Electrofusion: Nuclear Reprogramming of Somatic Cells by Cell Hybridization with Pluripotential Stem Cells

This chapter examines nuclear programming of somatic cells by cell hybridization with pluripotential stem cells. This chapter describes a practical procedure for electrofusion to produce hybrid cells between pluripotential stem cells and committed somatic cells without the use of virus or chemicals to mediate the fusion. One is required to culture exponentially growing ES cells on the inactivated PEFs with changes of culture medium once or twice a day. Carry out subculturing of the ES cells every 2 days by a 1:4 split. Trypsinize ES cells and remove excess trypsin quickly. Add 3 ml of ES medium to inactivate the trypsin and dissociate the cells into a single-cell suspension by gentle pipetting. Plate them on a new gelatin-coated 60-mm culture dish. Use the automatic operation switch to initiate AC followed by DC. AC is utilized to induce an inhomogeneous or divergent electric field, resulting in cell alignment and pearl chain formation.