Abstract
Polydimethylsiloxane (PDMS) chips have proven to be suitable environments for the growth of several filamentous organisms. However, depending on the specimen, the number of investigations concerning their growth and cell differentiation is limited. In this work, we monitored the developmental pattern of the brown alga Ectocarpus inside PDMS lab-on-chips. Two main methods of inoculation of the lab-on-chip were tested, i.e., via the direct injection of spores into the chamber as well as through the insertion of sporophyte filaments. The resulting growth rate, growth trajectory, cell differentiation, and cell branching were monitored and quantified for 20 days inside 25 or 40 μm parallel channels under standard light and temperature conditions. With growth rates of 2.8 μm⋅h–1, normal growth trajectories and cell differentiation, as well as branching occurring inside the microfluidic environment, the main development steps were shown to be similar to those observed in non-constrained in vitro conditions. Additionally, the labelling of Ectocarpus cell wall polysaccharides using calcofluor for cellulose detection and immunolocalisation with monoclonal antibodies for alginates showed the expected patterns when compared to open space growth evaluated with either epifluorescence or confocal microscopy. Overall, this article describes the experimental conditions for observing and studying the basic unaltered processes of brown algal growth using microfluidic technology which provides the basis for future biochemical and biological researches.
Highlights
Because of their highly polarised shape, filamentous organisms tend to raise many questions concerning their growth and differentiation, such as if their growth is restricted to the apical cell, which corresponds to a case of localised growth, or if it is shared by all the cells composing the filament as seen with diffuse growth patterns
While Ectocarpus was shown to grow on PDMS surfaces immersed in seawater (Evariste et al, 2012), we studied how its filaments cope with the restricted and constrained spatial environment provided by a microfluidic chip
We have shown that microfluidic chips with 44 parallel channels as narrow as 25 μm are suitable for studying Ectocarpus tip growth and dynamics of cell rounding
Summary
Because of their highly polarised shape, filamentous organisms tend to raise many questions concerning their growth and differentiation, such as if their growth is restricted to the apical cell, which corresponds to a case of localised growth, or if it is shared by all the cells composing the filament as seen with diffuse growth patterns. The reasons for using microfluidic devices to study small specimens are numerous and reflect the need of investigations to spatially constrain these cultured materials, for example by limiting the height and width of the available environment in the chip path in order to simplify long-term monitoring and improve the resolution of microscopy-based observations (Shamsudhin et al, 2016; Kozgunova and Goshima, 2019; Zhou et al, 2021) It can allow the separation of filaments which would otherwise grow too densely (Bascom et al, 2016) or guide them to grow perpendicular to a measuring device, such as a force sensor, positioned at the exit of the chip (Burri et al, 2018). In addition to the guidance of small organisms, microfluidic chips can be used for automated flows of controlled chemical compounds (Agudelo et al, 2013) as well as combined with microelectrodes to enable the generation of applied electrical pulses (Agudelo et al, 2016), both of which can be perceived as cues for the activation or repression of downstream signalling pathways in living specimens
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