Requirements for growth of Epstein-Barr virus-transformed cells at low cell densities

We investigated the requirements for growth of Epstein-Barr virus-transformed cells at low cell densities in a homotypic system: only Epstein-Barr virus-transformed cells or their products were used to supply feeder activity. The cloning efficiency was increased markedly under conditions favouring close cell-to-cell contact: culture in round-bottomed rather than in flat-bottomed wells or the addition of irradiated Epstein-Barr virus-transformed cells. Further enhancement was obtained by the addition of the tumour promoter phorbol-myristate acetate. Part of this effect was also induced by supernatants of Epstein-Barr virus-transformed cells, in particular when the latter had been stimulated with phorbol ester. Thus, under limiting dilution conditions, Epstein-Barr virus-transformed cells were found to be dependent upon autologous cell-mediated and soluble proliferation-stimulating signals. In such a homotypic feeder system, the cloning efficiency could be enhanced up to eight-fold; a 100% cloning efficiency could not be achieved. Evidence is presented that the residual deficit in cloning efficiency is inherent to these cell lines.