Live cell imaging enables an observation of cell behavior over a period of time and is a growing field in modern cell biology. Quantitative analysis of the spatio-temporal dynamics of heterogeneous cell populations in three-dimensional (3D) microenvironments contributes a better understanding of cell-cell and cell-matrix interactions for many biomedical questions of physiological and pathological processes. However, current live cell imaging and analysis techniques are frequently limited by non-physiological 2D settings. Furthermore, they often rely on cell labelling by fluorescent dyes or expression of fluorescent proteins to enhance contrast of cells, which frequently affects cell viability and behavior of cells. In this work, we present a quantitative, label-free 3D single cell tracking technique using standard bright-field microscopy and affordable computational resources for data analysis. We demonstrate the efficacy of the automated method by studying migratory behavior of a large number of primary human macrophages over long time periods of several days in a biomimetic 3D microenvironment. The new technology provides a highly affordable platform for long-term studies of single cell behavior in 3D settings with minimal cell manipulation and can be implemented for various studies regarding cell-matrix interactions, cell-cell interactions as well as drug screening platform for primary and heterogeneous cell populations.