Simultaneous cloning and selection of hybridomas and transfected cell lines in semisolid media

Selection and cloning are essential but often laborious and time-consuming steps during the generation of hybridomas and genetically modified cell lines that produce monoclonal antibodies or other proteins with desired properties. Methods for the simultaneous selection and cloning of hybridomas and transfected cell lines (e.g., CHO-S cells) in semisolid methylcellulose-based media have been developed. By using semisolid selection media, the cells that survive the selection process proliferate and form colonies of cells that remain physically separated from other colonies. Each colony thus originates from a single hybridoma or transfected cell and can be isolated and characterized separately. This approach avoids the isolation of multiple identical clones and the loss of useful clones due to overgrowth by other faster-growing, but possibly nonproducing clones, which are major problems of conventional procedures in liquid media. In this chapter, protocols are described for the generation of mouse hybridomas by fusion of spleen cells from immunized mice with myeloma cells and the subsequent selection and cloning of hybridomas in semisolid selection media. Protocol are also described for selection and cloning of transfected cell lines using semisolid antibiotic-containing selection media, as well as strategies to optimize selection and cloning in serum-containing, serum-free, and chemically defined selection media.