Hybridomas are exposed to many threats, such as contamination with bacteria and fungi, loss of chromosomes coding for antibody production, overgrowth by nonsecreting mutants, and cell death resulting from overgrowth. Therefore, newly established hybridomas should be frozen and stored in liquid nitrogen at −196�C as soon as possible. Alternatively, they can be stored in the vapor phase above the liquid nitrogen where the temperature will be between −120� and −195�C. Generally, eucaryotic cells are very sensitive to the freezing procedure, and programmable freezing devices are available that lower the temperature from 0� to −30�C and from −30� to −100�C at different time rates. However, in our experience, hybridoma cells will survive by simply incubating the vials at −20�C for 2 h followed by overnight incubation at −70�C. The vials are, then transferred to liquid nitrogen for long-time storage. Cells frozen in this way have survived for more than 5 yr.
