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Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery ofStentor coeruleus(SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery onStentoras our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.

 

Stentor coeruleus , a single-cell ciliated protozoan, is a model organism for wound healing and regeneration studies. Despite Stentor 's large size (up to 2 mm in extended state), microdissection of Stentor remains challenging. In this work, we describe a hydrodynamic cell splitter, consisting of a microfluidic cross junction, capable of splitting Stentor cells in a non-contact manner at a high throughput of ∼500 cells per minute under continuous operation. Introduction of asymmetry in the flow field at the cross junction leads to asymmetric splitting of the cells to generate cell fragments as small as ∼8.5 times the original cell size. Characterization of cell fragment viability shows reduced 5-day survival as fragment size decreases and as the extent of hydrodynamic stress imposed on the fragments increases. Our results suggest that cell fragment size and composition, as well as mechanical stress, play important roles in the long-term repair of Stentor cells and warrant further investigations. Nevertheless, the hydrodynamic splitter can be useful for studying phenomena immediately after cell splitting, such as the closure of wounds in the plasma membrane which occurs on the order of 100–1000 seconds in Stentor . 

Abstract Background Wound healing is one of the defining features of life and is seen not only in tissues but also within individual cells. Understanding wound response at the single-cell level is critical for determining fundamental cellular functions needed for cell repair and survival. This understanding could also enable the engineering of single-cell wound repair strategies in emerging synthetic cell research. One approach is to examine and adapt self-repair mechanisms from a living system that already demonstrates robust capacity to heal from large wounds. Towards this end, Stentor coeruleus , a single-celled free-living ciliate protozoan, is a unique model because of its robust wound healing capacity. This capacity allows one to perturb the wounding conditions and measure their effect on the repair process without immediately causing cell death, thereby providing a robust platform for probing the self-repair mechanism. Results Here we used a microfluidic guillotine and a fluorescence-based assay to probe the timescales of wound repair and of mechanical modes of wound response in Stentor . We found that Stentor requires ~ 100–1000 s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~ 8–80 μm 2 /s, faster than most other single cells reported in the literature. Further, we characterized three distinct mechanical modes of wound response in Stentor : contraction, cytoplasm retrieval, and twisting/pulling. Using chemical perturbations, active cilia were found to be important for only the twisting/pulling mode. Contraction of myonemes, a major contractile fiber in Stentor , was surprisingly not important for the contraction mode and was of low importance for the others. Conclusions While events local to the wound site have been the focus of many single-cell wound repair studies, our results suggest that large-scale mechanical behaviors may be of greater importance to single-cell wound repair than previously thought. The work here advances our understanding of the wound response in Stentor and will lay the foundation for further investigations into the underlying components and molecular mechanisms involved. 

When granular materials, colloidal suspensions, and even animals and crowds exit through a narrow outlet, clogs can form spontaneously when multiple particles or entities attempt to exit simultaneously, thereby obstructing the outlet and ultimately halting the flow. Counterintuitively, the presence of an obstacle upstream of the outlet has been found to suppress clog formation. For soft particles such as emulsion drops, clogging has not been observed in the fast flow limit due to their deformability and vanishing interparticle friction. Instead, they pinch off each other and undergo break up when multiple drops attempt to exit simultaneously. Similar to how an obstacle reduces clogging in a rigid particle system, we hypothesize and demonstrate that an obstacle could suppress break up in the two-dimensional hopper flow of a microfluidic crystal consisting of dense emulsion drops by preventing the simultaneous exit of multiple drops. A regime map plotting the fraction of drops that undergo break up in a channel with different obstacle sizes and locations delineates the geometrical constraints necessary for effective break up suppression. When optimally placed, the obstacle induced an unexpected ordering of the drops, causing them to alternate and exit the outlet one at a time. Droplet break up is suppressed drastically by almost three orders of magnitude compared to when the obstacle is absent. This result can provide a simple, passive strategy to prevent droplet break up and can find use in improving the robustness and integrity of droplet microfluidic biochemical assays as well as in extrusion-based three-dimensional printing of emulsion or foam-based materials.