Ex vivo tissue modelling informs drug selection for rare cancers.

The identification and therapeutic targeting of actionable gene mutations across many cancer types has resulted in improved response rates in a minority of patients. The identification of actionable mutations is usually not sufficient to ensure complete nor durable responses, and in rare cancers, where no therapeutic standard of care exists, precision medicine indications are often based on pan-cancer data. The inclusion of functional data, however, can provide evidence of oncogene dependence and guide treatment selection based on tumour genetic data. We applied an ex vivo cancer explant modelling approach, that can be embedded in routine clinical care and allows for pathological review within 10 days of tissue collection. We now report that ex vivo tissue modelling provided accurate longitudinal response data in a patient with BRAFV600E -mutant papillary thyroid tumour with squamous differentiation. The ex vivo model guided treatment selection for this patient and confirmed treatment resistance when the patient's disease progressed after 8 months of treatment.

Bacteriophage therapy reduces Staphylococcus aureus in a porcine and human ex vivo burn wound infection model.

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically.

Challenges in Psoriasis Research: A Systematic Review of Preclinical Models.

INTRODUCTION: Psoriasis is a chronic inflammatory skin disease with variable clinical presentation, multifactorial etiology and an immunogenetic basis. Several studies demonstrate that it results from a dysregulated interaction between skin keratinocytes, immune cells, and the environment that leads to a persistent inflammatory process modulated by cytokines and T cells. The development of new treatment options requires increased understanding of pathogenesis. However, the successful implementation of effective drugs requires well-characterized and highly available preclinical models that allow researchers to quickly and reproducibly determine their safety and efficacy. METHODS: A systematic search on PubMed and Scopus databases was performed and assessed to find appropriate articles about psoriasis models applying the key words previously defined. The PRISMA guidelines were employed. RESULTS: A total of 45 original articles were selected that met the selection criteria. Among these, there are articles on in vivo, in vitro, and ex vivo models, with the in vitro model being the majority due to its ease of use. Within animal models, the most widely used in recent years are chemically induced models using a compound known as imiquimod. However, the rest of the animal models used throughout the disease's research were also discussed. On the other hand, in vitro models were divided into two and three dimensions. The latter were the most used due to their similarity to human skin. Lastly, the ex vivo models were discussed, although they were the least used due to their difficulty in obtaining them. CONCLUSIONS: Therefore, this review summarizes the current preclinical models (in vivo, in vitro, and ex vivo), discussing how to develop them, their advantages, limitations, and applications. There are many challenges to improve the development of the different models. However, research in these in vitro model studies could reduce the use of animals. This is favored with the use of future technologies such as 3D bioprinting or organ-on-a-chip technologies.

Human Precision-Cut Liver Slices: A Potential Platform to Study Alcohol-Related Liver Disease.

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.

Miniaturized Bioengineered Models for Preterm Fetal Membrane Healing.

INTRODUCTION: The reason for the absence of fetal membrane (FM) healing after a fetoscopic intervention is still unknown. We hypothesize that the lack of robust miniaturized models to study preterm FM functions is currently hampering the development of new treatments for FM healing. Specifically, miniaturized models to study preterm FM healing with minimal amounts of tissue are currently lacking. METHODS: In this study, we collected FMs from planned cesarean deliveries and developed different ex vivo models with an engineered biomaterial to study FM healing. Then, the effect of platelet-derived growth factor BB (PDGF-BB) on the migration of cells from preterm and term FMs was evaluated. RESULTS: FMs could be viably cultured ex vivo for 14 days. In a model of punctured FMs, migration of cells into FM defects was less pronounced than migration out of the tissue into the biomaterial. In a miniaturized model of preterm cell migration, PDGF-BB promoted migration of preterm amnion cells into the biomaterial. DISCUSSION AND CONCLUSION: By using a novel miniaturized model of preterm tissue, we here successfully demonstrate that PDGF-BB can promote preterm FM cell migration of microtissues encapsulated in a three-dimensional environment.

Patient-Derived Multiple Myeloma 3D Models for Personalized Medicine-Are We There Yet?

Despite the wide variety of existing therapies, multiple myeloma (MM) remains a disease with dismal prognosis. Choosing the right treatment for each patient remains one of the major challenges. A new approach being explored is the use of ex vivo models for personalized medicine. Two-dimensional culture or animal models often fail to predict clinical outcomes. Three-dimensional ex vivo models using patients' bone marrow (BM) cells may better reproduce the complexity and heterogeneity of the BM microenvironment. Here, we review the strengths and limitations of currently existing patient-derived ex vivo three-dimensional MM models. We analyze their biochemical and biophysical properties, molecular and cellular characteristics, as well as their potential for drug testing and identification of disease biomarkers. Furthermore, we discuss the remaining challenges and give some insight on how to achieve a more biomimetic and accurate MM BM model. Overall, there is still a need for standardized culture methods and refined readout techniques. Including both myeloma and other cells of the BM microenvironment in a simple and reproducible three-dimensional scaffold is the key to faithfully mapping and examining the relationship between these players in MM. This will allow a patient-personalized profile, providing a powerful tool for clinical and research applications.

Models of Osteoarthritis: Relevance and New Insights.

Osteoarthritis (OA) is a progressive and disabling musculoskeletal disease affecting millions of people and resulting in major healthcare costs worldwide. It is the most common form of arthritis, characterised by degradation of the articular cartilage, formation of osteophytes, subchondral sclerosis, synovial inflammation and ultimate loss of joint function. Understanding the pathogenesis of OA and its multifactorial aetiology will lead to the development of effective treatments, which are currently lacking. Two-dimensional (2D) in vitro tissue models of OA allow affordable, high-throughput analysis and stringent control over specific variables. However, they are linear in fashion and are not representative of physiological conditions. Recent in vitro studies have adopted three-dimensional (3D) tissue models of OA, which retain the advantages of 2D models and are able to mimic physiological conditions, thereby allowing investigation of additional variables including interactions between the cells and their surrounding extracellular matrix. Numerous spontaneous and induced animal models are used to reproduce the onset and monitor the progression of OA based on the aetiology under investigation. This therefore allows elucidation of the pathogenesis of OA and will ultimately enable the development of novel and specific therapeutic interventions. This review summarises the current understanding of in vitro and in vivo OA models in the context of disease pathophysiology, classification and relevance, thus providing new insights and directions for OA research.

Precision cut lung slices: an ex vivo model for assessing the impact of immunomodulatory therapeutics on lung immune responses.

Chronic inflammatory diseases of the respiratory tract, such as chronic obstructive pulmonary disease (COPD) and asthma, are severe lung diseases that require effective treatments. In search for new medicines for these diseases, there is an unmet need for predictive and translatable disease-relevant in vitro/ex vivo models to determine the safety and efficacy of novel drug candidates. Here, we report the use of precision cut lung slices (PCLS) as a potential ex vivo platform to study compound effects in a physiologically relevant environment. PCLS derived from an elastase-challenged mouse model display key characteristics of increased inflammation ex vivo, which is exacerbated further upon challenge with LPS, mimicking the immune insult of a pathogen triggering disease exacerbation. Such LPS-induced inflammatory conditions are significantly abrogated by immunomodulatory agents targeting specific inflammatory signaling pathways in the absence of cytotoxic effects in lung slices. Thus, an ex vivo model of PCLS with a simulated pathogenic insult can replicate proposed in vivo pharmacological effects and thus could potentially act as a valuable tool to investigate the underlying mechanisms associated with lung safety, therapeutic efficacy and exacerbations with infection.

Investigational Antiviral Therapy Models for the Prevention and Treatment of Congenital Cytomegalovirus Infection during Pregnancy.

Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation, lifelong disease, and, in severe cases, fetal or neonatal death. Placental infection with HCMV is the major mechanism of mother-to-child transmission (MTCT) and fetal injury. Thus, any pharmaceutical antiviral interference to reduce viral load may reduce placental damage, MTCT, and fetal disease. However, there is currently no licensed HCMV antiviral for use during pregnancy. In this study, aciclovir and the HCMV-specific antivirals letermovir, maribavir, and cidofovir were compared with ganciclovir for antiviral effects in model systems of pregnancy, including first-trimester TEV-1 trophoblast cell cultures and third-trimester ex vivo placental explant histocultures. HCMV-infected trophoblasts at 7 days postinfection (dpi) showed an EC50 of 21 µM for aciclovir, 0.0007 µM for letermovir, 0.11 µM for maribavir, and 0.29 µM for cidofovir, relative to 0.42 µM for ganciclovir. Antivirals added at 10 µM showed no cytotoxic effects and did not affect trophoblast cell proliferation (P > 0.9999). Multiple-round HCMV replication measured at 7 dpi showed letermovir, maribavir, and cidofovir treatment inhibited immediate early, early, and true late viral protein expression as assayed on Western blots. Antiviral treatment of HCMV-infected placental explants showed significant inhibition (P < 0.05) of viral replication with letermovir (83.3%), maribavir (83.6%), cidofovir (89.3%), and ganciclovir (82.4%), but not aciclovir (P > 0.9999). In ex vivo model systems, recently trialed HCMV antivirals letermovir and maribavir were effective at inhibiting HCMV replication. They partly fulfil requirements for use as safe and effective therapeutics during pregnancy to control congenital HCMV. Clinical trials of these newer agents would assist assessment of their utility in pregnancy.

Time-Resolved Quantification of Nanoparticle Uptake, Distribution, and Impact in Precision-Cut Liver Slices.

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue.

Electrophysiological characterisation of atrial volume receptors using ex vivo models of isolated rat cardiac atria.

NEW FINDINGS: What is the central question of this study? What ex vivo preparation of the rat's cavoatrial junction is efficient for characterising atrial mechanoreceptors? What is the main finding and its importance? Of four different ex vivo preparations, static pressure, flow, open and euthermic, the optimal preparation was the euthermic one and involved direct recording from the right cardiac vagal branch with a Langendorff style perfusion at 37°C. Type A receptors were most common, and appeared insensitive to stretch and sensitive to atrial contraction. Type B and intermediate receptors were not isolated at 20°C but were observed closer to 37°C. The findings may suggest that type A and B receptors utilise different molecular transduction mechanisms. ABSTRACT: Atrial volume receptors are a family of afferent neurons whose mechanically sensitive endings terminate in the atria, particularly at the cavoatrial junctions. These mechanosensors form the afferent limb of an atrial volume receptor reflex that regulates plasma volume. The prevailing functional classification of atrial receptors arose as a result of in vivo recordings in the cat and dog and were classified as type A, B or intermediate according to the timing of peak discharge during the cardiac cycle. In contrast, there have been far fewer studies of the common small laboratory mammals such as the rat. Using several ex vivo rat cavoatrial preparations, a total of 30 successful single cavoatrial mechanosensory recordings were obtained. These experiments show that the rat possesses type A, B and intermediate atrial mechanoreceptors as described for larger mammals. Recording these cavoatrial receptors proved challenging from the main vagus, but direct recording from the cardiac vagal branch greatly increased the yield of mechanically sensitive single units. In contrast to type A units, type B atrial mechanoreceptor activity was never observed at room temperature but required elevation of temperature to a more physiological range in order to be detected. The adequate stimulus for these receptors remains unclear; however, type A atrial receptors appear insensitive to direct atrial stretch when applied using a programmable positioner. The findings may suggest that type A and type B atrial receptors utilise different molecular transduction mechanisms.

Ex vivo models for research in extracorporeal membrane oxygenation: a systematic review of the literature.

With ongoing progress of components of extracorporeal membrane oxygenation including improvements of oxygenators, pumps, and coating materials, extracorporeal membrane oxygenation became increasingly accepted in the clinical practice. A suitable testing in an adequate setup is essential for the development of new technical aspects. Relevant tests can be conducted in ex vivo models specifically designed to test certain aspects. Different setups have been used in the past for specific research questions. We conducted a systematic literature review of ex vivo models of extracorporeal membrane oxygenation components. MEDLINE and Embase were searched between January 1996 and October 2017. The inclusion criteria were ex vivo models including features of extracorporeal membrane oxygenation technology. The exclusion criteria were clinical studies, abstracts, studies in which the model of extracorporeal membrane oxygenation has been reported previously, and studies not reporting on extracorporeal membrane oxygenation components. A total of 50 studies reporting on different ex vivo extracorporeal membrane oxygenation models have been identified from the literature search. Models have been grouped according to the specific research question they were designed to test for. The groups are focused on oxygenator performance, pump performance, hemostasis, and pharmacokinetics. Pre-clinical testing including use of ex vivo models is an important step in the development and improvement of extracorporeal membrane oxygenation components and materials. Furthermore, ex vivo models offer valuable insights for clinicians to better understand the consequences of choice of components, setup, and management of an extracorporeal membrane oxygenation circuit in any given condition. There is a need to standardize the reporting of pre-clinical studies in this area and to develop best practice in their design.

Nanoparticle Properties for Delivery to Cartilage: The Implications of Disease State, Synovial Fluid, and Off-Target Uptake.

A major hurdle limiting the ability to treat and cure osteoarthritis, a common and debilitating disease, is rapid joint clearance and limited cartilage targeting of intra-articular therapies. Nanoscale drug carriers have the potential to improve therapeutic targeting and retention in the joint after direct injection; however, there still lacks a fundamental understanding of how the physicochemical properties of nanoparticles (NPs) influence localization to the degenerating cartilage and how joint conditions such as disease state and synovial fluid impact NP biodistribution. The goal of this study was to assess how physicochemical properties of NPs influence their interactions with joint tissues and, ultimately, cartilage localization. Ex vivo models of joint tissues were used to study how poly(lactide- co-glycolide) (PLGA) and polystyrene (PS) NP size, charge, and surface chemistry influence cartilage retention under normal and disease-mimicking conditions. Of the particles investigated, PLGA NPs surface-modified with a quaternary ammonium cation had the greatest retention within cartilage explants; however, retention was diminished 2- to 2.9-fold in arthritic tissue and in the presence of synovial fluid. Interactions with synovial fluid induced changes to NP surface properties and colloidal stability in vitro. The impact of NP charge on "off-target" synoviocyte uptake was also dependent on synovial fluid interactions. The results suggest that the design of nanocarriers for targeted drug delivery within the joint cannot be based on a single parameter such as zeta potential or size, and that the fate of injected delivery systems will likely be influenced by the disease state of the joint and the presence of synovial fluid.

Fungal infection models: Current progress of ex vivo methods.

Experimental alternative ex vivo models that simulate infectious processes in vivo are of fundamental importance for the evaluation of new drugs, since in some cases, their execution does not depend on the approval of an ethics committee in research. Although studies using alternative infectious models to evaluate the efficacy of antifungal molecules have been increasingly described and reported, there is no critical consensus that establishes the most appropriate ones regarding the type of infection. Numerous studies contemplate ex vivo protocols of fungal infections on nails, corneas, dentinal tubules and skin and reveal counterpoints and concordances not yet finely confronted. In this minireview, we propose a critical analysis of the main ex vivo models of fungal infections for the evaluation of new antifungal candidates for both topical and systemic use, as opposed to the advantages and disadvantages of the traditional in vivo models employed in preclinical research.

Cartilage responses to inflammatory stimuli and adipose stem/stromal cell-derived conditioned medium: Results from an ex vivo model.

Introduction: Osteoarthritis (OA), a chronic inflammatory joint disorder, still lacks effective therapeutic interventions. Consequently, the development of convenient experimental models is crucial. Recently, research has focused on the plasticity of Mesenchymal Stem/stromal Cells, particularly adipose-derived ones (ASCs), in halting OA progression. This study investigates the therapeutic potential of a cell-free approach, ASC-derived conditioned medium (CM), in reversing cytokine-induced OA markers in an ex vivo model of human cartilage explants. Methods: 4 mm cartilage punches, derived from the femoral heads of patients undergoing total hip replacement, were treated with 10 ng/ml TNFα, 1 ng/ml IL-1ß, or a combination of both, over a 3-day period. Analysis of OA-related markers, such as MMP activity, the release of NO and GAGs, and the expression of PTGS2, allowed for the selection of the most effective inflammatory stimulus. Subsequently, explants challenged with TNFα+IL-1ß were exposed to CM, consisting of a pool of concentrated supernatants from 72-h cultured ASCs, in order to evaluate its effect on cartilage catabolism and inflammation. Results: The 3-day treatment with both 10ng/ml TNFα and 1ng/ml IL-1ß significantly increased MMP activity and NO release, without affecting GAG release. The addition of CM significantly downregulated the abnormal MMP activity induced by the inflammatory stimuli, while also mildly reducing MMP3, MMP13, and PTGS2 gene expression. Finally, SOX9 and COL2A1 were downregulated by the cytokines, and further decreased by CM. Conclusion: The proposed cartilage explant model offers encouraging evidence of the therapeutic potential of ASC-derived CM against OA, and it could serve as a convenient ex vivo platform for drug screening.

Salivary Organotypic Tissue Culture: An Ex-vivo 3D Model for Studying Radiation-Induced Injury of Human Salivary Glands.

An organotypic tissue culture model can maintain the cellular and molecular interactions, as well as the extracellular components of a tissue ex vivo. Thus, this 3D model biologically mimics in vivo conditions better than commonly used 2D culture in vitro models. Here, we provide a detailed workflow for generating live 3D organotypic tissue slices from patient-derived freshly resected salivary glandular tissues. We also cover the processing of these tissues for various downstream applications like live-dead viability/cytotoxicity assay, FFPE sectioning and immunostaining, and RNA and protein extraction with a focus on the salivary gland radiation injury model. These procedures can be applied extensively to various solid organs and used for disease modeling for cancer research, radiation biology, and regenerative medicine.

Implantation accuracy of novel polyimide stereotactic electroencephalographic depth electrodes-a human cadaveric study.

Introduction: Stereoelectroencephalography (sEEG) is a minimally invasive procedure that uses depth electrodes stereotactically implanted into brain structures to map the origin and propagation of seizures in epileptic patients. Implantation accuracy of sEEG electrodes plays a critical role in the safety and efficacy of the procedure. This study used human cadaver heads, simulating clinical practice, to evaluate (1) neurosurgeon's ability to implant a new thin-film polyimide sEEG electrode according to the instructions for use (IFU), and (2) implantation accuracy. Methods: Four neurosurgeons (users) implanted 24 sEEG electrodes into two cadaver heads with the aid of the ROSA robotic system. Usability was evaluated using a questionnaire that assessed completion of all procedure steps per IFU and user errors. For implantation accuracy evaluation, planned electrode trajectories were compared with post-implantation trajectories after fusion of pre- and postoperative computer tomography (CT) images. Implantation accuracy was quantified using the Euclidean distance for entry point error (EPE) and target point error (TPE). Results: All sEEG electrodes were successfully placed following the IFU without user errors, and post-implant survey of users showed favorable handling characteristics. The EPE was 1.28 ± 0.86 mm and TPE was 1.61 ± 0.89 mm. Long trajectories (>50 mm) had significantly larger EPEs and TPEs than short trajectories (<50 mm), and no differences were found between orthogonal and oblique trajectories. Accuracies were similar or superior to those reported in the literature when using similar experimental conditions, and in the same range as those reported in patients. Discussion: The results demonstrate that newly developed polyimide sEEG electrodes can be implanted as accurately as similar devices in the marker without user errors when following the IFU in a simulated clinical environment. The human cadaver ex-vivo test system provided a realistic test system, owing to the size, anatomy and similarity of tissue composition to that of the live human brain.

Deciphering the links: Fragmented polystyrene as a driver of skin inflammation.

Nano- and microplastics (NMPs), ubiquitous in the environment, pose significant health risks. We report for the first time a comprehensive study using in-vitro, in-vivo, and ex-vivo models to investigate the penetration and inflammatory effects of fragmented polystyrene (fPS) on human skin, including the analysis of both penetration depth and fPS amounts that penetrate the skin. Human keratinocyte (HaCaT) and human dermal fibroblast (HDF) cells exposed to fPS exhibited notable internalization and cytotoxicity. In a 3D human skin model, fPS particles penetrated the dermal layer within one hour, with an average maximum penetration of 4.7 µg for particles smaller than 2 µm. Similarly, mouse dorsal skin and human abdominal skin models confirmed fPS penetration. RNA sequencing revealed substantial upregulation of inflammatory genes, including IL-1α, IL-1ß, IL-18, IL-6, IL-8, ICAM-1, FOS, and JUN, following fPS exposure. These findings were validated at both the mRNA and protein levels, indicating a robust inflammatory response. Notably, the inflammatory response in both the 3D human skin and mouse models increased in a dose-dependent manner, underscoring the toxicological impact of fPS on skin health. This study provides crucial insights into the mechanisms through which NMPs affect human health and underscores the need for further research to develop effective mitigation strategies.

Development of a vestibular schwannoma tumor slice model for pharmacological testing.

BACKGROUND: Our goal was to develop a 3D tumor slice model, replicating the individual tumor microenvironment and for individual pharmaceutical testing in vestibular schwannomas with and without relation to NF2. METHODS: Tissue samples from 16 VS patients (14 sporadic, 2 NF2-related) were prospectively analyzed. Slices of 350 µm thickness were cultured in vitro, and the 3D tumor slice model underwent thorough evaluation for culturing time, microenvironment characteristics, morphology, apoptosis, and proliferation rates. Common drugs - Lapatinib (10 µM), Nilotinib (20 µM), and Bevacizumab (10 µg/ml) - known for their responses in VS were used for treatment. Treatment responses were assessed using CC3 as an apoptosis marker and Ki67 as a proliferation marker. Standard 2D cell culture models of the same tumors served as controls. RESULTS: The 3D tumor slice model accurately mimicked VS ex vivo, maintaining stability for three months. Cell count within the model was approximately tenfold higher than in standard cell culture, and the tumor microenvironment remained stable for 46 days. Pharmacological testing was feasible for up to three weeks, revealing interindividual differences in treatment response to Lapatinib and intraindividual variability in response to Lapatinib and Nilotinib. The observed effects were less pronounced in tumor slices than in standard cell culture, indicating the model's proximity to in vivo tumor biology and enhanced realism. Bevacizumab had limited impact in both models. CONCLUSION: This study introduces a 3D tumor slice model for sporadic and NF2-related VS, demonstrating stability for up to 3 months, replication of the schwannoma microenvironment, and utility for individualized pharmacological testing.

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation.

A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by "felinizing" a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.