ES Cell Culture and Manipulation
Culturing ES cells
High glucose DMEM (-pyruvate, -glutamine)
20% Heat-inactivated Fetal calf serum (can vary by cell type, be sure!!)
1X Non-essential amino acids
1/100 volume ß-ME stock (stock is 7µl ß-ME in 10ml DMEM)
1:1000 dilution of LIF (add 500 µl 720LIFD cell conditioned media to 500ml media)
Thawing frozen vials of ES cells
Mic's Notes: Typically, one vial contains one confluent plate of cells, frozen down. Depending on how well the freezing works one vial is typically enough for 4-12 plates. For quick recovery, plate out less. For maximum expansion plate out more. 10 plates usually is borderline, go 1:12 ONLY if you know that the recovery will be very high.
Plating ES cells
See above for notes on splitting ES cells, frozen. Generally, ES cells are split 1:6-1:10. This depends on how "confluent" the plate is. Since ES cells grow as discrete colonies, you should NEVER get a plate that is confluent in ES cells the way fibroblasts are. You wish to split them when a colony is approximately 1-2 times the diameter of a stretched out fibroblast (STO cell). If the plate is densely covered with colonies, they may be split 1:10, or slightly greater. If there are a lot of colonies, split them 1:6-1:8. For a plate that has only a few colonies (12-20), allow them to get slightly larger than normal, and then split 1:4. To split, simply add 1ml. of the appropriate dilution of cells to a 100cm. plate containing a feeder layer, and 4 ml. of ES cell media.
Splitting ES cells
When ES cells are "confluent" and ready to split (see above) feed them with fresh media an hour or two before splitting. Aspirate media and wash cells once briefly with 1X trypsin-EDTA, aspirate. Add 0.5ml 1X trypsin-EDTA (for one well of a 6-well, 2 ml. for a 100cm plate). Then, add .5 ml. pre-warmed (37°C) trypsin to a 6-well, or 3ml. to a 100cm. and put at 37°C for 5-6 min. You wish to minimize this time, so monitor the cells carefully. When cells are loosened add 0.5ml ES cell media to the well with a 5 ml pipette. Attach a sterile yellow tip to the end of a 10 ml sterile plactic pipette and pipette cells up and down several times -- ES cells are sticky and this is necessary to achieve a single-cell suspension to prevent differentiation after plating. For a 100cm. plate, transfer the cells to a Centrifuge tube containing an equal volume of media:trypsin. Pipette back and forth at least three times using a sterile yellow tip attached to the bottom of a 10ml. pipette. Transfer cells to a l5ml centrifuge tube, add another 5 ml ES cell media and spin 2 min at 2000 rpm. For a 100cm. plate, add another trypsin volume's worth of ES cell media to the centrifuge tube, and mix. This may require the use of a 50 ml centrifuge tube. Spin 2 minutes at 2000 rpm. Aspirate and resuspend cells in ES cell media, plate onto fresh feeder cells (remember to be careful about the split ratio. ES cells will grow slowly, or not at all, if they are not dense enough. Unless the plate that you are splitting from is extremely dense, split cells no more than 1:10)
Freezing ES cells
Freeze as for STO/SNL cells, ES freezing media is regular ES media with 20% FBS and 10% DMSO. It is very important that the cells be cooled slowly, to prevent the formation of ice-crystals within the cells. This can be accomplished one of two ways. One way, is to freeze the cells at -20°C for an hour, and then transfer them to a -70°C overnight. The next day, move them to liquid nitrogen. An alternate way to freeze cells, if to use a cell-culture cooler (simply a thick styrofoam box, with a few holes poked in it). Place the cells inside the cell-culture cooler, and place at -70°C overnight. The next day, transfer the cells to liquid nitrogen.
Transformation of ES cells
Preparation of targeting DNA
Digest 100µg of targeting construct DNA with an enzyme to linearize Check 0.5µl on a gel to make sure it's completely digested. Extract DNA 2X with phenol/chlorofom. Add .15 volumes 2M NaOAc and 2 volumes EtOH to precipitate (the DNA will probably come right out of solution so freezing isn't needed). Pellet the DNA, wash with 70% EtOH and dry the pellet. Resuspend the DNA in 100µl sterile H20 or TE (you should be working in the hood here). Check 0.5 µl DNA on a gel, and make sure that the concentration is know.
Preparation of cells for transformation
Picking ES cell clones
One or two days before picking colonies prepare 24-well plates of feeders. You can also use alternate protocols that utilize 96-well plates, small tissue culture dishes, or 6 well plates, if you prefer. To pick clones you will need: fresh trypsin-EDTA, a P-200 pipetman and sterile yellow tips (preferably barrier, or plugged tips), a sterile 96well round-bottom plate, plenty of ES cell media, and an inverted microscope in a hood. Aspirate the REF media from the 24-wells and add 2ml of ES cell media to each well. Add 40 µl trypsin-EDTA to as many wells of the 96-well plate as you plan to pick clones. Working with one 10cm plate at a time, wash the plate with 2ml trypsin and then add another 2ml. Using the P-200 pick up a small amount trypsin (~5µ1) from the edge of the plate then pick up a well isolated colony in a minimal amount of trypsin. You can dislodge the colony slightly with the pipette tip. Transfer the colony to a well of the 96-well plate and number this well so you don't mix clones together. Repeat for each colony to be picked from this plate. Pick all sizes of colonies but you will have to work fast before the plate dries out. If clones become too loose and start to move around you can reduce trypsin to lml or skip the trypsin rinse. After picking the first 1/3 of the clones they must be broken up into a single-cell suspension -- this is very important because any clumps of cells will begin to differentiate, often within the first few days! Use the P-200 with yellow tips to pipette each clone many times (10-20X, try to keep number of bubbles down) and check individually under the 4X microscope to be sure no clumps are left. Transfer each disaggregated clone to a numbered 24-well feeder (the 2ml media will neutralize the trypsin). Notes: 1.) Change yellow tips for each clone at each step, you don't want to cross contaminate them 2.) The ES cells can take more pipetting and longer times in trypsin than you would expect, however work quickly and with one plate at a time so trypsin exposure doesn't become excessive 3.) I usually check all clones visually under the scope ~ every 2 days and if some are growing clumpy they may need to be split 1:1 before they are actually confluent.
Expansion of ES cell clones
Change the ES cell media of each picked clone the following day (now you only add lml per well) and every 1-2 days until the clones begin to become confluent (as evidenced by yellowing media) in ~5 days. When a clone is confluent it is trypsinized and split to one well of a fresh 24-well feeder and a gelatinized well of a 6-well plate, be sure to make the 24-well feeders ahead of time. Trypsinize by rinsing with 0.5ml trypsin-EDTA, aspirate and add another 200µl trypsin for ~10 minutes. Add 800µl ES media with a lml pipette with a yellow tip and pipette several times to break up cells. Transfer to a sterile l.5ml eppendorf tube and centrifuge ~2min at 3000rpm. Aspirate media and use a P-200 to resuspend pellet in 200,u1 ES media, transfer to feeder and gelatin wells (~140µl to feeder well and 60µ1 to gelatin). Feed the 24-well feeder plate the following day and by the next day the cells should be yellowing the media and are ready to be frozen (see freezing protocol below). I don't usually refeed the gelatin plate, just scrape the cells for DNA in several days when media is yellow. Note: If you have picked many clones the above method may be impossible to follow time-wise. A way to shorten the protocol is to eliminate the centrifuge step and just add the lml trypsin/ES mix to the two plates (700µ1 to feeders and 300µl to gelatin). Even this small amount of trypsin may make the clones grow a bit slower initially but if you change the media early the next day they seem to do OK and this saves a lot of time.
Freezing of ES cell clones in 24-well plates
When the 24-well feeder plates of clones begin yellowing the media they are ready to be frozen (~2 days). Make ES cell freezing media ahead of time and chill in fridge or on ice Also prechill a small styrofoam box at -80°C (the boxes NEB ships enzymes in are a perfect size, or you can hollow out the styrofoam trays from 15ml centrifuge tubes and make a sort of box out of them. This box is identical to the ES cell freezing box described above). Aspirate the media from a 24-well plate of clones and add 400µ1 prechilled freezing media to each well Wrap the plate well in double parafilm; if freezing multiple plates place on ice until all are done. As quickly as possible move plates into prechilled box in -80° freezer. Freeze plates several hours or overnight at -80° then move box to a -135°C freezer for long-term storage (i.e. liquid nitrogen, if possible). Cells can be kept long-term at -80° but it should be in a freezer that is rarely opened since the worst possible thing is repeated freeze-thaw!
Scraping ES cells from gelatin for DNA
When cells are confluent and media is good and yellow scrape cells to make DNA. Aspirate media and carefully rinse each well 2 times with lml of incomplete PBS. Add another lml PBS and scrape cells with a rubber policeman, transfer to an eppendorf tube and freeze at -20°C until ready to extract DNA.
Gelatin treatment of Tissue-culture plates
Make a 0.1% solution of gelatin (Sigma # G-1890) in Milli-Q water and autoclave, cool. Add enough gelatin to each plate, or well of a plate, to cover the bottom and let sit at least 5 minAspirate and add STO/SNL media.
Extraction of DNA from ES cells
Rapid Preparation of DNA from ES cells in 24-well tissue culture dishes
This simple method based on a protocol described by Miller et al. (1988) involves salting out cellular proteins with a saturated NaCl solution. It does not require extraction with phenol. Sufficient DNA can be obtained by this method for screening by Southern blot analysis (3-5 µg)
Materials And Equipment
Caution: Wear a mask while weighing SDS.
1. Aspirate medium from each well containing nearly confluent ES cells and rinse the cells once in PBS. Resuspend the cells in 200 yl of Iysis buffer. Incubate the dish for several hours to overnight at 55°C.
2. Transfer to 1.5-ml eppendorf tubes. After digestion with lysis buffer is complete, add 100 µl of saturated NaCl to each tube and shake vigorously (do not vortex, to avoid shearing fragile genomic DNA).
3. Centrifuge the tubes at 3000g in a microfuge for 15 minutes. Transfer the supernatant containing DNA to a fresh 1.5-ml polypropylene tube and add 2 volumes of ethanol at room temperature. Invert the tube several times until the DNA precipitates and then remove it with the disposable tip of a 200-µl pipetteman or with a glass rod. Rinse the pellet in 70% ethanol and resuspend it in 50 µl of TE. Allow the pellet to dissolve at room temperature for several hours.
Preparation of LIF Conditioned Media
The 720 LIFD cell line has been transformed with a plasmid that causes them to secrete a high level of LIF. For unknown reasons, this causes the cells to grow very slowly. They are also more easily contaminated (in my hands) than normal STO cells. Also, the cells will look sick, compared to normal STO cells. They tend to be slightly smaller, and much more vacoulated.
GIBCO a-MEM (-l-glutamine)(720LIFDs are fussy about the brand of media, they don't like Sigma)
10% Heat-inactivated calf serum
DO NOT add l-glutamine, the cells don't like it!
Growing 720LIFD cells
Grow cells on gelatinized 10cm tissue culture plates (see STO/SNL protocol for gelatinizing). Change media every 2 days, the cells will grow slowly, and look messed up. This is normal for this cell line. Split cells as soon as they become confluent.
Harvesting LIF conditioned media
When cells are almost confluent (~80-90%) change the media. The following day collect the conditioned media in a 50ml centrifuge tube(s)Spin at 2000 rpm for 5 min to remove any cells. Filter through a 0.2µ filter and aliquot into cryovials or parafilmed-centrifuge tubes. Store the stock LIF at -80°C. You can usually add fresh media to these cells and harvest a second batch of LIF the next day. Throw these cells out when you are done as, they have now been confluent for ~2 days. LIF conditioned media CAN be stored short term (a few moths) at -20°C.
Plate 1 x 104 feeder-free ES cells in each of the wells of a gelatinized 6-well in ES media without LIF. At plating add LIF at 1:100, 1:500, 1:1000, 1:2500, 1:5000 and 1:10,000 dilutions from stock. Check for differentiation over a 1 week period. Select the lowest dilution than that which allows any amount of visible differentiation. Usually 1:2500 or 1:5000 is best(If the ES cells are becoming confluent in less than 1 week you may need to plate a smaller number)
Notes: The cells can occasionally be reselected in 0.1mM methotrexate to make sure they are still stably transformed for the LIF transgene. When growing in methotrexate the calf serum must be dialyzed.
Care and Handling of Feeder Layer Cells
STO/SNL cells are a derivative of the standard STO cell line. They are transformed with a LIF producing plasmid (low-level). They grow quite well, and look slightly more vacoulated than regular STO cells. This line was made available to use from the Bradfield lab.
Add all components to DMEM, sterile filter if desired
Thaw frozen vial of cells
Add all components to DMEM, sterile filter if desired
Thaw frozen vial of cells
Splitting STO/SNL cells
Freezing STO/SNL cells
Trypsinize either regular or MitoC-treated STO/SNL cells as aboveAfter centrifuging resuspend cells in freezing media (STO/SNL media with 20% NCS and 10% DMSO)Volume should be approximately lml per plate of cells, aliquot to cryovials adding lml per vialFreeze vials immediately at -20°C for ~2 hr, then at -80°C overnight, transfer to liquid N2 the next morning.
Growth-Arrest for Feeder Layers
Split cells as described above. However, instead of plating, transfer the cells (in media) to a sterile centrfuge tube. Parafilm the tube, and place the cells in a small ice-bucket. Bring the cells down to the gammacell (you MUST be certified to use the gammacell. See Rex for information about certification). Place the cells in the sample cavity (dont forget to sign in) and irradiate for 40 minutes. Wipe off the tube with 70% ethanol, and place the tube back in the hood. Plate the cells, using 1 full plate of cells for 1 plate of feeders. One 10cm2 plate makes enogh feeder cells for another 10cm2 plate, 2 24-well plates, or 2 96-well plates. Check the cells and change media (to ES cell media) the next day. It is not unusual to see some death, but normally the cells spread out enough that this does not cause problems. You want the cells to cover the entire plate, confluency is preffered. You do not wish plated ES cells to be exposed to open surfaces on the plate (although they generally only stick to the feeder cells anyway).
Plating MitoC treated feeder cells
A confluent 10 cm plate of STO/SNL cells will make: 2 10 cm plates, 2 6-well plates, 1 24-well of feedersAdd the appropriate MitoC-treated cells to gelatinized wells in STO/SNL mediaAllow 12 hours for cells to attach before using feeders. If after 12 hours feeder cells appear too thin more may be added.