OBJECTIVE: The most common approach for hESC derivation is elimination of trophoblast cells from the blastocyst by immunosurgery (patent # 5843780). But, in this first step the human embryo has already been exposed to animal derived products such as antibodies and complement used to identify and destroy the trophoblast cells. As an alternative, the isolation of the inner cell mass (ICM) from blastocyst stage embryos can be achieved with intricate and complex laser-assisted techniques (patent #7294508). To address this issue, we developed simple and efficient microsurgical methods to establish hESC lines in animal component free conditions.DESIGN: The efficiencies of different methods of establishment of hESC from embryos tested by PGD were compared.MATERIALS AND METHODS: ICM from blastocysts were isolated by immunosurgery with the help of anti-human antibody and quinea pig complement. The microsurgical methods include the dissection of trophoblast cells using glass needles and micromanipulation tools. The ICM was cultured on the mitomycin-inactivated embryonic fibroblasts in K-DMEM supplemented with Serum Replacement. We introduced this technique for the derivation of the first hESC lines carrying genetic mutations derived after PGD (Galat et al., 2004).RESULTS: The efficiency of establishing hESC lines by either surgical method was comparable: 47.0 % for microsurgery and 53.3 % for immunosurgery, but microsurgery has the advantage of maintaining animal free conditions. The efficiency of whole blastocyst plating was substantially lower since actively growing trophoblast cells obstruct the development of ICM (16.7%). Recently, single cell embryo biopsy techniques were also established as a method of hESC derivation without embryo destruction (Chung et al., 2008). The embryos in the present study underwent biopsy 3 times; both 1st and 2nd polar bodies and then one blastomere were removed for genetic testing. Our data confirm that properly performed embryo biopsy has little or no effect on subsequent development of the human embryo to the blastocyst stage (70.7 %) of embryos manipulated in this way successfully develop to blastocyst.CONCLUSIONS: The efficiency of establishing hESC lines by either immunosurgery or microsurgery is comparable. Microsurgery has the advantage of maintaining animal free conditions. Properly performed embryo biopsy has little or no effect on subsequent development of the human embryo to the blastocyst stage. OBJECTIVE: The most common approach for hESC derivation is elimination of trophoblast cells from the blastocyst by immunosurgery (patent # 5843780). But, in this first step the human embryo has already been exposed to animal derived products such as antibodies and complement used to identify and destroy the trophoblast cells. As an alternative, the isolation of the inner cell mass (ICM) from blastocyst stage embryos can be achieved with intricate and complex laser-assisted techniques (patent #7294508). To address this issue, we developed simple and efficient microsurgical methods to establish hESC lines in animal component free conditions. DESIGN: The efficiencies of different methods of establishment of hESC from embryos tested by PGD were compared. MATERIALS AND METHODS: ICM from blastocysts were isolated by immunosurgery with the help of anti-human antibody and quinea pig complement. The microsurgical methods include the dissection of trophoblast cells using glass needles and micromanipulation tools. The ICM was cultured on the mitomycin-inactivated embryonic fibroblasts in K-DMEM supplemented with Serum Replacement. We introduced this technique for the derivation of the first hESC lines carrying genetic mutations derived after PGD (Galat et al., 2004). RESULTS: The efficiency of establishing hESC lines by either surgical method was comparable: 47.0 % for microsurgery and 53.3 % for immunosurgery, but microsurgery has the advantage of maintaining animal free conditions. The efficiency of whole blastocyst plating was substantially lower since actively growing trophoblast cells obstruct the development of ICM (16.7%). Recently, single cell embryo biopsy techniques were also established as a method of hESC derivation without embryo destruction (Chung et al., 2008). The embryos in the present study underwent biopsy 3 times; both 1st and 2nd polar bodies and then one blastomere were removed for genetic testing. Our data confirm that properly performed embryo biopsy has little or no effect on subsequent development of the human embryo to the blastocyst stage (70.7 %) of embryos manipulated in this way successfully develop to blastocyst. CONCLUSIONS: The efficiency of establishing hESC lines by either immunosurgery or microsurgery is comparable. Microsurgery has the advantage of maintaining animal free conditions. Properly performed embryo biopsy has little or no effect on subsequent development of the human embryo to the blastocyst stage.