In bioproduction processes, cellular heterogeneity can cause unpredictable process outcomes or even provoke process failure. Still, cellular heterogeneity is not examined systematically in bioprocess research and development. One reason for this shortcoming is the applied average bulk analyses, which are not able to detect cell-to-cell differences. In this study, we present a microfluidic tool for mammalian single-cell cultivation (MaSC) of suspension cells. The design of our platform allows cultivation in highly controllable environments. As a model system, Chinese hamster ovary cells (CHO-K1) were cultivated over 150h. Growth behavior was analyzed on a single-cell level and resulted in growth rates between 0.85 and 1.16day-1 . At the same time, heterogeneous growth and division behavior, for example, unequal division time, as well as rare cellular events like polynucleation or reversed mitosis were observed, which would have remained undetected in a standard population analysis based on average measurements. Therefore, MaSC will open the door for systematic single-cell analysis of mammalian suspension cells. Possible fields of application represent basic research topics like cell-to-cell heterogeneity, clonal stability, pharmaceutical drug screening, and stem cell research, as well as bioprocess related topics such as media development and novel scale-down approaches. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.