How to Perform a Microfluidic Cultivation Experiment—A Guideline to Success

Täuber, Sarah; Schmitz, Julian; Blöbaum, Luisa; Fante, Niklas; Steinhoff, Heiko; Grünberger, Alexander

doi:10.3390/bios11120485

Cited by 9 publications

(5 citation statements)

References 89 publications

(126 reference statements)

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“…Due to its ease of fabrication and its biocompatible properties, polydimethylsiloxane (PDMS)-based microfluidics is of increasing relevance for developmental biology [ 14 ] and has been widely applied for the investigation of various small organisms ranging from plant specimens [ 15 , 16 , 17 ] to animal models [ 18 , 19 , 20 , 21 , 22 ]. Furthermore, microfluidic-based cultivation has recently been reported as a suitable tool for the study of the filamentous growth of the brown alga Ectocarpus silicosus [ 23 ].…”

Section: Resultsmentioning

confidence: 99%

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices

et al. 2022

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The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen’s health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device’s operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen’s cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

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Section: Resultsmentioning

confidence: 99%

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices

et al. 2022

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“…Then inlets and outlets for the fluid and the gas flow connections were manually punched using a punching tool (ϕ = 0.75 mm, World Precision Instruments, USA). Single PDMS chips were finally bonded to glass substrates (D263 ® Bio, 39.5 mm × 34.5 mm × 0.175 mm, Schott AG, Germany) after an O 2 plasma treatment (Femto Plasma Cleaner, Diener Electronics, Germany) for 25 s. For full fabrication details, the reader is referred to Gruenberger et al ( 2013 ) and Täuber et al ( 2021 ).…”

Section: Materials and Equipmentmentioning

confidence: 99%

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

et al. 2023

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Microfluidic cultivation devices that facilitate O2 control enable unique studies of the complex interplay between environmental O2 availability and microbial physiology at the single-cell level. Therefore, microbial single-cell analysis based on time-lapse microscopy is typically used to resolve microbial behavior at the single-cell level with spatiotemporal resolution. Time-lapse imaging then provides large image-data stacks that can be efficiently analyzed by deep learning analysis techniques, providing new insights into microbiology. This knowledge gain justifies the additional and often laborious microfluidic experiments. Obviously, the integration of on-chip O2 measurement and control during the already complex microfluidic cultivation, and the development of image analysis tools, can be a challenging endeavor. A comprehensive experimental approach to allow spatiotemporal single-cell analysis of living microorganisms under controlled O2 availability is presented here. To this end, a gas-permeable polydimethylsiloxane microfluidic cultivation chip and a low-cost 3D-printed mini-incubator were successfully used to control O2 availability inside microfluidic growth chambers during time-lapse microscopy. Dissolved O2 was monitored by imaging the fluorescence lifetime of the O2-sensitive dye RTDP using FLIM microscopy. The acquired image-data stacks from biological experiments containing phase contrast and fluorescence intensity data were analyzed using in-house developed and open-source image-analysis tools. The resulting oxygen concentration could be dynamically controlled between 0% and 100%. The system was experimentally tested by culturing and analyzing an E. coli strain expressing green fluorescent protein as an indirect intracellular oxygen indicator. The presented system allows for innovative microbiological research on microorganisms and microbial ecology with single-cell resolution.

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“…Culturing cells and bacteria in a microfluidic system, particularly in the long term, can also lead to other problems, including fluid leakage [165], clogging [166], and unwanted accumulation of bubbles in the channels. Although some of these problems are manageable through a careful selection of materials or protocols, bubble accumulation is a frequent obstacle that is extremely difficult to avoid in most PDMS microfluidic systems [167].…”

Section: Polyfermsmentioning

confidence: 99%

Designs and methodologies to recreate in vitro human gut microbiota models

et al. 2022

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The human gut microbiota is widely considered to be a metabolic organ hidden within our bodies, playing a crucial role in the host’s physiology. Several factors affect its composition, so a wide variety of microbes residing in the gut are present in the world population. Individual excessive imbalances in microbial composition are often associated with human disorders and pathologies, and new investigative strategies to gain insight into these pathologies and define pharmaceutical therapies for their treatment are needed. In vitro models of the human gut microbiota are commonly used to study microbial fermentation patterns, community composition, and host-microbe interactions. Bioreactors and microfluidic devices have been designed to culture microorganisms from the human gut microbiota in a dynamic environment in the presence or absence of eukaryotic cells to interact with. In this review, we will describe the overall elements required to create a functioning, reproducible, and accurate in vitro culture of the human gut microbiota. In addition, we will analyze some of the devices currently used to study fermentation processes and relationships between the human gut microbiota and host eukaryotic cells. Graphic abstract

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How to Perform a Microfluidic Cultivation Experiment—A Guideline to Success

Cited by 9 publications

References 89 publications

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices

Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

Designs and methodologies to recreate in vitro human gut microbiota models

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