microfluidic-picoliter-bioreactor-for-microbial-single-cell-analysis

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Forschungszentrum Juelich GmbH

Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigus-induced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen-mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus.

Protease inhibitors prevent plasminogen-mediated, but not pemphigus vulgaris-induced, acantholysis in human epidermis.

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

Single-cell analysis in microfluidic systems has opened up new possibilities in biotechnological research enabling us to deal with large eukaryotic cells and even small bacteria. In particular, transient investigations in laminar flow or diffusive environments can be performed to unravel single cell behaviour. Up to now, most systems have been limited with respect to precise cell inoculation and sampling methods. Individual cell selection and manipulations have now been made possible by combining laser tweezers with microfluidic cell cultivation environments specifically tailored for micrometre-sized bacteria. Single cells were optically seeded into various micrometre-sized growth sites arranged in parallel. During cultivation, single-cell elongation, morphology and growth rates were derived from single cells and microcolonies of up to 500 cells. Growth of irradiated bacteria was not impaired by minimizing the exposed laser dosage as confirmed by exceptional growth rates. In fact, Escherichia coli exhibited doubling times of less than 20min. For the first time, a filamentous Escherichia coli WT (MG1655) was safely relocated from its growing microcolony by laser manipulations. The cell was transferred to an empty cultivation spot allowing single-cell growth and morphology investigations. Contrary to previous discussions, the filamentous E. coli exhibited normal cell morphology and division after a few generations. This combination of optical tweezers and single-cell analysis in microfluidics adds a new degree of freedom to microbial single-cell analysis.

Christopher Probst

Institute of Bio- and Geosciences,
IBG-1: Biotechnology,
Institute of Bio- and Geosciences, IBG-1: Biotechnology

Forschungszentrum Juelich GmbH

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

Christopher Probst

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Bio- and Geosciences,IBG-1: Biotechnology,Institute of Bio- and Geosciences, IBG-1: Biotechnology

Forschungszentrum Juelich GmbH

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

Institute of Bio- and Geosciences,
IBG-1: Biotechnology,
Institute of Bio- and Geosciences, IBG-1: Biotechnology