is drawn out, passes through
After stem cell media washes twice, the cell drawn out is put on feeder layer with ovum needle is picked up.
(2) blastaea 30s to 1min full embryo culture method: is digested with chain protease, it is seen that blastaea mucoprotein is sloughed, oolemma becomes
It is thin when will disappear, it is washed 3 times with picking up ovum needle and blastaea is sucked out being transferred in stem cell media.Oolemma is blown and beaten when washing falls it
It falls, with two 1mL syringe needle compounding practices if not falling off, peels off blastaea oolemma.The embryo for peelling off oolemma is straight
It connects and is placed on feeder layer, make its natural adherent growth.
(3) mechanical phonograph recorder separation: blastaea first is digested with chain protease, removes the mucoprotein and oolemma of embryo, method is same
(2).Then, to the greatest extent may be used in the glass dish containing stem cell media with 2 1mL syringe needle separation ICM and trophocyte
The trophocyte around ICM can be removed.The ICM separated with trophoderm is sucked out, washs 3 times in stem cell media, so
After be transferred directly on feeder layer, it is also possible to ICM is cut into two pieces by syringe needle, is then transferred on feeder layer.
The influence for using different separation methods adherent to rabbit ICM and original clone is formed is counted, as a result such as 1 institute of table
Show.The results show that the ICM adherent rate and primary colonies formation rate that micro- partition method obtains are minimum, respectively 37.03%,
25.93%.The formation rate highest of ICM adherent rate and primary colonies that mechanical phonograph recorder separation obtains, respectively 87.93%,
70.69%, the ICM adherent rate that is obtained with micro- partition method, full embryo culture method, primary colonies formation rate significant difference (P <
0.05).Therefore, follow-up test selection mechanical phonograph recorder separation handles blastaea.
The influence that the different separation methods of table 1 are adherent to rabbit ICM and original clone is formed
The had digestive transfer culture of 5 rbESCs of embodiment
The 3rd~7 day after blastaea separation, Clone formation situation is observed under inverted microscope, and carry out secondary culture.
Fig. 2 is primary colonies forming process and differentiation situation, the results show that the incubation time of primary colonies is too long, meeting
Cause cell growth in part in clone overstocked, under the microscope it is observed that the cell at these positions is crowded, deformation, divides
Change.And counted the time (table 2) of primary colonies formation and primary colonies cell starts time (table 3) of crowded differentiation, specifically
It is as follows:
This test random observation 46 adherent ICM form the process of primary colonies, this 46 ICM after culture altogether
38 primary colonies formed.38 ICM have 4 ICM in the 3rd day formation primary colonies of culture, have 20 ICM cultivating
The 4th day formation primary colonies, have 12 ICM in the 5th day formation primary colonies of culture, last 2 ICM are the 6th of culture
It forms primary colonies.Wherein the formation rate of the 4th day primary colonies is significantly higher than other number of days (P < 0.05), is secondly the 5th
It, this ICM for sharing 84.2% for two days forms primary colonies.Weighted calculation primary colonies form required average cultivated days
It is 4.31 days.
The time that 2 primary colonies of table are formed
Respectively there is 1,14,21,2 primary colonies cell to start daily within the 4th day, the 5th day, the 6th day, the 7th day in culture
The phenomenon that existing crowded differentiation.Wherein 92.1% primary colonies concentrate on ICM culture there is showing for crowded differentiation within the 5th, 6 day
As weighted calculation average time is 5.63 days.
3 primary colonies cell of table starts the time of crowded differentiation
Therefore, clone's generation time of follow-up test carries out for 4.31 days to 5.63 days preferably after ICM inoculation.
The cell in old hole will continue to cultivate after passage, wherein remaining part stem cell may regrow and become gram
It is grand.If clone originally grows fine, cell is undifferentiated, and the cell in each hole can pass on 2 times.Cell reaches P1 instead of
Afterwards, passed on for the state of cell, when the colony morphology in a hole maintains preferably, only a small amount of cell differentiation when, can be with picking up
Ovum needle, which is chosen, dedifferentes cell, is digested, is passed on to the cell in entire hole using enzyme digestion after PBS cleaning；When a hole
In clone only have a small amount of cellular morphology to maintain preferably, to be passed on picking up ovum needle picking and going out the preferable cell of form.It is basic to pass
There are 3 kinds for method, is passed on according to the growing way of cell, quantity according to 1:2~1:4.Method is as follows.
(1) Mechanical Method: the preferably undifferentiated clone of picking form under the microscope is gently cut clone cell with ovum needle is picked up
At fritter, new feeder layer culture dish is directly chosen.
(2) trypsin digestion: discarding stem cell media, is washed 2 times using PBS, and the 200 appropriate pancreatin of μ L are added in every hole, puts
Enter incubator and digests 2~3min.The new culture medium of 1mL is added and terminates digestion, gently uniformly, all liq is transferred to for piping and druming
1.5mL EP pipe.It is centrifuged 1000rpm 3min, discards supernatant, portion's cell precipitation of keeping on file.Appropriate stem cell media is added, moves
Liquid rifle, which gently blows and beats 2~3 times, is resuspended cell precipitation, liquid is transferred in 24 orifice plates of new feeder layer by cell quantity or
12 orifice plates rock mixing, are put into incubator and cultivate.
(3) ReLeSR digestion method: discarding stem cell media, is washed 2 times using PBS, appropriate ReLeSR is added, be put into culture
1min is digested in case.Siphon away extra digestive juice after 1min, a small amount of liquid only stayed not have cell, be put into incubator hatching 3~
5min.0.5mL stem cell media is added, is placed under inverted microscope and observes, has such as observed part clone still without departing from hole
Bottom is gently struck plate and is shaken off, and liquid is transferred to 1.5mL EP pipe with liquid-transfering gun.The PBS of 0.5mL is added to clean digestion hole, it will
PBS is also transferred to EP pipe.Subsequent centrifugation, resuspension, inoculation step are the same as above-mentioned trypsin digestion.In addition, can also be under the microscope
First picking form is preferably cloned, and is transferred in 96 orifice plates containing 100 μ L ReLeSR liquid, place incubator in digestion 3~
5min.It is gently that clone's piping and druming is loose using 200 μ L rifles after having digested, the EP that liquid is transferred to 1.5mL is managed, 900 μ L are supplemented
Stem cell media, subsequent centrifugation are resuspended, inoculation step also same trypsin digestion.
The result shows that primary colonies can directly be digested with ReLeSR, ReLeSR can make stem cell preferentially be detached from culture dish, and
Primary trophocyte and feeder cells continuation are adherent, as shown in Figure 3.Therefore, subsequent experimental selects ReLeSR digestion method
Pass on rbESCs clone.
6 different culture medium of embodiment is adherent to rabbit ICM, original clone is formed, the influence of had digestive transfer culture
The influence that the culture medium for counting different is adherent to rabbit ICM and original clone is formed, the results are shown in Table 4.
The influence that the different culture medium of table 4 is adherent to rabbit ICM and original clone is formed
Primary colonies forming process and had digestive transfer culture process under different culture medium condition of culture are observed, as a result such as Fig. 4
Shown in~Fig. 7:
Fig. 4 is the primary colonies of mTesR culture medium culture, the results showed that, in the processed no feeder layer ware of Matrigel
In, using mTeSR culture medium culture ICM, the cell grown is loose, cannot form primary colonies；When there is feeder layer, mTeSR
Culture can form cloning colony, but cell homogeneity is poor in colony, and the cell of surrounding differentiation is more.
Fig. 5 is the colony morphology of KB, KBL, CXBL culture medium culture, the results showed that, the rbESCs clone in KB culture medium
It is flat, the good big colony of homogeneous can be formed, has limbus with feeder layer, stem cell morphology is obvious, can be seen clearly that cell is in
Circle has the characteristics that small in size, core is big；The rbESCs that KBL culture medium obtains has significantly small in size, nucleocytoplasmic ratio is high to do
Cell morphological characteristic, clone-internal cell homogeneity is good, and clone edge is brighter, and three-dimensional sense is stronger, configuration and KB culture medium
In clone it is similar；And the rbESCs that CXBL culture medium is turned out has the colony morphology of similar mESCs convex growth, Ke Longji
It falls in " island ", three-dimensional sense is strong, and edge refractivity is strong.
Fig. 6 is the clone of different cultivating systems, the results showed that, using ReLeSR digestion method to each cultivating system culture
RbESCs is passed on, and it was more than 12 generations that the rbESCs under KB, KBL cultivating system, which has cell strain culture, at present, and CXBL cultivates body
RbESCs under system can be passaged to for 14 generations.
Fig. 7 is the clone that grows after the rbESCs pancreatin had digestive transfer culture in CXBL cultivating system, the results showed that, rbESCs warp
It crosses after pancreatin digestion at after unicellular, still with the ability of Clone formation；And the rbESCs of KB, KBL cultivating system passes through pancreatin
After digestion, directly breaks up, clone can not be formed.
The acquisition of the cDNA template of 7 rbESCs of embodiment
(1) collection of rbESCs: prepare the mixed liquor of 4 DEG C of PBS and 1%BSA, it is small that several 50 μ L are made on culture dish
Drop, with ovum needle gently picking cell clone is picked up, clone is put into droplet, is quickly rinsed in drop 3 times, then with pick up ovum needle general gram
It is grand to put into the PCR pipe containing 10 μ L cell pyrolysis liquids.When cloning a fairly large number of in hole, standby enzyme digestion collects cell,
Cell is resuspended with appropriate cell pyrolysis liquid after centrifugation, dispenses cell suspension into each PCR pipe by the amount of 10 μ L.What is be collected into is thin
Born of the same parents directly carry out micro reversion or are put into -80 DEG C of refrigerators and freeze.
(2) cell cracking: wink from the liquid in PCR pipe, with 75 DEG C of incubation 15min of PCR instrument.
(3) remove template DNA: 0.1 μ L RNase Inhibitor, 1.3 10 × ROD of μ L is added in each PCR pipe
Buffer, 1 μ L DNase I, wink is from 37 DEG C of incubation 40min of PCR instrument.
(4) terminate DNase I reaction: EDTA, each 1 μ L of dNTP, Random Primer is added in each PCR pipe, and wink is from use
65 DEG C of incubation 10min of PCR instrument.
(5) micro reversion: 4 μ L5 × FS Buffer, 2 μ L DTT, 0.5 μ L RNase Inhibitor are added in every hole
RRI, 0.25 μ L Reverse transcriptase SSII, wink from, put and inverted into PCR instrument, program be 25 DEG C of 10min, 42
DEG C 90min, 95 DEG C of 10min, -80 DEG C of product preservations.
8 PCR of embodiment is reacted with qPCR
(1) PCR: each reagent is added into PCR pipe according to the form below 5, carries out PCR amplification with 20 μ L reaction systems.Reaction condition
For 98 DEG C of initial denaturations 5min, 98 DEG C of denaturation 30s, according to the annealing temperature annealing 30s of design of primers, 72 DEG C extend (by every 1kb production
Object extension of time is that extension of time is arranged in 10s), denaturation, annealing, the recurring number extended are 35 circulations, last 72 DEG C of extensions
5min。
5 PCR reaction system of table
(2) qPCR: each reagent is added into PCR pipe according to the form below 6, and qPCR response procedures are 95 DEG C of initial denaturation 5min, and 95 DEG C
It is denaturalized 15s, 60 DEG C of annealing 50s, 40 denaturation, annealing circulations.Each sample is repeated 3 times, using 2-△△CT method calculates gene
Expression.It maps to the data obtained GraphPad Prism 5, carries out statistics credit with SPSS Statistics software
Analysis.
6 qPCR reaction system of table
(3) under different condition of culture rbESCs multipotency expression conditions
As shown in Figure 8, the results showed that, in each culture medium group, the rbESCs of CXBL culture medium culture its OCT4,
High, the wherein table of OCT4, SOX2, NANOG, C-MYC of mrna expression amount ratio KB, KBL group of SOX2, NANOG, KLF4, C-MYC
It is significantly higher than KB, KBL culture medium group (P < 0.05) up to amount.The rbESCs of KBL culture medium culture its OCT4, SOX2, KLF4's
Mrna expression amount and KB culture medium group without significant difference, but the expression quantity of its NANOG, C-MYC compared with KB culture medium group height and
Significant difference (P < 0.05).
(4) rbESCs clones AP Activity determination
Alkaline phosphatase (AP) dyeing is carried out to the clone of CXBL culture medium to show as shown in figure 9, result is in bluish violet
RbESCs clone obtained is positive in AP.
(5) the RT-PCR detection of rbESCs multipotency gene
As shown in Figure 10, the results showed that, rbESCs expresses versatility gene OCT4, SOX2, NANOG, KLF4, C-MYC.
9 immunofluorescence of embodiment
1, test procedure
(1) it is cleaned cell 3 times with PBS, 4% paraformaldehyde, the fixed 30min of room temperature is added；
(2) fixer is discarded, PBS is cleaned cell 3 times, 1%Triton X-100 is added, at room temperature permeabilization 30min；
(3) permeabilization liquid is discarded, PBS is cleaned cell 3 times, and 1%BSA is added, closes 1h at room temperature；
(4) confining liquid is discarded, PBS is cleaned cell 3 times, and corresponding primary antibody dilution is added, and covers ware bottom, 4 DEG C were incubated for
Night；
(5) second days, the primary antibody dilution in hole is siphoned away, PBS is cleaned cell 5 times, each 3min, and it is anti-that corresponding primary antibody is added
Former secondary antibody diluent is protected from light is incubated for 40min at room temperature；
(6) secondary antibody diluent is sucked, PBS is cleaned cell 5 times, each 3min, and 10 μ g/mL Hoechst dye liquors, room is added
It is protected from light under temperature and is incubated for 10min；
(7) Hoechst dye liquor is sucked, PBS is cleaned cell 2 times, and appropriate PBS is added, and observes luciferase expression situation.
2, result
The identified by immunofluorescence result of rbESCs versatility gene is as shown in figure 11, the clone that CXBL cultivating system is turned out
The marker gene FGF5 for expressing OCT4, SOX2, NANOG, E-CADHERIN and Primed state stem cell, is not detected
The expression of SSEA1, SSEA4.
The experiment of 10 vitro differentiation of embodiment
1, test procedure
(1) unicellular differentiating: rbESCs is digested to unicellular, addition culture medium termination, 1000rpm 3min with pancreatin
Centrifugation.Supernatant is discarded, cell is resuspended using ordinary cells culture solution, appropriate cell is inoculated in the ware of no feeder layer, every other day
Liquid is changed, 7~10 days observation cellular morphologies are cultivated.
(2) embryoid body makes: digestion centrifugation step is added 200 μ L Nostoc commune Vanch liquid and cell is resuspended, in ware lid with (1)
The droplet of 40 μ L or so is made of cell suspension, PBS is added at ware bottom, and cell suspension is inverted culture.Liquid is changed using picking up ovum needle every other day,
Culture 1~2 week.
2, result
(1) the differentiation qualification result of rbESCs is as shown in figure 12.The result shows that cell can adherent growth, and differentiate not
With the cell of form: epithelioid cell, neural-like cells, fibroblast-like cells, bounce cardiac-like muscle cell.Immunohistochemistry identification
It expresses epithelial cell marker gene: CK18, nerve cell marker gene: TUBB3.
(2) embryoid body that rbESCs is formed is as shown in figure 13.It is cultivated the result shows that unicellular droplet is inverted, after culture
D2 observes cell aggregation, and D7 forms embryoid body, and embryoid body is inoculated into ware and is cultivated, can equally be formed epithelioid cell, at
Fibroblast-like cell, neural-like cells.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle
Within the scope of.
Claims (9)
1. a kind of for being separately cultured the composition of rabbit embryonic stem cell, which is characterized in that the composition is by basic fibroblast
Growth factor bFGF, LIF ELISA LIF, GSK3 inhibitor C HIR99021 and β-catenin specific inhibitor XAV-
939 compositions.
2. composition according to claim 1, which is characterized in that bFGF, LIF, CHIR99021, XAV- in the composition
939 final concentration ratio is (4~6 ng/mL): (9~11 ng/mL): (2.5~3.5 μM): (1.5~2.5 μM).
3. a kind of for being separately cultured the culture solution of rabbit embryonic stem cell, which is characterized in that every 50mL culture solution is by following components
Composition: 37 mL KnockOut DMEM culture solutions, 7.5 mL serum substitutes, 2.5 mL fetal calf serums, 0.5 mL blueness strepto-
Element, 0.5 mL nonessential amino acid, 0.5 mL glutamic acid, 0.5 mL L-Glutamine, 0.5 mL beta -mercaptoethanol, 0.5 mL
Nucleosides, the bFGF of final concentration of 4~6 ng/mL, the LIF of final concentration of 9~11 ng/mL, final concentration of 2.5~3.5 μM
CHIR99021, final concentration of 1.5~2.5 μM of XAV-939.
4. culture solution according to claim 3, which is characterized in that bFGF, LIF, CHIR99021, XAV- in the culture solution
939 final concentration is respectively 4 ng/mL, 10 ng/mL, 3 μM, 2 μM.
5. a kind of method for being separately cultured rabbit embryonic stem cell, which comprises the steps of: use mechanical phonograph recorder separation
Rabbit blastaea is handled, the isolated entire ICM of each blastaea is inoculated into the feeding containing any culture solution of claim 3 or 4
It supports in layer, in 37 DEG C, 5% CO2, cultivate under conditions of relative humidity 95%, originally culture 4.31~5.63 days；It uses later
ReLeSR digestion method carries out clone's passage to get rabbit embryonic stem cell, and gained rabbit embryonic stem cell in vitro culture can be passaged to 14
Generation.
6. method according to claim 5, which is characterized in that first to be digested with chain protease the step of the mechanical phonograph recorder separation
Blastaea removes the mucoprotein and oolemma of embryo；ICM is separated with trophocyte again, ICM suction is then transferred to raising
In layer.
7. method according to claim 5, which is characterized in that the processing time of the ReLeSR digestion method is 4~6 min.
8. method according to claim 5, which is characterized in that the step of the ReLeSR digestion method are as follows: it is dry to discard rabbit embryonic
Cell culture fluid is washed 2 times with PBS, and appropriate ReLeSR is added and carries out 1 min of digestion；Extra digestive juice is siphoned away, a small amount of liquid is stayed
Cell is not crossed, 3~5 min of hatching in incubator are put into；0.5 new mL rabbit embryonic stem cell culture solution is added and terminates digestion, it will
Liquid is transferred to EP pipe；Add the PBS of 0.5 mL to clean digestion hole, PBS is also transferred to EP pipe；It is centrifuged 1000 rpm, 3 min,
Supernatant is discarded, portion's cell precipitation of keeping on file；It is added appropriate rabbit embryonic stem cell culture solution, cell precipitation is resuspended in piping and druming, by 1~
2×105Liquid is transferred in 24 orifice plates of new feeder layer or 12 orifice plates by/mL cell quantity, is rocked mixing, is put into incubator
Middle culture carries out had digestive transfer culture next time.
9. method according to claim 5, which is characterized in that the step of the ReLeSR digestion method are as follows: under the microscope first
Picking form is preferably cloned, and is transferred in 96 orifice plates containing 100 μ L ReLeSR liquid, place incubator in digestion 3~
5min；It is gently that clone's piping and druming is loose after having digested, liquid is transferred to EP pipe, supplements 900 μ L rabbit embryonic stem cell cultures
Liquid；It is centrifuged 1000 rpm, 3 min, discards supernatant, portion's cell precipitation of keeping on file；Appropriate rabbit embryonic stem cell culture solution is added, blows
Beating is resuspended cell precipitation, by 1~2 × 105Liquid is transferred in 24 orifice plates of new feeder layer or 12 holes by/mL cell quantity
Plate rocks mixing, is put into incubator and cultivates, and carries out had digestive transfer culture next time.
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| CN1217746A (en) * | 1996-04-29 | 1999-05-26 | 勒芬研究与发展公司 | Pluripotent rabbit embryonic stem (ES) cell lines and their use in generation of chimeric rabbits |
| WO2013159349A1 (en) * | 2012-04-27 | 2013-10-31 | Curegenix Inc. | Method for producing cardiomyocytes |
| 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 |
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2018
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| CN1217746A (en) * | 1996-04-29 | 1999-05-26 | 勒芬研究与发展公司 | Pluripotent rabbit embryonic stem (ES) cell lines and their use in generation of chimeric rabbits |
| WO2013159349A1 (en) * | 2012-04-27 | 2013-10-31 | Curegenix Inc. | Method for producing cardiomyocytes |
| 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 |
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