Conditioned media from cultured cells have long been used to increase the cloning efficiency, growth rate and antibody secretion of hybridomas. The potential sources of conditioned media include cultured splenocytes, peritoneal macrophages, and macrophage, endothelial or fibroblast cell lines. The conditioned media from these cultures contain a variety of cytokines that act as growth factors for hybridomas. However, the constituents of conditioned media are largely undefined. Conditioned media may contain cell metabolites, cellular contaminants, and other undefined components that may interfere with the bio-analysis of hybridoma-derived antibodies.

Further, the levels of cytokines and other components in each preparation may vary from lot to lot. We have investigated the ability of recombinant cytokine formulations to replace conditioned media at different stages of murine hybridoma development. Several cytokine formulations have been identified that match or exceed the ability of commercial conditioned media preparations to improve the outgrowth and cloning efficiency of newly created hybridomas, and to increase proliferation and antibody secretion of established hybridoma clones.

The formulations also improve the recovery of viable hybridomas after cryopreservation and improve cell viability in high- density bioreactor cultures. The recombinant cytokine formulations can be prepared with high lot-to-lot consistency, and without the presence of cell metabolites, cellular contaminants, and other undefined components that may interfere with the bio-analysis of hybridoma-derived antibodies

Summary

We have identified formulations of recombinant cytokines and growth factors that can replace the growth and cloning properties of conditioned media. The formulations meet or exceed the ability of a conditioned media to improve outgrowth and cloning efficiency of newly created hybridomas. The formulations also improve the viability, proliferation, recovery after cryopreservation and antibody secretion of existing clones created using HCF conditioned media

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Authors

Liisa Alajoki and John S. Kenney