The sequencing of the human genome and the discovery that synthetic
siRNA between 19mer and 22mer could silence genes led to the development of siRNA
libraries capable of targeting all known genes within a genome. The emergence of high
throughput genetic screens represent a powerful unbiased approach for identifying new
targets and may fundamentally change biological research by increasing the speed with
which disease mechanisms and potential drug targets can be identified. High-throughput
RNAi screens are typically performed using two-dimensional monolayer cell culture
models due to ease, convenience, and high cell viability. Although conventional two
dimensional cell culture systems have improved our understanding of basic cell biology,
the morphology and physiology of cells grown as monolayers in dish cultures differ
substantially from the morphology and physiology of cells grown in vivo within a
complex three-dimensional microenvironment. There is now a growing realization that
3D cell culture models are superior in biological studies. Three dimensional cell culture
models can boost the physiological relevance of cell-based assays and advance the
quantitative modeling of biological systems, from cells to organisms. These models
exhibit a high degree of structural complexity and homeostasis, analogous to the
complexity and homeostasis of tissues and organs. In this chapter we discuss 3D cell
culture models and describe a three dimensional spheroid cell culture system and the
standard operating procedure for its successful use in high throughput RNAi screens.
Keywords: 3D cell culture, high content image analysis, high Throughput
screening, matrix-free nanoculture plates, RNAi, spheroid cell culture.
