On Day 6 of incubation (Wednesday) the embryo explants are ready for disaggregation. Each disaggregation is done individually. {This is the single hardest and touchiest part of the entire procedure and it normally takes ALL DAY}
For each embryo to be disaggregated, prepare a single receiving well of a 4-well PMEF plate. Replace the PMEF-medium with ES cell culture medium. Recipe for ES cell culture medium
Components:
ES cell culture medium/50 ml:
(17) For each well that will receive a disaggregated embryo explant, aspirate the PMEF-medium and replace with 1.0ml ES cell culture medium. {Do this first and allow the receiving plates to equilibrate in the incubator for a little while prior to use}
Prior to each individual disaggregation prepare the following: Pull 1 or 2 pasteur pipets. Go for a fairly wide bore and straight line (no kinks). {The explants are sticky and a wide bore prevents trapping}
Pull a small collection of embryo handling pipets, but use 6-inch stock. {I like these to have a little longer back end, in case I suck up too hard: 1° disaggregations can get rough and these explants are very precious}
Put a 50microliter drop of trypsin (Trypsin (1x), SAFC Biosciences [thru Sigma], 59428C-500ML) on a 35mm dish. Cover with sterile filtered mineral oil.
(18) Wash an individual embryo explant twice with PBS. Leave the second wash on the well.
(19) Visualize the explant under the tissue culture microscope (4x objective). Score around the explant with a pasteur pipet. Gently transfer the explant with an underlying fragment of monolayer into the drop of trypsin under the oil. Return the original 4-well plate to the incubator. {The explants look VERY DIFFERENT from conventional ICMs: much smaller and sometimes they consist of more than one rounded mass}
(20) Place the dish in the incubator and incubate for 5 min. {Prepare the pipets and trypsin you will need for the next explant during this time}
(21) Remove the trypsin dish and a fresh 4-well with ES cell culture medium from the incubator. Label the receiving well with the embryo's number and 1°.
(22) Take a long embryo handling pipet with a bore that is no wider than the explant. Suck up some ES cell culture medium from the receiving well. Visualize the trypsinized explant under the microscope and expel a small amount of FBS medium into the drop of trypsin, swirling it around the explant.
(23) Take the embryo handling pipet and suck up the trypsinized explant back and forth several times. The explant will start to disaggregate. This will often take a lot of suction, as the explant is very resistant to disaggregation. Reduce the explant into several smaller clumps of cells, but do not attempt to reduce it completely to single cells. {In fact, you probably couldn't break it down to single cells, even if you tried}
(24) Transfer the clumps of cells to the receiving well, gently distributing the clumps all over the monolayer, as visualized under the microscope. Return plate to incubator.
(25) Repeat these steps for each individual embryo explant. Alternate the culture and receiving plates so that no 4-well plate stays out of the incubator for a long time. Return all 1° disaggregations to the incubator. {Day 6}
(26) The next day (Day 7), replace the ES cell culture medium with 1.0ml SR-medium.
(27) Leave primary disaggregations undisturbed for 4-5 days. Monitor daily.
(28) Around Day 11 you should start to see clear nests of ES cells in some of the 1° wells. These can be passaged (see below). However, do not discard anything yet. Keep and eye on the primary disaggregations for 4-5 more days, as some of them will often begin to display ES cells. Passage these later lines as appropriate. After a week, if nothing shows up, discard the primaries.
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