CN109609446B

CN109609446B - Culture solution and method for isolated culture of rabbit embryonic stem cells

Info

Publication number: CN109609446B
Application number: CN201811354111.7A
Authority: CN
Inventors: 邓彦飞; 农恬颖; 陈凤; 杨素芳; 石德顺
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2020-11-03
Anticipated expiration: 2038-11-14
Also published as: CN109609446A

Abstract

The invention discloses a culture solution and a method for isolated culture of rabbit embryonic stem cells. The culture solution comprises a composition for separating and culturing rabbit embryonic stem cells, wherein the composition consists of bFGF, LIF, CHIR99021 and XAV-939. The culture solution is added with bFGF, LIF, CHIR99021 and XAV939 to maintain the pluripotency of rbESCs, the cultured rbESCs are similar to mouse embryonic stem cell clone in shape, grow in an island-shaped bulge, have strong stereoscopic impression and partial primitive state pluripotency, and can still form monoclone after being subjected to trypsinization and passage. In addition, the cultured rbESCs can express higher-level stem cell pluripotency related genes, have the characteristic differentiation capacity of the Na-meive-state stem cells and the Primed-state stem cells, can be subjected to long-term in-vitro subculture in a culture solution, and can be passaged to 14 generations.

Description

Culture solution and method for isolated culture of rabbit embryonic stem cells

Technical Field

The invention relates to the technical field of cell engineering, in particular to a culture solution and a method for separating and culturing rabbit embryonic stem cells.

Background

Rabbits are a common laboratory animal, which is easy to breed, inexpensive, short in breeding cycle, large in litter size, and has long been used in biomedical research. The rabbit volume is bigger than the mouse, and the experiment operation is convenient, so that the operation is easier and more effective to obtain a large sample. In biomedicine, rabbits are widely used in biomedical acute experiments, endocrine experiments, substance metabolism experiments, pharmacological experiments, and the like. Rabbits are evolutionarily closer to humans than rodents for human disease applications. Human disease presents a more similar etiology in rabbits than in mice, transgenic rabbits are excellent animal models of inherited and acquired human disease, and rabbits are largely used as experimental models for the treatment of human disease: such as diabetes, hypertrophic cardiomyopathy, hypertension, atherosclerosis, hyperlipidemia, lipoprotein metabolism disorder, retinal degeneration, lung injury, and inflammatory diseases. Another advantage of rabbits is their use as a classical laboratory species in reproductive technology. The rabbit has obvious superiority as an experimental animal in the aspects of superovulation, in-vitro fertilization, sperm injection, semen cryopreservation, ovum and embryo cryopreservation, prokaryotic microinjection, nuclear transfer cloning and the like. Therefore, the research on Rabbit Embryonic stem cells (rbESCs) derived from Rabbit blastocyst ICM is also considered, and if the rbESCs can be obtained for the research on human diseases, the production of transgenic rabbits, the test of stem cell therapy and the research of gene targeting technology, the research has important significance. However, as the research progresses, the isolation and culture system of rbESCs still needs to be studied intensively.

At present, mechanical methods, immunosurgery methods, microseparation methods, and the like are commonly used for isolating the ICM. WANG et al cultured rbcESCs that could be passaged for a long time by using parthenogenetic activated embryos, and found that the separation effect of the immunosurgical method was not good when the ICM was separated. Japan scientist Honda uses micromanipulator technique to provide a better method for ICM separation. Among Stem cell studies of various species, mouse Embryonic Stem Cells (mESCs) have been studied more. The mESCs can be stably passaged for a long time only by adding LIF, and clone with transparent edges and stereoscopic impression is kept. In contrast, rbESCs, human Embryonic Stem Cells (hESCs) are difficult to culture and establish. The culture medium of rbESCs and hESCs is usually added with bFGF, so that the clones are obtained with a high probability, and the cultured rbESCs and hESCs show a flat clone shape, cannot be digested by pancreatin and need to be carefully passaged. At present, although many laboratories isolated and cultured the rbESCs and successfully established the system, the rbESCs were also a small part of the success obtained after a large number of experiments, and the differences of operation methods and culture conditions in different laboratories are large, and the obtained rbESCs also have differences. Even if the same medium and the same culture temperature are used, the manipulation technique and the individual embryo difference cannot be controlled. Most rbESCs can not be stably passed, and are gradually differentiated in the culture process, only a few of rbESCs can be cultured for a long time, some laboratories report that the rbESCs can pass dozens of generations, and some laboratories report that the rbESCs can pass only a few generations.

In the studies reported so far, there is still a big debate about the composition of the culture system of rbESCs. PierreOsteil et al found that Serum Replacement (KOSR) was essential for the culture of ESCs, and that media without KOSR supplemented with Fetal Bovine Serum (FBS) alone could not produce rbESCs. AP Cattunda et al believe that LIF prevents differentiation better, first LIF, LIFR, gp130 expression occurs when rabbit embryos develop into blastocysts, and second myocardial-like cells occur after LIF is removed when rbESC is cultured. Honda considers that an Acitvin/Nodal-SMAD2/3 signal channel is important for maintaining the dryness of the rbESCs, and the existence or nonexistence of LIF does not influence the expression of pluripotent genes such as OCT4 and NANOG of the rbESCs, and does not influence the clone morphology and AP staining of the rbESCs. Phosphorylated STAT3 protein levels in rbESCs also did not change with LIF concentrations, as did phosphorylated STAT3 protein in the absence of LIF. Intawcha P, et al, consider addition of LIF necessary to increase the number of primary clones and long-term passage after LIF removal would lead to cell differentiation. Subsequent researches of the Intawich P show that the addition of bFGF with proper concentration in a culture medium can improve the activity of ERK1/2 and AKT in rbESCs and promote the maintenance and self-renewal of the pluripotency of the rbESCs. LIF and bFGF are added into the culture medium at the same time, so that the STAT3/AKT/ERK level of the rbESCs is greatly improved, and the maintenance of the pluripotency of the rbESCs is facilitated. LIF and bFGF contributed to the maintenance of clonal morphology of rbESCs even under feeder-free culture conditions. Xue F combines and cultures rbESCs by different feeding layers and LIFs from different sources, and establishes a rbESC cell line after finding that the rabbit homologous LIF and the rabbit homologous feeding layer are used together. Pierre Osteil et al think that LIF/JAK signal conduction is unnecessary for self-renewal in rbESCs, but the rbESCs clone cultured by adding LIF has certain chimeric ability and can be more effectively planted in ectoderm when being used as a chimeric embryo

The characteristics of the state ESCs. Pierre Osteil et al also compared the efficiency of culturing the rbESCs line in the medium supplemented with LIF, bFGF, and in the medium supplemented with no growth factors. The results show that the culture medium added with bFGF establishes the series effectThe highest rate.

In conclusion, although both bFGF and LIF are currently used in the culture of rabbit embryonic stem cells, the obtained embryonic stem cells have reduced pluripotency and cannot maintain the morphological characteristics and biological characteristics specific to the stem cells for a long period of time. Therefore, there is a need to develop a new culture system and method for rabbit embryonic stem cells to achieve long-term subculture of rbESCs in vitro and maintain their morphological and biological characteristics.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a composition for separating and culturing rabbit embryonic stem cells, wherein the bFGF, LIF, CHIR99021 and XAV-939 in the composition can enable the expression level of stem cell pluripotency related genes in cultured rbESCs to be higher, and the composition has the functions of promoting the expression of stem cell pluripotency related genes in the cultured rbESCs

The characteristics of the stem cells in the states and Primed states realize the long-term subculture in vitro and have differentiation capacity.

The invention also aims to provide a culture solution for separating and culturing rabbit embryonic stem cells, which comprises the composition.

Another object of the present invention is to provide a method for isolated culture of rabbit embryonic stem cells.

In order to achieve the purpose, the invention is realized by the following scheme:

a composition for separating and culturing rabbit embryonic stem cells comprises basic fibroblast growth factor (bFGF), Leukemia Inhibitory Factor (LIF), inhibitor CHIR99021 of GSK3 and specific inhibitor XAV-939 of beta-catenin.

Wherein, the small molecular compounds CHIR99021(GSK3 inhibitor) and XAV-939 (beta-catenin specific inhibitor) are related inhibitors on the Wnt/beta-catenin pathway. CHIR99021 inhibits GSK3 to promote the accumulation of beta-catenin, and CHIR99021 is a key additive for culturing mouse embryonic stem cells and maintaining the original state pluripotency state. XAV939 has the function of stabilizing Axin in the beta-catenin protein degradation compound, wherein the Axin can inhibit beta-catenin from entering cell nucleus and inhibit the expression of beta-catenin induced differentiation genes. Therefore, the addition of CHIR99021 and XAV939 on the basis of bFGF (basic fibroblast growth factor) and LIF (leukemia inhibitory factor) is beneficial to maintaining the pluripotency of rbESCs, so that the specific morphological characteristics and biological characteristics of stem cells are maintained for a long time.

Preferably, the final concentration ratio of bFGF, LIF, CHIR99021 and XAV-939 in the composition is (4-6 ng/mL): (9-11 ng/mL): (2.5-3.5. mu.M): (1.5-2.5. mu.M).

The invention also claims a culture solution for separating and culturing the rabbit embryonic stem cells, wherein each 50mL of the culture solution comprises the following components: 37mL of KnockOut DMEM culture solution, 7.5mL of serum substitute, 2.5mL of fetal bovine serum, 0.5mL of streptomycin, 0.5mL of non-essential amino acid, 0.5mL of glutamic acid, 0.5mL of L-glutamine, 0.5mL of beta-mercaptoethanol, 0.5mL of nucleoside, bFGF at a final concentration of 4-6 ng/mL, LIF at a final concentration of 9-11 ng/mL, CHIR99021 at a final concentration of 2.5-3.5 mu M, and XAV-939 at a final concentration of 1.5-2.5 mu M.

Compared with the KBL group (without adding CHIR99021 and XAV939), the cultured rbESCs have the following advantages: (1) the shape is similar to the cloning of mouse embryonic stem cells, the cells grow in an island-shaped bulge, and the stereoscopic impression is strong; (2) has partial original state pluripotency, and can still form monoclone after being digested and passaged by pancreatin; (3) the cultured rbESCs can express higher-level stem cell pluripotency related genes; (4) also has

The characteristic differentiation capacity of the stem cells in the status and Primed status can differentiate into beating myocardial cells; (5) can be subjected to long-term in vitro subculture in the culture solution, and can be subcultured to 14 generations.

Preferably, the final concentrations of bFGF, LIF, CHIR99021 and XAV-939 in the culture solution are 4ng/mL, 10ng/mL, 3. mu.M and 2. mu.M respectively.

The invention also requests to protect a method for separating and culturing rabbit embryonic stem cells, which comprises the following steps: mechanically separating rabbit blastocysts, inoculating the whole ICM isolated from each blastocyst to a feeder layer containing the above culture mediumIn the presence of 5% CO at 37 deg.C₂Culturing under the condition of relative humidity of 95%, and performing primary culture for 4.31-5.63 days; and then cloning and passaging by adopting a ReLeSR digestion method to obtain the rabbit embryonic stem cells, wherein the obtained rabbit embryonic stem cells can be subjected to in vitro culture to be passaged to 14 generations.

The invention optimizes the processing method of blastocysts, the passage time of cloning, the passage method of cloning, a culture system and the like:

(1) the optimized blastocyst processing method is a mechanical separation method, the mechanical separation method is adopted to process the blastocyst, the Cell adherence rate and the clone formation rate of an Inner Cell Mass (ICM) respectively reach 87.93 percent and 70.69 percent, and the method is obviously higher than a micro-separation method and a whole embryo culture method.

(2) The optimized colony passage time is preferably from 4.31 days to 5.63 days after ICM inoculation because 84.2% of ICM cells form primary colonies on days 4-5 of inoculation onto the feeder layer, with an average time to primary colony formation of 4.31 days; 92.1% of the ICM cells started differentiating on days 5-6 of the feeder layer, with an average time of 5.63 days.

(3) The optimized clone passage method is a ReLeSR digestion method, the ReLeSR digestion method can selectively enable undifferentiated clone cells to be separated from a culture dish and digested into small cell clusters with uniform sizes, the number of clones formed after passage is large, the clones can be uniformly spread and grown, and the passage effect is superior to that of a mechanical method and a trypsin digestion method.

Preferably, the mechanical treatment method comprises the steps of digesting the blastocyst with pronase to remove mucin and zona pellucida from the embryo; the ICM is then separated from the trophoblast cells and the ICM is aspirated and transferred to a feeder layer.

Preferably, the treatment time of the ReLeSR digestion method is 4-6 min.

As a preferred embodiment, the steps of the ReLeSR digestion process are: discarding the rabbit embryonic stem cell culture solution, washing with PBS for 2 times, adding appropriate amount of ReLeSR, and digesting for 1 min; sucking away redundant digestive juice, leaving a small amount of liquid to submerge cells, and putting the cells into an incubator to incubate for 3-5 min; digestion was stopped by adding new 0.5mL rabbit embryonic stem cell culture medium and the medium was transferred to EPA tube; the digestion wells were washed with 0.5mL PBS, which was also transferred to the EP tube; centrifuging at 1000rpm for 3min, removing supernatant, and collecting bottom cell precipitate; adding a proper amount of rabbit embryonic stem cell culture solution, blowing to resuspend the cell precipitate, and performing 1-2 × 10⁵The liquid is transferred to a 24-well plate or a 12-well plate with a new feeder layer, is shaken and mixed evenly, is put into an incubator for culture, and is subjected to the next digestion passage.

As another preferred embodiment, the steps of the ReLeSR digestion method are: firstly, selecting clones with good shapes under a microscope, transferring the clones to a 96-well plate containing 100 mu L of ReLeSR liquid, and placing the clones in an incubator for digestion for 3-5 min; after digestion, the clones are blown loose gently, the liquid is transferred to an EP tube, and 900 mu L of rabbit embryonic stem cell culture solution is supplemented; centrifuging at 1000rpm for 3min, removing supernatant, and collecting bottom cell precipitate; adding a proper amount of rabbit embryonic stem cell culture solution, blowing to resuspend the cell precipitate, and performing 1-2 × 10⁵The liquid is transferred to a 24-well plate or a 12-well plate with a new feeder layer, is shaken and mixed evenly, is put into an incubator for culture, and is subjected to the next digestion passage.

As an alternative embodiment, the method for separating and culturing the rabbit embryonic stem cells comprises the following specific steps: taking the mating time of the female rabbits as 0 day, taking the female rabbits mating for 2 days to carry out living embryo washing operation, and culturing the washed embryos in vitro. On day 3 of mating, the fibroblast feeder layer made of ICR fetal rat tissue was thawed. On day 4 of mating, the feeder cell medium was changed to the formulated CXBL stem cell medium (as specified in example 1 below) and embryos that developed to the blastocyst stage were taken and the mucin, zona pellucida of the blastocyst was digested in the streptokinase droplets. After digestion of mucin, zona pellucida, blastocysts were transferred to glass dishes containing CXBL stem cell culture medium. Under a body type microscope, two 1mL syringes are used, the needle points are matched with each other, trophoblast cells around the inner cell mass of the blastocyst are stripped, the inner cell mass of the blastocyst is separated out and then inoculated on a feeder layer. And after the inner cell mass is inoculated on a feeder layer, on average, 4.31d forms clone, before the cloned cells grow to a certain number and are not greatly differentiated, cells are digested for 4-6 min by a ReLeSR reagent, the cells are collected after a culture medium is added, the cells are subjected to centrifugation for passage, and the cells are transferred to a new culture medium containing CXBL stem cells of the feeder layer for continuous culture.

Compared with the prior art, the invention has the following beneficial effects:

the morphology of the rbESCs cultured by the culture solution provided by the invention and the separation culture method is similar to that of mouse embryonic stem cell clone, the rbESCs grow in an island-shaped bulge, the stereoscopic impression is strong, the rbESCs have partial original state pluripotency, and monoclonals can be formed after trypsinization passage. In addition, the cultured rbESCs can express higher-level stem cell pluripotency related genes and have the stem cell pluripotency related genes

The stem cells in the status and Primed status have characteristic differentiation capacity, and can be subjected to long-term in vitro subculture in the culture solution, and can be subcultured to 14 generations.

Drawings

FIG. 1 is a photograph of a blastocyst of a rabbit under an inverted microscope at a magnification of 10X 20 and a scale bar of 100. mu.m.

FIG. 2 shows the primary colony formation process and differentiation; wherein A, B, C, D, E, F represents D2, D3, D4, D5, D6 and D6 after ICM culture, and the arrowed regions indicate colony crowding; g is the clone with the center showing differentiation, and the arrowed pointing region indicates the clone differentiation.

FIG. 3 shows clones of rbESCs passaged by ReLeSR digestion; wherein, A: cells being digested by ReLeSR; b: the cells remaining after passage were digested by ReLeSR; C. d: clonal colonies formed after passage by the ReLeSR digestion method.

FIG. 4 is a primary clone cultured in mTesR medium; wherein, A: no feeding layer is provided; b: a breeding layer is arranged; from left to right, there were ICM-cultured D1, D2, D4 and D5, respectively.

FIG. 5 shows the clone morphology cultured in KB, KBL, CXBL medium; wherein, A: KB medium cultured clones; b: cloning by KBL culture medium; c: CXBL medium cultured clones.

FIG. 6 shows cloning of different culture systems; wherein, A: KB medium was cultured to 12 generation clones; b: culturing in KBL culture medium to 12 generation clone; c: CXBL medium was cultured to 14 clones.

FIG. 7 is a clone that grows out of rbESCs after trypsinization passage in a CXBL culture system; wherein, A: d1 after passage; b: after passage D3.

FIG. 8 shows the expression of the multipotent genes of rbESCs under different culture conditions.

FIG. 9 shows the result of the determination of the activity of the RBESCs clone AP.

FIG. 10 shows the RT-PCR detection results of the rbESCs pluripotent genes; wherein, lane 1: rbESCs; lane 2: REF; lane 3: BLACK.

FIG. 11 shows immunofluorescence identification results for the rbESCs pluripotency genes; wherein, from left to right, respectively represent fluorescence, nuclear staining and normal light.

FIG. 12 shows the differentiation identification of rbESCs; wherein, A: epithelial-like cells; B. e: a neural-like cell; c: a fibroblast-like cell; d: beating cardiomyocyte-like cells.

FIG. 13 shows embryoid bodies formed by rbESCs; wherein, A: suspended embryoid bodies; b: an adherent embryoid body; from left to right, epithelial-like cells, fibroblast-like cells, and nerve-like cells grow around the embryoid body.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 preparation of rbESCs culture System

50mL of medium KB contains the following components:

medium KBL: LIF (final concentration 10ng/mL) was added based on KB medium.

Culture medium CXBL: on the basis of KBL medium, CHIR99021 (final concentration 3. mu.M) and XAV-939 (final concentration 2. mu.M) were added.

EXAMPLE 2 preparation of feeder layer

ICR mice were naturally mated overnight, and the next morning a vaginal plug was scored as 0.5 days. Pregnant mice 13 days after mating were sacrificed by cervical dislocation. The pregnant mouse is placed in a posture with the abdomen facing upwards and alcohol is sprayed on the abdomen for disinfection. The abdominal cavity is cut open according to the U shape, and the uterus is taken out. Spraying a proper amount of alcohol on the uterus, and soaking and washing the uterus for 3-5 times by using a proper amount of PBS (containing 3% streptomycin) solution containing high-power antibiotics to remove bloodstains. Cutting open uterus in a super clean bench, stripping fetal rat, fetal membranes, amniotic fluid and partial placenta which wrap the fetal rat out of the uterus, soaking the fetal rat in 70% alcohol for 20-30 s, and then soaking and washing the fetal rat in PBS (containing 1% streptomycin) for 3-5 times. The fetal membranes are cut open and the fetal rat is removed. The head, tail, limbs and internal organs of the fetal rat were carefully removed with ophthalmic scissors and forceps, and the remaining tissue was rinsed 3 times with PBS and transferred to a new 1.5mL EP tube. The tissue is cut into minced shape as much as possible by an ophthalmic scissors, a common culture medium with the volume about 3 times of that of the tissue block is added, and the mixture is blown into tissue block suspension. And sucking a proper amount of suspension liquid and dropping the suspension liquid to the bottom of a culture dish with the diameter of 60mm, and uniformly spreading the suspension liquid by using the gun head of a liquid transfer gun to uniformly distribute tissue blocks at the bottom of the dish. Place the dish on its side, aspirate excess fluid, and place the next dish to repeat the procedure. Petri dish at 37 ℃ with 5% CO₂The culture box is used for inverted culture for 2-4 h (so that the tissue block adheres to the wall more firmly). During inversion, the liquid on the tissue mass was checked every half hour, and if the liquid was too dry, it was added appropriately. After inversion, 1mL of normal medium was carefully added to each dish and the plates were incubated upright. The solution was changed the next day, 2mL of normal medium was added, and after a little more fibroblasts had climbed out of the tissue mass the third day, medium was added to 4 mL.

After the cells were full approximately 1: 3, passage is carried out. And (3) freezing and storing part of cells at P2, culturing the rest cells until the cells reach P3 or P4, adding mitomycin C according to the volume of a culture medium, treating for 3 hours, washing for 5 times by PBS, and freezing or subculturing to form a feeder layer. When the feeding layer is needed, the feeding layer is prepared one day in advance. Adding a proper amount of Matrigel to cover the bottom of a culture dish, placing an incubator for incubation 3Thawing or passaging mitomycin C treated mouse fibroblast at 4 × 10min⁵/cm²The right and left densities were plated onto Matrigel-treated dishes (excess Matrigel was aspirated off during plating) and the MEF medium was changed to the corresponding embryonic stem cell medium (mTeSR medium) the next day.

Example 3 superovulation of female rabbits and embryo retrieval

(1) Superovulation of female rabbits: selecting female rabbits with slightly pink pudendum at 4:00 pm, injecting PMSG 35IU into the inner thigh, checking the female rabbits pudendum after 48h, and mating when the female rabbits become bright red or bright red. The female rabbit is put into a male rabbit cage for natural mating, and the male rabbit climbs and generates a marked twitch action to fall to the ground, namely the mating is successful once. And (5) lifting the female rabbit out, and slightly throwing the buttocks to further flow the semen of the male rabbit into the pudendum. Injecting LH 35IU into the inner thigh muscle of the female rabbit, recording as 0 day after mating, in order to prevent the female rabbit from mating once and not successfully fertilizing, the female rabbit is continuously combined with the male rabbit, and the female rabbit is placed back to the original cage after mating is successfully carried out for three times.

(2) Manufacturing a culture tray: the culture plate was prepared one day after mating of the female rabbits. The bottom of the external dish of a disposable cell culture dish with the diameter of 35mm is averagely divided into 4 sectors by a marking pen, and each sector is marked with numbers from 1 to 4 in sequence. Each sector was made up of 3 drops of 35. mu.L embryo culture medium microdroplets, overlaid with 2.5mL paraffin oil, placed at 38 ℃ in 5% CO₂Equilibration was performed in an incubator.

(3) Obtaining an embryo: taking the female rabbits 2 days after mating for embryo washing operation of living bodies. During operation, the anesthetic is injected intravenously from the ear margin of the female rabbit, after the drug effect is stable, the hairs in the abdominal cavity are scraped, and the hair cleaning part is coated with iodophor and alcohol in sequence for disinfection. The skin layer and the abdominal cavity layer are cut by a scalpel according to the abdominal midline at the approximate position of the ovary of the female rabbit, and the incision length is about 5 cm. After the abdominal cavity is opened, the uterus, fallopian tube and ovary are found. During embryo washing, one person inserts a steel needle with a flat needle head from a uterus part 1cm away from the junction of the uterine tube, the needle head penetrates through the junction of the uterine tube and is inserted into a part of the oviduct, and the other person inserts and fixes the scalp needle by aligning a plastic thin tube of the scalp needle with the ovarian umbrella part. 10mL of preheated CCM is injected at the end of the steel needle, the other end of the thin tube collects the flushed liquid, and the embryo in the liquid is picked out by an egg picking needle under a stereoscopic microscope. Embryos are washed 3 times in medium, impurities are removed, and finally the embryos are transferred to microdrops of a culture tray for culture.

At 48h after mating, the in vivo embryos have developed to 16 cell stage, and the obtained 16 cell stage embryos are cultured in a culture dish, and the embryos develop into blastula after two days of culture. As shown in FIG. 1, ICM of rabbit blastocyst is evident, and unlike other animal embryos, the zona pellucida is coated with a thick layer of mucin.

Example 4 isolation and culture of rbESCs

Feeder layer cells were prepared on the third day after mating of female rabbits, and all feeder layers were seeded on 24-well plates. Blastocysts were isolated on the fourth day after mating of female rabbits, and the feeder medium was replaced with the corresponding rbESCs medium 3h before isolation.

(1) A microscopic separation method: under a stereoscopic microscope, blastocysts were transferred into droplets of pronase by an egg pick-up needle, and mucin was digested by gentle pipetting for 20 s. The mucin-removed blastocysts were transferred to small drops (covered with paraffin oil) in a petri dish using an egg pick-up needle and placed on a micromanipulation stage. The blastocyst is fixed by a fixing needle, the injection needle is inserted into the blastocyst, the cells of the ICM are sucked out, and after the cells are washed twice by stem cell culture medium, the sucked out cells are placed on a feeding layer by an egg picking needle.

(2) The whole embryo culture method comprises the following steps: digesting blastocyst with pronase for 30 s-1 min, when blastocyst mucin is eliminated and zona pellucida is thinned and about to disappear, sucking out blastocyst with ovum picking needle, transferring into stem cell culture medium, and washing for 3 times. The zona pellucida is blown to drop during washing, and if the zona pellucida does not drop, two 1mL syringe needles are used for matching operation, and the blastocyst zona pellucida is stripped. Directly placing the embryo stripped of the zona pellucida on a feeding layer to make the embryo naturally grow adherent to the wall.

(3) Mechanical separation method: the blastocyst is first digested with pronase to remove mucin and zona pellucida from the embryo in the same manner as in (2). Next, the ICM and trophoblast cells were separated in a glass dish containing stem cell culture medium using 21 mL syringe needles to remove as much trophoblast cells around the ICM as possible. The ICM separated from the feeder layer is aspirated, washed 3 times in stem cell culture medium, and then transferred directly to the feeder layer, or the ICM may be cut into two pieces with a needle and then transferred to the feeder layer.

Statistics were made on the effect of different isolation methods on the adhesion of the rabbit ICM and the formation of the original clones, and the results are shown in Table 1. The results show that the ICM adherence rate and the primary clone formation rate obtained by the microdisolation method are the lowest, and are respectively 37.03 percent and 25.93 percent. The ICM adherence rate and the primary clone formation rate obtained by the mechanical separation method are respectively 87.93% and 70.69%, and the difference between the ICM adherence rate and the primary clone formation rate obtained by the micro-separation method and the whole embryo culture method is obvious (P is less than 0.05). Therefore, subsequent experiments chose mechanical separation methods for processing blastocysts.

TABLE 1 Effect of different isolation methods on the adherence of ICM to rabbits and the formation of primary clones

Example 5 digestive passage of rbESCs

And 3-7 days after the blastocyst is separated, observing the clone formation condition under an inverted microscope, and carrying out subculture.

FIG. 2 shows the formation process and differentiation of primary clones, and the results show that too long culture time of primary clones can result in dense growth of some cells in the clones, and the cells at these sites can be observed under microscope to be crowded and deformed, and differentiation occurs. And the time for the formation of the primary clone (table 2) and the time for the onset of the crowded differentiation of the primary clone cells (table 3) were counted as follows:

the experiment randomly observed the formation of primary clones by 46 adherent ICMs, which coincided with 38 primary clones formed by the 46 ICMs after culture. 38 ICMs, 4 ICMs forming primary clones on day 3 of culture, 20 ICMs forming primary clones on day 4 of culture, 12 ICMs forming primary clones on day 5 of culture, and the last 2 ICMs forming primary clones on day 6 of culture. The formation rate of primary clones was significantly higher on day 4 than on the other days (P < 0.05), followed by day 5, which had 84.2% ICM formed primary clones. The average number of days of culture required for the formation of primary clones was calculated as a weighted number of 4.31 days.

TABLE 2 time to Primary clone formation

The phenomenon of crowded differentiation of 1, 14, 21, 2 primary clone cells began to appear every day on the 4 th, 5 th, 6 th, and 7 th days of culture. Among them, 92.1% of the primary clones concentrated in the crowded differentiation on

days

5 and 6 of ICM culture, and the average time was 5.63 days by weight calculation.

TABLE 3 time to onset of crowded differentiation of primary clonal cells

Therefore, the cloning passage time for the subsequent experiments is preferably from 4.31 days to 5.63 days after the ICM inoculation.

After passaging, the cells in the old wells are cultured, and the residual stem cells may grow again to become clones. If the original clone grew well and the cells did not differentiate, the cells in each well could be passaged 2 times. After the cells are transferred to the P1 generation, the cells are passed aiming at the state of the cells, when the clone shape in one hole is better maintained and only a small amount of cells are differentiated, an ovum picking needle can be used for picking out the differentiated cells, and the cells in the whole hole are digested and passed by an enzyme digestion method after being washed by PBS; when only a small amount of clone in one hole has better cell morphology maintenance, the cells with better morphology are picked out by an egg picking needle for passage. The basic passage method comprises 3 passages according to the growth vigor and the number of the cells of 1: 2-1: 4. The method is as follows.

(1) Mechanical method: under microscope, selecting undifferentiated clone with good shape, cutting the cloned cell into small pieces by using ovum picking needle, and directly picking to new culture dish of feeder layer.

(2) A trypsin digestion method: and (3) discarding the stem cell culture medium, washing the stem cell culture medium for 2 times by using PBS (phosphate buffer solution), adding a proper amount of pancreatin of 200 mu L into each hole, and putting the mixture into an incubator for digestion for 2-3 min. Digestion was stopped by adding 1mL of fresh medium, gently pipetting the mixture evenly, and all the liquid was transferred to a 1.5mL EP tube. Centrifuging at 1000rpm for 3min, discarding supernatant, and collecting bottom cell precipitate. Adding a proper amount of stem cell culture medium, lightly blowing and beating for 2-3 times by a liquid transfer gun to resuspend cell sediments, transferring liquid into a 24-pore plate or a 12-pore plate with a new feeder layer according to the number of cells, shaking and uniformly mixing, and putting into an incubator for culture.

(3) ReLeSR digestion method: the stem cell culture medium is discarded, washed for 2 times by PBS, added with a proper amount of ReLeSR, and placed in an incubator for digestion for 1 min. Sucking away redundant digestive juice after 1min, only leaving a small amount of liquid to submerge cells, and putting the cells into an incubator to incubate for 3-5 min. 0.5mL of stem cell culture medium was added and placed under an inverted microscope for observation, if some of the colonies were observed not to break away from the well bottom, the plate was gently tapped off, and the liquid was transferred to a 1.5mL EP tube using a pipette. The digestion wells were washed with 0.5mL of PBS and the PBS was also transferred to the EP tube. The subsequent centrifugation, resuspension and inoculation steps are the same as those of the trypsinization method. In addition, the clone with good morphology can be picked under a microscope, transferred to a 96-well plate containing 100 mu L of ReLeSR liquid, and placed in an incubator for digestion for 3-5 min. After digestion, the clones were blown loose using a 200 μ L gun, the liquid was transferred to a 1.5mL EP tube, supplemented with 900 μ L of stem cell medium, and the subsequent centrifugation, resuspension, and inoculation steps were also identical to those of trypsinization.

The results indicate that primary clones can be digested directly with ReLeSR, which causes stem cells to preferentially detach from the dish, while primary trophoblast cells and feeder cells continue to adhere, as shown in FIG. 3. Therefore, subsequent experiments selected clones from rbESCs passaged by ReLeSR digestion.

Example 6 Effect of different media on adhesion of ICM, Primary clone formation, digestion passages in rabbits

The effect of different media on the adherence of rabbit ICM and the formation of primary clones was counted and the results are shown in Table 4.

TABLE 4 Effect of different media on adhesion and Primary clone formation of rabbits ICM

The primary clone formation process and the digestion passage process under different culture medium culture conditions are observed, and the results are shown in fig. 4-7:

FIG. 4 is a primary clone cultured on mTesR medium, showing that when ICM was cultured on a Matrigel-treated feeder-free petri dish using mTesR medium, the outgrowth of cells was loose and failed to form a primary clone; when a feeder layer is available, mTeSR culture can form clone-like colonies, but the colonies have poor cell homogeneity and many cells differentiate around.

FIG. 5 shows the clone morphology of KB, KBL and CXBL culture medium, and the results show that the rbESCs in KB culture medium are flat, can form large homogeneous colonies, have obvious edges with a feeder layer, have obvious stem cell morphology, can clearly see that the cells are round, and have the characteristics of small volume and large nucleus; the rbESCs obtained by the KBL culture medium have obvious stem cell morphological characteristics of small volume and high nuclear-to-cytoplasmic ratio, cells inside the clone have good homogeneity, the clone edge is bright, the stereoscopic impression is strong, and the overall morphology is similar to that of the clones in the KB culture medium; and the rbESCs cultured by the CXBL culture medium have a clone shape similar to the protruding growth of mESCs, and the clone colony is in an island shape, has strong stereoscopic impression and strong edge refractivity.

FIG. 6 shows the cloning of different culture systems, and the results show that when the RBESCs cultured by each culture system are subcultured by ReLeSR digestion method, cell strains of the RBESCs under KB and KBL culture systems are cultured for more than 12 generations, and the RBESCs under CXBL culture systems can be subcultured to 14 generations.

FIG. 7 shows clones grown after trypsinization passage of rbESCs in a CXBL culture system, and the results show that the rbESCs still have the capacity of clone formation after being trypsinized into single cells; however, the rbESCs in KB and KBL culture systems are directly differentiated after being digested by pancreatin, and cannot form clones.

Example 7 retrieval of cDNA templates from rbESCs

(1) Collection of rbESCs: a mixture of 4 ℃ PBS and 1% BSA was prepared, and prepared into several 50. mu.L droplets on a petri dish, and the cell clones were gently picked up with an egg pick-up needle, placed in the droplets, quickly rinsed 3 times in the droplets, and then placed into a PCR tube containing 10. mu.L of cell lysate with the egg pick-up needle. When the number of clones in the hole is large, collecting cells by an enzyme digestion method, suspending the cells by using a proper amount of cell lysate after centrifugation, and subpackaging the cell suspension into each PCR tube according to the amount of 10 mu L. The collected cells are directly subjected to micro-inversion or put into a refrigerator at the temperature of minus 80 ℃ for freezing storage.

(2) Cell lysis: the liquid in the PCR tube was instantaneously detached and incubated for 15min at 75 ℃ using a PCR instrument.

(3) Removing the template DNA: mu.L of RNase Inhibitor, 1.3. mu.L of 10 XRODBuffer, 1. mu.L of DNase I were added to each PCR tube, flash separated, and incubated with PCR instrument at 37 ℃ for 40 min.

(4) Termination of the DNase I reaction: each PCR tube was added with 1. mu.L each of EDTA, dNTP, Random Primer, and subjected to flash dissociation and incubation at 65 ℃ for 10min using a PCR apparatus.

(5) Micro-inversion: add 4. mu.L of 5 XFS Buffer, 2. mu.L of DTT, 0.5. mu.L of RNase inhibitor RRI, 0.25. mu.L of Reverse transcriptase SSII to each well, flash-separate, place in PCR instrument for inversion, program 25 ℃ 10min, 42 ℃ 90min, 95 ℃ 10min, product-80 ℃ for storage.

Example 8 PCR and qPCR reactions

(1) And (3) PCR: each reagent was added to a PCR tube according to the following Table 5, and PCR amplification was carried out in a 20. mu.L reaction system. The reaction conditions are pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at the annealing temperature designed according to the primers for 30s, extension at 72 ℃ (the extension time is set according to the extension time of 10s per 1kb product), the cycle number of denaturation, annealing and extension is 35 cycles, and finally extension at 72 ℃ is 5 min.

TABLE 5 PCR reaction System

(2) qPCR: the reagents were added to the PCR tubes according to the following Table 6, and the qPCR reaction procedure was pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 50s, and denaturation and annealing for 40 cycles. Each sample was repeated 3 times with 2^-△△The CT method calculates the expression of the gene. The data obtained were plotted using GraphPad Prism 5 and statistically analyzed using SPSS Statistics software.

TABLE 6 qPCR reaction System

(3) Expression of pluripotent genes of rbESCs under different culture conditions

As shown in FIG. 8, the results showed that the mRNA expression levels of OCT4, SOX2, NANOG, KLF4, C-MYC were higher in the RBESCs cultured in CXBL medium than in the KB, KBL medium group in each medium group, wherein the expression levels of OCT4, SOX2, NANOG, C-MYC were significantly higher in the KB, KBL medium group (P < 0.05). The mRNA expression level of the RBESCs cultured by the KBL culture medium is not obviously different from that of the KB culture medium group in the OCT4, SOX2 and KLF4, but the expression level of NANOG and C-MYC is higher and is obviously different from that of the KB culture medium group (P < 0.05).

(4) Detection of the Activity of the rbESCs clone AP

Clones of CXBL medium were stained with Alkaline Phosphatase (AP), and as shown in fig. 9, the results were bluish purple, indicating that the obtained rbESCs clones were AP-positive.

(5) RT-PCR detection of rbESCs pluripotent genes

As shown in FIG. 10, the results showed that rbESCs expressed the pluripotency genes OCT4, SOX2, NANOG, KLF4, C-MYC.

Example 9 immunofluorescence

1. Test procedure

(1) Washing cells with PBS for 3 times, adding 4% paraformaldehyde, and fixing at room temperature for 30 min;

(2) discarding the fixative, washing the cells with PBS for 3 times, adding 1% Triton X-100, and permeabilizing at room temperature for 30 min;

(3) removing the permeabilization solution, washing the cells with PBS for 3 times, adding 1% BSA, and blocking at room temperature for 1 h;

(4) removing the blocking solution, washing the cells with PBS for 3 times, adding corresponding primary antibody diluent, covering the bottom of the dish, and incubating overnight at 4 ℃;

(5) on the next day, the primary antibody diluent in the wells is aspirated away, the cells are washed with PBS for 5 times, 3min each time, a secondary antibody diluent corresponding to the primary antigen is added, and incubation is carried out for 40min at room temperature in a dark place;

(6) absorbing the secondary antibody diluent, washing the cells for 5 times by PBS (phosphate buffer solution), adding 10 mu g/mL Hoechst dye solution for 3min each time, and incubating for 10min at room temperature in a dark place;

(7) the Hoechst staining solution was aspirated, the cells were washed with PBS 2 times, and appropriate amount of PBS was added to observe the fluorescent expression.

2. Results

The immunofluorescence identification result of the rbESCs pluripotency genes is shown in FIG. 11, the clone cultured by the CXBL culture system expresses OCT4, SOX2, NANOG, E-CADHERIN and the marker gene FGF5 of Primed-state stem cells, and the expression of SSEA1 and SSEA4 is not detected.

Example 10 in vitro differentiation assay

1. Test procedure

(1) Single cell differentiation: the rbESCs were digested into single cells with pancreatin, quenched by addition of medium and centrifuged at 1000rpm for 3 min. And (3) discarding the supernatant, using a common cell culture solution to resuspend the cells, inoculating a proper amount of cells in a dish without a feeding layer, changing the solution every other day, and culturing for 7-10 days to observe the cell morphology.

(2) Preparing a pseudo-embryo body: the digestion and centrifugation steps are the same as those in (1), 200 mu L of common culture solution is added to resuspend the cells, the cell suspension is made into droplets of about 40 mu L on a dish cover, PBS is added to the bottom of the dish, and the cell suspension is inversely cultured. And (5) replacing the liquid by using an egg picking needle every other day, and culturing for 1-2 weeks.

2. Results

(1) The differentiation identification results of rbESCs are shown in FIG. 12. The results show that cells can grow wall-attached and differentiate into cells of different morphologies: epithelial-like cells, nerve-like cells, fibroblast-like cells, beating myocardial-like cells. Immunohistochemical identification of the expression epithelial cell marker gene: CK18, neural cell marker gene: TUBB 3.

(2) The formation of embryoid bodies by rbESCs is shown in FIG. 13. The results show that when the single cell is cultured in a micro-drop inversion way, cell aggregation is observed after the culture of D2, D7 forms an embryoid body, and the embryoid body is inoculated into a dish for culture, and can also form epithelial-like cells, fibroblast-like cells and nerve-like cells.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A composition for separating and culturing rabbit embryonic stem cells is characterized by consisting of basic fibroblast growth factor (bFGF), Leukemia Inhibitory Factor (LIF), GSK3 inhibitor CHIR99021 and beta-catenin specific inhibitor XAV-939;

the final concentration ratio of bFGF, LIF, CHIR99021 and XAV-939 in the composition is (4-6 ng/mL): (9-11 ng/mL): (2.5-3.5. mu.M): (1.5-2.5. mu.M).

2. A culture solution for separating and culturing rabbit embryonic stem cells is characterized in that every 50mL of the culture solution consists of the following components: 37mL of LKnockOutDMEM culture solution, 7.5mL of serum substitute, 2.5mL of fetal bovine serum, 0.5mL of streptomycin, 0.5mL of non-essential amino acid, 0.5mL of glutamic acid, 0.5mL of glutamine, 0.5mL of beta-mercaptoethanol, 0.5mL of nucleoside, bFGF at a final concentration of 4-6 ng/mL, LIF at a final concentration of 9-11 ng/mL, CHIR99021 at a final concentration of 2.5-3.5 mu M, and XAV-939 at a final concentration of 1.5-2.5 mu M.

3. The culture solution of claim 2, wherein the final concentrations of bFGF, LIF, CHIR99021 and XAV-939 in the culture solution are 4ng/mL, 10ng/mL, 3. mu.M and 2. mu.M, respectively.

4. A method for separating and culturing rabbit embryonic stem cells is characterized by comprising the following steps: mechanically separating rabbit blastocysts, inoculating the whole ICM isolated from each blastocyst into a feeder layer containing the culture medium of claim 2 or 3, and incubating the feeder layer at 37 deg.C and 5% CO₂Culturing under the condition of relative humidity of 95%, and performing primary culture for 4.31-5.63 days; then performing gram by adopting a ReLeSR digestion methodAnd (5) carrying out augmentation and passage to obtain the rabbit embryonic stem cells.

5. The method of claim 4, wherein the mechanical separation comprises the steps of digesting the blastocyst with pronase to remove mucins and zona pellucida from the blastocyst; the ICM is then separated from the trophoblast cells and the ICM is aspirated and transferred to a feeder layer.

6. The method according to claim 4, wherein the ReLeSR digestion process is carried out for a period of 4-6 min.

7. The method of claim 4, wherein the ReLeSR digestion comprises the steps of: discarding the rabbit embryonic stem cell culture solution, washing with PBS for 2 times, adding appropriate amount of ReLeSR, and digesting for 1 min; sucking away redundant digestive juice, leaving a small amount of liquid to submerge cells, and putting the cells into an incubator to incubate for 3-5 min; adding new 0.5mL rabbit embryonic stem cell culture solution to stop digestion, and transferring the solution to an EP tube; the digestion wells were washed with 0.5mL PBS, which was also transferred to the EP tube; centrifuging at 1000rpm for 3min, removing supernatant, and collecting bottom cell precipitate; adding appropriate amount of rabbit embryonic stem cell culture fluid, blowing to resuspend the cell precipitate, and mixing at a ratio of 1 × 10⁵～2×10⁵The liquid is transferred to a 24-well plate or a 12-well plate with a new feeder layer, is shaken and mixed evenly, is put into an incubator for culture, and is subjected to the next digestion passage.

8. The method of claim 4, wherein the ReLeSR digestion comprises the steps of: firstly, selecting a clone with a good form under a microscope, transferring the clone into a 96-well plate containing 100 mu LReLeSR liquid, and placing the clone into an incubator for digestion for 3-5 min; after digestion, the clones are blown loose gently, the liquid is transferred to an EP tube, and 900 mu L of rabbit embryonic stem cell culture solution is supplemented; centrifuging at 1000rpm for 3min, removing supernatant, and collecting bottom cell precipitate; adding appropriate amount of rabbit embryonic stem cell culture fluid, blowing to resuspend the cell precipitate, and mixing at a ratio of 1 × 10⁵～2×10⁵Perml cell number the liquid is transferred to a 24-well plate or 12-well plate with a fresh feeder layer and mixed by shakingMixing, culturing in incubator, and performing next digestion passage.