Our understanding of important biological phenomena such as invasion, migration, and apoptosis has advanced greatly through the prudent use of in vitro models based on isolated cells in culture. Established cell lines are readily manipulated using simple molecular biological techniques and their abundance as homogenous populations allows the rapid accumulation of statistically significant data. The study of human trophoblast function in vitro has been hampered by the difficulties associated with obtaining and culturing primary human trophoblasts including the paucity of material and contamination with other cell types. This chapter describes a cheap and simple method for the production of human trophoblast cell lines using poly-L-ornithine. It details the production, isolation and initial characterization of these cells and provides simple tips on how to store and maintain a bank of cells for future needs.