Fungi-on-a-Chip: microfluidic platforms for single-cell studies on fungi

Abstract

This review highlights new advances in the emerging field of 'Fungi-on-a-Chip' microfluidics for single-cell studies on fungi and discusses several future frontiers, where we envisage microfluidic technology development to be instrumental in aiding our understanding of fungal biology. Fungi, with their enormous diversity, bear essential roles both in nature and our everyday lives. They inhabit a range of ecosystems, such as soil, where they are involved in organic matter degradation and bioremediation processes. More recently, fungi have been recognized as key components of the microbiome in other eukaryotes, such as humans, where they play a fundamental role not only in human pathogenesis, but also likely as commensals. In the food sector, fungi are used either directly or as fermenting agents and are often key players in the biotechnological industry, where they are responsible for the production of both bulk chemicals and antibiotics. Although the macroscopic fruiting bodies are immediately recognizable by most observers, the structure, function, and interactions of fungi with other microbes at the microscopic scale still remain largely hidden. Herein, we shed light on new advances in the emerging field of Fungi-on-a-Chip microfluidic technologies for single-cell studies on fungi. We discuss the development and application of microfluidic tools in the fields of medicine and biotechnology, as well as in-depth biological studies having significance for ecology and general natural processes. Finally, a future perspective is provided, highlighting new frontiers in which microfluidic technology can benefit this field.

Keywords: Fungi-on-a-Chip microfluidic technology; arbuscular mycorrhizal fungi; fungal biology; fungal highways; single-cell microscopy; yeast.

Figure 1.

Schematic illustrating yeast cell immobilization strategies. The illustration shows cross-sections through a microchannel to describe different methods of immobilizing yeast cells in a microfluidic device. (A) Cells fixed by in situ polymerization of a hydrogel. (B) Yeast cells fixed by affinity binding using Concanavalin A. (C) Spatial segmentation of cells into microcolonies via passive trapping. (D) Pressure-based trapping of cells between the channel ceiling and bottom. When flow pressure is high (+p), the channel widens and cells enter into the microchamber. Upon release of pressure (-p), the channel shrinks to a size comparable to that of the cell diameter, thus immobilizing the cells.

Figure 2.

Examples of applications of microfluidic immobilization of yeast cells by in situ immobilization and affinity binding. (A) Device for studying pheromone chemotropism in α- and A-yeast cells. Cells are trapped in alginate gel and subjected to asymmetric pheromone conditions. Image reproduced with modifications from Vo et al. (2020) with permission from AIP Publishing (Licence Number: 5271490260805). (B) Y-device, coated with concanavalin A (Con A) to help α-cells adhere to the channel. A gradient of α-factor was created, followed with the aid of Dextran-3000-TRITC as a tracking dye. Cells in five different areas, A–E, were imaged. Image reproduced with modifications from Moore et al. (2008) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/). (C) Example of five-channel device with computer-controlled 3-way valves to subject Con A-immobilized yeast cells to pulsed treatments with the protein kinase A (PKA) inhibitor 1-NM-PP1. A 63X microscope objective was used to monitor Msn2-mCherry translocation dynamics and gene expression in single cells. Image reproduced with modifications from Hansen and O’Shea (2013) with permission from John Wiley and Sons (Licence Number: 5271490732299).

Figure 3.

Exemplar applications of microfluidic immobilization of yeast cells by compartmentalization into microcolonies and active, pressure-based trapping. (A) Schematic illustration of a device having eight independently addressable rows each containing 15 microchemostats, highlighting the high degree of parallelization. Yeast microcolonies (here S. pombe) can be imaged and studied in parallel as well as subjected to different media conditions. The bright-field image shows three microchambers filled to confluence with yeast cells. Image reproduced with modifications from Nobs and Maerkl (2014) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/). (B) Microfluidic platform for trapping single yeast cells (here S. cerevisiae) under pressure-based expandable micropads (30 × 15 µm), used to study cell ageing. During cell loading, the micropads are slightly lifted due to the hydrodynamic pressure created by the flow. Upon release of pressure, the micropads resume their original height, thus trapping cells. Smaller daughter cells budding off from the mother cells are automatically washed away during dynamic cultivation with a slow constant flow. Microscope image showing some yeast cells (mother cells) trapped underneath the micropads and some smaller cells (daughter cells) being flushed out. Image reproduced with modifications from Lee et al. (2012) with permission from National Academy of Sciences. Scale bars represent 20 µm.

Figure 4.

Overview of channel features used to trap single yeast cells. The above schematic depicts a cross-section taken through a microfluidic channel containing yeast traps, as well as top-down views of four different trap designs (A)–(D). Electron micrographs of semicircular (A) and square (B) three-pillar trap designs reproduced with modifications from Ryley and Pereira-Smith (2006) with permission from John Wiley and Sons (Licence Number: 5271491246941). (C) Microscopy image of an oval two-pillar trap design reproduced with modifications from Crane et al. (2014) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/). (D) Microscopy image showing a channel-based trap design reproduced with modifications from Rowat et al. (2009) with permission from National Academy of Sciences. Scale bars represent 5 µm.

Figure 5.

Single-cell trapping of yeast cells using on-chip valve and slipstream techniques. (A) The microfluidic trapping device allows a valve-controlled two mode operation, a dynamic growth study, as well as end point/time point analysis of lysed cells. Image reproduced with modifications from Stratz et al. (2018) with permission from John Wiley and Sons (Licence Number: 5271500367493). (B) A microdevice for contactless trapping utilizing the slipstream effect. Image reproduced with modifications from Duran et al. (2020) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/).

Figure 6.

Droplet-based microfluidic methods. Schematics illustrating droplet generation using (A) flow focussing or (B) T-junction techniques. (C) Image showing the principle of fluorescence-activated droplet sorting as described in Wang et al. (2019), which was used to sort yeast genotypes with high amylase activity from a randomized library. Image reproduced with modifications from Wang et al. (2019) with permission from the National Academy of Sciences.

Figure 7.

Microfluidic devices for blood cleansing tasks. (A) A dialysis-like microfluidic device employed to filter fungal pathogens from blood via magnetic opsonins beads specific to the targeted pathogen. The particles with the attached pathogen are then extracted from the blood into an aqueous parallel waste collection stream using a magnet. The illustration is based on Yung et al. (2009). (B) Microdevice used to filter pathogens from blood relying on margination. As observed in blood vessels, red blood cells tend to migrate to the channel centre resulting in other blood components such as platelets or microbes being pushed towards the channel walls. Subsequently, they can be removed by a sudden widening of the microchannel. Image reproduced with modifications from Hou et al. (2012) with permission from AIP Publishing (Licence Number: 5271501114499). (C) A device, termed the Inertial Fungal Focuser (IFF), used to remove fungal pathogens from blood via inertial focussing. Image reproduced with modifications from Fuchs et al. (2019) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/). Scale bars represent ca. 100 µm.

Figure 8.

Microfluidic devices to study preinfection aspects of fungal pathogens. (A) Microdevice used to analyze surface adhesion of pathogenic yeasts under different shear-stress conditions. Shown are the principles of cell adhesion in yeasts, involving adhesin proteins under sheer stress (top) and the device design featuring arrays of parallel microchambers (bottom). (B) Under-oil microfluidic device for studying pathogenic fungal biofilm dynamics. The three-phase system consists of the PDMS substrate with micropatterned glass channels, an oil layer covering the channels and the aqueous medium or cell suspension, which is commonly injected into the oil phase. Images reproduced with modifications from Li et al. (2020) and Reinmets et al. (2019), respectively, with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/).

Figure 9.

Lung-on-a-Chip device for studying fungal lung infections. (A) Schematic illustrating a microfluidic device used to mimic the human bronchiole. (B) Microscopy image of the experimental set up. (C) Channels coated with cells stained with Hoechst (blue, nuclear stain), anti-CD31 antibody (green, endothelial tight junction marker), and anti-EpCAM antibody (red, epithelial cell–cell adhesion maker). Scale bars are 500 µm. Images reproduced with modifications from Barkal et al. (2017) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/).

Figure 10.

Microfluidic platforms for the study of fungal behaviour in artificial microenvironments mimicking aspects of the natural habitat. (A) Device used to study the behaviour of filamentous fungi upon facing special confinement on a subcellular level. Scale bar represents 20 µm. (B) Microdevice for studying space searching strategies in different fungal species. Scale bars represent 100 µm. (C) Microchip used to investigate chemotropism and uptake of lipophilic molecules (e.g. benzo[a]pyrene, BaP) by filamentous fungi. Image reproduced with modifications from Baranger et al. (2021) with permission from Elsevier (Licence Number: 5271511231788). (D) Microfluidic platform simulating several aspects of soil communities, featuring different channel designs filled with soil particles, soil bacteria, microfauna, and filamentous fungi, as well as being saturated with water or with air. Scale bars represent 20 µm. Images in (A), (B), and (D) reproduced with modifications from Fukuda et al. (2021), Hopke et al. (2021), and Mafla-Endara et al. (2021), respectively, with permission from the Creative Commons Attribution licence (www.creativecommons.org/licenses/by/4.0/).

Figure 11.

Microfluidic devices for measuring hyphal and cellular tip force. (A) A channel design containing a dead-end channel with a force sensing pillar. Upon deflection of the pillar, values for the force exerted by the hyphal tip can be calculated. Image reproduced with modifications from Sun et al. (2018) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/). (B) Microwells precisely fitting single cells of S. pombe. Upon cell fission and growth, the elastic PDMS wells are deformed and forces exerted can be deduced. (C) Microscopic image of trapped yeast cells with clearly visible deformation of the wells upon cell growth. Scale bar represents 10 µm. Images (B) and (C) reproduced with modifications from Minc et al. (2009) with permission from Elsevier (Licence Number: 5271520737780).

Figure 12.

Microfluidic devices for investigating hyphal interactions. (A) Image of the bacterial–fungal interaction (BFI) device, inoculated with an agar plug containing fungal mycelium. Scale bar represents 5 mm. (B) Bacillus subtilis (green) attaching to Coprinopsis cinerea hyphae (red), coinoculated in the BFI device. Scale bar representing 50 µm. (C) Time series showing the collapse of a C. cinerea cell and subsequent loss of its cellular contents, caused by B. subtilis. Scale bar representing 25 µm. Images (A)–(C) reproduced with modifications from Stanley et al. (2014) with permission from Oxford University Press (Licence Number: 5297210130322). (D) Schematic design of a device for studying fungal–nematode interactions. Scale bar represents 500 µm. (E) Proposed model for fungal response upon nematode predation. Bulk hyphae switch cytoplasmic bulk flow direction every 2–3 h in order to redistribute nutrients (e.g. 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), green hexagons). Red indicates the spatial distribution of the C. cinerea defence response upon predation. Images (D) and (E) reproduced with modifications from Schmieder et al. (2019) with permission from Elsevier (Licence Number: 5297260732132). (F) Schematic design of the fungal–fungal interaction (FFI) device (top). Scale bar represents 1 mm. (Bottom) Phase contrast image of hyphae of C. rosea and F. graminearum approaching each other in the interaction zone. Scale bar represents 250 µm. (G) Time series showing the detection of GFP within the mycelium of C. rosea (left). The fluorescence signal within these hyphae was found to increase and decrease compared to control channel (right). Images (F) and (G) reproduced with modifications from Gimeno et al. (2021) with permission from the Creative Commons Attribution license (www.creativecommons.org/licenses/by/4.0/).