Blastocyst 3.5 days Generating ES Cells 30 cells Harvest blastocysts on day 3.5 by flushing from the uterine lumen with Inner Cell mass Plate out blastocysts in individual 10 mm dishes (128 cell stage) on mitotically inactive MEF or STO feeder cells to provide liF and other factors 1-totipotent After 1-2 days, blastocysts hatch(A) and attach to the dish by migration of 2-tissue culture the trophectoderm TE), while the nner cell mass(ICM)grows(B) 3-Transfectable After about 96-120 hours in culture (C, D), the ICM can be dislodged from the TE layer, washed and transplanted to microdrops of medium containing trypsin to disperse the 4-Selection clumps 5- Differentiation Transfer disaggregated contents to a fresh feeder cell tissue culture well and inspect daily for signs of In vitro differentiation. Primary cell colonies are clearly visible asBlastocyst 3.5 days Inner Cell Mass (128 cell stage) 1-totipotent 2-tissue culture 3-Transfectable 4-Selection 5- Differentiation In vitro Harvest blastocysts on day 3.5 by flushing from the uterine lumem with M2 medium Plate out blastocysts in individual 10 mm dishes on mitotically inactive MEF or STO feeder cells to provide LIF and other factors After about 96-120 hours in culture (C, D), the ICM can be dislodged from the TE layer, washed and transplanted to microdrops of medium containing trypsin to disperse the clumps After 1-2 days, blastocysts hatch (A) and attach to the dish by migration of the trophectoderm (TE), while the inner cell mass (ICM) grows (B) Transfer disaggregated contents to a fresh feeder cell tissue culture well and inspect daily for signs of differentiation. Primary cell colonies are clearly visible as distinct clumps Generating ES Cells ~30 cells