Blastocyst culture
Following in vitro fertilization (IVF) treatment, human pronuclear (PN) stage embryos were first cultured to the eight cell stage. At day 3, embryos
were graded according to the scale of Gardner et al. After assessment of number of divisions and cell morphology, good quality embryos were reserved for IVF treatment. The surplus embryos donated to this study with informed consent were transferred to droplets of medium G2.3 for further culturing up to the blastocyst stage. Culture medium G-2.3 (Vitrolife, Sweden) was supplemented with 5 mg/ml human serum albumin (Vitrolife) and 1000 U/ml leukaemia inhibitory factor (LIF, Chemicon, USA). The protocols used in this research follow the stem cell guidelines issued by the Ministry of Science and Technology and the Ministry of Health of China and were approved by the Ethics Committee of the Sun Yat-sen University.
Preparation of feeder layer
Mouse embryonic feeder cells (MEF, Cyagen Biosciences, USA) were isolated from 13-14 dpc of KM strain mice and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco Invitrogen, USA); supplemented with 2 mmol/L glutamine, 0.1 mmol/L ?mercaptoethanol and 1% nonessential amino acid (Sigma, USA); 10% foetal calf serum (HyClone, USA). Within 5 passages, the MEF cells were treated with 55 Gy ?íray irradiation and washed by phosphate buffered saline (PBS) five times before transference to dishes treated with 0.1% gelatin coated, tissue culture of suitable density.