Isolation and culture of ES cell lines of blastocyst origin
Isolation and culture of ES cell lines of blastocyst origin
Mouse embryonic stem cells (ES cells) can be used not only as a model to study the regulatory mechanisms of mammalian development, but also as a vector for genetic studies.
Operation method
Isolation and culture of ES cell lines of blastocyst origin
Materials and Instruments
Equipment: Move The basic process of isolation and culture of ES cell lines can be divided into the following steps: 1. Preparation of reagents (1) MEF culture medium: 450 ml DMEM + 50 ml FBS + 5 ml 100xpenicillin and streptomycin, stored at 4 ℃, effective for 3~4 weeks. (2) Lineage-building culture medium: 400 ml DMEM／F12 + 100 ml KOSR + 5 ml 200 mmol/L (100x) glutamine + 5-ml (100x) b-mercaptoethanol + 50 μl LIF, filtered to remove bacteria, stored at 4 ℃, effective for 3～4 weeks. (3) Embryonic stem cell culture medium: knockout DMEM + 20% Knockout SR + penicillin (100 U/ml) / streptomycin (100 μg / ml) + 2 mmol / L L-glutamine + 1xminimal essential medium nonessential amino acids + 100 μmol／L β-mercaptoethanol (100x concentrated reservoir, 7 μl β-mercaptoethanol in 10 ml PBS) + 1000 U／ml recombinant mouse leukemia inhibitory factor. (4) Cell freezing solution: 50 ml FBS (50%) + 40 ml DMEM (40%) + 10 ml DMSO (10%). It should be freshly prepared on the same day. 2. Preparation of feeder layer (1) Cultivation of primary cells: ①Dislocate the E13.5 pregnant rats, sterilize the abdominal surface with 70% ethanol, tear the skin transversely at the midline of the abdomen, and turn it to both sides to reveal the abdominal wall. ② The abdominal organs were exposed by longitudinal dissection along the midline with sterile scissors, and the uterus was removed from the mother, placed on a 10 cm Petri dish containing 10 ml of DPBS, returned to the ultra-clean table and washed repeatedly for 3-4 times, and then transferred into new DPBS. ③ In the new DPBS, the uterus will be cut and the fetal membranes will be torn to remove the fetal rats, and then washed with PBS for 2 times. ④ Remove the fetal head, viscera, limbs, and cut the fetal rat's trunk into pieces less than 1 mm3 with ophthalmic scissors, add 2 ml 0.05% trypsin-EDTA and collect it into a 15 ml centrifuge tube, and incubate it at 37 ℃ for 20 min while shaking it slightly to promote the digestion of the cells; ⑤ Add 5 ml of DMEM + 10% FBS to terminate the digestion. ⑥ Collect the digested solution, centrifuge at 1200 r/min for 5 min, discard the supernatant, and resuspend the cells with fresh pre-warmed DMEM + 10% FBS. (vii) The cell suspension was spread in 2x10 plates in 100 mm dishes, and 10 ml of fresh DMEM + 10% FBS medium was added to the plates, and the incubation was continued overnight. (8) On the morning of the 2nd day, change the solution, discard the unaffixed cells, and continue the culture. ⑨ When the primary cell confluence into a piece, accounting for 80% to 90% of the culture dish when the passaging culture. (2) Mouse fibroblast passaging culture: ① When the fibroblasts grow to 80% ~ 90% confluence, discard the medium, add DPBS rinse again, discard. ② Add 0.05% trypsin-EDTA and put into the incubator for about 3 minutes to digest the cells. ③When the cell gap increases and the cytosol tends to become rounded, add DMEM + 10% FBS medium to abort the digestion, blow repeatedly to form a single-cell suspension, and centrifuge at 1200 r/min for 5 minutes. ④ Discard the supernatant, add a little medium to resuspend the precipitate, and re-inoculate in a new petri dish according to the ratio of 1:3. (3) Production of feeder layer cells: ① Use mouse fibroblasts of P1 or P2 generation to make feeder layer, the cell viability of P3 to P5 generation is obviously reduced, not able to support the process of ES lineage construction, so it is not favorable to make feeder layer. ② Mouse fibroblast cultures grown to 90%～95% confluence were discarded, and DMEM+10% FBS medium containing 10 μg/ml mitomycin C (final concentration 10 μg/ml, no. 107409; Roche, Basel, Switzerland) was added to block mitosis. (iii) After mitomycin C treatment for 2.5 h, the culture medium containing mitomycin C was discarded, washed twice with DPBS, added 0.05% trypsin-EDTA, and put into the incubator for digestion for about 3 min. ④When the cells were digested to the point that the cell bodies tended to become rounded, DMEM + 10% FBS medium was added to abort the digestion, and the cells were repeatedly blown into a single-cell suspension, which was then centrifuged at 1200 r/min for 5 minutes. ⑤ Discard the supernatant, add a little medium to resuspend the precipitate, freeze it or spread it in 35 mm culture dishes inoculated with gelatin at a density of 1x10. Note: ＊Mitomycin C is easily degraded by light and should be stored in a dark place; Mitomycin C is a highly toxic substance, so please take precautions when using it. (4) Coating of culture plates: ①Make gelatin solution: weigh the appropriate amount of gelatin (no. G1890, Sigma) dissolved in Milli-Q water to prepare 0.1% gelatin solution, autoclaved and stored at room temperature. ② The petri dish to be coated should be completely covered with gelatin solution until the bottom of the dish is covered, and then put into the incubator at 37 ℃ for 1 hour. ③ Discard the remaining gelatin in the dish before use and air-dry it in an ultra-clean bench for at least 30 minutes. ④ Wash it once with cell culture medium or PBS before use. 3. Embryonic Stem Cell Lineage Establishment (1) Preparation of feeder layer and preparation of blastocyst inoculation: ① On the day before inoculation of blastocysts, prepare the feeder layer according to the above method, usually in 12-well or 4-well plates. ② Replace the culture medium of the feeder layer with embryonic stem cell culture medium 1 to 3 hours before inoculation. (2) Acquisition of blastocysts and inoculation: E3.5 pregnant mice were used for the acquisition of blastocysts for embryonic stem cell establishment lines. ①Dislocation method was used to execute the E3.5 pregnant mice, and the abdominal surface was sterilized with 70% ethanol. The skin was torn horizontally at the position of the abdominal midline, and then turned over to both sides to reveal the abdominal wall. ② The abdominal organs were exposed by longitudinal dissection along the midline with sterile scissors, and the uterus was removed from the mother's body and immediately placed into a 35 mmI dish L containing Knockout DMEM (no. 10829-18, Gibco) + 1 μl of HEPES (15630, Gibeo) (HEPES/DMEM), and the excess fat was removed. (iii) The fat-eliminated uterus was placed in a 35 mm dish, 2 ml of pre-warmed HEPES/DMEM was added, and the uterus was flushed with a 2 ml syringe containing HEPES/DMEM with a 0.6 mm needle that had been de-tipped. ④ Observe and collect the blastocysts under a stereomicroscope. ⑤ Transfer the blastocysts with a glass pipette onto the feeder layer that has been replaced with the embryonic stem cell culture medium, put it back into the incubator, and wait for 6 days for observation. 4. Transmission culture and freezing of embryonic stem cell lines (1) First passaging culture: ① Prepare appropriate amount of feeder layer cells in 24-well plate one day in advance according to the number of clones to be passaged. ② Change the culture medium of feeder layer to ES culture medium 2 hours before the passaging. ③Add 25 μl of 0.25% trypsin to the 96-well plate according to the number of clones and set aside. ④ Discard the culture medium of the cells to be passaged, wash it once with DPBS, and add 2 ml PBS to the dish. ⑤ Pick the ES sample clone from the dish with a 10 μl pipette and put it into a 96-well plate with 0.25% trypsin. ⑥ After digestion for 5 min in an incubator at 37 ℃, add 200 μl of ES culture solution to terminate digestion. ⑦ Repeatedly blow to digest into single cells and transfer into a 24-well plate lined with feeder layer and add 1 ml of ES culture solution. Please make sure that there is only one clone in each well of the 24-well plate. (3) Routine passaging culture: ① Prepare the appropriate amount of feeder layer cells in the petri dish/plate one day in advance according to the amount of cells to be passaged. ② Change the culture medium of feeder layer to ES culture medium 2 hours before passaging. ③ Rinse the ES to be passaged or frozen twice with PBS. ④ Add trypsin37 ℃ and digest for 3 to 5 minutes until the cells are rolled up. ⑤ Add ES culture medium and blow repeatedly until it becomes a single cell. ⑥ Centrifuge the cells at 1200 r/min for 5 min, remove the supernatant and suspend with ES culture medium. ⑦ Add the suspended cell suspension on top of the prepared feeder layer and place it in a 5% CO2 incubator at 37 ℃. (8) The cells were usually passaged at a ratio of 1:2 or 1:3, and the culture medium was changed to fresh culture medium once a day after passaging. Usually ES cells are passaged once every 3-5 days. (4) ES freezing: ① The ES to be frozen is rinsed twice with PBS. ② Add trypsin and digest at 37 ℃ for 3 to 5 minutes until the cells are rolled up. ③ Add ES culture solution and blow repeatedly until it becomes a single cell. ④Centrifuge at 1200 r/min for 5 minutes, remove the supernatant, suspend with cell cryopreservation solution, and load into cryotubes. ⑤ Place the cryotube in a freezer box in a -80 ℃ refrigerator and allow it to cool down slowly at a rate of -1 ℃/min. (6) Cells can be stored temporarily in -80 ℃ refrigerator or put into liquid nitrogen tank for long-term storage. (5) ES recovery: ① Put the cells to be resuscitated from the liquid nitrogen tank quickly into the 37 ℃ water bath and shake until half of the ice crystals disappear. ② Slowly add 0.5 ml MEF culture medium into the freezing tube and add the cell suspension into a 15 ml centrifuge tube containing 5 ml MEF culture medium. ③ Centrifuge at 1200 r/min for 5 min and discard the supernatant. 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① Ultra-clean bench, water bath, culture flasks, centrifuge,
② Blood cell counting plate,
③CO
2
incubator,
④ Inverted phase contrast microscope (Leica, IMIRB), stereo microscope (NikonSMZ800)
⑤ Pointed tweezers, pointed scissors, centrifuge tubes, pipettes, pipettes, Petri dishes,
⑥ 4-well plate, 24-well plate, V-bottom 96-well plate
⑦Filter
Reagents:
①Materials: Pregnant mice
②70% ethanol
③DMEM
④Fetal bovine serum
⑤Knockout serum (Gibco, 10828-028); ⑥Nonessential amino-acid solution (Invitrogen, 11140-050)
⑤Knockout serum (Gibco, 10828-028); ⑥Nonessential amino-acid solution (Invitrogen, 11140-050).
Trypsin, 0.25% (1x) with EDTA 4Na, liquid (Invitrogen, 25200-072).
⑧Gelatin (Sigma, C1890-100G);
⑨ DMSO (Sigma, D2650), 2-Mercaptoethanol (Sigma, M7522), 1-Glutamine, nonanimal source
(Sigma, G8540)
⑩ PBS, HEPES,>99.5%, Mitomycin-c (Sigma, M0503)