Use of the fibroblast model in the study of cellular senescence

In this chapter, we present standard procedures for the culture of human cells that exhibit a finite proliferative capacity (replicative life-span). The use of a cell culture model has the advantage of providing a controlled environment to study a wide variety of cellular phenomena. It also has the inherent limitation of isolating cells from the regulatory elements that might be provided by other types of cells in vivo. Nevertheless, cell culture models have been crucial to our current understanding of mechanisms of growth, differentiation, development, and neoplasia and numerous other disease states. In this chapter we present procedures for human fibroblast culture including serumfree cultivation of cells, which is necessary when the cellular environment must be fully defined. In addition, we present procedures for the determination of replicative life-span, saturation density, and assessment of replicative capacity from labeled thymidine incorporation in fibroblasts. The methods described here have been well tested and provide highly reproducible results (1, 2).