Applications In Cell Biology Research Articles

Current Advances in Corneal Stromal Stem Cell Biology and Therapeutic Applications.

Corneal stromal stem cells (CSSCs) are of particular interest in regenerative ophthalmology, offering a new therapeutic target for corneal injuries and diseases. This review provides a comprehensive examination of CSSCs, exploring their anatomy, functions, and role in maintaining corneal integrity. Molecular markers, wound healing mechanisms, and potential therapeutic applications are discussed. Global corneal blindness, especially in more resource-limited regions, underscores the need for innovative solutions. Challenges posed by corneal defects, emphasizing the urgent need for advanced therapeutic interventions, are discussed. The review places a spotlight on exosome therapy as a potential therapy. CSSC-derived exosomes exhibit significant potential for modulating inflammation, promoting tissue repair, and addressing corneal transparency. Additionally, the rejuvenation potential of CSSCs through epigenetic reprogramming adds to the evolving regenerative landscape. The imperative for clinical trials and human studies to seamlessly integrate these strategies into practice is emphasized. This points towards a future where CSSC-based therapies, particularly leveraging exosomes, play a central role in diversifying ophthalmic regenerative medicine.

Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture.

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes. The advantages of this approach include its efficiency in producing cellular heterogeneity, particularly in non-adherent 3D cultures, and its ability to create hypoxic spheroids, making it a suitable model for studying cancer. Moreover, the hanging drop method has proven valuable in investigating various aspects such as tissue structure, signaling pathways, immune activation of cancer cells, and notably, cell proliferation. Researchers have utilized the hanging drop method to explore the dynamics of cell proliferation, studying the effects of mesenchymal stem cells (MSC) secretome on cancer cells. The method's application involves co-culturing different cell lines, assessing spheroid formations, and quantifying their sizes over time. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell growth and viability within a three-dimensional co-culture paradigm.

2 in 1 Vectors Improve in Planta BiFC and FRET Analysis.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes, and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2-in-1 cloning approach, and their application in plant cell biology: ratiometric bimolecular fluorescence complementation (rBiFC), FRET acceptor photobleaching (FRET-AB), and fluorescent lifetime imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Principles of Indirect Co-culture Method Using Transwell.

The co-culture method is a simple type of cell culture method used to evaluate the effects of communication between various types of cells in an in vitro setting. In the co-culture method, two or more eukaryotic cell types, or eukaryotic and prokaryotic cells, are cultured together. The co-culture method reflects in vivo cell behaviors and thereby emerges as a pivotal technique with diverse applications in cancer research and cell biology. Two categories of co-culture methods (indirect methods and direct methods) are well known. Direct co-culture methods allow physical contact between the various cell types (juxtacrine signaling). In indirect methods, cells are physically separated into two different populations (for example, using a Transwell) that allow communication only via secretory factors (paracrine signaling). Herein, we focus on the principles of the indirect co-culture method. Nowadays, this method is used to explore the effects of mesenchymal stem cell (MSC) secretome on cancer cells. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell proliferation, invasion, apoptosis, and polarity.

Generalized Michaelis-Menten rate law with time-varying molecular concentrations.

The Michaelis-Menten (MM) rate law has been the dominant paradigm of modeling biochemical rate processes for over a century with applications in biochemistry, biophysics, cell biology, systems biology, and chemical engineering. The MM rate law and its remedied form stand on the assumption that the concentration of the complex of interacting molecules, at each moment, approaches an equilibrium (quasi-steady state) much faster than the molecular concentrations change. Yet, this assumption is not always justified. Here, we relax this quasi-steady state requirement and propose the generalized MM rate law for the interactions of molecules with active concentration changes over time. Our approach for time-varying molecular concentrations, termed the effective time-delay scheme (ETS), is based on rigorously estimated time-delay effects in molecular complex formation. With particularly marked improvements in protein-protein and protein-DNA interaction modeling, the ETS provides an analytical framework to interpret and predict rich transient or rhythmic dynamics (such as autogenously-regulated cellular adaptation and circadian protein turnover), which goes beyond the quasi-steady state assumption.

Thermoresponsive Poly(N,N'-dimethylacrylamide)-Based Diblock Copolymer Worm Gels via RAFT Solution Polymerization: Synthesis, Characterization, and Cell Biology Applications.

RAFT solution polymerization is used to polymerize 2-hydroxypropyl methacrylate (HPMA). The resulting PHPMA precursor is then chain-extended using N,N'-dimethylacrylamide (DMAC) to produce a series of thermoresponsive PHPMA-PDMAC diblock copolymers. Such amphiphilic copolymers can be directly dispersed in ice-cold water and self-assembled at 20 °C to form spheres, worms, or vesicles depending on their copolymer composition. Construction of a pseudo-phase diagram is required to identify the pure worm phase, which corresponds to a rather narrow range of PDMAC DPs. Such worms form soft, free-standing gels in aqueous solution at around ambient temperature. Rheology studies confirm the thermoresponsive nature of such worms, which undergo a reversible worm-to-sphere on cooling below ambient temperature. This morphological transition leads to in situ degelation, and variable temperature 1H NMR studies indicate a higher degree of (partial) hydration for the weakly hydrophobic PHPMA chains at lower temperatures. The trithiocarbonate end-group located at the end of each PDMAC chain can be removed by treatment with excess hydrazine. The resulting terminal secondary thiol group can form disulfide bonds via coupling, which produces PHPMA-PDMAC-PHPMA triblock copolymer chains. Alternatively, this reactive thiol group can be used for conjugation reactions. A PHPMA141-PDMAC36 worm gel was used to store human mesenchymal stem cells (MSCs) for up to three weeks at 37 °C. MSCs retrieved from this gel subsequently underwent proliferation and maintained their ability to differentiate into osteoblastic cells.

Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α5β1 Integrin and Syndecan-4 Crosstalk.

Cells regulate adhesion to the fibrillar extracellular matrix (ECM) of which fibronectin is an essential component. However, most studies characterize cell adhesion to globular fibronectin substrates at time scales long after cells polarize and migrate. To overcome this limitation, a simple and scalable method to engineer biomimetic 3D fibrillar fibronectin matrices is introduced and how they are sensed by fibroblasts from the onset of attachment is characterized. Compared to globular fibronectin substrates, fibroblasts accelerate adhesion initiation and strengthening within seconds to fibrillar fibronectin matrices via α5β1 integrin and syndecan-4. This regulation, which additionally accelerates on stiffened fibrillar matrices, involves actin polymerization, actomyosin contraction, and the cytoplasmic proteins paxillin, focal adhesion kinase, and phosphoinositide 3-kinase. Furthermore, this immediate sensing and adhesion of fibroblast to fibrillar fibronectin guides migration speed, persistency, and proliferation range from hours to weeks. The findings highlight that fibrillar fibronectin matrices, compared to widely-used globular fibronectin, trigger short- and long-term cell decisions very differently and urge the use of such matrices to better understand in vivo interactions of cells and ECMs. The engineered fibronectin matrices, which can be printed onto non-biological surfaces without loss of function, open avenues for various cell biological, tissue engineering and medical applications.

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

Suppression of auto-fluorescence from high-resolution 3D polymeric architectures fabricated via two-photon polymerization for cell biology applications

A 460 800 Pixels CMOS Capacitive Sensor Array With Programmable Fusion Pixels and Noise Canceling for Life Science Applications

Capacitive sensor array (CSA) plays an important role in life science applications, such as cell monitoring, DNA detection, or drug screening. This paper presents a 480×960 time-sharing TDC (ts-TDC) architecture of global shutter CSA. It provides an opportunity to merge interfacing, read-out and sampling circuits in pixel and dynamically adjust the electrode size according to the characteristic of biosample. Each pixel size is within 2.25×2.25 μm2. The sensitivity of the proposed CSA is 6.896 codes/fF, and 43 fps can be adopted for real-time monitoring. Experiment results show that noise canceling significantly improves the sensing performance. Moreover, sensitivity can be optimized by given fusion-pixel pattern, making our proposal very flexible and suitable for cell biology applications and personalized medicine.

Micro 3D Printing Elastomeric IP-PDMS Using Two-Photon Polymerisation: A Comparative Analysis of Mechanical and Feature Resolution Properties.

The mechanical properties of two-photon-polymerised (2PP) polymers are highly dependent on the employed printing parameters. In particular, the mechanical features of elastomeric polymers, such as IP-PDMS, are important for cell culture studies as they can influence cell mechanobiological responses. Herein, we employed optical-interferometer-based nanoindentation to characterise two-photon-polymerised structures manufactured with varying laser powers, scan speeds, slicing distances, and hatching distances. The minimum reported effective Young's modulus (YM) was 350 kPa, while the maximum one was 17.8 MPa. In addition, we showed that, on average, immersion in water lowered the YM by 5.4%, a very important point as in the context of cell biology applications, the material must be employed within an aqueous environment. We also developed a printing strategy and performed a scanning electron microscopy morphological characterisation to find the smallest achievable feature size and the maximum length of a double-clamped freestanding beam. The maximum reported length of a printed beam was 70 µm with a minimum width of 1.46 ± 0.11 µm and a thickness of 4.49 ± 0.05 µm. The minimum beam width of 1.03 ± 0.02 µm was achieved for a beam length of 50 µm with a height of 3.00 ± 0.06 µm. In conclusion, the reported investigation of micron-scale two-photon-polymerized 3D IP-PDMS structures featuring tuneable mechanical properties paves the way for the use of this material in several cell biology applications, ranging from fundamental mechanobiology to in vitro disease modelling to tissue engineering.

Cross-species comparison of mouse and human preimplantation development with an emphasis on lineage specification.

Human embryogenesis still remains largely unexplored. This review helps identify some of our current gaps in knowledge pertaining to preimplantation development, which may have implications for understanding fundamental aspects of human development, assisted reproductive technologies, and stem cell biology. Preimplantation development is arguably one of the most critical stages of embryogenesis. Beginning with the formation of the totipotent zygote post-fertilization, a series of cell divisions, and a complex coordination of physical cues, molecular signals and changes in gene expression lead to the formation of the blastocyst, a structure capable of implanting into the uterine wall. The blastocyst is composed of more specified cellular lineages, which will give rise to every tissue of the developing organism as well as the extra-embryonic lineages which support fetal growth. While the mouse has been used as a model to understand the events of preimplantation development for decades, in recent years, an expanding body of work has been conducted using the human embryo. These studies have identified some crucial species differences, particularly in the transcriptional and spatio-temporal expression of lineage markers and responses to cell signaling perturbations. This review compares recent findings on preimplantation development in mouse and human, with a focus on the specification of the first cellular lineages. Highlighting differences and noting mechanisms that require further examination in the human embryo is of critical importance for both the accurate translation of results from the mouse model and our overall understanding of mammalian development. We further highlight the latest advancement in reproductive research, the development of the 3D stem cell-based models known as 'blastoids'. The knowledge discussed in this review has major clinical implications for assisted reproductive technologies such as in vitro fertilization and for applications in stem cell biology.

See the Unseen: Red-Emissive Carbon Dots for Visualizing the Nucleolar Structures in Two Model Animals and In Vivo Drug Toxicity.

Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cellor an organism. Here, bright red-emissive carbon dots (termed CPCDs) with excitation-independent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine. The CPCDs can achieve wash-free, real-time, long-term, and high-quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals-zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish-related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag+ ), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Methods for studying mammalian aquaporin biology.

Aquaporins (AQPs), transmembrane water-conducting channels, have earned a great deal of scrutiny for their critical physiological roles in healthy and disease cell states, especially in the biomedical field. Numerous methods have been implemented to elucidate the involvement of AQP-mediated water transport and downstream signaling activation in eliciting whole cell, tissue, and organ functional responses. To modulate these responses, other methods have been employed to investigate AQP druggability. This review discusses standard in vitro, in vivo, and in silico methods for studying AQPs, especially for biomedical and mammalian cell biology applications. We also propose some new techniques and approaches for future AQP research to address current gaps in methodology.

Triboelectric Nanogenerator Array as a Probe for In Situ Dynamic Mapping of Interface Charge Transfer at a Liquid-Solid Contacting.

Contact between water droplets with hydrophobic surfaces is a common phenomenon at functional interfaces, and it has been extensively studied. However, quantifying the charge transfer between the liquid-solid interfacial contacting, especially for the charge density distribution throughout the movement of liquid droplet on a dielectric surface, remains to be investigated. Here, we developed a pixeled droplet triboelectric nanogenerator (pixeled droplet-TENG) array with high-density electrode array as a probe for measuring the charge transfer at a liquid-solid interface when a water drop moves on the hydrophobic surface. To intuitively observe the charge transfer between the liquid-solid interface, we "imaged" the transferred charges along movement trajectory of a water droplet as it slides along a tilted solid surface at a spatial resolution of 0.4 mm and time sensitivity of 0.02 s. Our study shows that the transferred charges are not uniformly distributed along the path, which is possibly due to the two-step model of electron transfer and ion adsorbed on the solid surface, and thus the formation of an electric double layer will inevitably shield the net surface on the solid surface. Our study presents a probe technology with potential applications in surface chemistry, physics, material science, and cell biology.

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.

Preprint Highlight: ALIBY: ALFA nanobody-based toolkit for imaging and biochemistry in yeast.

Many commonly used protein epitope tags are optimized for one or a few biological applications. The recently developed ALFA tag performs well across a broad range of cell biological applications including localization, biochemical, and functional studies but has not been available in budding yeast. This study engineers a modular plasmid-based platform to introduce ALFA tag on proteins of interest in budding yeast for detection via fluorescently tagged ALFA nanobodies. The toolkit offers excellent performance for quantitative live imaging, spatiotemporal analyses of proteins, protein kinetics, and immunoprecipitation studies, all with the same tag. These materials will be of broad interest to yeast biologists for simplified imaging and biochemical analyses. This preprint has been assigned the following badges: New Methods, New Materials. Read the preprint on bioRxiv ( Akhuli et al., 2022 ): https://doi.org/10.1101/2022.07.18.500560 .

ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in Yeast.

ABSTRACTSpecialized epitope tags continue to be integral components of various biochemical and cell biological applications such as fluorescence microscopy, immunoblotting, immunoprecipitation, and protein purification. However, until recently, no single tag could offer this complete set of functionalities on its own. Here, we present a plasmid-based toolkit named ALIBY (ALFA toolkit for imaging and biochemistry in yeast) that provides a universal workflow to adopt the versatile ALFA tag/NbALFA system within the well-established model organism Saccharomyces cerevisiae. The kit comprises tagging plasmids for labeling a protein of interest with the ALFA tag and detection plasmids encoding fluorescent-protein-tagged NbALFA for live-cell imaging purposes. We demonstrate the suitability of ALIBY for visualizing the spatiotemporal localization of yeast proteins (i.e., the cytoskeleton, nucleus, centrosome, mitochondria, vacuole, endoplasmic reticulum, exocyst, and divisome) in live cells. Our approach has yielded an excellent signal-to-noise ratio without off-target effects or any effect on cell growth. In summary, our yeast-specific toolkit aims to simplify and further advance the live-cell imaging of differentially abundant yeast proteins while also being suitable for biochemical applications.IMPORTANCE In yeast research, conventional fluorescent protein tags and small epitope tags are widely used to study the spatiotemporal dynamics and activity of proteins. Although proven to be efficient, these tags lack the versatility for use across different cell biological and biochemical studies of a given protein of interest. Therefore, there is an urgent need for a unified platform for visualization and biochemical and functional analyses of proteins of interest in yeast. Here, we have engineered ALIBY, a plasmid-based toolkit that expands the benefits of the recently developed ALFA tag/NbALFA system to studies in the well-established model organism Saccharomyces cerevisiae. We demonstrate that ALIBY provides a simple and versatile strain construction workflow for long-duration live-cell imaging and biochemical applications in yeast.

Imageseg: An R package for deep learning‐based image segmentation

Abstract Convolutional neural networks (CNNs) and deep learning are powerful and robust tools for ecological applications, and are particularly suited for image data. Image segmentation (the classification of all pixels in images) is one such application and can, for example, be used to assess forest structural metrics. While CNN‐based image segmentation methods for such applications have been suggested, widespread adoption in ecological research has been slow, likely due to technical difficulties in implementation of CNNs and lack of toolboxes for ecologists. Here, we present R package imageseg which implements a CNN‐based workflow for general purpose image segmentation using the U‐Net and U‐Net++ architectures in R. The workflow covers data (pre)processing, model training and predictions. We illustrate the utility of the package with image recognition models for two forest structural metrics: tree canopy density and understorey vegetation density. We trained the models using large and diverse training datasets from a variety of forest types and biomes, consisting of 2877 canopy images (both canopy cover and hemispherical canopy closure photographs) and 1285 understorey vegetation images. Overall segmentation accuracy of the models was high with a Dice score of 0.91 for the canopy model and 0.89 for the understorey vegetation model (assessed with 821 and 367 images respectively). The image segmentation models performed significantly better than commonly used thresholding methods, and generalized well to data from study areas not included in training. This indicates robustness to variation in input images and good generalization strength across forest types and biomes. The package and its workflow allow simple yet powerful assessments of forest structural metrics using pretrained models. Furthermore, the package facilitates custom image segmentation with single or multiple classes and based on colour or grayscale images, for example, for applications in cell biology or for medical images. Our package is free, open source and available from CRAN. It will enable easier and faster implementation of deep learning‐based image segmentation within R for ecological applications and beyond.

Bioorthogonal Photoreactive Surfaces for Single-Cell Analysis of Intercellular Communications.

Methods to construct single-cell pairs of heterogeneous cells attract attention because of their potential in cell biological and medical applications for analyzing individual intercellular communications such as immune and nerve synaptic interactions. Photoactivatable substrate surfaces for cell anchoring are promising tools to achieve single-cell pairing. However, conventional surfaces that photoactivate a single type of cell anchoring moiety restrict the combination of cell pair types and their applications. We developed a photoresponsive material comprising a bioorthogonal photoreactive moiety and non-cell adhesive hydrophilic polymer. The material-coated surface allows conjugation with various cell anchoring molecules in response to light at specific timings and consequently achieves light-induced anchoring of a variety of cells at defined regions. Using the platform surface, an array of cancer cell and natural-killer (NK) cell pairs was constructed on a flat substrate surface and the dynamic morphological changes of the cancer cells were monitored by cytotoxic interaction with NK cells at a single-cell level. The photoreactive surface is a useful tool for image-based investigation of the communications between a variety of cell types.