Donated human embryos are cultured according to standard IVF protocols to the blastocyst stage. The method of immunosurgery is performed as described by Solter and Knowles (9). It involves the removal of the zona pellucida followed by incubation of the embryo in antibody solution that binds to the proteins of trophectoderm cells but not to the inner cell mass cells; then lysing trophectoderm cells with guinea pig compliment.
1. Remove the zona pellucida by incubation in 0.5% pronase solution in embryo culture medium (see Note 5) for 30-40 s under microscope observation. When zona pellucida start to dissolve, remove embryos from the pronase solution and wash at least 7-10 times in embryo culture media.
2. Incubate the embryos in human anti-placental alkaline phosphatase antibody diluted 1:10 in embryo culture medium for 40 min. After incubation, wash the embryos five to seven times in embryo culture medium.
3. Incubate the embryos in guinea pig complement diluted 1:4 in embryo culture medium (embryos may collapse during this procedure).
4. Wash the collapsed embryos five to seven times in embryo culture medium using a pulled Pasteur pipet with a fire polished 100- to 120-|im diameter until lysing trophoblast cells are removed.
5. Plate the intact ICM onto an inactivated mouse embryonic fibroblast (MEF) monolayer in hES medium (for MEF monolayer preparation see Subheading 3.2.).
6. Change 50% hES medium every other day in ICM cultures. Start passaging 7-10 d after ICM growth will be observed (for further passaging of ICM, see Subheading 3.4.2.).
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