The focus of this research award is improved methodology for the scale-up of animal and insect cell cultures. Three projects are now in progress which involve collaborations with industrial and academic laboratories. Diagnosis of Cell Stress in Large-Scale Bioreactors. The goal of this project is to develop techniques for identifying specific sources of cell stress in large-scale bioreactors through analysis of intracellular protein synthesis. Recent results have shown a set of intracellular proteins whose increased synthesis rate is indicative of heightened hydrodynamic stress. The goal of future work is to identify and quantify intracellular proteins associated with other types of environmental stress. This information is to be ultimately used for the rational scale-up of animal cell cultures. Scale-Up of Genetically-Engineered Insect Cells. An insect cell expression system has recently been developed for the production of genetically-engineered proteins of therapeutic and agricultural interest. In this project, the potential of this expression system will be assessed. Recent results have identified the conditions necessary for the growth of these cells in a sparged bioreactor. Future work will examine environmental conditions necessary for optimum cell growth and product formation in large-scale bioreactors. Metabolic Engineering of Mammalian Cells. Normal human cells have a limited life span in cell culture. This problem is being addressed through the incorporation of a specific class of genes, called oncogenes, into human cells. Recent results demonstrate that one of these genes can increase the life span of human kidney cells at least five-fold. Future research is directed toward further characterization of these kidney cell strains, toward the reduction of mammalian cell growth factor requirements through oncogene incorporation, and toward an extension of this approach to other important cell types.