Preparation of embryonic fibroblast (EF) cells
“Good” EF cells are one of the keys in maintaining totipotence of ES cells.
Although other cell lines such as STO cells or high concentrations of LIF
have been used to substitute for EF cells in ES cell culture, we found EF
cells give very satisfactory and consistent results. If you prefer using
STO cells as feeders, make sure you obtain them from good sources.
EF cells are cultured in HEPES-buffered (pH 7.3) DMEM supplemented with
10% fetal calf serum and antibiotics (see below for the details of medium
preparation). EF cells are isolated from mouse embryos at embryonic
days 12-14. Briefly, EF cells are prepared with the following procedure:
(1) Isolate embryos and wash once in HEPES saline, dissect individual
embryos by removing the head and vicerating soft tissues (e.g. liver,
heart and other viscera) and wash the carcasses twice in HEPES buffer.
(2) Mince the embryo carcasses into fine pieces in a small volume of
trypsin/EDTA solution (just cover all the embryos), add more trypsin/EDTA
solution (2 ml trypsin solution in total for 10 embryos) and mix well
with the embryo tissues and incubate the mixture at 37°C for 30 min.
(3) Add 10 ml DMEM plus 10% FCS and transfer digested tissues into a
50 ml tube and dissociate the tissues by vigorous pipetting up and down.
(4) Allow the large pieces of tissue debris to settle down and transfer
the supernatant into a clean tube. Add another 10 ml medium and pipet
the tissues to obtain more dissociated EF cells and repeat this step
three or more times. (5) Combine all the supernatant and plate out the
cell suspension into T75 (growth area is 75 cm2 ) flasks
(about 1 embryo per flask); freeze one confluent flask of EF cells into
two vials. To prepare mitotically inactive feeder cells, primary EF cells
are expanded for several passages and treated with mitomycin C (Sigma)
at 10 µg/ml for 2-4 hrs. or subjected to gamma-irradiation
(2000-3000 rads) (see below for protocol for EF cell expansion).
Aliquots of mitotically inactivated EF cells are frozen at -70°C for
several months or in liquid nitrogen for years.
**Do not over-expand the EF cells. We use morphological criteria for
scoring good feeders. The cells should have longitudial or long spindle
shape (skinny) and grow compact. When individual cells become enlarged
with a flat appearance (fatty), they are senescent and no good. The
density of EF cells in culture is important. If the density is too low,
the EF cells easily become senescent.
Expansion, irradiation, and freezing of EF cells:
- Plate 1 vial primary embryonic fibroblast cells in one T75 flask with 20 ml DMEM, 10% FCS (1 vial primary fibroblast is derived from about 1/2 embryo).
- Split 1 T75 flask of cells into 2-3 T75 flasks.
- Expand each T75 flask of cells into one T175 flask (growth area is 175 cm2 ). Each T175 flask of cells can be further split into 2-3 T175 flasks. [** It is important to note that the number of passages is not important. The most IMPORTANT thing is to monitor their growth and morphology.]
- Trypsinize and pool all the cells, spin down and resuspend in 40-50 ml of DMEM containing 10% FCS
- Irradiate cells (gamma-irradiation, 2000-3000 rads)
- Spin down and resuspend the cells in DMEM, 10% FCS. Normally, cells from one T175 are frozen into two vials (0.5 ml/vial). So, the cells from one T175 flask are resuspended in 0.5 ml of DMEM. Add equal volume of 2X freezing medium, mix and freeze for overnight in 0.5 ml aliquot at -80°C and then store in liquid nitrogen.
[Feeder cells from each vial are sufficient for 85 cm2 growth area. Since the recovery and plating efficiency may not be 100%, this amount of feeder cells is normally plated onto 1 X T75 or 3 X T25 flasks. You may adjust these numbers according to the plating efficiency. I have complied a list of growth areas for commonly used flasks and plates in the section for Solution Preparation (see below).]
Thawing and culturing EF and ES cells:
- Day 1: Coat T25 flask with 0.2% gelatin (3-4 ml, 5 min, at RT.). Thaw EF cells rapidly in 37°C water bath. Transfer the cells to a 15 ml tube with 5 ml EF cell-medium, Spin down at 1000 rpm for 5 min. Resuspend cells in appropriate volume. Transfer the cells into T25 flask (end volume 5-6 ml)
- Day 2: Thaw ES cells rapidly in 37°C water bath. Transfer the cells into a 15 ml tube with 5 ml ES cell-medium, Spin down for 5′ at 1000 rpm. Resuspend cells in 6 ml ES cell medium. Transfer the cells into a T25 flask that contain feeder cells
- As EF cells settle dowm much faster than ES cells, you may thaw and culture both EF and ES cells at the same time. But if you do not know whether your EF cells quality and their density, it is better to plate out EF cells one day before culturing ES cells]
- Re-feed DAILY with 6 ml ES cell-medium. NEVER LET ES CELLS OVER-GROW. Split the cells when confluent. Do not wait for another day. ES cells won’t wait for you.
Passaging of ES cells
Prepare fresh flasks at least one day before splitting ES cells: gelatin-coated and plated feeders; alternatively, the feeders can be plated out with ES cells on the same day since the feeder cells normally seed down on the plate before ES cells do.
- Wash T25 flask with 3-4 ml HEPES
- Add 0.5 ml trypsin that has been warmed at 37°C
- Incubate 5-7 minutes at 37°C. Monitor how well the cells are trypsinized under the scope. Depending on particular batch of trypsin, the time required for trypsinization may vary. Do not over-trypsinize the cells.
- Add 6 ml ES cell-medium
- Triturate the ES cells to single-cell suspension with a pipet
- Transfer to a 15 ml tube
- Spin down for 5′ at 1000 rpm
- Resuspend cells in appropriate volume ES cell-medium
- Transfer cells to appropriate flasks, usually 4-5 times the original surface
Freezing
The cell pellet from a confluent T25 flask is resuspended in 1 ml of medium, add 1 ml of 2X freezing medium, mix, and aliqot 0.5 ml. Freeze overnight at -80°C and then transfer into liquid nitrogen freezer
Preparation of DNA for electroporation
*Linearize vector DNA (per electroporation) with appropriate enzyme. Use 2-5-fold excess restriction enzyme, digest during 1 hour. Check DNA digest on agarose gel. Phenol/chloroform/iso-amyl-alcohol (24:24:1) extraction. Chloroform/iso-amyl-alcohol (49:1) extraction. Transfer supernatant from last extraction to screw tube in hood. Add 1/10 volume 3 M NaOAc and 2 volumes 100% ethanol. From this point on, treat as tissue culture sterile. Incubate for 15-30′ on dry ice and spin: 10′ max. rpm. Wash pellet with 70% ethanol in hood. Dissolve the pellet in H2O in hood (final concentration 1µg/µl). Let DNA dissolve at R.T.
Electroporation of ES cells
- Day before electroporation: prepare 10 cm dishes coated and plated feeders, 8-10 ml medium per plate
- Prepare: A confluent T25 flask will give rise to approximately 10X106 ES cells. Prepare cell suspension at 20 x 106/ml. The cuvette (Bio-Rad, 0.4 cm electrode) used for electroporation will hold approximately 0.8 ml each. We normally plate out 2-4 x106 ES cells onto a 10 cm plate for selection after electroporation.
- Electroporation
- Wash T25 flask with 3-4 ml HEPES
- Add 0.5 ml trypsin
- 5-7′ 37°C
- Add 5 ml ES cell-medium
- Resuspend cells well with 5 ml ES cell-medium and transfer to a 15 ml tube
- Spin: 5’1000 rpm
- Wash cells once with 5 ml electroporation buffer and resuspend the cells in electroporation buffer
- Add DNA (25-50 µg/ml) to the cells and mix well
- Transfer the cells to electroporation cuvet
- Electroporate, 400 V, 25 µF or 800 V, 3 µF (time constant usually 0.4 – 0.5 msec)
- Incubate for 10 min at R.T.
- Plate out the cells onto 10 cm feeder plates (2-4 X106/plate)
- Apply selection 24 hours after electroporation: Neo (G418): 50 mg/ml (active form) = 250 x stock
- **Before making up G418 stock, it is important to calculate the percentage of the active form of G418 in the crude powder for that specific lot, e.g. 740 mg active form/g
- Puro: 2 mg/ml = 1000 x stock
Hygro: 100 mg/ml = 500-1000 x stock
- Re-feed cells daily (with selection medium)
- Pick colonies on day 7-9 after electroporation.
Picking ES Clones, Expanding Them, and Freezing Expanded Clones in 96 Well Plates
Day 1
Materials for Day 1:
>
- 10 cm plates containing ES colonies ready for picking
- HEPES wash
- 96 well V bottom plates for trypsinizing colonies
- P20 pipetman with tips in racks
- 12 place multiwell micropipetor
- 0.25% trypsin in HEPES
- 96 well flat bottom plates containing preplated feeders
- ES cell media
- EF cell media
- Feed colonies on plates 1-2 hours before picking them.
- Add to every well in the 96 well V bottom plates, 10 µl HEPES.
- Wash colonies on 10 cm dish with HEPES.
- Pick colonies with P20, picking up to 10 µl HEPES with the colony
- Transfer colony to a well in the 96 well dish containing HEPES (the volume of HEPES is 20 µl now).
- Pick 12- 24 colonies this way.
- Add to the picked colonies 20 µl of trypsin with the 12-place pipetor.
- Incubate at 37°C for 5 minutes.
- Add 100 µl ES media to trypsinized colonies with the 12 place pipetor.
- Pipet cells up and down several times to get single cell suspensions.
- Transfer the cells to a 96 well flat bottom plate containing feeders in 100 µl ES medium, feed the ES cells in 96-well plate daily with 100 µl ES media for 2-3 days. Normally the cells will become confluent after 2-3 days in culture
Day 2-3
ES cells are trypsinized as described above. One third (1/3) of the cells are transferred into a 96-well plate that contain EF cells. Two third (2/3) of the cells will be transferred into a gelatinized 24 well plate (for DNA) without feeders in the presence of EF cell media. Feed the ES cells in 96-well plate daily with 100 µl ES media. 2-3 days later, this plate will be processed for freezing.There is no need to refeed the cells that are expanded for DNA. I usually add 1 ml of EF cell medium in the beginning and wait until the color of medium becomes yellow, which indicates that cells are ready for DNA preparation. [If there are no sufficient ES cells grown in the well for DNA preparation, you could increase ES cells by simply trypsinizing the existing ES cells and growing them in the same well.]
Freezing Cells on Day 4-6
Materials for Day 4-6:
- HEPES
- 0.25% trypsin in HEPES
- ES media
- 2X freezing solution:
- 20% v/v DMSO
- 20% v/v FCS
- 60% v/v DMEM
- Parafilm
- Hybridization bags
- Wash cells in 96 well plate with hepes wash.
- Add 20 µl trypsin, 37°C 5 minutes.
- Add 30 µl ES media, pipet up and down to get a single cell suspension.
- Add 50 µl 2X freezing solution, pipet up and down 2X to mix.
- Wrap edge of plate in parafilm, seal in hybridization bag, freeze at -70°C.
DNA isolation and analysis
- Remove medium from 24-well dish
- Add 0.5 ml lysis buffer (add proteinase-K fresh)
- Incubate at least 4 hours, back in usual incubator or until all wells are lysed (Lysed cells may be in incubator, together with growing colonies for days)
- Put plates onto a horizontal rotator (tape down, be careful)
- Rotate 30′
- Add 0.5 ml isopropanol/well
- Rotate until DNA precipitate is completely dehydrated
- Transfer DNA to 100 µl TE using a yellow tip. Try to avoid to take two much liquid containing the isopropanol that may inhibit restriction enzyme digestion or PCR
- Dissolve overnight at 55°C on a rocker
- Typically 15 µl is used for a restriction digest, using 50 units restriction enzyme.
We normally set up restriction digestion or PCR in a 96-well plate with the aid of 12-channel pipetor
- For Southern blotting analysis, the digested genomic DNA is separated on a 0.7 % agarose gel in TAE at 100V running for about 5 hours
- The DNA is denatured and transferred to a Zetabind membrane using alkaline transfer procedure for 3-4 hr
- The DNA is crosslinked to the filter by using the Stratagene UV crosslinker (auto-crosslink step)
- Hybridizations are done overnight in Church buffer (typically in 5-10 ml of hybridization buffer at 65°C)
Recovery of positive ES cell clones
- After identifying positive clones by Southern blotting or PCR analysis, ES cells will be recovered. To recover ES cells,thaw the cells by adding warm media directly into wells that contain positive ES cells.
- Transfer thawed cells to a tube containing enough feeders for a single well of a 48 well dish.[By mxing with EF cells, the recovery of ES cells is increased.]
- Spin down cells (by co-spinning feeders and ES cells you minimize loss of the small number of ES cells you’ve frozen).
- Resuspend cells in ES medium, transfer to a gelatinized well of a 48 well plate.
Solutions and growth factor
0.2% gelatin
Plates for ES cell culture are coated with 0.2% gelatin (Sigma) which is made by dissolving gelatin into water, and the solution is autoclaved and stored at 4°C. You may want to filter the solution through 0.2 micron filter
DMEM
See additional protocol for making DMEM and how to adjust osmolarity to 290 mmol/Kg
ES cell-medium
|
Final conc |
| 450 ml DMEM |
|
| 75 ml FCS |
15% |
| 5 ml non-essential amino acids (0.01 M) |
0.1 mM |
| 5 ml Pen/Strep |
|
| 4 µl β-mercaptoethanol (Sigma) |
0.1 mM |
| 0.125-0.25 ml of LIF (106 Units/ml) |
250 – 500 Units/ml
1000 Units/ml when growing without feeders |
LIF is obtained from Gibco/BRL in 10×106 units/vial. Reconstitute in DMEM +10% FCS (or EF cell medium). Aliquot 0.5 ml and store at 4°C.
2x freezing medium (1x = 10% DMSO, 10% FCS in DMEM)
Mix 60 ml DMEM well with 20 ml DMSO. Medium will become warm (exothermic), allow to cool before adding 20 ml of FCS. Filter and aliquot 5 ml
Electroporation buffer
- 20 mM HEPES (pH7.0)
- 137 mM NaCl
- 5 mM KCl
- 0.7 mM Na2HPO4
- 6 mM glucose
- 0.1 mM β-mercaptoethanol [add fresh before use, if older than 1 month; To make 10 mM stock (100X), add 7 µl of β-mercaptoethanol into 10 ml of H2O]
- Store at 4°C
Lysis buffer
- 100 mM Tris-HCl pH 8.5
- 5 mM EDTA
- 0.2 % SDS
- 200 mM NaCl
- 100 µg/ml Proteinase-K (add fresh)
Trvpsin (high concentration) (0.25 % trypsin, 1 mM EDTA in HEPES)
- 5 ml of 2.5% trypsin
- 45 ml of HEPES
- 100 µl of 0.5 M EDTA
- Filter sterilize and freeze in aliquots at -20°C
HEPES (HEPES-buffered saline
- 121 mM NaCl
- 5.4 mM KCl
- 0.44 mM KH2PO4
- 0.30 mM Na2HPO4
- 5.56 mM glucose
- 20 mM HEPES (pH 7.3)
- Phenol red.
- Osmolarity: 290 mmol/kg
Volumes and growth area for commonly used plastic flasks and plates
|
cm2 |
Medium |
Wash |
Trypsinize |
| T25 |
25 |
5 ml |
3-4 ml |
0.5 ml |
| T75 |
75 |
15 ml |
10 ml |
1 ml |
| T175 |
175 |
50 ml |
20 ml |
2 ml |
| 10cm dish |
60 |
8 ml |
4 ml |
1 ml |
| 24 wells |
2 |
1 ml |
0.5 ml |
2 drops |
| 12 wells |
3.8 |
2 ml |
1 ml |
3 drops |
| 96 wells |
0.32 |
0.15 ml |
0.2 ml |
20µl |
| 48 wells |
1 |
1 ml |
1 ml |
0.1 ml |
| 4 wells |
1 |
1 ml |
1 ml |
0.1 ml |
| 6 wells |
10 |
4 ml |
2 ml |
4 drops |
