Mouse embryonic stem cell culture experiment
Mouse embryonic stem cell culture experiment
Mouse embryonic stem cell culture can be: (1) cell preservation; (2) used for stem cell research; (3) used for cell physiology, morphology and other research; (4) animal cloning.
Operation method
in vitro assimilation
Principle
Embryonic stem cells can be differentiated into various cell types in vitro and in vivo. Our in vitro differentiation method favors neural precursor cells by selection within the minimal amount of medium (step 3), expansion in the presence of bFGF (step 4) and final differentiation in step 5. Cells were cultured throughout at 37°C, 5% CO2, and 100% humidity. Differentiation towards neuronal cells is generally induced in vitro, but can also be induced using low concentrations of RA.
Materials and Instruments
Mouse Move I. ES medium Caveat 1. It is necessary to use high purity water for cell culture. 2. It is necessary to operate the experiment in a sterile environment. Common Problems 1. In the absence of added LIF, the clone formation rate and AKP staining positivity of ES cells cultured on the feeder layer were significantly higher than that of ES cells cultured without feeder layer. 2. when LIF was added up to l000IU/ml, there was a master-slave relationship between exogenous LIF and stromal LIF produced by the feeder layer in maintaining the undifferentiated state. 3. When there is no or insufficient exogenous LIF, the differentiation inhibition produced by the feeder layer plays a major role in differentiation inhibition. For more product details, please visit Aladdin Scientific website.
Gelatin PBS DMEM Horse serum L-glutamine HEPES β-mercaptoethanol PEST LIF
Culture Plates Tubes Water Baths Centrifuges 6-well Plates 24-well Plates
Cell culture was maintained in the presence of LIF.
II.EB medium
Formation of embryoid bodies after removal of LIF using bacterial petri dishes took 4 days.
1. Disperse and purify the cells (see Cell Passage section above).
2. 2 hours after purification transfer cells into 50 ml Falcon tubes containing ES medium, count cells to take appropriate volume into 15 ml tubes and centrifuge for 3 minutes.
3. Resuspend the cells in 2 ml of ESB medium and blow at least 10 times to make a single cell suspension.
4. a 15 cm bacterial culture dish is inoculated with 4 to 5x106 cells (1 fully fused tissue culture dish is usually sufficient to inoculate 4 bacterial culture dishes of the same size).
5. After 2 days the medium is changed and the blastomeres are transferred into conical tubes and left for 3 to 5 minutes to allow the cells to settle. Discard the supernatant, resuspend the cells in fresh medium and inoculate in a new bacterial culture dish.
6. On day 4 of incubation in EB medium, the cells are transferred to uncoated tissue culture dishes (this is the cell transplantation step). 1 bacterial culture dish is placed in 1 tissue culture dish, and the embryoid bodies are adhered to the same size plate one day prior to step 3.
7. It takes 4 days for the embryos to form. Cultivate the embryos in EB medium for 1 more day to adhere to the surface of the tissue culture dish. This day is considered to be the demarcation between steps 2 and 3.
III. ITSFn medium
Selection of neural precursor cells in minimal medium
1. Change the medium to ITSFn medium one day after embryoid inoculation in a tissue culture dish.
2. not all embryoid bodies have adhered by this time, so be careful when removing the medium so that most of the embryoid bodies remain in the dish.
3. keep the cells cultured in ITSFn for about 10 days, changing the medium as needed - about every other day.
4. Observe the cell morphology, with neural-like cells appearing around days 4 through 7. When neural-like cells can be identified, switch to step 4. The step changeover should occur a few days after the first clear sign of neural precursor cells, usually on days 6 through 10 of step 3.
IV. N3 medium + bFGF
Expansion was performed by culturing neural precursor cells in medium containing 10 ng/ml bFGF.
1. PBS was washed and 1-2 ml of trypsin was added.
2. Incubate at 37°C for 5 min.
3. Terminate tryptic enzyme activity with 4 ml of EB medium, transfer the cells into a conical tube and leave for 3-5 minutes Remove the cell mass and transfer the supernatant into a new centrifuge tube for centrifugation.
4. Resuspend the cells in N3 medium containing bFGF.
5. Inoculate cells on coverslips coated with poly-L-ornithine/fibronectin (it is best to place coverslips in 24-well plates or 6-well plates, 5 coverslips per well in 6-well plates 4 if plastic to maximize the number of samples possible). 24-well plates are inoculated with 3. 5x105/well or 6-well plates with 1. 7x106/well.
6. Change the medium after 2 days.
V. N3 medium
Differentiation of neural precursor cells by withdrawal of bFGF.
1. Cells were inoculated on coverslips for 4 days after which the medium was changed to N3 medium without bFGF.
2. Change the medium as needed (approximately every other day).
3. Fix the cells after 10 to 15 days of differentiation.
4. Remove culture medium.
5. wash with PBS, add 4% formalin, leave at room temperature for 30 minutes, wash with PBS 2 times store in PBS.
6. use PBS containing 0.01% azide Na for long term storage.
VI. Preparation of transplanted cells
Cells were transplanted 4 days after the formation of the blastomeres (corresponding to the end of step 2 in the in vitro differentiation process where cells were transplated into tissue culture plates). As described in the previous section on in vitro differentiation, the blastomeres begin to form 4 days prior to transplantation. Usually one more day is needed to transplant the blastomeres into a 10 cm dish.
1. Transfer the blastomeres to a 15 ml conical tube and leave for 3 to 5 minutes to allow the blastomeres to settle. It is better to centrifuge the cells for 3 minutes. Leaving them to settle will remove more individual cells (including dead cells) that can be centrifuged.
2. Remove the supernatant by resuspending the blastomeres in 1xPBS without calcium and magnesium ions.
2. Centrifuge for 3 minutes.
3. Remove supernatant and add 1x trypsin (1 ml to a 10 cm dish).
4. 37°C water bath for 5 min.
5. Add 5 ml of EB medium and carefully blow about 10 times.
6. It is important to handle the cells carefully and not to blow vigorously when performing cell passages to disperse the cells.
7. Centrifuge for 2 minutes.
8. Aspirate the supernatant and resuspend the cells in 500 ul EB medium.
9. Blow carefully 5 times with a smooth Pasteur pipette.
10. Centrifuge 2 times.
11. Aspirate the supernatant to resuspend the cells in 100 ul EB medium.
VII. Caprylyl nicotinate labeling of ES cells for transplantation
1. Prepare a 10 ug/ml hexosidine nicotinate stain (bisbenzimide, 118 in the freezer).
2. add 1 mg (the smallest amount you can weigh) to 5 ml of EB medium (made to 200 ug/ml).
3. dilute the solution to 10 ug/ml (100 ul 200 ug/ml solution and 1.9 ml EB medium).
4. ES cell suspension was prepared by resuspending the cells in 2 ml of nicotinic acid hashkolide staining solution instead of EB medium as described previously.
5. Place the suspension at room temperature (or on ice) for 30 min.
6. Centrifuge for 2 minutes.
7. Aspirate the supernatant to resuspend the cells in 2 ml of EB medium.
8. Repeat once.
9. Centrifuge for 2 minutes.
10. Aspirate the supernatant and resuspend the cells in 100 ul EB medium and place on ice.