小鼠ES细胞培养protocol
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General Concerns
1. It is strongly recommended that ES cultures be monitored daily. Mouse ES cells require far more attention than most other tissue culture cells. They are unlikely to be recovered once allowed to differentiate.
2. Those actions that will likely cause Mouse ES cells to differentiate include but are not limited to:
1. Plating too sparsely (<0.5x105 cells per cm2).
2. Plating too densely (>4x105 cells per cm2).
3. Failure to change the media when needed. Strive to keep the pH indicator red to red-orange at all times, never yellow.
4. Allowing the cells to get too confluent.
5. Allowing the culture to consist of a few large colonies rather than several smaller colonies.
6. Allowing the colonies to get too large.
7. Failing to add the appropriate amount of LIF (Leukimia Inhibitory Factor) to the culture media.
3. Mouse ES cells maintain their pluripotent state best when seeded as a single cell suspension at a rate of approximately 1.5x105 - 4x105 cells per cm2.
4. It is likely you will have to split your cells every two to four days.
5. Generally Mouse ES cells are split no less than 1:4 and no more than 1:10. Please refer to the Photo Gallery for visual guidelines of cell density and differentiation.
Thawing Mouse ES cells
1. Using the predetermined surface area that the cryotube of Mouse ES cells will cover (see surface area table), decide which size plate you will be seeding the cells onto. Label the plates accordingly. NOTE: the area should already be coated with feeder cells.
2. Place 5ml of ES cell media into a 15ml conical tube.
3. Remove the cryotube from the freezer and place it IMMEDIATELY into the 37°C water bath making sure not to submerge the cryotube above the base of the cap.
4. Carefully monitor the media in the cryotube until there is only a small crystal of ice present. At this time take the cryotube into the hood.
5. Using a 1ml pipette, carefully pipette the media up and down two to three times ensuring that any cells settled to the bottom of the cryotube are resuspended (the ice crystal will have melted by now).
6. Place the Mouse ES cells into the 5ml of ES cell media you have placed in the 15ml conical tube and mix gently.
7. Pellet the cells by centrifugation at approximately 340xg for 3 minutes. Be careful not to spin the cells to forcefully as this will damage them.
8. Carefully aspirate the supernatant.
9. Resuspend the pellet in the appropriate amount of media and aliquot to the predetermined surface area.
Feeding Mouse ES cells
1. Mouse ES cells will generally require feeding every day.
2. Carefully aspirate the old media from the plate by placing your pipette along the side of the plate, well, or flask while tilting.
3. If you detect a large amount of dead cells or debris on the plate (as is commonly the case the day after thawing) you can gently rinse the cells with PBS and aspirate. CAUTION: Mouse ES cells can easily be washed off of the feeder layer.
4. Apply fresh ES cell media to the plate by placing your pipette along the side of the plate, well or flask and gently dispensing the media.
Splitting Mouse ES cells
1. Mouse ES cells generally should not be split at ratios lower than 1:4 or higher than 1:10. Cells should be split when they reach ~80% confluency. Please see the photographs in the photo gallery taken at 48 hours, of Mouse ES cells plated at 4x105 cells per cm2 for an approximation of their appearance.
2. Aspirate the media from the tissue culture dish.
3. Gently rinse the dish with PBS to remove any residual serum (the presence of serum will inactivate trypsin).
4. Add enough trypsin (0.25% Trypsin in 0.1% EDTA) to just cover the surface area (for example: 1ml of trypsin will cover a 100mm diameter dish).
5. Place the dish in the 37°C, 5% CO2, humidified incubator for 3-5 minutes or until cells release from the bottom of the dish (this should be visible with the naked eye). Beware that extended exposure of Mouse ES cells to trypsin is toxic. Keep the time to a minumum.
6. Remove the dish from the incubator and observe the cells under the microscope. Ensure that colonies have released from the plate and have separated into single cells. You may have to apply some gentle agitation in order to break up the colonies.
7. Continue with the trypsinization until approximately 70% of the cells are single cells.
8. In the hood, add 6-10 times the volume of ES cell media as trypsin (for example: if you added 1ml of trypsin you now need to add 6-10ml of ES cell media).
9. Pipette the cells up and down a few times while rinsing the dish in order to ensure that all the culture is in a single cell suspension.
10. Collect the cells into a clean tube.
11. Centrifuge at no more than 340xg for approximately 3 minutes to pellet the cells.
12. Resuspend the cells in the desired volume of ES cell media and aliquot to new dishes. Make sure the cells are evenly dispersed across the surface area of the dish. Do this by gently moving the dish back and forth and side to side, never swirl in a circular manner, as this will cause the cells to accumulate around the perimeter of the dish.
Freezing Mouse ES cells
1. Mouse ES cells are generally frozen down and thawed from and to the same surface area provided the area was confluent at the time of freezing. For example: an 80% confluent 100mm dish (having 55cm2 of surface area) can be frozen down into 1ml and thawed back to a total surface area of 55cm2.
2. Mouse ES cells can be frozen at any time, although generally cells are frozen down when they would normally need to be split (~80% confluency).
3. To freeze ES cells; first typsinize the cells into a single cell suspension. Pellet the cells by centrifugation as above. Freeze the cells down in a 4:6 ratio of ES cell media to ES cell freezing media. For example: first resuspend the ES cell pellet in 0.4ml ES cell media. Add 0.6ml ES cell freezing media for a final volume of 1ml. Mix, and place 1ml into a labeled cryotube.
4. Place the cryotubes containing the cells to be frozen into a storage box, place the box into a cooler, place the cooler in a ?0°C freezer and leave them overnight. Transfer the cryotubes to cryostorage at -140C (typically under liquid nitrogen). This process allows the cells to undergo a slow decline in temperature until frozen.
Corning or Costar Flask size Growth Area (cm2)
T25 25
T75 75
T150 150
T162 162
T175 175
T225 225
Treated Tissue Culture Dish Size (mm) Growth Area (cm)2
35mm 8
60mm 21
100mm 55
150mm 148
245mm 500
Plate Size Growth Area (cm2) for single well Growth Area(cm2) for entire plate
6 well 9.5 57
12 well 3.8 45.6
24 well 1.9 45.6
48 well 0.95 45.6
ES Cell Media Recipes
ES Media ES Freeze Media
Component 1 liter 500ml 250ml 500ml 250 ml 100 ml
DMEM 800ml 400ml 200ml
FCS 150ml 75ml 37.5ml 410ml 205ml 82ml
L-Glu 20ml 10ml 5ml
NEAA 10 5 2.5
MaPyr 10 5 2.5
P/S 10 5 2.5
b-me 7µlW 3.5µlW 1.75µlW
LIF † † †
DMSO 90ml* 45ml* 18ml*
Note: FCS needs to be heat inactivated for ES freeze media
Heat inactivation is accomplished by incubating FCS at 56°C for 30 minutes followed by a gradual cooling to room temperature.
* When making ES freeze media, always place the heat inactivated FCS into the bottletop filter prior to the addition of DMSO.
† LIF should always be added to ES cell media. If using commercial LIF the exact amount will be indicated with the product. If using non-commercial LIF the amount will have to be determined empirically by titration.
W Never add b-mercaptoethanol directly to a bottletop filter. Only add it in conjunction with other fluids.
Abbreviations:
DMEM - Dulbecco抯 modification of eagle抯 medium, FCS - fetal calf serum, L-Glu - L-glutamine, NEAA - non-essential amino acids, NaPyr - sodium pyruvate, P/S - penicillin streptomycin, b-me - beta mercaptoethanol, LIF - leukemia inhibitory factor, DMSO - dimethyl sulfoxide
Some notes on strain background
After 20 generations of brother sister matings, on average at least 98.6% of the loci in each mouse are homozygous. The following information concerning strain background is taken from: J A Beck, S Lloyd, M Hafezparast, M Lennon-Pierce, J T Eppig, M F W Festing & E M C Fisher Geneologies of Mouse Strains, Nature Genetics 24, 23?5 (2000) (an excellent reference).
1. It is strongly recommended that ES cultures be monitored daily. Mouse ES cells require far more attention than most other tissue culture cells. They are unlikely to be recovered once allowed to differentiate.
2. Those actions that will likely cause Mouse ES cells to differentiate include but are not limited to:
1. Plating too sparsely (<0.5x105 cells per cm2).
2. Plating too densely (>4x105 cells per cm2).
3. Failure to change the media when needed. Strive to keep the pH indicator red to red-orange at all times, never yellow.
4. Allowing the cells to get too confluent.
5. Allowing the culture to consist of a few large colonies rather than several smaller colonies.
6. Allowing the colonies to get too large.
7. Failing to add the appropriate amount of LIF (Leukimia Inhibitory Factor) to the culture media.
3. Mouse ES cells maintain their pluripotent state best when seeded as a single cell suspension at a rate of approximately 1.5x105 - 4x105 cells per cm2.
4. It is likely you will have to split your cells every two to four days.
5. Generally Mouse ES cells are split no less than 1:4 and no more than 1:10. Please refer to the Photo Gallery for visual guidelines of cell density and differentiation.
Thawing Mouse ES cells
1. Using the predetermined surface area that the cryotube of Mouse ES cells will cover (see surface area table), decide which size plate you will be seeding the cells onto. Label the plates accordingly. NOTE: the area should already be coated with feeder cells.
2. Place 5ml of ES cell media into a 15ml conical tube.
3. Remove the cryotube from the freezer and place it IMMEDIATELY into the 37°C water bath making sure not to submerge the cryotube above the base of the cap.
4. Carefully monitor the media in the cryotube until there is only a small crystal of ice present. At this time take the cryotube into the hood.
5. Using a 1ml pipette, carefully pipette the media up and down two to three times ensuring that any cells settled to the bottom of the cryotube are resuspended (the ice crystal will have melted by now).
6. Place the Mouse ES cells into the 5ml of ES cell media you have placed in the 15ml conical tube and mix gently.
7. Pellet the cells by centrifugation at approximately 340xg for 3 minutes. Be careful not to spin the cells to forcefully as this will damage them.
8. Carefully aspirate the supernatant.
9. Resuspend the pellet in the appropriate amount of media and aliquot to the predetermined surface area.
Feeding Mouse ES cells
1. Mouse ES cells will generally require feeding every day.
2. Carefully aspirate the old media from the plate by placing your pipette along the side of the plate, well, or flask while tilting.
3. If you detect a large amount of dead cells or debris on the plate (as is commonly the case the day after thawing) you can gently rinse the cells with PBS and aspirate. CAUTION: Mouse ES cells can easily be washed off of the feeder layer.
4. Apply fresh ES cell media to the plate by placing your pipette along the side of the plate, well or flask and gently dispensing the media.
Splitting Mouse ES cells
1. Mouse ES cells generally should not be split at ratios lower than 1:4 or higher than 1:10. Cells should be split when they reach ~80% confluency. Please see the photographs in the photo gallery taken at 48 hours, of Mouse ES cells plated at 4x105 cells per cm2 for an approximation of their appearance.
2. Aspirate the media from the tissue culture dish.
3. Gently rinse the dish with PBS to remove any residual serum (the presence of serum will inactivate trypsin).
4. Add enough trypsin (0.25% Trypsin in 0.1% EDTA) to just cover the surface area (for example: 1ml of trypsin will cover a 100mm diameter dish).
5. Place the dish in the 37°C, 5% CO2, humidified incubator for 3-5 minutes or until cells release from the bottom of the dish (this should be visible with the naked eye). Beware that extended exposure of Mouse ES cells to trypsin is toxic. Keep the time to a minumum.
6. Remove the dish from the incubator and observe the cells under the microscope. Ensure that colonies have released from the plate and have separated into single cells. You may have to apply some gentle agitation in order to break up the colonies.
7. Continue with the trypsinization until approximately 70% of the cells are single cells.
8. In the hood, add 6-10 times the volume of ES cell media as trypsin (for example: if you added 1ml of trypsin you now need to add 6-10ml of ES cell media).
9. Pipette the cells up and down a few times while rinsing the dish in order to ensure that all the culture is in a single cell suspension.
10. Collect the cells into a clean tube.
11. Centrifuge at no more than 340xg for approximately 3 minutes to pellet the cells.
12. Resuspend the cells in the desired volume of ES cell media and aliquot to new dishes. Make sure the cells are evenly dispersed across the surface area of the dish. Do this by gently moving the dish back and forth and side to side, never swirl in a circular manner, as this will cause the cells to accumulate around the perimeter of the dish.
Freezing Mouse ES cells
1. Mouse ES cells are generally frozen down and thawed from and to the same surface area provided the area was confluent at the time of freezing. For example: an 80% confluent 100mm dish (having 55cm2 of surface area) can be frozen down into 1ml and thawed back to a total surface area of 55cm2.
2. Mouse ES cells can be frozen at any time, although generally cells are frozen down when they would normally need to be split (~80% confluency).
3. To freeze ES cells; first typsinize the cells into a single cell suspension. Pellet the cells by centrifugation as above. Freeze the cells down in a 4:6 ratio of ES cell media to ES cell freezing media. For example: first resuspend the ES cell pellet in 0.4ml ES cell media. Add 0.6ml ES cell freezing media for a final volume of 1ml. Mix, and place 1ml into a labeled cryotube.
4. Place the cryotubes containing the cells to be frozen into a storage box, place the box into a cooler, place the cooler in a ?0°C freezer and leave them overnight. Transfer the cryotubes to cryostorage at -140C (typically under liquid nitrogen). This process allows the cells to undergo a slow decline in temperature until frozen.
Corning or Costar Flask size Growth Area (cm2)
T25 25
T75 75
T150 150
T162 162
T175 175
T225 225
Treated Tissue Culture Dish Size (mm) Growth Area (cm)2
35mm 8
60mm 21
100mm 55
150mm 148
245mm 500
Plate Size Growth Area (cm2) for single well Growth Area(cm2) for entire plate
6 well 9.5 57
12 well 3.8 45.6
24 well 1.9 45.6
48 well 0.95 45.6
ES Cell Media Recipes
ES Media ES Freeze Media
Component 1 liter 500ml 250ml 500ml 250 ml 100 ml
DMEM 800ml 400ml 200ml
FCS 150ml 75ml 37.5ml 410ml 205ml 82ml
L-Glu 20ml 10ml 5ml
NEAA 10 5 2.5
MaPyr 10 5 2.5
P/S 10 5 2.5
b-me 7µlW 3.5µlW 1.75µlW
LIF † † †
DMSO 90ml* 45ml* 18ml*
Note: FCS needs to be heat inactivated for ES freeze media
Heat inactivation is accomplished by incubating FCS at 56°C for 30 minutes followed by a gradual cooling to room temperature.
* When making ES freeze media, always place the heat inactivated FCS into the bottletop filter prior to the addition of DMSO.
† LIF should always be added to ES cell media. If using commercial LIF the exact amount will be indicated with the product. If using non-commercial LIF the amount will have to be determined empirically by titration.
W Never add b-mercaptoethanol directly to a bottletop filter. Only add it in conjunction with other fluids.
Abbreviations:
DMEM - Dulbecco抯 modification of eagle抯 medium, FCS - fetal calf serum, L-Glu - L-glutamine, NEAA - non-essential amino acids, NaPyr - sodium pyruvate, P/S - penicillin streptomycin, b-me - beta mercaptoethanol, LIF - leukemia inhibitory factor, DMSO - dimethyl sulfoxide
Some notes on strain background
After 20 generations of brother sister matings, on average at least 98.6% of the loci in each mouse are homozygous. The following information concerning strain background is taken from: J A Beck, S Lloyd, M Hafezparast, M Lennon-Pierce, J T Eppig, M F W Festing & E M C Fisher Geneologies of Mouse Strains, Nature Genetics 24, 23?5 (2000) (an excellent reference).
