In nature and in technical systems, microbial cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., existence of a starvation zone, and quantity, e.g., average residence time in this zone. For strain development and process design, cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically emulating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for cultivation of single cells and small cell clusters under dynamic environmental conditions (dynamic microfluidic single-cell cultivation (dMSCC)). This system enables to control nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). Resulting changes in cellular physiology, such as the colony growth rate and cell morphology were analysed and revealed significant differences with growth rate and cell length between the different conditions. dMSCC also allows to apply defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the cultivation of cells under complex changing environmental conditions.

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 are the applied average bulk analyses, which are not able to detect cell-to-cell differences. In this work we present a microfluidic tool for single-cell cultivation of mammalian suspension cells (MaSC). The design of our platform allows long-term cultivation at highly controllable environments. As model system CHO K1 cells were cultivated over 150 h. Growth behavior was analyzed on single-cell level and resulted in growth rates between 0.85 - 1.16 1/d, which are comparable to classical cultivation approaches such as shake flask and lab-scale bioreactor. At the same time, heterogeneous growth and division behavior, e.g., 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 studies, clonal stability, pharmaceutical drug screening and stem cell research, as well as bioprocess related topics such as media development and novel scale-down approaches.

We present a new microfluidic trapping concept to retain randomly moving suspension cells inside a cultivation chamber. In comparison to previously published complex multilayer structures, we achieve cell retention by a thin PDMS barrier, which can be easily integrated into various PDMS-based cultivation devices. Cell loss during cultivation is effectively prevented while diffusive media supply is still ensured.