Bioreactors have proven useful for a vast amount of applications. Besides classical large-scale bioreactors and fermenters for prokaryotic and eukaryotic organisms, micro-bioreactors, as specialized bioreactor systems, have become an invaluable tool for mammalian 3D cell cultures. In this systematic review we analyze the literature in the field of eukaryotic 3D cell culture in micro-bioreactors within the last 20 years. For this, we define complexity levels with regard to the cellular 3D microenvironment concerning cell−matrix-contact, cell−cell-contact and the number of different cell types present at the same time. Moreover, we examine the data with regard to the micro-bioreactor design including mode of cell stimulation/nutrient supply and materials used for the micro-bioreactors, the corresponding 3D cell culture techniques and the related cellular microenvironment, the cell types and in vitro models used. As a data source we used the National Library of Medicine and analyzed the studies published from 2000 to 2020.