Establishment of a single-step hybridoma cloning protocol using an automated cell transfer system: comparison with limiting dilution.

An easy-to-standardize single-step protocol of hybridoma cloning has been established using a recently introduced, commercially available cell transfer system. By controlling the volume of air within a sealed glass micropipette by means of a Peltier device, single cells are gently collected or ejected. The transfer of cells from a source dish to the wells of a target microplate is controlled by a microprocessor. Since collection as well as expulsion of cells is done under microscopic control seeding of single cells can be guaranteed. Monoclonality is therefore reproducibly achieved in a single step, reducing the time required for cloning enormously, and conserving man-power and material. Since the automated transfer of cells is time-saving and easy-to-standardize, it substantially facilitates cloning of hybridoma. The present protocol therefore represents an alternative to limiting-dilution cloning as well as to other previously introduced techniques of single-cell cloning. It is easily adapted to a wide spectrum of other cell types and can therefore be used in many other applications involving single cell manipulation.