Biofabrication technologies, recently developed, offer the potential to create 3-D tissue constructs, thereby opening pathways for investigating cell growth and developmental processes. The structures presented here hold considerable potential in depicting a cellular environment wherein cells are able to interact with their cellular neighbors and their local microenvironment, providing a much more physiologically accurate representation. Converting from 2D to 3D cellular research necessitates the translation of commonly used cell viability assessment methods from 2D cell culture techniques to the assessment of viability in 3D tissue models. Critical for understanding how tissue constructs react to drug treatment or other stimuli, cell viability assays assess the health of the cells. As 3D cellular frameworks become the new norm in biomedical engineering, this chapter details methods for evaluating cell viability both qualitatively and quantitatively within these 3D constructs.
A common feature of cellular analyses is the measurement of proliferative activity within a cell population. Live and in vivo monitoring of cell cycle progression is possible using the FUCCI system. Cellular cell cycle phases (G0/1 or S/G2/M) are identifiable using fluorescence imaging of nuclei, utilizing the mutually exclusive activation of fluorescently labeled cdt1 and geminin proteins in individual cells. The generation of NIH/3T3 cells harboring the FUCCI reporter system, accomplished through lentiviral transduction, is described, along with their application in three-dimensional cell culture models. This adaptable protocol can be utilized with other cell lines.
Monitoring calcium flux via live-cell imaging provides insight into the dynamic and multi-modal nature of cellular signaling. The temporal and spatial shifts of calcium concentration stimulate specific downstream pathways, and by methodically cataloging these events, we can examine the communication methods used by cells internally and in their interactions with other cells. Consequently, calcium imaging's popularity and utility are directly linked to its dependence on highly-detailed optical data measured by fluorescence intensity. Within fixed regions of interest, monitoring temporal changes in fluorescence intensity is easy during the execution on adherent cells. In spite of this, the perfusion of non-adherent or barely adhering cells results in their mechanical displacement, impeding the temporal resolution of variations in fluorescence intensity. We offer here a simple and affordable gelatin protocol to keep cells stable during solution changes that occur during the recording process.
The mechanisms of cell migration and invasion are instrumental in both the healthy functioning of the body and the progression of disease. Hence, procedures aimed at assessing the migratory and invasive capabilities of cells are important for elucidating normal cellular processes and the underlying mechanisms of disease. Selleck Bortezomib This report details the common transwell in vitro methods utilized for the study of cellular migration and invasion. Within the transwell migration assay, cell chemotaxis is measured as cells traverse a porous membrane, which is placed between two compartments containing media with a chemoattractant gradient. An extracellular matrix is integral to the transwell invasion assay, situated atop a porous membrane, enabling the chemotaxis of invasive cells, a characteristic of tumor cells.
For previously non-treatable diseases, adoptive T-cell therapies, a powerful type of immune cell therapy, represent a groundbreaking treatment approach. Immune cell therapies, while aiming for targeted action, can nonetheless induce severe and potentially life-threatening side effects due to the cells' non-specific distribution throughout the body, affecting tissues beyond the intended tumor cells (off-target/on-tumor effects). A potential means of reducing undesirable side effects and improving the infiltration of tumors is the precise targeting of effector cells, such as T cells, to the specific tumor region. Cells can be spatially guided by employing superparamagnetic iron oxide nanoparticles (SPIONs) for magnetization, which can then be controlled by external magnetic fields. To effectively utilize SPION-loaded T cells in adoptive T-cell therapies, the preservation of cell viability and functionality post-nanoparticle loading is essential. We describe a flow cytometry procedure for determining single-cell viability and functional attributes, such as activation, proliferation, cytokine release, and differentiation.
The movement of cells is a fundamental aspect of many physiological events, encompassing the intricate details of embryonic development, the construction of tissues, the actions of the immune system, the occurrence of inflammation, and the progression of cancerous processes. This document outlines four in vitro assays, methodically detailing cell adhesion, migration, and invasion processes and their corresponding image data quantification. Employing these methods, two-dimensional wound healing assays, along with two-dimensional individual cell-tracking experiments visualized through live cell imaging, are combined with three-dimensional spreading and transwell assays. The optimized assays will, critically, allow for a physiological and cellular understanding of cell adhesion and motility. This knowledge will enable the rapid screening of specific therapeutic agents impacting adhesion, the development of innovative approaches in diagnosing pathophysiological processes, and the discovery of novel molecules associated with cancer cell migration, invasion, and metastasis.
Traditional biochemical assays offer a comprehensive approach to investigating the ways in which a test substance alters cellular behavior. Despite this, present assays provide only a single measurement, focusing on a single parameter at a time, while potentially incorporating interferences related to labels and fluorescent illumination. Selleck Bortezomib The cellasys #8 test, a microphysiometric assay for real-time cellular analysis, resolves the previously identified constraints. Within 24 hours, the cellasys #8 test not only identifies the effects of a test substance, but also quantifies recovery effects. A multi-parametric read-out within the test facilitates the real-time observation of metabolic and morphological transformations. Selleck Bortezomib A detailed introduction to the materials, along with a step-by-step procedure, is presented in this protocol to facilitate adoption by scientists. The standardized, automated assay presents novel avenues for biological mechanism study, new therapeutic approach development, and serum-free media formulation validation to scientists.
Fundamental to preclinical drug development, cell viability assays are indispensable tools for studying cellular characteristics and overall health following in vitro drug sensitivity analyses. Optimizing your selected viability assay is critical for generating reproducible and replicable results, in conjunction with using appropriate drug response metrics (including IC50, AUC, GR50, and GRmax), allowing for the identification of promising drug candidates for further in vivo investigation. A rapid, economical, user-friendly, and highly sensitive approach, the resazurin reduction assay, was utilized to examine the phenotypic characteristics of the cells. Through the employment of the MCF7 breast cancer cell line, we provide a detailed, step-by-step protocol for optimizing drug sensitivity screenings using the resazurin assay.
The structure of cells is fundamental to their activity, which is particularly apparent in the highly organized and functionally specialized skeletal muscle cells. The microstructure's structural variations exert a direct influence on performance parameters, such as isometric and tetanic force generation, in this scenario. Second harmonic generation (SHG) microscopy enables noninvasive, three-dimensional visualization of the microarchitecture of the actin-myosin lattice within living muscle cells, circumventing the need for introducing fluorescent labels into the samples. In this resource, we present instruments and step-by-step instructions to help you acquire SHG microscopy data from samples, allowing for the extraction of characteristic values representing cellular microarchitecture from the specific patterns of myofibrillar lattice alignments.
To study living cells in culture, digital holographic microscopy is an ideal choice; it avoids the need for labeling and yields high-contrast, quantitative pixel information from computationally generated phase maps. A comprehensive experiment necessitates instrument calibration, cell culture quality assessment, the selection and setup of imaging chambers, a defined sampling procedure, image acquisition, phase and amplitude map reconstruction, and subsequent parameter map post-processing to derive insights into cell morphology and/or motility. The following steps detail results observed from imaging four distinct human cell lines, each depicted below. Several approaches to post-processing are explained, all for the purpose of monitoring the individual cells and their collective behavior in cell populations.
The cell viability assay, neutral red uptake (NRU), can be used to evaluate cytotoxicity induced by compounds. The process relies on the ability of living cells to sequester the weak cationic dye neutral red within their lysosomes. The degree of xenobiotic-induced cytotoxicity is characterized by a concentration-dependent reduction in neutral red uptake, as compared to cells exposed to the appropriate vehicle control. Hazard assessment within in vitro toxicology research frequently employs the NRU assay. Accordingly, this procedure has been integrated into regulatory suggestions, such as the OECD test guideline TG 432, which outlines an in vitro 3T3-NRU phototoxicity assay for measuring the cytotoxic effects of compounds in the presence or absence of ultraviolet light. A study investigates the cytotoxicity of acetaminophen and acetylsalicylic acid.
Lipid membrane phase states, especially phase transitions, are demonstrably linked to alterations in membrane mechanical properties, such as permeability and bending modulus. Differential scanning calorimetry (DSC), a common method for characterizing lipid membrane transitions, often proves unsuitable for analyzing many biological membranes.