Spiral ganglia neurons (SGNs) impairment can cause deafness. One important therapeutic approach involves utilizing stem cells to restore impaired auditory circuitry. Nevertheless, the inadequate implementation of research methodologies poses a challenge in accurately assessing the functionality of derived cells within the circuit. Here, we describe a novel method for converting human embryonic stem cells (hESCs) into otic neurons (ONs) and assess their functional connectivity using an optogenetic approach with cells or an organotypic slice of rat cochlear nucleus (CN) in coculture. Embryonic stem cell-derived otic neurons (eONs) exhibited SGN marker expression and generated functional synaptic connection when cocultured with cochlear nucleus neurons (CNNs). Synapsin 1 and VGLUT expression are found in the cochlear nucleus of brain slices, where eONs projected processes during the coculture of eONs and CN brain slices. Action potential spikes and INa+/IK+ of CNNs increased in tandem with light stimulations to eONs. These findings provide further evidence that eONs may be a candidate source to treat SGN-deafness.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7 days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5 Hz, 1 Hz, and 2 Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24 h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7 d. Pacing rates above 1 Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7 d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1 d and 7 d, while Ca2+ transient duration was prolonged after 7 d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7 d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Reproductive processes are dynamic and involve extensive morphological remodeling and cell-cell interactions. Live imaging of organs enhances our understanding of how biological processes occur in real time. Slice culture is a type of organ culture where thick slices are collected from an organ and cultured for several days. Slice culture is a useful and easy-to-implement technique for live imaging of reproductive events at cellular resolution. Here we describe a pipeline of live imaging on slice culture to visualize the process of urethra closure in mouse embryonic penis as a proof of principle. In combination with genetic reporter mice, nuclear stains, and exposure experiments, we demonstrate the feasibility of slice culture on a reproductive organ. We also provide a step-by-step protocol and troubleshooting guide to facilitate the adoption of slice culture with live imaging in other reproductive organs. Lastly, we discuss potential utilities and experiments that could be implemented with slice culture in reproductive sciences.

Alcohol-related liver disease (ALD) encompasses a range of pathological conditions that are complex to study at the clinical and preclinical levels. Despite the global burden of ALD, there is a lack of effective treatments, and mortality is high. One of the reasons for the unsuccessful development of novel therapies is that experimental studies are hindered by the challenge of recapitulating this multifactorial disorder in vitro, including the contributions of hepatotoxicity, impaired lipid metabolism, fibrosis and inflammatory cytokine storm, which are critical drivers in the pathogenesis of ALD in patients and primary targets for drug development. Here, we present the unique characteristics of the culture of human precision-cut liver slices (PCLS) to replicate key disease processes in ALD. PCLS were prepared from human liver specimens and treated with ethanol alone or in combination with fatty acids and lipopolysaccharide (FA + LPS) for up to 5 days to induce hepatotoxic, inflammatory and fibrotic events associated with ALD. Alcohol insult induced hepatocyte death which was more pronounced with the addition of FA + LPS. This mixture showed a significant increase in the cytokines conventionally associated with the prototypical inflammatory response observed in severe ALD, and interestingly, alcohol alone exhibited a different effect. Profibrogenic activation was also observed in the slices and investigated in the context of slice preparation. These results support the versatility of this organotypic model to study different pathways involved in alcohol-induced liver damage and ALD progression and highlight the applicability of the PCLS for drug discovery, confirming their relevance as a bridge between preclinical and clinical studies.

We are witnessing the revival of the CAM model, which has already used been in the past by several researchers studying angiogenesis and anti-cancer drugs and now offers a refined model to fill, in the translational meaning, the gap between in vitro and in vivo studies. It can be used for a wide range of purposes, from testing cytotoxicity, pharmacokinetics, tumorigenesis, and invasion to the action mechanisms of molecules and validation of new materials from tissue engineering research. The CAM model is easy to use, with a fast outcome, and makes experimental research more sustainable since it allows us to replace, reduce, and refine pre-clinical experimentation (\"3Rs\" rules). This review aims to highlight some unique potential that the CAM-assay presents; in particular, the authors intend to use the CAM model in the future to verify, in a microenvironment comparable to in vivo conditions, albeit simplified, the angiogenic ability of functionalized 3D constructs to be used in regenerative medicine strategies in the recovery of skeletal injuries of critical size (CSD) that do not repair spontaneously. For this purpose, organotypic cultures will be planned on several CAMs set up in temporal sequences, and a sort of organ model for assessing CSD will be utilized in the CAM bioreactor rather than in vivo.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

Postnatal bone fractures of the growth plate (GP) are often associated with regenerative complications such as growth impairment. In order to understand the underlying processes of trauma-associated growth impairment within postnatal bone, an ex vivo rat femur slice model was developed. To achieve this, a 2 mm horizontal cut was made through the GP of rat femur prior to the organotypic culture being cultivated for 15 days in vitro. Histological analysis showed disrupted endochondral ossification, including disordered architecture, increased chondrocyte metabolic activity, and a loss of hypertrophic zone throughout the distal femur. Furthermore, altered expression patterns of Col2α1, Acan, and ColX, and increased chondrocyte metabolic activity in the TZ and MZ at day 7 and day 15 postinjury were observed. STEM revealed the presence of stem cells, fibroblasts, and chondrocytes within the injury site at day 7. In summary, the findings of this study suggest that the ex vivo organotypic GP injury model could be a valuable tool for investigating the underlying mechanisms of GP regeneration post-trauma, as well as other tissue engineering and disease studies.

The basic idea behind the use of 3-dimensional (3D) tools in biomedical research is the assumption that the structures under study will perform at the best in vitro if cultivated in an environment that is as similar as possible to their natural in vivo embedding. Tissue slicing fulfills this premise optimally: it is an accessible, unexpensive, imaging-friendly, and technically rather simple procedure which largely preserves the extracellular matrix and includes all or at least most supportive cell types in the correct tissue architecture with little cellular damage. Vibrating microtomes (vibratomes) can further improve the quality of the generated slices because of the lateral, saw-like movement of the blade, which significantly reduces tissue pulling or tearing compared to a straight cut. In spite of its obvious advantages, vibrating microtome slices are rather underrepresented in the current discussion on 3D tools, which is dominated by methods as organoids, organ-on-chip and bioprinting. Here, we review the development of vibrating microtome tissue slices, the major technical features underlying its application, as well as its current use and potential advances, such as a combination with novel microfluidic culture chambers. Once fully integrated into the 3D toolbox, tissue slices may significantly contribute to decrease the use of laboratory animals and is likely to have a strong impact on basic and translational research as well as drug screening.

Head and neck squamous cell carcinoma (HNSCC) outcomes remain stagnant, in part due to a poor understanding of HNSCC biology. The importance of tumor heterogeneity as an independent predictor of outcomes and treatment failure in HNSCC has recently come to light. With this understanding, 3D culture systems, including patient derived organoids (PDO) and organotypic culture (OTC), that capture this heterogeneity may allow for modeling and manipulation of critical subpopulations, such as p-EMT, as well as interactions between cancer cells and immune and stromal cells in the microenvironment. Here, we review work that has been done using PDO and OTC models of HNSCC, which demonstrates that these 3D culture models capture in vivo tumor heterogeneity and can be used to model tumor biology and treatment response in a way that faithfully recapitulates in vivo characteristics. As such, in vitro 3D culture models represent an important bridge between 2D monolayer culture and in vivo models such as patient derived xenografts.