Microfluidic systems offer precisely controlled experimental conditions for efficient study of mammalian cells, ranged from a single cell to matrix Based three-dimensional (3D) in vitro culture models. Droplet-based microfluidic system is a robust and highly reproducible device enabling encapsulation of a cell or cells within well-confined microenvironment. Recently, such droplet-based cell culture models have drawn much attention due to their unique properties such that conventional culture systems couldn't provide. The encapsulation of cells in specifically designed aqueous phase of a microfluidic system can provide profound understand of cell to cell and cell to extracellular matrix interactions, also can be used to regulate various cell behaviors. A droplet-based cell culture system allows better control over confinement for culturing, maintaining, and analyzing cells, such as high-throughput screening. In this review, we discuss recent researches on microdroplet-based 3D cell culture models, and advanced applications of microfluidic systems.