Repeatability and reproducibility of microscopic examination of adhesive tape strip cytology slides for the quantification of Malassezia spp. in canine skin.

BACKGROUND: The optimal microscopic magnification and number of optical fields of adhesive tape strip cytological slides that should be examined when searching for Malassezia yeasts on canine skin are unknown. OBJECTIVES: To determine the optimal magnification and the minimum number of optical fields that should be examined to maximise intraobserver repeatability and interobserver reproducibility. MATERIALS AND METHODS: Seven experienced examiners counted, twice, the number of yeasts in 10, 20, 30, 40 and 50 optical fields of 40 slides at ×400 and ×1000 magnification. RESULTS: The number of yeasts per unit surface area was significantly higher at ×1000 compared to ×400 magnification. Repeatability and reproducibility for counting the yeasts was very poor. CONCLUSIONS AND CLINICAL RELEVANCE: Adhesive tape strip cytological slides should be examined microscopically for Malassezia spp. at ×1000 magnification. The repeatability of this examination for counting the yeasts is poor.

Contexte - Le grossissement microscopique optimal et le nombre de champs optiques des lames cytologiques de bandes adhésives à examiner lors de la recherche de levures Malassezia sur la peau de chien sont inconnus. Objectifs - Déterminer le grossissement optimal et le nombre minimal de champs à examiner pour maximiser la répétabilité intra-observateur et la reproductibilité inter-observateur. Matériels et méthodes - Sept examinateurs expérimentés ont compté, deux fois, le nombre de levures dans 10, 20, 30, 40 et 50 champs de 40 lames aux grossissements ×400 et ×1 000. Résultats - Le nombre de levures par unité de surface était significativement plus élevé au grossissement ×1 000 par rapport au grossissement ×400. La répétabilité et la reproductibilité du comptage des levures étaient très médiocres. Conclusions et pertinence clinique - Les lames cytologiques de bandes adhésives doivent être examinées au microscope pour Malassezia spp. à un grossissement ×1 000. La répétabilité de cet examen de comptage des levures est faible.

Introducción- se desconoce el aumento microscópico óptimo y el número de campos ópticos de los portaobjetos citológicos en tiras de cinta adhesiva que deben examinarse al buscar levaduras Malassezia en la piel canina. Objetivos- determinar el aumento óptimo y el número mínimo de campos ópticos que deben examinarse para maximizar la repetibilidad intraobservador y la reproducibilidad interobservador. Materiales y métodos- siete examinadores experimentados contaron dos veces el número de levaduras en campos ópticos de 10, 20, 30, 40 y 50 de 40 portaobjetos con aumentos de x ×400 y ×1000. Resultados- el número de levaduras por unidad de superficie fue significativamente mayor con un aumento de ×1000 en comparación con un aumento de ×400. La repetibilidad y reproducibilidad para contar las levaduras fue muy pobre. Conclusiones y relevancia clínica - Los portaobjetos citológicos en tiras de cinta adhesiva deben examinarse microscópicamente para detectar Malassezia spp. con un aumento de ×1.000. La repetibilidad de este examen para contar las levaduras es pobre.

Contexto - A ampliação microscópica ideal e o número de campos ópticos das lâminas citológicas de fita adesiva que devem ser examinados nas pesquisas de leveduras do gênero Malassezia em cães são desconhecidos. Objetivos - Determinar a magnificação ideal e o número mínimo de campos ópticos que devem ser examinados para maximizar a repetibilidade intraobservador e a reproducibilidade interobservador. Materiais e métodos - Sete examinadores experientes contaram duas vezes o número de leveduras em 10, 20, 30, 40 e 50 campos ópticos de 40 lâminas nas magnificações de x400 e x1000. Resultados - O número de leveduras por unidade de área de superfície foi significativamente maior em x1000 em comparação com a ampliação de x400. A repetibilidade e a reprodutibilidade para a contagem de leveduras foi muito pobre. Conclusões e relevância clínica - Lâminas de citologia por fica adesiva devem ser examinadas microscopicamente para Malassezia spp a uma magnificação de x1.000. A repetibilidade deste exame para contagem de leveduras foi pobre.

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Quantitatively Controlled Intercellular Mitochondrial Transfer by Cell Fusion-Based Method Using a Microfluidic Device.

Quantitative control of mitochondrial transfer is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA) because it enables precise modulation of heteroplasmy. Furthermore, single mitochondrion transfer from a mtDNA mutation-accumulated cell to a mtDNA-less (ρ0) cell potentially achieves homoplasmy of mutated mtDNA. Here we describe the method for quantitative control of mitochondrial transfer including achieving single mitochondrion transfer between live single cells using a microfluidic device.

Elevation of Intra-Cellular Calcium in Nucleus Pulposus Cells with Micro-Pipette-Guided Ultrasound.

Modulation of intra-cellular calcium by ultrasound offers a possible means for therapeutic applications. One such possibility is the modulation of nucleus pulposus cells as a preventive measure for inter-vertebral disc degeneration. We report a cellular stimulation device (micro-pipette ultrasound) using a glass micro-pipette as a waveguide to deliver ultrasound through the pipette tip and to elevate intra-cellular calcium in nucleus pulposus cells. The device generates two relevant stimuli at the cellular level: ultrasound propagation throughout the cell and acoustic streaming on the apical side. Ultrasound is radiated from a tip of a few microns, and its amplitude is proportional to the input voltage; acoustic streaming can be controlled by the duty factor. The novelty of the device is to impose a unique cellular loading: shear stress on cell apical surfaces combined with compressional waves propagating through the cells. G protein-coupled receptors and acid-sensing ion channel 3 were shown to play a role in calcium elevation by micro-pipette ultrasound in nucleus pulposus cells. Our results demonstrate that micro-pipette ultrasound can be an effective tool to elevate intra-cellular calcium levels in different cells, facilitating the identification of different mechanoreceptors in action.

An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells.

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.

Hydrogel Micropost Arrays with Single Post Tunability to Study Cell Volume and Mechanotransduction.

The extracellular matrix varies considerably in mechanical properties at the microscale. It remains unclear how cells respond to these properties, in part, due to lack of tools to create precisely defined microenvironments in a discrete manner. Here, freeform stereolithography is leveraged to control the placement and elastic modulus of individual hydrogel microposts that serve as discrete matrix signals to interface with cells. Mesenchymal stromal cells (MSCs) located in the interstitial spaces between microposts above a base layer are analyzed. Cell volume is higher when MSCs interact with more microposts. MSCs show higher strain energy when they interact simultaneously with 4-kPa and 20-kPa microposts than with mechanically homogeneous micropost arrays. MSCs are sensitive to pharmacological inhibition of Rho-associated protein kinase in 4-kPa arrays, but resistant when presented together with 20-kPa arrays. Yes-associated protein (YAP) activity increases with higher cell volume and elastic modulus of microposts. Surprisingly, YAP activity becomes less variable with higher cell volume and decreases with higher average force and strain energy per post when MSCs interact with both 4-kPa and 20-kPa microposts simultaneously. Together, these results describe a material system for systematically investigating how the placement and intrinsic properties of discrete matrix signals impact cell volume and mechanotransduction.

Studying cell wall mechanics using an automated confocal micro-extensometer.

Recently there has been a lot of interest in quantifying mechanical properties and responses to mechanical stress. This type of data can provide insight into how growth is regulated, the processes that enable it to occur and how stresses that build up during development feedback onto development itself. However, quantifying mechanical properties of plant cell walls is difficult as the material is heterogeneous, anisotropic and shows complex time-dependent properties as well as being subject to the complex geometries of plant tissues. It is therefore necessary to have a range of methods to enable the quantification of these properties at different resolutions and time-scales. Here we provide a guide to quantifying mechanical properties in Arabidopsis thaliana hypocotyls using a tensile testing device an automated confocal micro-extensometer (ACME). In contrast to indentation methods, tensile testing provides information on the tissue as a whole and in the plane of the sample. We also detail how to adapt the method to use it for quantifying responses to mechanical stress.

Building a smart FNA cart: When Google meets cytology.

BACKGROUND: The appropriate management of a fine needle aspiration (FNA) supply cart and equipment set up is essential to ensure the smooth and optimal operation of a busy FNA clinic. We applied Lean strategies such as value stream mapping (VSM), the 5S method (Sort, Set in order, Shine, Standardize, Sustain), and Kanban to remove waste and improve patient flow in an FNA clinic. METHODS: The workflow analysis suggested that existent problems such as suboptimal inventory management and unavailability of standard operating procedures (SOPs) caused a 10% to 85% increase in total procedure time. To improve inventory management, we created a 2-bin Kanban system. We used the "Scan to Web" app and a Google Drive form to create a cost-effective electronic inventory management system. We distributed the essential SOPs in the format of video clips using our YouTube channel and leveraged barcode technology to access the links. RESULTS: Upon completion of our process improvement project, we succeeded to eliminate the stock-out events and maintain a process cycle efficiency of 87%. The 5S audit checklist result increased from 6% to 100% implementation, which is consistent with focused improvement. The developed inventory system enabled us to track the supply usage, forecast demands, and improve the accuracy of orders. CONCLUSIONS: Lean methods such as VSM, 5S, and Kanban combined with open source technologies can be implemented to ensure material availability, track inventory, and provide immediate access to SOPs on demand. The developed system also led to increased efficiency and improved flow, as well as responsiveness to changes in demand.

A 3D Microfabricated Scaffold System for Unidirectional Cell Migration.

The present study demonstrates unidirectional cell migration using a novel 3D microfabricated scaffold, as revealed by the uneven sorting of cells into an area of 1 mm × 1 mm. To induce unidirectional cell migration, it is important to determine the optimal arrangement of 3D edges, and thus, the anisotropic periodic structures of micropatterns are adjusted appropriately. The cells put forth protrusions directionally along the sharp edges of these micropatterns, and migrated in the protruding direction. There are three advantages to this novel system. First, the range of applications is wide, because this system effectively induces unidirectional migration as long as 3D shapes of the scaffolds are maintained. Second, this system can contribute to the field of cell biology as a novel taxis assay. Third, this system is highly applicable to the development of medical devices. In the present report, unique 3D microfabricated scaffolds that provoked unidirectional migration of NIH3T3 cells are described. The 3D scaffolds could provoke cells to accumulate in a single target location, or could provoke a dissipated cell distribution. Because the shapes are very simple, they could be applied to the surfaces of various medical devices. Their utilization as a cell separation technology is also anticipated.

Three-tissue microphysiological system for studying inflammatory responses in gut-liver Axis.

The interaction between the gut and the liver, often known as the gut-liver axis, play crucial roles in modulating the body's responses to the xenobiotics as well as progression of diseases. Dysfunction of the axis can cause metabolic disorders as well as obesity, diabetes, and fatty liver disease. During the progression of such diseases, inflammatory responses involving the immune system also play an important part. In this study, we developed a three-tissue microphysiological system (MPS) that can accommodate three different cell types in separated compartments connected via fluidic channels in a microfluidic device. Using computational fluid dynamics, geometry of fluidic channels and flow conditions were optimized for seeding and culturing different cell types in the three-tissue MPS. Caco-2 (gut), RAW264.7 (immune), and HepG2 (liver) cells were seeded and cultured in the chip. Stimulation of the gut cells in the MPS with lipopolysaccharide (LPS) resulted in induction of inflammatory response and production of nitric oxide (NO) in all connected chambers. The anti-inflammatory effect of luteolin was demonstrated. Our study demonstrates that the three-tissue MPS can recapitulate the inflammatory responses involving the gut, liver and immune cells.

Spatially selective manipulation of cells with single-beam acoustical tweezers.

Acoustical tweezers open major prospects in microbiology for cells and microorganisms contactless manipulation, organization and mechanical properties testing since they are biocompatible, label-free and have the potential to exert forces several orders of magnitude larger than their optical counterpart at equivalent power. Yet, these perspectives have so far been hindered by the absence of spatial selectivity of existing acoustical tweezers - i.e., the ability to select and move objects individually - and/or their limited resolution restricting their use to large particle manipulation only and/or finally the limited forces that they could apply. Here, we report precise selective manipulation and positioning of individual human cells in a standard microscopy environment with trapping forces up to ~200 pN without altering their viability. These results are obtained with miniaturized acoustical tweezers combining holography with active materials to synthesize specific wavefields called focused acoustical vortices designed to produce stiff localized traps with reduced acoustic power.

Magnetic Patterning of Vorticella convallaria in a Microfluidic Device.

We describe an inexpensive magnetic cell patterning method as a tool for protozoologists. The ciliate Vorticella convallaria is useful for various biofluidics applications. Here, we show that V. convallaria will ingest metal beads and that permanent magnets can be used to pattern cells in Petri dishes or a microfluidic device. Patterning is reversibly achieved by placing magnets at the point of desired cell attachment. Analogous magnetic manipulation could be performed using other phagocytic cells.

Self-organization and multi-line transport of human spermatozoa in rectangular microchannels due to cell-cell interactions.

The journey of sperm navigation towards ovum is one of the most important questions in mammalian fertilisation and reproduction. However, we know very little about spermatozoa propagation in a complex fluidic, chemical and topographic environment of a fertility tract. Using microfluidics techniques, we investigate the influence of cell-cell interactions on spermatozoa swimming behavior in constrained environment at different concentrations. Our study shows that at high enough cell concentration the interaction between boundary-following cells leads to formation of areas with preferential direction of cell swimming. In the microchannel of a rectangular cross-section, this leads to formation of a "four-lane" swimming pattern with the asymmetry of the cell distribution of up to 40%. We propose that this is caused by the combination of cell-cell collisions in the corners of the microchannel and the existence of morphologically different spermatozoa: slightly asymmetric cells with trajectories curved left and the symmetric ones, with trajectories curved right. Our findings suggest that cell-cell interactions in highly folded environment of mammalian reproductive tract are important for spermatozoa swimming behavior and play role in selection of highly motile cells.

High-throughput cell migration assay under combinatorial chemical environments by a novel 24-well-plate based device.

The quantitative studies of cell proliferation and migration under different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a new device to pattern several types of cells in 24-well-plate and demonstrated its' application in cancer cell proliferation and migration assay. The new device combined 3D-printed-silica-part for multi cell types loading with PDMS-through-hole-layer-part for cell micro-patterning which was matched with commercial 24-well-plate. This 24-well-plate based device is flexible and feasible in many applications and can be used in one piece or multi pieces. Besides the application for two types of cells proliferation and migration assay in one chemical condition, as a demonstration, the migration behaviors of four types of cells under 24 types of EGF + bFGF combinatorial conditions were studied. We believed this device could be widely used in clinical searching for new anti-cancer therapeutics and other related studies.

Organic Electrochemical Transistors for Real-Time Monitoring of In Vitro Silver Nanoparticle Toxicity.

Nanomaterials are being widely used in medical applications and consumer products such as cosmetics, fabrics, and food packaging, although their impact on health and the environment is yet to be understood. Strategies enabling reliable and reproducible safety assessment of nanomaterials are needed because predicting their toxic effects is challenging as there is no simple correlation between their properties and the interaction with living systems. Here, the real-time monitoring of toxic effects induced by nanoparticles on cells using organic electrochemical transistors (OECTs) is reported. Noteworthy, OECTs are able to assess the coating-dependent toxicity of nanoparticles on both barrier and non-barrier tissue cells and, moreover, to monitor the cell health status as a function of exposure time, allowing useful insight on the interaction processes between nanomaterials and cells. These results demonstrate that OECTs are effective devices for real-time cell monitoring and in vitro assessment of nanomaterial toxicity.

Well Plate Integrated Topography Gradient Screening Technology for Studying Cell-Surface Topography Interactions.

New high-throughput technologies for cell-material interaction studies provide researchers with powerful tools to speed up research in the field of biomaterial-cell interactions. However, sharing technologies is often difficult due to the necessity of specific knowledge and experiences. Engineered surfaces can elucidate effects of surface topography on cell behavior, which is of critical value for gaining control over cellular processes. Here, the translation of a gradient-based high-throughput cell screening approach for aligned nano/micro topographies interacting with cells is presented. An aligned topography 96-well plate is created by upscaling of highly specific gradient technology. The resulting cell culture dishes are compatible with general laboratory and imaging equipment, and the platform allows for studying cell behavior with regard to adhesion and alignment. The challenge lies in increasing the dimensions of the previous 1 × 1 cm gradient topography substrate, to be able to cover the span of a 96-well plate and translate it into a standardized cell-screening tool. Adhesion experiments of human bone marrow derived mesenchymal stem cells confirm the standardization, compatibility, and usability of the technology. In the process of using multi-system imaging and analysis, it becomes apparent that future challenges need to include universally applied data analysis approaches.

Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs.

Label-free characterization of different kinds of cells using optoelectrokinetic-based microfluidics.

We report a novel, to the best of our knowledge, method to rapidly characterize different kinds of cells and drug-treated cancer cells using a label-free biomarker of self-rotation in an optoelectrokinetics (OEK)-based microfluidic platform. OEK incorporates optics and electrokinetics into microfluidics, thereby offering a contact-free, label-free, and rapid approach to the cellular manipulation community. Self-rotational behaviors of four different kinds of cells were experimentally investigated by the frequency-sweeping of an AC bias potential in an optically induced nonuniform and irrotational electric field. The results revealed that these kinds of cells displayed a Gaussian distribution versus the AC frequency as well as different self-rotational speeds under the same conditions. Furthermore, the peak self-rotational speed varied from one kind of cell to another, with that of cancer cells higher than that of normal cells. In addition, MCF-7 cells treated by various concentrations of drug showed remarkably different self-rotational speeds. This finding suggests a high potential of developing a new label-free biomarker to rapidly distinguish different kinds of cancer cells and quantitatively monitor the response of cancer patients to various treatments.

Assessing mitochondrial respiratory bioenergetics in whole cells and isolated organelles by microplate respirometry.

Mitochondria are responsible for the generation of ATP by oxidative phosphorylation; however, these multifaceted organelles regulate many other key cellular functions as well, such as calcium homeostasis, apoptosis, and biosynthesis of steroid hormones, heme and phospholipids. In order to carry out these functions, mitochondria establish physical and functional connections with other organelles such as the plasma membrane, lipid droplets/vesicles, peroxisomes, endosomes, and the endoplasmic reticulum. Dysregulation of any of the aforementioned processes or inter-organelle contacts can lead to mitochondrial dysfunction and subsequent changes in oxygen consumption and ATP production. Seahorse technology has become a critical tool for quantification of mitochondrial oxygen consumption and can help differentiate primary mitochondrial disorders from disorders where alterations in mitochondrial metabolism are consequences of a prior, upstream insult. In this chapter, we describe the application of Seahorse technology for assaying mitochondrial respiration in whole cells, permeabilized cells and isolated mitochondria. We leave it to the researcher's discretion to determine which of these approaches will generate the most physiologically relevant data based on the model system and research question at hand.

The Antibody-Free Recognition of Cancer Cells Using Plasmonic Biosensor Platforms with the Anisotropic Resonant Metasurfaces.

It is vital and promising for portable and disposable biosensing devices to achieve on-site detection and analysis of cancer cells. Although traditional labeling techniques provide an accurate quantitative measurement, the complicated cell staining and high-cost measurements limit their further development. Here, we demonstrate a nonimmune biosensing technology. The plasmonic biosensors, which are based on anisotropic resonant split ring resonators in the terahertz range, successfully realize the antibody-free recognition of cancer cells. The dependences of Δf and the fitted phase slope on the cancer cell concentration at different polarizations give new perspective in hexagonal radar maps. The results indicate that the lung cancer cell A549 and liver cancer cell HepG2 can be distinguished and determined simply based on the enclosed shapes in the radar maps without any antibody introduction. The minimum concentration of identification reduces to as low as 1 × 104 cells/mL and such identification can be kept valid in a wide range of cell concentration, ranging from 104 to 105. The construction of two-dimensional extinction intensity cards of corresponding cancer cells based on the wavelet transform method also supplies corresponding information for the antibody-free recognition and determination of two cancer cells. Our plasmonic metasurface biosensors show a great potential in the determination and recognition of label-free cancer cells, being an alternative to nonimmune biosensing technology.