Hydrodynamic dissection of Stentor coeruleus in a microfluidic cross junction.
Lab on a chip
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.
View details for DOI 10.1039/d2lc00527a
View details for PubMedID 35971861
Microfluidic Surgery in Single Cells and Multicellular Systems.
Microscale surgery on single cells and small organisms has enabled major advances in fundamental biology and in engineering biological systems. Examples of applications range from wound healing and regeneration studies to the generation of hybridoma to produce monoclonal antibodies. Even today, these surgical operations are often performed manually, but they are labor intensive and lack reproducibility. Microfluidics has emerged as a powerful technology to control and manipulate cells and multicellular systems at the micro- and nanoscale with high precision. Here, we review the physical and chemical mechanisms of microscale surgery and the corresponding design principles, applications, and implementations in microfluidic systems. We consider four types of surgical operations: (1) sectioning, which splits a biological entity into multiple parts, (2) ablation, which destroys part of an entity, (3) biopsy, which extracts materials from within a living cell, and (4) fusion, which joins multiple entities into one. For each type of surgery, we summarize the motivating applications and the microfluidic devices developed. Throughout this review, we highlight existing challenges and opportunities. We hope that this review will inspire scientists and engineers to continue to explore and improve microfluidic surgical methods.
View details for DOI 10.1021/acs.chemrev.1c00616
View details for PubMedID 35049287
Microfluidic guillotine reveals multiple timescales and mechanical modes of wound response in Stentor coeruleus.
2021; 19 (1): 63
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 ~100-1000s to close bisection wounds, depending on the severity of the wound. This corresponds to a healing rate of ~8-80mum2/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.
View details for DOI 10.1186/s12915-021-00970-0
View details for PubMedID 33810789
Fomite Transmission, Physicochemical Origin of Virus-Surface Interactions, and Disinfection Strategies for Enveloped Viruses with Applications to SARS-CoV-2.
2021; 6 (10): 6509–27
Inanimate objects or surfaces contaminated with infectious agents, referred to as fomites, play an important role in the spread of viruses, including SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The long persistence of viruses (hours to days) on surfaces calls for an urgent need for effective surface disinfection strategies to intercept virus transmission and the spread of diseases. Elucidating the physicochemical processes and surface science underlying the adsorption and transfer of virus between surfaces, as well as their inactivation, is important for understanding how diseases are transmitted and for developing effective intervention strategies. This review summarizes the current knowledge and underlying physicochemical processes of virus transmission, in particular via fomites, and common disinfection approaches. Gaps in knowledge and the areas in need of further research are also identified. The review focuses on SARS-CoV-2, but discussion of related viruses is included to provide a more comprehensive review given that much remains unknown about SARS-CoV-2. Our aim is that this review will provide a broad survey of the issues involved in fomite transmission and intervention to a wide range of readers to better enable them to take on the open research challenges.
View details for DOI 10.1021/acsomega.0c06335
View details for PubMedID 33748563
Fabrication of 3D Micro-Blades for the Cutting of Biological Structures in a Microfluidic Guillotine.
2021; 12 (9)
Micro-blade design is an important factor in the cutting of single cells and other biological structures. This paper describes the fabrication process of three-dimensional (3D) micro-blades for the cutting of single cells in a microfluidic "guillotine" intended for fundamental wound repair and regeneration studies. Our microfluidic guillotine consists of a fixed 3D micro-blade centered in a microchannel to bisect cells flowing through. We show that the Nanoscribe two-photon polymerization direct laser writing system is capable of fabricating complex 3D micro-blade geometries. However, structures made of the Nanoscribe IP-S resin have low adhesion to silicon, and they tend to peel off from the substrate after at most two times of replica molding in poly(dimethylsiloxane) (PDMS). Our work demonstrates that the use of a secondary mold replicates Nanoscribe-printed features faithfully for at least 10 iterations. Finally, we show that complex micro-blade features can generate different degrees of cell wounding and cell survival rates compared with simple blades possessing a vertical cutting edge fabricated with conventional 2.5D photolithography. Our work lays the foundation for future applications in single cell analyses, wound repair and regeneration studies, as well as investigations of the physics of cutting and the interaction between the micro-blade and biological structures.
View details for DOI 10.3390/mi12091005
View details for PubMedID 34577648
The Pediatric Temporal-spatial Deviation Index: quantifying gait impairment for children with cerebral palsy.
Developmental medicine and child neurology
AIM: To develop an easily-administered metric to quantify gait impairment in children and to assess its use in children with cerebral palsy (CP).METHOD: The Pediatric Temporal-spatial Deviation Index (TDI) was developed from gait data collected from 75 typically developing children (37 males, 38 females; mean age 9y 4mo; interquartile range [IQR] 8-10y) and 17 children diagnosed with spastic CP (nine males, eight females; mean age 9y 9mo; IQR 9-11y), inGross Motor Function Classification System (GMFCS) levels I to III, aged 7 to 11years. Children walked on a pressure-sensitive mat. Children with CP also completed 3D gait analysis. The Kaiser-Meyer-Olkin test of sampling adequacy was used for temporal-spatial feature selection. Principal components obtained from temporal-spatial gait parameters quantified deviation from typically developing gait. Deviation was normalized to a Pediatric TDI score mean (standard deviation [SD]) of 100 (10). The Pediatric TDI for children with CP was compared to 3D motion capture-based Gait Deviation Index (GDI).RESULTS: The Pediatric TDI was significantly lower for children with CP compared to typically developing children (p<0.001), correlated with average GDI (r=0.610, p=0.009), and demonstrated sensitivity (0.78) and specificity (0.88) to gait function, assessed with GDI.INTERPRETATION: The Pediatric TDI is an easily administered, revealing gait metric that can be used in children with CP in pediatric clinics and for research. Detection of gait abnormalities in the clinic can expedite diagnosis and treatment. What this paper adds The Pediatric Temporal-spatial Deviation Index (TDI) is a single-score index of gait deviation, based on nine parameters. The Pediatric TDI was more revealing than single temporal-spatial gait parameters. The Pediatric TDI is quick and simple to administer in the clinic.
View details for DOI 10.1111/dmcn.14271
View details for PubMedID 31206183
- Kirigami and the Caspar-Klug construction for viral shells with negative Gauss curvature PHYSICAL REVIEW E 2019; 99 (2)