The efficient production of industrially relevant biomolecules can only be achieved if a developed bioprocess is successfully transferred from the laboratory scale to the industrial scale [1]. Increasing bioreactor volumes results in increasing mixing times, which leads to the formation of concentration gradients (Figure 1a) and thus to fluctuations in the microenvironment of microorganisms [2]. These gradients represent a major challenge when scaling up bioprocesses and potentially lower production yields and impact the robustness of bioprocesses and might be responsible for process failures 3, 4, 5. Therefore, an analysis of these gradients and their effect on cellular physiology is essential. The effect of bioprocess-relevant gradients, for example, substrate concentration, dissolved oxygen (DO), and pH values, is typically investigated using scale-down bioreactors (Figure 1b) 2, 6, 7. Thus far, these scale-down simulations are carried out in 1–10-L bioreactors 8, 9, with all results being averages of the whole population, and the single-cell behavior of each individual cell is typically not monitored [10].

Scale-down bioreactors can be categorized into two main classes: pulse feeding bioreactors and multimass compartment bioreactors 11, 12. These systems are most often used to analyze single- and multiparameter gradients, for example, substrate and DO concentrations, to investigate how the gradients affect growth, product formation, and other metabolic traits. Despite these advantages, state-of-the-art scale-down bioreactors have several limitations (Figure 1d): (i) different process parameter gradients cannot be decoupled (e.g. DO and glucose concentrations or pH and osmolarity) and (ii) the frequency and amplitude of the environmental fluctuations are strongly dependent on the tested parameter. Temporal resolution is often restricted to minute as well as larger-second timescales. Several of these shortcomings can be addressed with the help of microfluidic single-cell cultivation (MSCC) systems (Figure 1c).

In this opinion piece, we will first introduce MSCC systems. We will show how these systems have been used to study the cellular behavior of cells under fluctuating environmental conditions. Then, we will discuss the directions and challenges of using MSCC systems to perform bioprocess scale-down studies at the single-cell level.