Development and application of a cultivation platform for mammalian suspension cell lines with single-cell resolution.

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 150 h. Growth behavior was analyzed on a single-cell level and resulted in growth rates between 0.85 and 1.16 day-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.

Reproduction of Large-Scale Bioreactor Conditions on Microfluidic Chips.

Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g., nutrient concentration, dissolved oxygen, temperature, and pH. These inhomogeneities can influence the cell physiology and metabolism, e.g., decelerate cell growth and product formation. Microfluidic systems offer new opportunities to study such effects in great detail by examining responses to varying environmental conditions at single-cell level. However, the possibility to reproduce large-scale bioreactor conditions in microscale cultivation systems has not yet been systematically investigated. Hence, we apply computational fluid dynamics (CFD) simulations to analyze and compare three commonly used microfluidic single-cell trapping and cultivation devices that are based on (i) mother machines (MM), (ii) monolayer growth chambers (MGC), and (iii) negative dielectrophoresis (nDEP). Several representative time-variant nutrient concentration profiles are applied at the chip entry. Responses to these input signals within the studied microfluidic devices are comparatively evaluated at the positions of the cultivated cells. The results are comprehensively presented in a Bode diagram that illustrates the degree of signal damping depending on the frequency of change in the inlet concentration. As a key finding, the MM can accurately reproduce signal changes that occur within 1 s or slower, which are typical for the environmental conditions observed by single cells in large-scale bioreactors, while faster changes are levelled out. In contrast, the nDEP and MGC are found to level out signal changes occurring within 10 s or faster, which can be critical for the proposed application.

A microfluidic photobioreactor for simultaneous observation and cultivation of single microalgal cells or cell aggregates.

Microalgae are an ubiquitous and powerful driver of geochemical cycles which have formed Earth's biosphere since early in the evolution. Lately, microalgal research has been strongly stimulated by economic potential expected in biofuels, wastewater treatment, and high-value products. Similar to bacteria and other microorganisms, most work so far has been performed on the level of suspensions which typically contain millions of algal cells per millilitre. The thus obtained macroscopic parameters average cells, which may be in various phases of their cell cycle or even, in the case of microbial consortia, cells of different species. This averaging may obscure essential features which may be needed for the correct understanding and interpretation of investigated processes. In contrast to these conventional macroscopic cultivation and measuring tools, microfluidic single-cell cultivation systems represent an excellent alternative to study individual cells or a small number of mutually interacting cells in a well-defined environment. A novel microfluidic photobioreactor was developed and successfully tested by the photoautotrophic cultivation of Chlorella sorokiniana. The reported microbioreactor facilitates automated long-term cultivation of algae with controlled temperature and with an illumination adjustable over a wide range of photon flux densities. Chemical composition of the medium in the microbioreactor can be stabilised or modulated rapidly to study the response of individual cells. Furthermore, the algae are cultivated in one focal plane and separate chambers, enabling single-cell level investigation of over 100 microcolonies in parallel. The developed platform can be used for systematic growth studies, medium screening, species interaction studies, and the thorough investigation of light-dependent growth kinetics.

Microbial single-cell growth response at defined carbon limiting conditions.

Growth is one of the most fundamental characteristics of life, but detailed knowledge regarding growth at nutrient limiting conditions remains scarce. In recent years progress in microfluidic single-cell analysis and cultivation techniques has given insights into many fundamental growth characteristics such as growth homeostasis, aging and cell division of microbial cells. Using microfluidic single-cell cultivation technologies we examined how single-cell growth at defined carbon conditions, ranging from strongly limiting conditions (0.01 mmol L-1) to a carbon surplus (100 mmol L-1), influenced cell-to-cell variability. The experiments showed robust growth of populations at intermediate concentrations and cell-to-cell variability was higher at low and high carbon concentrations, among an isogenic population. Single-cell growth at extremely limiting conditions led not only to significant variability of division times, but also to an increased number of cells that did not pursue growth. Overall, the results demonstrate that cellular behaviour shows robust, Monod-like growth, with significant cell-to-cell heterogeneity at extreme limiting conditions, resembling natural habitats. Due to this significant influence of the environment on cellular physiology, more carefulness needs to be given future microfluidic single-cell experiments. Consequently, our results lay the foundation for the re-interpretation and design of workflows for future experiments aiming at an improved understanding of cell growth mechanisms.

A microfluidic co-cultivation platform to investigate microbial interactions at defined microenvironments.

Interspecies interactions inside microbial communities bear a tremendous diversity of complex chemical processes that are by far not understood. Even for simplified, often synthetic systems, the interactions between two microbes are barely revealed in detail. Here, we present a microfluidic co-cultivation platform for the analysis of growth and interactions inside microbial consortia with single-cell resolution. Our device allows the spatial separation of two different microbial organisms inside adjacent microchambers facilitating sufficient exchange of metabolites via connecting nanochannels. Inside the cultivation chambers cell growth can be observed with high spatio-temporal resolution by live-cell imaging. In contrast to conventional approaches, in which single-cell activity is typically fully masked by the average bulk behavior, the small dimensions of the microfluidic cultivation chambers enable accurate environmental control and observation of cellular interactions with full spatio-temporal resolution. Our method enables one to study phenomena in microbial interactions, such as gene transfer or metabolic cross-feeding. We chose two different microbial model systems to demonstrate the wide applicability of the technology. First, we investigated commensalistic interactions between an industrially relevant l-lysine-producing Corynebacterium glutamicum strain and an l-lysine auxotrophic variant of the same species. Spatially separated co-cultivation of both strains resulted in growth of the auxotrophic strain due to secreted l-lysine supplied by the producer strain. As a second example we investigated bacterial conjugation between Escherichia coli S17-1 and Pseudomonas putida KT2440 cells. We could show that direct cell contact is essential for the successful gene transfer via conjugation and was hindered when cells were spatially separated. The presented device lays the foundation for further studies on contactless and contact-based interactions of natural and synthetic microbial communities.

Quantitative measurements in single-cell analysis: towards scalability in microbial bioprocess development.

Single-cell analysis in microfluidic cultivation devices bears a great potential for the development and optimization of industrial bioprocesses. High parallelization allows running a large number of cultivation experiments simultaneously even under quick alteration of environmental conditions. For example, the impact of changes in media composition on cell growth during classical batch cultivation can be easily resolved. A missing link for the scalability of microfluidic experiments is, however, their complete characterization via conventional performance indicators such as product titer and productivity. While existing mass spectrometry technology is not yet sufficiently coupled with microfluidics, optical methods like enzymatic assays or fluorescence sensors are promising alternatives but require further improvement to generate quantitative measurements of extracellular metabolites.

Quantitative modelling of nutrient-limited growth of bacterial colonies in microfluidic cultivation.

Nutrient gradients and limitations play a pivotal role in the life of all microbes, both in their natural habitat as well as in artificial, microfluidic systems. Spatial concentration gradients of nutrients in densely packed cell configurations may locally affect the bacterial growth leading to heterogeneous micropopulations. A detailed understanding and quantitative modelling of cellular behaviour under nutrient limitations is thus highly desirable. We use microfluidic cultivations to investigate growth and microbial behaviour of the model organism Corynebacterium glutamicum under well-controlled conditions. With a reaction-diffusion-type model, parameters are extracted from steady-state experiments with a one-dimensional nutrient gradient. Subsequently, we employ particle-based simulations with these parameters to predict the dynamical growth of a colony in two dimensions. Comparing the results of those simulations with microfluidic experiments yields excellent agreement. Our modelling approach lays the foundation for a better understanding of dynamic microbial growth processes, both in nature and in applied biotechnology.

Coarse-graining bacteria colonies for modelling critical solute distributions in picolitre bioreactors for bacterial studies on single-cell level.

Microfluidic single-cell bioreactors have found widespread application to investigate growth and gene expression of microbial model organisms, but yet there are few attempts to systematically characterize different design and cultivation concepts. Quantitative measurements of critical solute concentrations, e.g. limiting nutrients, are not yet feasible within the typical volumes in the range of picolitres. A way to gain new insights about the mass transport within those volumes is by simulation, but the complex geometry resulting from the multitude of cells within a colony leads to time and resource consuming computational challenges. In this work, six different concepts for the model representation of cellular microcolonies within microfluidic monolayer growth chamber devices are compared. The Gini coefficient is proposed as new measure for inhomogeneity within cellular colonies. An example cell colony is represented by a single point source, a cylindrical volume with homogeneous reaction rates with and without adjusted diffusion coefficient, as point sources for each single cell and as rod-shaped, diffusion blocking, three-dimensional cells with varying shapes. Simulated concentration profiles across the chambers depended strongly on the chosen cell representation. The representation with the lowest degree of abstraction, three-dimensional cells, leads to complex geometries and high computational effort, but also gives a conservative and therefore preferable estimate for the cultivation conditions within a given cultivation chamber geometry. Interestingly, the cylindrical volume with adjusted diffusion coefficient gives similar results but requires far less computational effort. Therefore, it is proposed to use the three-dimensional cells for detailed studies and to determine parameters for the cylindrical volume with adjusted diffusion coefficient, which can then be used for experimental design, screening of parameter spaces, and similar applications.

Modeling and CFD simulation of nutrient distribution in picoliter bioreactors for bacterial growth studies on single-cell level.

A microfluidic device for microbial single-cell cultivation of bacteria was modeled and simulated using COMSOL Multiphysics. The liquid velocity field and the mass transfer within the supply channels and cultivation chambers were calculated to gain insight in the distribution of supplied nutrients and metabolic products secreted by the cultivated bacteria. The goal was to identify potential substrate limitations or product accumulations within the cultivation device. The metabolic uptake and production rates, colony size, and growth medium composition were varied covering a wide range of operating conditions. Simulations with glucose as substrate did not show limitations within the typically used concentration range, but for alternative substrates limitations could not be ruled out. This lays the foundation for further studies and the optimization of existing picoliter bioreactor systems.