Embryonic stem cell in vitro culture and directed induction of differentiation experiments
Embryonic stem cell in vitro culture and directed induction of differentiation experiments
Feeder cells are necessary for embryonic-derived stem cells including embryonic cancer cells (EC), embryonic stem cells (ES), and primordial germ cells (EG) to maintain a symmetrically dividing and undifferentiated state in vitro. The mechanism of action is not fully understood. For mouse embryonic stem cells, LIF secreted by feeder cells is one of the reasons for their pluripotency; therefore, in some mouse cell cultures, either feeder cells or LIF is sufficient. For human embryonic stem cell culture, feeder cells are still necessary. Two types of cells are commonly used as feeder cells for embryonic stem cell cultures: primary cultured embryonic murine fibroblasts (MEFs) or mouse fibroblast STO lines (derived from inbred SIM mice, thioguanine and ouabain-resistant). They were treated with y-ray irradiation or mitomycin c to inhibit their mitotic activity, and then inoculated into gelatin-coated culture dishes as feeder cells for embryonic stem cell culture. Author: Xuedao Pei, this experiment is from "Stem Cell Experiment Guide".
Operation method
In vitro culture and targeted induction of embryonic stem cell differentiation Move Embryonic stem cell isolation and culture Feeder Cell Culture and Feeder Layer Preparation Feeder cells are necessary for embryonic stem cells, including embryonic carcinoma cells (EC), embryonic stem cells (ES), and primordial germ cells (EG), to remain symmetrically dividing and undifferentiated in vitro. The mechanism of action is not fully understood. For mouse embryonic stem cells, LIF secreted by feeder cells is one of the reasons for their pluripotency, and therefore, in some mouse cell cultures, either feeder cells or LIF is sufficient. For human embryonic stem cell culture, feeder cells are still necessary. Two types of cells are commonly used as feeder cells for embryonic stem cell cultures: primary cultured embryonic murine fibroblasts (MEFs) or mouse fibroblast STO lines (derived from inbred SIM mice, thioguanine and ouabain-resistant). The cells were irradiated with y-rays or treated with mitomycin c to inhibit their mitotic activity, and then inoculated into gelatin-coated dishes as feeder cells for embryonic stem cell culture. Isolation and culture of embryonic mouse fibroblasts MEF can be isolated from any strain of mouse, with more than 100 embryos taken from 1 or 2 pregnant females. 1) Reagents and materials (1) Female rats of 13-14 days of gestation, defined as 1 day pregnant on the day of the bolus. (2) Sterile dissecting instruments: 2 ophthalmic scissors, 2 ophthalmic valves, 3 pairs of glass dishes, 1 valvate, 1 tissue cutter. (3) Ca2+ and Mg2+ free PBS, 0.05% trypsin solution/0.53 mol/L EDTA, 10mg/ml Nase solution. (4) MEF growth medium: high sugar type DMEM with 10% fetal bovine serum. (5) 35 mm, 60 mm and IOOmm tissue culture dishes. 2 ) Separation method The pregnant female mice were decapitated, the abdominal cavity was opened, the uterus was removed and placed in a glass Petri dish with PBS, the uterus was cut along the longitudinal axis of the uterus with ophthalmic scissors, the embryos were removed, the extra-embryonic tissues such as placenta and amniotic membrane were removed, and the blood was rinsed out with PBS. The embryo head and viscera were removed with forceps. The embryo was washed twice with PBS to remove blood and residual visceral tissue. Use ophthalmic scissors to cut the residual embryos, transfer the paste to a 20 ml flat-bottomed graduated test tube, add 5 ml of trypsin solution and 4OOul of DNase crude, so as not to release DNA that causes the solution to be too viscous. Incubate in a water bath at 37℃ for 30 min, or place at 4℃ overnight, shake, and then pour the upper cell suspension into an IOml centrifuge tube containing IOml MEF growth medium and mix well. The cells were centrifuged at 1000 r/min for 6 min and collected. The cells were then suspended in 15 ml of growth medium and counted using a hemocyte counter plate. Ten 15-day-old fetal mice yielded 5xl0 7 to IxlO 8 cells. Each IxlO 6 cells were suspended in 10 ml of MEF growth medium and inoculated into an IOOmm dish. Cells were wall-plated immediately after inoculation and replaced with fresh MEF growth medium 24 h later. About 2--4 days later, the cells were full grown, the culture medium was aspirated, washed twice with PBS, and 6--7 ml of trypsin was added to each dish, and incubated at room temperature for 3--5 min. 10 ml of trypsin was added to each dish of trypsin-digested cells. Add 10 ml of growth medium to each trypsin-digested cell dish. Add 10 m! of growth medium to each trypsin-digested cell dish, blow gently to disperse the cells, then transfer the suspension to a 50 ml centrifuge tube, and combine the cells from two dishes into one tube. The cells are collected by centrifugation, suspended in culture medium and distributed 1:3 into three IOO m m dishes. After about 3 days the cells are full grown and can be collected and frozen in liquid nitrogen. 3) Quality of MEF Primary embryonic murine fibroblasts isolated by the method described above were impure, with fibroblast-like cells as the main component, but there were also neuronal-like cells, cardiomyocytes, and some cells of unknown type. The heterogeneous cells decreased after transmission, and with the increase of the number of generations, the cell composition tended to be single, but the proliferation ability of the cells decreased, and the cells basically ceased to proliferate after 3--5 generations. The feeder cells used in the experiment are preferred to be passed on for less than 3 generations. However, the proliferative capacity of cells is relative, and it is more related to the culture medium, especially the quality of serum has a great influence on it. In addition, the gestational age of the embryonic mouse used for isolation also affects the quality and purity of the isolated cells. Cells older than 16 days of age are very heterogeneous, and usually 13 to 14 days of age are better. Freezing and Recovery 1) Freezing (2) 1.8 mI freezing tube, 1.2 ml freezing tube. (3) 80%~90% confluent cells were washed once with culture medium/pBS, and 6~7m l of 0.05% trypsin was added to each IOO m m dish and incubated for 3~5 min at room temperature, and IOmI of culture medium containing 10% serum was added to each dish to abort the digestion of trypsin. Then transfer the cell suspension to a 50 ml centrifuge tube and centrifuge at 1000 r/m i n for 5 min; the cell precipitates were suspended in cryopreservative, and lml of substitutionally pre-cooled cryopreservative was added to each IOO m m dish of cells. (4) Add Imi cell suspension to each freezing tube, tighten the lid, equilibrate at 4 ¾ for 30 min, let it rest at -20℃ for 60~90 min, and let it rest in liquid nitrogen vapor (about -70℃) for 2 h, then transfer it to liquid nitrogen for long-term storage. 2 ) Resuscitation One IOOOml beaker and 5 ○○ mI of autoclaved distilled water. Warm up the sterilized distilled water bath to 37--40 ℃ and pour it into the beaker; take out the cell cryopreservation tubes from liquid nitrogen, immediately invade them into warm water and stir them rapidly until the ice crystals are melted. Transfer the melted cell suspension to a conical centrifuge tube containing an equal volume of culture medium, centrifuge for 5 min at lOOOr/m i n, suspend the cell precipitate with culture medium, and inoculate the cells from each cryotube into an IOO m m petri dish. Cells were inoculated into an IOO m m dish for each frozen tube. Preparation of feeder cells In order to prevent MEF cells from proliferating more than ES cells, they were thawed and frozen to stop mitosis, and then inoculated in culture dishes suitable for ES cell growth. In this paper, two methods were used to inhibit the mitosis of MEF cells, i.e., Y-ray irradiation or addition of mitomycin C (l0ng/ml) for 2.5-3 h. The cells were then inoculated in culture dishes suitable for the growth of ESCs. Mitomycin C treatment 1 ) Reagents and Materials (1) l0 ug/m l mitomycin C solution: 2mg of mitomycin C powder was dissolved in IOml PBS, filtered and sterilized, and 0.5m I was dispensed into each tube and frozen at -20℃ in DD to avoid light. Before use, add 9.5 ml of DME liquid per 0.5 ml, the concentration of DME M is (2) 0.1% gelatin solution: add 5 g gelatin to 500 ml of water for injection, high pressure sterilization, concentration of 1%, 4 ℃ storage. For use, take 100 ml, add 90 ml of PBS, the concentration is 0.1%, stored at 4 ℃. (3) 35 cm, 60 cm, IOOcm and other types of tissue culture dishes. 2 ) Operation method For 80%-90% confluent cells, aspirate the culture solution, add mitomycin C solution (100 mm/6 ml, 60 mm/2 ml, 35 mm/lml) and incubate at 37 ¾ for 2-3 h. Aspirate the mitomycin C solution and wash twice with 6 ml of PBS. Radiation exposure Cultured cells are sent to the cobalt source room in relatively clean (sterile preferably) containers, such as aluminum or enamel boxes, and irradiated with a dose of 25-35 Gy, which will inactivate the mitotic activity of the cells, while the cells can still survive for a certain period of time. After irradiation, the cells can be digested and inoculated into gelatin-coated petri dishes in the same way as above. Y-ray irradiation is a simple and efficient method if a cobalt source is readily available, such as close to the laboratory. We usually isolate once, culture a large number of cells, irradiate them together, and then freeze them. For use, the cells are thawed and inoculated directly into gelatin-coated dishes, and the culture medium is changed on the second day for embryonic stem cell culture. The irradiation dose should be flexibly controlled, according to our experience, the irradiation dose of cells with strong proliferation should be large, for example, the irradiation dose of primary cells should not be less than 30 Gy; the irradiation dose of cells with 3-5 generations of progeny should be 20 Gy, if the dose is too large, the cells will die in large numbers after 3 days. Embryonic stem cells are derived from the inner cell mass (ICM) of the blastocyst, which can proliferate for a long period of time and have many differentiation potentials. Since the success of mouse embryonic stem cell culture in 1981, a large number of studies have found that embryonic stem cells can proliferate indefinitely, maintain a normal karyotype, and differentiate into all tissue cell types in vivo and in vitro, including functional germ cells. Mouse embryonic stem cells are important models for transgenic, embryonic development, cell differentiation and other studies. The difficulty of embryonic stem cell culture lies in the fact that the culture system should not only meet the needs of cell proliferation, but also keep the cells undifferentiated during long-term culture. This paper firstly introduces the isolation and culture of mouse embryonic stem cells. Isolation and culture of inner cell mass There are two methods for the separation of endocytosis: microscopic separation and immunosurgical methods. The microscopic method involves the mechanical separation of the endocytosis under direct microscopic visualization. The immunosurgical method is based on the principle that macromolecules such as immunoglobulins cannot pass through the blastocyst trophoblast ectoderm into the blastocyst lumen, and therefore, using immune-mediated cytotoxicity, the trophoblast ectodermal cells can be selectively destroyed, whereas the cells within the blastocyst lumen, the inner cell mass, are unaffected. In this paper, we will describe the isolation of the inner cell mass by immunological methods. 1 ) Materials and reagents (1) Rabbit anti-mouse serum: melt at room temperature, inactivate the complement by 56T water bath for 30 m i n , filter and sterilize, store at -20℃. (2) Guinea pig serum: 1 adult guinea pig (North Medical Animal Center), draw about 5 ml of blood from the heart, slowly inject it into a conical centrifuge tube, centrifuge at 2000r/m i n for 15 min, suck out the upper layer of serum, filter and sterilize the serum, dispense it in 0.2 ml/tube, and store it at -20 ℃. (3) 0.5% Streptomyces protease: I O m g melted in 2m l of saline filtration and sterilization, 0.2 ml/tube, -20 ℃ storage. (4) ES cell culture medium: high sugar DME medium containing 20% FCS, 1% non-essential amino acids, O.lmmol/L mercaptoethanol, 2 mmol/L glutamine, 50U/ml penicillin, 50U/ml streptomycin, 1000U/ml LIF. (5) 129Sv (129/SvpaslcoCrIBR) pregnant rats. 2 ) Operation Separations were carried out in paraffin oil-covered 3 0 0 droplets; incubation was carried out at 3 7 °C with 5% CO2; antibodies and guinea pig sera were diluted with DM EM culture medium. Pregnant 129S V rats that had been bolused for 3.5 days were decapitated and killed by dissecting the abdomen and removing the uterine horns. The embryos were flushed out with 3 7 ¾ pre-warmed serum-containing culture medium. The embryos were digested with 0.5% streptavidin for approximately 5 m i n to remove the zona pellucida. After removal of zona pellucida, the embryos are transferred to DMEM culture medium containing 10% FCS for 3 h. The exotrophoblasts are removed by immunosurgery. 3 ) Results Seven mouse blastocysts (Fig. 3.2) were post-desquamated blastocysts, which were incubated in antiserum and then transferred to complement for about 5 min. The outer trophoblast cells were swollen (Fig. 3.3), and after incubation for 10-25 m in, complete inner cell clusters could be separated by gently blowing with a fine pipette (Fig. 3_4). The inner cell clusters were walled within 24 h after inoculation, and four of them grew ES-like cells, which were in the form of colonies (Figure 3.3). ES-like Cell Amplification The inner cell mass will be attached to the wall within 24 hours after inoculation, and small cells with typical large nuclei and obvious nucleoli can be seen to grow out in 3--5 days. The cell mass with such cells will be removed with a fine glass pipette, digested with 0.01% trypsin for 2 minutes, and then blown by a fine pipette, and then the broken single cells and small cell mass will be inoculated onto the new cultured cells, and the culture medium will be changed every day. The culture medium was changed every day, and the culture medium was the same as the above culture medium of inner cell mass. Typical stem cell colonies could be seen after 3-4 days, and after 5-6 days, the colonies could be seen after 3-4 days. single-cell cloning The cells with normal karyotype were digested with trypsin to break up into single cells, inoculated into 35 mm dishes, incubated in the incubator for Ih, changed the liquid, gently blew down the adherent cells, and transferred into another dish, and then pipetted the single cells into 96-well plates lined with feeder cells under direct visualization of a dissecting microscope. The culture medium was changed every other day, and obvious colonies appeared after about 5~7 days. Trypsin digestion was performed to break up the cells into single cells, which were inoculated into 35 mm dishes and the culture medium was changed daily. The cells were passaged and frozen every 3 days. Results: A total of 192 wells were inoculated and 64 single-cell colonies were grown, with a cloning rate of 33%. Among them, 5 had abnormal karyotype. The colonies with normal karyotype were selected and expanded, and once the expansion was normal, the cell line was established. Identification 1) Materials and reagents (2) Hypotonic solution: 0.075mol/L KCl. (2) Hypotonic solution: 0.075mol/L potassium chloride solution, placed in a 37℃ incubator. (3) Fixing solution: methanol: acetic acid = 3:1, freshly prepared at the time of use. (4) Digestive solution: 0.2% pancreatic enzyme, freshly prepared at the time of use. (5) Gimsa staining solution. (6) Antibodies: rat anti-SSEA-l (MC-480), SSEA-3 (MC-631), SSEA-4 (MC-813-70); mouse anti-TRA-1-60 (MAB4360). TRA-1-81 (MAB4381), OCT-4 (MAB4305). (7) FITC-labeled goat anti-rat IgG (ZF-0312). (8) Alkaline phosphatase buffer: 100 mmol/L NACL, 5 mmol/L MgCl2, lOOmmol/LTris .HCKpH9.5). (9) Alkaline phosphatase chromogenic substrate: NBT/BCIP (SK-5400). 2) Methods and results of surface marker and karyotype identification (1) Karyotype identification: The conventional G-band method was used. Cells in the proliferative index phase (3 days after the generation) were replaced with fresh culture medium, added with colchicine, and returned to the incubator for 2 h. The colchicine-containing culture medium was aspirated, and 0.05% pancreatic enzyme/0.53 mmol/LEDTA was added, and the cells were digested for 3--5 min at room temperature, and then an equal volume of serum-containing medium was added to terminate the digestion. Terminate digestion, Pasteur pipette blowing, the cell suspension l 〇〇〇r/min centrifugation 6 min, remove the supernatant, add 4m l hypotonic solution to the precipitate, pipette blowing and mixing, 37 ℃ water bath for 8 min; add lm l of fixing solution, blowing gently and mixing; 1500r/min centrifugation IOmin, discarding the supernatant, the precipitate was added to 4m l of fixing solution, blowing gently and mixing; centrifugation, repeat the fixing 1 time; discard the supernatant, the precipitate was added to 4m l of fixing solution, blowing and mixing; centrifugation, fixing once. centrifugation, repeat the fixation for 1 time; discard the supernatant, add 0.5--lm l of starch into the fixative for suspension; drop the cell suspension onto a clean slide immersed in 0.5Z distilled water; dry the slides at 6.5 ℃ for 2~4 h; the slides are put into the digestive solution to be digested for 8--10s, and then add saline to terminate the digestion; Gimsa staining solution is added to the slides to be digested. The slides were digested in digestive solution for 8-10 s, and the digestion was terminated by adding saline. (2) Alkaline phosphatase assay: cytochemical method was used. Cells were cultured in 35 m m dishes, washed with PBS for one time, fixed with 4% paraformaldehyde for 30 min at room temperature; washed with PBS for three times, each time for 5 min; added Iml magnesium-containing Tris-HCl buffer (p H 9.5), and 5Ial each of NBT/BCIP (VeCtor), mixed well, and incubated at room temperature and protected from light until the color was developed; rinsed with tap water to terminate the staining; rinsed with tap water to terminate the staining. The reaction was terminated by rinsing with tap water. (3) SSEA-I staining: Immunohistochemical two-step method was used. Mouse anti-MC-480(SSEA-1) was purchased from the University of Iowa (USA), and the color kit EliVision™ was purchased from Fuzhou Maixin Bio-Technology Development Company, according to the steps provided in the kit. Cells in 4-well plates were fixed with 4% paraformaldehyde and washed three times with PBS for 5 min each time; 30003% hydrogen peroxide was added to each well and incubated for IOmin at room temperature; washed three times with PBS; anti-SSEA-I was added at a dilution of 1:200, and incubated overnight at 4℃; washed with PBS, and 300 jil of enhancer polymer was added and incubated for 30 min at room temperature; washed three times with PBS. Diaminobenzidine (DAB) was used for color development. (4) Results: Normal 40XY karyotype. The cell surface markers alkaline phosphatase and SSEA-I were positive (Figure 3.8 and Figure 3.9). 3 ) Identification of in vitro differentiation and results (1) Identification method: About IO7 embryonic stem cells were suspended in 0.1~0.2 ml of PBS solution and injected into the subcutis of 4-week-old nude mice. After 4~5 weeks, the tumor blocks were removed, fixed in 4% paraformaldehyde, embedded in paraffin, sliced, stained with HE, and observed under the microscope. (2) Results: Two weeks after subcutaneous injection of cells into nude mice, the mass could be touched and enlarged progressively. Histological identification of the mass revealed that it contained cells from three germ layers, such as squamous epithelial tissue, nerve tissue, cartilage, columnar epithelium and other tissues. This confirms the ability of the cultured cells to differentiate into 3 germ layers under in vivo conditions. The isolation and culture of human embryonic stem cells include: separation of inner cell mass, identification of embryonic stem cells, expansion of embryonic stem cells and so on. Successful isolation and culture of human embryonic stem cells must have the following key conditions: (1) good quality blastocysts; (2) effective separation of inner cell mass; (3) reliable and stable culture system; (4) reliable and effective transmission. Separation and culture of inner cell mass In the isolation and culture of mouse embryonic stem cells, it has been demonstrated that the immunosurgery (immunosurgery) method can be used to isolate and culture the inner cell mass. Preparation of Antisera and Complement 1) Serum Preparation Antisera against human choriocarcinoma cells were prepared by immunizing rabbits with human choriocarcinoma cell line JEG-3 cells (Figure 3.10). The method was as follows: one Japanese white male rabbit, weighing 2.0 kg. The method was as follows: One Japanese male rabbit with large ears, weighing 2.0 kg. JEG-3 cells, about 107+, were suspended in 3-5 ml of PBS and injected into a vein at the ear margin of the rabbit. The cells were injected into a vein at the ear margin of the rabbit once a day for three consecutive times. After the last injection, 2 ml of blood was drawn from the marginal vein of the ear on the 15th day, 6 ml of blood from the heart on the 17th day, and 12 ml of blood from the heart on the 20th day, and then the rabbits were put to death. The blood samples were kept at room temperature for 30 m in, centrifuged at 250 r/m in for 15 min, and the serum was aspirated, bathed in water at 56℃ for 30 min, inactivated the complements, filtered to remove the bacteria, and dispensed in 0.5 ml tubes, and stored at -20 ¾. 2) Complement Preparation Guinea pig serum was used as complement. Two 3-month-old guinea pigs were used to draw blood from the heart, and each guinea pig was allowed to draw about 5~6ml of heart blood. The blood samples were left at room temperature for 30m in and then centrifuged at 2500r/m in for 15m in, and the serum was aspirated, filtered, sterilized, and dispensed in 0.5ml tubes and stored at -200 ℃. Separation of inner cell mass 1 ) Reagents 0.5% pronaseE (Merck): Prepare with G-2, dispense 0.5 ml/tube and store at -20 °C. 2 ) Methods Antisera and complement were diluted with DMEM solution. Antisera were diluted 1:50 and complement 1:100. The reactions were carried out in a 30ul droplet covered with paraffin oil. After 5--7 days, the aged blastocysts were transferred into 0.5% pronaseE droplet and observed under the dissecting microscope, and the zona pellucida disappeared after about 3--5mn; the blastocysts with the zona pellucida eliminated were transferred into DMEM droplet to remove the zona pellucida, and the zona pellucida was removed from the blastocysts. 3) Results Inner cell mass culture 1) Culture solution Knockout TM medium, containing 20% FBS with 1% non-essential amino acids, 0.1mmol/l mercaptoethanol, 2m mol/L glutamine, 50U/m l penicillin, 50U/m l streptomycin, this is the serum-containing culture medium; FBS was replaced with the same concentration of serum substitutes, and 4ng/m l of b F GF was added, this is the serum-free culture medium. 2 ) Culture method The day before the isolation of endocytosis, the cultured cells were prepared and inoculated into 0.1% gelatin-coated 4-well plates and incubated overnight. On the second day, the isolated inner cell clusters were inoculated into 4-well plates and cultured at 37 ℃, 6% C02 and 94% humidity. The culture medium was replaced with fresh culture medium every day. 3 ) Results All of the 15 inoculated endocytosis clusters were wall-affixed within 24 h, of which 14 were wall-affixed within 12 h and one was wall-affixed at 24 h. The results showed that the biological activity of the isolated endocytosis clusters was well preserved. This indicates that the isolated endocytotic clusters have preserved their biological activities. Four of them were inoculated onto freshly prepared MEF feeder cells, five were apoptotic, two had only trophoblast cells, and the other seven had small cell clusters with indistinct cell boundaries in the center of proliferating trophoblast cells (Fig. 3.12), three of which were first-generation differentiated, and the other four were proliferating vigorously, so that four embryonic stem cell lines, i.e., B 4, B 7, PKU 1, PKU 2, were cultured from the 14 endocytotic cell clusters. One inner cell mass was inoculated in the feeder layer of adult endometrial mesenchymal stromal cells and apoptosed after 4 days. Expansion of ES-like cells Transmission of human embryonic stem cells is difficult, and mechanical, collagenase, Dispase and EDTA methods have been reported in the literature. When the number of colonies is small, the mechanical method is more effective and reliable. When there are hundreds or even hundreds of colonies, the mechanical method is almost unusable. In this section, we have explored a variety of generation methods and established an effective method for mass expansion of E S cells. 1 ) Mechanical method Cut the cell colony into small pieces of about 200--300 cells each. Use a pipette to inoculate the cell clumps onto freshly reared cells. The cell clusters were walled within 12 h as were the inner cell cluste For more product details, please visit Aladdin Scientific website.
(1) MEF freezing medium: high sugar type DMEM, containing 10% serum, 10% DMSO, freshly prepared before use.
Cells are wall-affixed after 30--60 m i n . The next day, the culture medium is changed and dead cells are removed. The next day, the culture medium is changed and dead cells are removed. The cells can be passaged in 2--3 days or prepared as feeder cells.
Add 9.5 ml of DME solution to each 0.5 ml of DME concentration of 10ug/ml before use, wrapped in tin foil and protected from light, and stored at 4 ℃ for 2 weeks.
Wash twice with 6 ml of PBS to remove the residual mitomycin. Add 6 ml of trypsin and incubate at room temperature for 3~5 min, add an equal volume of serum-containing culture^ to abort digestion; centrifuge the cell suspension at 1000r/m in for 5 min; suspend the precipitate with culture medium, count, and inoculate the cells at a density of I05 cells/cm2 in 0.1% gelatin-coated Petri dishes (add gelatin solution and leave for 30 min at room temperature). Feeding cells
The inoculation density of feeder cells should be 50%-60% confluent, as shown in Figure 3.1. 
The procedure was as follows: transfer the embryos into a droplet of anti-mouse serum diluted at I:10 for 30 min; wash the DMEM droplet 3~5 times for 5 min each time; incubate in a droplet of guinea pig serum diluted at I:10 for I5--30 min; wash the DMEM droplet; and blow gently with a pipette with a tip diameter of 30~400 um to separate out the exotrophic cells. Then the cells were gently blown with a fine pipette with a tip diameter of 30~4.0 um, and the inner cell mass was separated and inoculated onto feeder cells, which were incubated at 37℃, 5%C 0 2 , 95% humidity, and the culture medium was changed every day. The culture medium was the aforementioned ES cell culture medium.
The cells were colonized, with clear margins, large nuclei, obvious nucleoli and high karyoplasmic ratios (Figure 3.5 and Figure 3.6). 
After 5-6 days, the colonies grew to 100~200p m in diameter. The typical colonies were mechanically removed, digested with 0.05% trypsin, broken into single cells, inoculated into new feeder cells, and the culture medium was changed every day, and multiple stem cell colonies could be seen after 2 days. Two cell lines with karyotype 40 XY were established by the smooth expansion of two cells numbered A and G (Fig. 3.7). The A cell line was easily differentiated during passaging and freeze-thawing, and was frozen up to 15 generations. The G cell line is stable in the process of transmitting and freeze-thawing, and single-cell cloning is carried out in the 11th generation, so the single-cell clonal cell line is named G11q. Transmitting and freezing part of the cells with trypsin is performed every 3~4 days. The freezing solution contains 90% stem cell culture medium and 10% DMSO.
(1) Colchicine (Sigma,C9754): Prepare a concentration of 5ug/ml with water, store at 4℃, dilute to 250ng/mI when using.
is a reliable and effective method to isolate the inner cell mass, so this method was used in this subsection to isolate the inner cell mass of human blastocyst. 
Antisera, complement as described above.
Wash the blastocysts; then transfer the blastocysts into antisera droplet and incubate for 30 min; then transfer them into complement droplet and incubate for 30 min; transfer the swollen blastocysts with ectotrophoblasts into DMEM droplet and wash them; and then gently blow the blastocysts with a fine pipette, then the inner cell mass can be separated (Fig. 3.11).
A total of 30 blastocysts were isolated, among which the first blastocyst (grade 6A A) had all inner cell mass and outer trophoblast cells lysed during isolation due to the immature isolation conditions, while the rest of the blastocysts with inner cell mass of grade B or above were isolated with a total of 15 intact inner cell masses.
