Establishment and characterization of cell clones from the Papilio cell line RIRI-PaDe-3 by a high-efficiency clonal method

Cell cloning is of great importance in keeping particular properties of cultured cells, and interesting cells can be selected by cloning from heterogeneous cell populations. In addition, continuous cell lines usually from primary culture are prone to heterologous constitution and genetic instability, so that supplementary cloning steps are necessary for achieving a homogenous cell population. In this study, limiting dilution culture and feeder layer culture were originally used for cloning RIRI-PaDe-3 cell line, but both failed. Afterward, we designed a cloning protocol which was composed of two steps: cells in semisolid medium with seeding density in the range of 3.05 x 10(5)-6.10 x 10(5) cells/mL formed colonies from monodispersed cell suspensions; 40 well-dispersed colonies were removed from the suspended state by using micromanipulator system and finally scaled up. To determine whether this method can isolate cell lines possessing characteristics different from the parent population, we made an evaluation of cells monoclonal in biological characteristics. Significant differences have been found among clones isolated from the RIRI-PaDe-3 insect cell line in cell morphology, chromosome numbers, and genetic background. Thus the indicated modified semisolid medium cloning protocol was advantageous to the convenient and genuine cloning from the previously heterogeneous population.