Quantifying microbial robustness in dynamic environments using microfluidic single-cell cultivation.

BACKGROUND: Microorganisms must respond to changes in their environment. Analysing the robustness of functions (i.e. performance stability) to such dynamic perturbations is of great interest in both laboratory and industrial settings. Recently, a quantification method capable of assessing the robustness of various functions, such as specific growth rate or product yield, across different conditions, time frames, and populations has been developed for microorganisms grown in a 96-well plate. In micro-titer-plates, environmental change is slow and undefined. Dynamic microfluidic single-cell cultivation (dMSCC) enables the precise maintenance and manipulation of microenvironments, while tracking single cells over time using live-cell imaging. Here, we combined dMSCC and a robustness quantification method to a pipeline for assessing performance stability to changes occurring within seconds or minutes. RESULTS: Saccharomyces cerevisiae CEN.PK113-7D, harbouring a biosensor for intracellular ATP levels, was exposed to glucose feast-starvation cycles, with each condition lasting from 1.5 to 48 min over a 20 h period. A semi-automated image and data analysis pipeline was developed and applied to assess the performance and robustness of various functions at population, subpopulation, and single-cell resolution. We observed a decrease in specific growth rate but an increase in intracellular ATP levels with longer oscillation intervals. Cells subjected to 48 min oscillations exhibited the highest average ATP content, but the lowest stability over time and the highest heterogeneity within the population. CONCLUSION: The proposed pipeline enabled the investigation of function stability in dynamic environments, both over time and within populations. The strategy allows for parallelisation and automation, and is easily adaptable to new organisms, biosensors, cultivation conditions, and oscillation frequencies. Insights on the microbial response to changing environments will guide strain development and bioprocess optimisation.

Efficient Microfluidic Downstream Processes for Rapid Antibody Hit Confirmation.

Microfluidics has been recently applied to better understand the spatial and temporal progression of the immune response in several species, for tool and biotherapeutic production cell line development and rapid antibody hit discovery. Several technologies have emerged that allow interrogation of large diversities of antibody-secreting cells in defined compartments such as picoliter droplets or nanopens. Mostly primary cells of immunized rodents but also recombinant mammalian libraries are screened for specific binding or directly for the desired function. While post-microfluidic downstream processes appear as standard steps, they represent considerable and interdependent challenges that can lead to high attrition rates even if original selections had been successful. In addition to next-generation sequencing recently described in depth elsewhere, this report aims at in detail explanations of exemplary droplet-based sorting followed by single-cell antibody gene PCR recovery and reproduction or single-cell sub-cultivation for crude supernatant confirmatory studies.

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS , is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS . In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial kS estimation, sl-MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl-MSCC lays the foundation for strain-specific kS estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.

Microbial lifelines in bioprocesses: From concept to application.

Bioprocesses are scaled up for the production of large product quantities. With larger fermenter volumes, mixing becomes increasingly inefficient and environmental gradients get more prominent than in smaller scales. Environmental gradients have an impact on the microorganism's metabolism, which makes the prediction of large-scale performance difficult and can lead to scale-up failure. A promising approach for improved understanding and estimation of dynamics of microbial populations in large-scale bioprocesses is the analysis of microbial lifelines. The lifeline of a microbe in a bioprocess is the experience of environmental gradients from a cell's perspective, which can be described as a time series of position, environment and intracellular condition. Currently, lifelines are predominantly determined using models with computational fluid dynamics, but new technical developments in flow-following sensor particles and microfluidic single-cell cultivation open the door to a more interdisciplinary concept. We critically review the current concepts and challenges in lifeline determination and application of lifeline analysis, as well as strategies for the integration of these techniques into bioprocess development. Lifelines can contribute to a successful scale-up by guiding scale-down experiments and identifying strain engineering targets or bioreactor optimisations.

Novel transfer learning schemes based on Siamese networks and synthetic data.

Transfer learning schemes based on deep networks which have been trained on huge image corpora offer state-of-the-art technologies in computer vision. Here, supervised and semi-supervised approaches constitute efficient technologies which work well with comparably small data sets. Yet, such applications are currently restricted to application domains where suitable deep network models are readily available. In this contribution, we address an important application area in the domain of biotechnology, the automatic analysis of CHO-K1 suspension growth in microfluidic single-cell cultivation, where data characteristics are very dissimilar to existing domains and trained deep networks cannot easily be adapted by classical transfer learning. We propose a novel transfer learning scheme which expands a recently introduced Twin-VAE architecture, which is trained on realistic and synthetic data, and we modify its specialized training procedure to the transfer learning domain. In the specific domain, often only few to no labels exist and annotations are costly. We investigate a novel transfer learning strategy, which incorporates a simultaneous retraining on natural and synthetic data using an invariant shared representation as well as suitable target variables, while it learns to handle unseen data from a different microscopy technology. We show the superiority of the variation of our Twin-VAE architecture over the state-of-the-art transfer learning methodology in image processing as well as classical image processing technologies, which persists, even with strongly shortened training times and leads to satisfactory results in this domain. The source code is available at https://github.com/dstallmann/transfer_learning_twinvae, works cross-platform, is open-source and free (MIT licensed) software. We make the data sets available at https://pub.uni-bielefeld.de/record/2960030.

Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of Corynebacterium glutamicum at oscillating pH values.

In large-scale bioreactors, gradients in cultivation parameters such as oxygen, substrate, and pH result in fluctuating cell environments. pH fluctuations were identified as a critical parameter for bioprocess performance. Traditionally, scale-down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strains and thus process performance. Here, we demonstrate the application of dynamic microfluidic single-cell cultivation (dMSCC) as a novel scale-down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model stress factor. A detailed comparison between two-compartment reactor (two-CR) scale-down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 (~8 min) and disturbed pH 6 (~2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two-CR 27%) compared to undisturbed cultivation at pH 7. Afterward, systematic experiments at symmetric and asymmetric pH oscillations, between pH ranges of 4-6 and 8-11 and different intervals from 1 to 20 min, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions of a putative large-scale bioprocess, which is difficult to conduct using two-CRs.

How to Perform a Microfluidic Cultivation Experiment-A Guideline to Success.

As a result of the steadily ongoing development of microfluidic cultivation (MC) devices, a plethora of setups is used in biological laboratories for the cultivation and analysis of different organisms. Because of their biocompatibility and ease of fabrication, polydimethylsiloxane (PDMS)-glass-based devices are most prominent. Especially the successful and reproducible cultivation of cells in microfluidic systems, ranging from bacteria over algae and fungi to mammalians, is a fundamental step for further quantitative biological analysis. In combination with live-cell imaging, MC devices allow the cultivation of small cell clusters (or even single cells) under defined environmental conditions and with high spatio-temporal resolution. Yet, most setups in use are custom made and only few standardised setups are available, making trouble-free application and inter-laboratory transfer tricky. Therefore, we provide a guideline to overcome the most frequently occurring challenges during a MC experiment to allow untrained users to learn the application of continuous-flow-based MC devices. By giving a concise overview of the respective workflow, we give the reader a general understanding of the whole procedure and its most common pitfalls. Additionally, we complement the listing of challenges with solutions to overcome these hurdles. On selected case studies, covering successful and reproducible growth of cells in MC devices, we demonstrate detailed solutions to solve occurring challenges as a blueprint for further troubleshooting. Since developer and end-user of MC devices are often different persons, we believe that our guideline will help to enhance a broader applicability of MC in the field of life science and eventually promote the ongoing advancement of MC.

Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale.

Scaling down bioproduction processes has become a major driving force for more accelerated and efficient process development over the last decades. Especially expensive and time-consuming processes like the production of biopharmaceuticals with mammalian cell lines benefit clearly from miniaturization, due to higher parallelization and increased insights while at the same time decreasing experimental time and costs. Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells. So far, growth characteristics of microfluidic cultivated cell lines were not systematically compared to larger cultivation scales; however, validation of a miniaturization tool against initial cultivation scales is mandatory to prove its applicability for bioprocess development. Here, we systematically investigate growth, morphology, and eGFP production of CHO-K1 cells in different cultivation scales ranging from a microfluidic chip (230 nl) to a shake flask (125 ml) and laboratory-scale stirred tank bioreactor (2.0 L). Our study shows a high comparability regarding specific growth rates, cellular diameters, and eGFP production, which proves the feasibility of MSCC as a miniaturized cultivation tool for mammalian cell culture. In addition, we demonstrate that MSCC provides insights into cellular heterogeneity and single-cell dynamics concerning growth and production behavior which, when occurring in bioproduction processes, might severely affect process robustness.

Growth Response and Recovery of Corynebacterium glutamicum Colonies on Single-Cell Level Upon Defined pH Stress Pulses.

Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum.

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.

Microfluidics for Biotechnology: Bridging Gaps to Foster Microfluidic Applications.

Microfluidics and novel lab-on-a-chip applications have the potential to boost biotechnological research in ways that are not possible using traditional methods. Although microfluidic tools were increasingly used for different applications within biotechnology in recent years, a systematic and routine use in academic and industrial labs is still not established. For many years, absent innovative, ground-breaking and "out-of-the-box" applications have been made responsible for the missing drive to integrate microfluidic technologies into fundamental and applied biotechnological research. In this review, we highlight microfluidics' offers and compare them to the most important demands of the biotechnologists. Furthermore, a detailed analysis in the state-of-the-art use of microfluidics within biotechnology was conducted exemplarily for four emerging biotechnological fields that can substantially benefit from the application of microfluidic systems, namely the phenotypic screening of cells, the analysis of microbial population heterogeneity, organ-on-a-chip approaches and the characterisation of synthetic co-cultures. The analysis resulted in a discussion of potential "gaps" that can be responsible for the rare integration of microfluidics into biotechnological studies. Our analysis revealed six major gaps, concerning the lack of interdisciplinary communication, mutual knowledge and motivation, methodological compatibility, technological readiness and missing commercialisation, which need to be bridged in the future. We conclude that connecting microfluidics and biotechnology is not an impossible challenge and made seven suggestions to bridge the gaps between those disciplines. This lays the foundation for routine integration of microfluidic systems into biotechnology research procedures.

Heterogeneity Studies of Mammalian Cells for Bioproduction: From Tools to Application.

Quantified process parameters in pharmaceutical production and in mammalian cell fermentation process development only represent culture average values and do not portray the heterogeneity and the individual behaviour of single cells. The first studies using well-established techniques like flow cytometry or novel lab-on-a-chip systems, such as droplet microfluidics or microfluidic single-cell cultivation, indicate a substantial level of cell-to-cell heterogeneity with important implications for biotechnological processes. Understanding the reasons, degree, and dynamics of cell-to-cell heterogeneity within bioprocesses will pave the way to developing more stable cell lines and more reproducible bioprocesses. We review single-cell cultivation and analytical methods, including their application in bioprocess technology.

Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions.

Quantitative single-cell cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-cell level. Novel microfluidic single-cell cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a "persister" strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic cultivation approaches. Failure and non-reproducible cultivations are thus most likely to be found in less controllable larger-scale cultivation workflows and as a result of environmental gradients within large-scale cultivations.

Ultrahigh-throughput functional profiling of microbiota communities.

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.