16 research outputs found
Recommended from our members
Cryo-printed microfluidics enable rapid prototyping for optical-cell analysis
This paper highlights an innovative, low-cost rapid-prototyping method for generating microfluidic chips with extraordinary short fabrication times of only a few minutes. Microchannels and inlet/outlet ports are created by controlled deposition of aqueous microdroplets on a cooled surface resulting in printed ice microstructures, which are in turn coated with a UV-curable acrylic cover layer. Thawing leaves an inverse imprint as a microchannel structure. For an exemplary case, we applied this technology for creating a microfluidic chip for cell-customized optical-cell analysis. The chip design includes containers for cell cultivation and analysis. Container shape, length, position, and angle relative to the main channel were iteratively optimized to cultivate and analyze different cell types. With the chip, we performed physiological analyses of morphologically distinct prokaryotic Corynebacterium glutamicum DM1919, eukaryotic Hansenula polymorpha RB11 MOX-GFP, and phototrophic Synechocystis sp. PCC 6803 cells via quantitative time-lapse fluorescence microscopy. The technology is not limited to rapid prototyping of complex biocompatible microfluidics. Further exploration may include printing with different materials other than water, printing on other substrates in-situ biofunctionalization, the inclusion of electrodes and many other applications
An inert continuous microreactor for the isolation and analysis of a single microbial cell
Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level
Microfluidic single-cell analysis in biotechnology: from monitoring towards understanding.
Dusny C, Grünberger A. Microfluidic single-cell analysis in biotechnology: from monitoring towards understanding. Current opinion in biotechnology. 2019;63:26-33.Our understanding of the microbial cell is based on averaged values from bulks. Microfluidic single-cell analysis holds the promise of understanding cellular processes from a single cell perspective. But what is needed to measure single-cell physiology and to disclose the consequences of individuality for biotechnology? Current single-cell research is not yet able to provide all the necessary insights, but innovative approaches now emerge that propel the field towards a better understanding of cellular processes via quantitative physiology. Here, we critically review novel single-cell technologies that enable us to control cellular input parameters such as environmental conditions and to measure intracellular processes, as well as novel approaches that enable for the first time to quantify non-averaged cell-specific rates and yields. Finally, we demonstrate how integrating microfluidic single-cell analysis into established population-based experimental workflows might unlock its full potential for biotechnology research in the future. Copyright © 2019 Elsevier Ltd. All rights reserved
The Metabolic Flux Probe (MFP)—Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis
Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics
Quantitative Physiology of Single Cells for Linking Phenotype and Environment
Dusny C, Grünberger A, Rosenthal K, Wiechert W, Schmid A. Quantitative Physiology of Single Cells for Linking Phenotype and Environment. Presented at the 24th Annual Conference of the German Society for Flow Cytometry, Dresden, Germany
An Inert Continuous Microreactor for the Isolation and Analysis of a Single Microbial Cell
Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level
From extracellular Environment to functional phenotype. Quantitative comparison of single cell physiology in static and steady environments
Dusny C, Grünberger A, Wiechert W, Kohlheyer D, Schmid A. From extracellular Environment to functional phenotype. Quantitative comparison of single cell physiology in static and steady environments. Presented at the 3rd BioProScale Symposium "Inhomogeneities in large-scale bioprocesses. System biology and process dynamics", Berlin, Germany
Technical bias of microcultivation environments on single cell physiology
Dusny C, Grünberger A, Probst C, Wiechert W, Kohlheyer D, Schmid A. Technical bias of microcultivation environments on single cell physiology. Lab on a chip. 2015;15(8):1822-1834.Microscale cultivation systems are important tools to elucidate cellular dynamics beyond the population average and understand the functional architecture of single cells. However, there is scant knowledge about the bias of different microcultivation technologies on cellular functions. We therefore performed a systematic cross-platform comparison of three different microscale cultivation systems commonly harnessed in single-cell analysis: microfluidic non-contact cell traps driven by negative dielectrophoresis, microfluidic monolayer growth chambers, and semi-solid agarose pads. We assessed the specific single-cell growth rates, division rates and morphological characteristics of single Corynebacterium glutamicum cells and microcolonies as a bacterial model organism with medical and biotechnological relevance under standardized growth conditions. Strikingly, the specific single-cell and microcolony growth rates, μmax, were robust and conserved for several cell generations with all three microcultivation technologies, whereas the division rates of cells grown on agarose pads deviated by up to 50% from those of cells cultivated in negative dielectrophoresis traps and monolayer growth chambers. Furthermore, morphological characteristics like cell lengths and division symmetries of individual cells were affected when the cells were grown on agarose pads. This indicated a significant impact of solid cultivation supports on cellular traits. The results demonstrate the impact of microcultivation technology on microbial physiology for the first time and show the need for a careful selection and design of the microcultivation technology in order to allow unbiased analysis of cellular behavior