Further passaging is done into ES cell culture medium on PMEFs. Following overnight incubation, the medium is replaced with SR medium. Feed daily thereafter, until confluent.
(29) Around day 11, wash an individual ES cell containing primary well with PBS.
(30) Apply 0.2ml of trypsin and return to incubator for 5 min.
(31) Apply 1.0ml of ES cell culture medium. Break up the well with a blue tip. Transfer entire contents of the well to the well of a fresh 4-well PMEF plate. {This 2° passaging step should seem very easy compared to the primary disaggregations}
(32) The next day, aspirate the medium and replace with SR-medium.
(33) Feed daily with SR-medium until confluent. At this point the well will contain an established individual ES cell line, and it should be monitored and passaged just like a normal ES cell line.
(34) Passage to 1 or 2 fresh 35mm PMEF dishes as appropriate. Employ ES cell culture medium for all trypsinization steps. Following overnight incubation, replace with SR-medium and feed daily until confluent.
(35) Generate at least ½ a confluent 35mm dish to create a ½ vial of cells for freezing. {That is an absolute minimum, the preference would be one or two full vials, each containing a confluent 35mm dish on PMEFs}
(36) At freezing take a small aliquot for DNA. Passage the aliquot onto gelatinized plastic with ES cell culture medium. Replace with SR-medium the next day and grow the DNA aliquot until confluent. Carefully observe the DNA aliquot. Cell-lines that will adapt off of feeders should retain normal ES cell morphology and should grow quickly. Cell-lines that will not adapt are very obvious when taken off of feeders, and should grow slowly.
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