Newly developed biofabrication techniques, which are capable of constructing 3-dimensional tissue models, can pave the way for novel cell growth and developmental modeling. These frameworks present considerable promise in depicting an environment where cells interact with neighboring cells and their microenvironment in a manner that is considerably more physiologically accurate. The shift from 2D to 3D cellular environments requires translating common cell viability analysis methods employed in 2D cell cultures to be appropriate for 3D tissue-based experiments. In order to better comprehend how drug treatments or other stimuli affect tissue constructs, cell viability assays are fundamental in evaluating the health of the cells. This chapter focuses on diverse assays for evaluating cell viability in 3D environments, both qualitatively and quantitatively, as 3D cellular systems become increasingly prominent in biomedical engineering.

The proliferative activity of a cellular population is one of the most frequently evaluated aspects in cellular studies. In vivo cell cycle progression can be observed live using the fluorescence ubiquitin cell cycle indicator (FUCCI) system. Nuclei fluorescence imaging enables the determination of individual cells' cell cycle phase (G0/1 or S/G2/M), directly related to the mutually exclusive actions of cdt1 and geminin, both tagged with fluorescent markers. This document describes the creation of NIH/3T3 cells carrying the FUCCI reporter system via lentiviral transduction and their practical application in three-dimensional cell culture studies. Applications of this protocol can be expanded to incorporate other cell lines.

The process of live-cell imaging of calcium flux offers a means of unveiling dynamic and multi-modal cell signaling. Changes in calcium concentration across time and space induce particular downstream processes; classifying these events allows us to dissect the language cells use for both self-communication and communication with other cells. Thus, calcium imaging's widespread use and range of applications are rooted in its utilization of high-resolution optical data, specifically quantifiable by fluorescence intensity. Adherent cells readily undergo this execution, as shifts in fluorescence intensity can be tracked over time within defined regions of interest. Although perfusion is necessary, non-adherent or weakly adherent cells experience mechanical displacement, hindering the precision of time-dependent fluorescence intensity variations. This protocol, leveraging gelatin's properties, details a simple and cost-effective method to maintain cell integrity during solution exchanges in recordings.

The processes of cellular migration and invasion are critical to both healthy bodily function and the manifestation 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. learn more This paper presents a description of frequently used transwell in vitro methods for studying cell migration and invasion. A chemoattractant gradient across a porous membrane, established by two separate compartments containing medium, initiates cell chemotaxis, defining the transwell migration assay. A porous membrane used in the transwell invasion assay is overlaid by an extracellular matrix, which selectively enables chemotaxis only by cells possessing invasive properties, for example, tumor cells.

Adoptive T-cell therapies, a highly innovative type of immune cell therapy, offer a potent and effective approach to previously untreatable diseases. While immune cell therapies are intended to be precise in their action, there is still the concern of substantial and life-threatening side effects because of the cells' widespread distribution, leading to the impact of the therapy on areas beyond the intended tumor (off-target/on-tumor effects). One way to both reduce adverse effects and improve tumor penetration is by specifically targeting the effector cells, for instance, T cells, to the intended tumor area. Spatial guidance of cells can be facilitated by magnetizing them with superparamagnetic iron oxide nanoparticles (SPIONs), thereby allowing manipulation by external magnetic fields. The application of SPION-loaded T cells in adoptive T-cell therapies depends on the cells retaining their viability and functionality following nanoparticle loading. A single-cell level analysis of cell viability and function, including activation, proliferation, cytokine release, and differentiation, is achieved using a flow cytometry protocol.

Cell migration, a fundamental mechanism in physiological functions, is crucial for embryogenesis, tissue construction, immune function, inflammatory processes, and the progression of cancer. Four in vitro assays demonstrate the successive stages of cell adhesion, migration, and invasion, with corresponding image data analysis. 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. Optimized assays will lead to a more complete understanding of cell adhesion and motility in physiological and cellular settings, thereby aiding the rapid screening of therapeutic agents for adhesion-related processes, the development of innovative methods for diagnosing pathophysiological conditions, and the study of new molecules involved in cancer cell migration, invasion, and metastasis.

Identifying the effects of a test substance on cells is critically facilitated by the array of traditional biochemical assays. Nonetheless, existing assays are limited to singular data points, providing a snapshot of just one parameter at a time, and possibly introducing artifacts due to labeling and fluorescent illumination. learn more We have dealt with these limitations by introducing the cellasys #8 test, which is a microphysiometric assay for the real-time analysis of cells. The cellasys #8 test, within a span of 24 hours, can detect the consequences of a test substance, and simultaneously evaluate the recovery processes. The test's multi-parametric read-out facilitates real-time monitoring of metabolic and morphological changes. learn more Scientists will find a thorough introduction to the materials, coupled with a meticulously crafted, step-by-step description, within this protocol to support its adoption. The automated and standardized assay provides scientists with a platform to explore the diverse applications of biological mechanism studies, develop new therapeutic interventions, and validate serum-free media formulations.

During the preclinical drug development process, cell viability assays are instrumental in evaluating the phenotypic properties and general well-being of cells after in vitro drug sensitivity experiments. In order to yield consistent and reproducible findings from your chosen viability assay, meticulous optimization is needed; alongside this, employing relevant drug response metrics (like IC50, AUC, GR50, and GRmax) is crucial for identifying candidate drugs suitable for further in vivo assessment. We leveraged the resazurin reduction assay, a rapid, cost-effective, straightforward, and sensitive method, in order to determine the phenotypic properties of the cells. By utilizing the MCF7 breast cancer cell line, we detail a comprehensive, step-by-step procedure for refining drug susceptibility screens using the resazurin assay.

Cellular architecture underpins cellular functionality, especially within the complex and functionally adapted skeletal muscle cells. Structural variations in the microstructure have a direct impact on performance parameters, exemplified by isometric and tetanic force production, in this instance. Noninvasive 3D detection of the actin-myosin lattice's microarchitecture in living muscle cells is achievable through second harmonic generation (SHG) microscopy, eliminating the requirement for sample alteration using fluorescent probes. 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.

In the study of living cells in culture, digital holographic microscopy presents a particularly advantageous imaging technique, as it eliminates the need for labeling and generates highly-detailed, quantitative pixel information from computed phase maps. A thorough experimental procedure includes instrument calibration, cell culture quality control, the selection and preparation of imaging chambers, a sampling protocol, image capture, phase and amplitude map reconstruction, and parameter map analysis to discern details about cell morphology and/or motility. The following steps detail results observed from imaging four distinct human cell lines, each depicted below. A thorough examination of various post-processing strategies is presented, with the specific objective of tracking individual cells and the collective behaviors of their populations.

For assessing the cytotoxicity caused by compounds, the neutral red uptake (NRU) assay for cell viability is employed. Living cells' absorption of neutral red, a weak cationic dye, within lysosomes underlies the principle of this method. Xenobiotic-induced cytotoxicity is reflected in a reduction of neutral red uptake, which is directly proportional to the concentration of xenobiotic, relative to cells treated with vehicle controls. In vitro toxicology applications frequently utilize the NRU assay for assessing hazards. Thus, this methodology has been adopted in regulatory recommendations, including OECD test guideline TG 432, outlining an in vitro 3T3-NRU phototoxicity assay to determine the cytotoxicity of compounds under ultraviolet irradiation or without. Acetaminophen and acetylsalicylic acid's cytotoxicity is quantified in an illustrative experiment.

The phase state of synthetic lipid membranes, and especially the transitions between phases, is well-established to drastically affect mechanical properties like permeability and bending modulus. The primary method for detecting lipid membrane transitions is differential scanning calorimetry (DSC); however, this technique proves insufficient for numerous biological membranes.