Subtle physiological patterns within the human organism, such as molecular fluctuations or systemic adaptations, often remain hidden from direct observation due to the inherent variability and noise within biological samples. The liver, a vital organ essential for systemic regulation and toxicological assessment, presents this challenge due to its heightened activity, which can influence enzyme dynamics and metabolic processes. Unlike direct observation, in vitro liver models offer a more precise means of understanding these trends, providing insights into the organ's (patho)physiology, and serving as a platform for toxicity evaluation. However, current liver models lack essential features required to faithfully replicate the liver's microenvironment, resulting in reduced accuracy in toxicity assessments. Furthermore, while researchers emphasize mechanistic insights, such as the molecular processes governing glucose metabolism and cellular energy production, clinicians focus on tangible health outcomes, like blood glucose levels in patients. This disconnect between the objectives and methodologies of basic researchers and clinicians amplifies this gap, hindering effective translational research. Addressing these challenges, a novel liver cell culture system that resembles the in vivo liver microenvironment with clinical instrumentation has been proposed to enhance current liver models, improving their capacity to emulate in vivo conditions. This study introduces a novel liver cell culture system, utilizing a 96-well plate format incorporating hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, and hepatocytes to replicate the liver microenvironment. The model integrates clinical instrumentation, specifically a biochemical analyzer to ensure biomarkers closely align with those observed in clinical diagnostics. This design enables researchers to fine-tune conditions that mimic the liver's microanatomy and physiological responses, enhancing its translational potential for toxicity assessments. The approach involves primary cell culture preparation, supernatant analysis through a clinical biochemistry analyzer, and cell viability assessment using the Membrane Potential Cell Viability Assay (MPCVA) method. Additionally, advanced imaging techniques and data analysis tools are incorporated to refine the model's capabilities and ensure greater translatability to clinical applications. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Developing a novel liver cell culture system that resembles the in vivo liver microenvironment Basic Protocol 2: Flow cytometry analysis of cell phases using a permeable DNA dye.
Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined "3D Flipwell system" using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH4). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 3D Flipwell construction, assembly, and collagen coating Basic Protocol 2: Flipwell cell seeding and cell culture Basic Protocol 3: Addition of bacterial culture to the Flipwell system Basic Protocol 4: Flipwell disassembly for scanning electron microscopy (SEM) studies Basic Protocol 5: Immunofluorescence antibody staining for confocal microscopy.
Energy is required for life, and organisms obtain their energy from fuel sources to enable both anabolic and catabolic processes. Some of this energy is radiated as heat, which can be quantified as a measure of metabolic rate. In some cases, environmental toxicants can alter metabolic energy in undesirable ways, and characterization of new pharmaceuticals can determine the efficacy of desirable metabolic rate manipulation or identify off-target adverse effects. Current methods to directly measure heat production in laboratory mice are expensive, can be laborious, and make it challenging to monitor animals in ways that are multiplexed, robust, and non-invasive. We present a set of protocols for assembling and deploying a simple, low-cost thermal camera to monitor and record thermogenic activity, modified from prior work. Parts used to build this system currently cost approximately $150 USD and, when assembled, can record mouse temperatures as well as ambient cage temperatures up to twice per second for extended periods. By using multiplexed cameras in a diurnal mouse incubator system, the thermogenic capacity of several mice can be simultaneously recorded and graphed. Exogenous agents and genotypes that alter metabolism can be readily identified with this technology. In this set of protocols, we describe the assembly of the thermal video camera device, its use, and related data capture and analysis methods. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assembling thermal camera for thermogenic stress test Basic Protocol 2: In vivo measurement of mouse temperature under different ambient conditions.
These protocols describe a detailed method to determine the DNA damage and F-actin and microtubule defects of metaphase II oocytes caused by hexavalent chromium, Cr(VI), an endocrine disrupting chemical (EDC). The protocol provides systematic steps to determine protein expression encoded by pluripotency proteins such as Oct4, Nanog, and Cdx2 during early embryonic development. Occupational or environmental exposure to EDCs has significantly increased infertility in both men and women. The urinary concentration of the EDC bisphenol A in patients undergoing in vitro fertilization (IVF) is directly related to decreased implantation rates and the number of metaphase II oocytes recovered. This protocol outlines crucial steps in assessing the structure of F-actin and microtubules, DNA damage, and repair mechanisms in metaphase II oocytes as well as pluripotency protein markers of early-stage embryos. IVF techniques to achieve fertility goals in both humans and animals are of paramount importance. The interplay between F-actin and microtubules is crucial for bipolar spindle assembly and correct partitioning of the nuclear genome in mammalian oocyte meiosis. EDCs induce DNA damage and impair DNA repair mechanisms, compromising oocyte quality. In human IVF, this results in failure to implant, early miscarriage, and live births with congenital disorders, thus decreasing success rates and increasing poor outcomes. The application of IVF protocols in rats to understand EDC-mediated defects in the cytoskeletal network of metaphase II oocytes is not well established. We present a newly defined rat IVF protocol and demonstrate outcomes using these protocols to determine the adverse effects of Cr(VI) on metaphase II oocytes. Basic Protocol 1 includes steps to superovulate rats, dissect ampullae, retrieve oocytes/eggs, perform immunofluorescence staining of cytoskeletal machinery (microtubules and F-actin), and assess expression of the DNA double-strand break marker γ-H2AX and the DNA repair protein RAD51 in control and Cr(VI)-exposed rats. Basic Protocol 2 describes methods for detecting the pluripotency proteins Oct4, Nanog, and Cdx2 during early embryonic development in control rats. © 2024 Wiley Periodicals LLC. Basic Protocol 1: In vivo EDC treatment of rats and immunostaining of treated oocytes Basic Protocol 2: In vitro fertilization and immunostaining of early-stage embryos.
One of the major concerns following cancer treatment is cardiotoxicity. Therefore, it is important to predict potential cardiotoxicity of cancer chemotherapeutics at the preclinical phase. Current models of cardiotoxicity testing involve either cell culture models or rodent models. We developed a simple invertebrate animal model for rapid screening of cardiotoxicity of cancer chemotherapeutics. Daphnia magna (water flea, a crustacean) has a transparent body and a large myogenic heart that can be easily monitored under a microscope. Using this model, we have previously described comparative cardiotoxicity of several kinase inhibitors that were approved for the treatment of multiple cancers. In this article, we describe the step-wise protocols for evaluating the heart rate and survival of D. magna with relevant information on troubleshooting. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Culturing and maintenance of D. magna Basic Protocol 2: Experimental design for evaluating heart rate of Daphnia Basic Protocol 3: Long-term effect on Daphnia survival upon drug exposure.
