Search Results (2,057)

The continual spermatogenesis throughout adulthood is ensured by a rare and unique cell group named spermatogonial stem cells (SSCs), which undergo self-renewal and/or differentiate into sperm cells. SSCs also become a promising genetic source for the protection of animal biodiversity. However, the isolation and culture of SSCs in vitro is still a big challenge and poorly explored in rabbits. The main objective of this study was to isolate, culture, and deeply characterize SSCs obtained from rabbit testes. Briefly, rabbit testicular tissue was mechanically and enzymatically dissociated, and obtained testicular somatic and germ cells were cultured for a short term in culture media supplemented with specific molecular factors maintaining SSC self-renewal and proliferation (GDNF, GFRα-1, FGF2, etc.). Immunofluorescent and PCR techniques were used for molecular profiling of cultured SSCs, while TEM analysis revealed their ultrastructure. After a few weeks, round and grape-like SSC colonies emerged, growing on the feeder cell layer. Rabbit SSCs showed positive staining for DBA, GFRA1, PLZF, RET, PGP9.5, DAZL, and DDX4. Increased expression of additional SSC markers was noticed using RT-qPCR and dd PCR (RET, PLZF, PGP9.5, DAZL, DDX4, CDH1, CD9, CD14, CD90, c-kit, ALDH, SSEA-4, SALL4, OCT4, and SOX2), while ultrastructure typical for primitive undifferentiated cells was observed under TEM. In conclusion, we successfully established a method for rabbit SSC isolation, culture, and phenotyping, which might facilitate their collection for further cryopreservation. However, the self-renewal, proliferative, and differentiation capacities of cultured SSCs still need to be confirmed through an in vivo SSC transplantation experiment. Full article

The sebaceous gland (SG) is a highly dynamic skin appendage that plays a central role in maintaining skin homeostasis through lipid production, immune modulation, and interactions with the skin microbiome. Dysregulation of SG function is implicated in several dermatological disorders, including acne and other inflammatory skin diseases. Over the past decades, a wide range of experimental models has been developed to investigate SG biology and pathology. These include in vivo animal models, ex vivo human skin explants, primary sebocyte cultures, and immortalized sebocyte cell lines, each offering distinct advantages and limitations. More recently, significant progress has been made in the development of advanced three-dimensional (3D) models of sebaceous gland biology, including sebocyte spheroids, SG organoids, human skin equivalents containing sebaceous components, and organotypic co-culture models, which better recapitulate the structural and functional complexity of SGs. In particular, the emergence of SG organoids derived from adult or pluripotent stem cells represents a breakthrough, enabling the study of sebocyte differentiation, lipid metabolism, and cell–cell interactions in a physiologically relevant context. Despite these advances, current models still face important challenges, including incomplete cellular maturation, limited representation of the immune and vascular components, and insufficient modeling of the native skin microenvironment. Future developments integrating bioengineering approaches, microfluidic platforms, and multi-cellular systems are expected to further enhance model complexity and translational relevance. This review summarizes current knowledge on experimental models developed to study SG and sebocyte physiology and pathology. Full article

Hepatic ischemia–reperfusion injury (IRI) is a major clinical challenge in liver surgery and transplantation, contributing to postoperative complications and graft dysfunction. The pathogenesis of hepatic IRI is complex and multifactorial, involving ischemia-induced metabolic consequences, oxidative stress, inflammatory responses, endothelial dysfunction, and the activation of immune pathways upon reperfusion. Despite extensive research efforts, the translation of preclinical findings into effective clinical interventions remains limited. This review provides a critical overview of the principal models used to investigate hepatic IRI. Conventional two-dimensional in vitro systems, including monoculture and co-culture models, offer controlled environments for mechanistic studies and high-throughput screening, but fail to fully reproduce the structural and cellular complexity of the liver microenvironment. Animal models, particularly those based on mice, rats, and pigs, remain essential for studying the systemic and multicellular aspects of hepatic IRI. Nevertheless, species-specific physiological differences, ethical concerns, high costs, and limited translational predictability represent significant limitations. In this context, three-dimensional liver models have emerged as promising alternatives capable of bridging the gap between in vitro systems and animal experimentation. By more accurately recapitulating tissue architecture, cell–cell interactions, and functional heterogeneity, 3D platforms offer enhanced physiological relevance and translational potential. We discuss the strengths and limitations of each experimental approach and highlight the role of advanced 3D models as complementary tools that may enable more accurate investigations of hepatic IRI and accelerate the development of effective therapeutic strategies. Full article

Aflatoxins (AFs) are highly carcinogenic mycotoxins produced by toxigenic fungi, posing serious threats to food safety, animal production, and human health. Microbial aflatoxin control has recently emerged as a promising, sustainable, and environmentally friendly strategy for reducing aflatoxin contamination. However, growing evidence suggests that reductions in AF levels within microbial treatment systems may arise not only from direct toxin degradation, but also from inhibition of fungal growth, adsorption or sequestration processes, and suppression of AF biosynthesis. In microbial co-culture systems involving viable toxigenic fungi, exogenous microorganisms and their metabolites can establish persistent ecological stress through nutrient competition, oxidative stress, and interspecies signaling. These stresses activate fungal cell wall integrity pathways, MAPK signaling cascades, and transcriptional regulatory networks, leading to membrane remodeling, alterations in lipid and energy metabolism, and redistribution of cellular resources. As a consequence, fungal physiology progressively shifts from a growth- and toxin-production-oriented state toward a survival- and defense-oriented state, resulting in impaired growth and reduced AF biosynthesis. This review proposes the internal disintegration framework, which conceptualizes AF suppression as a progressive loss of toxin-producing capacity caused by sustained microbial-induced physiological remodeling rather than solely by fungal growth inhibition or toxin degradation. It will provide new perspectives for developing precise, efficient, and eco-friendly dual-target control strategies. Full article

Background/Objectives: The oxidation of low-density lipoprotein (LDL) is a crucial step in atherogenesis. Beta-Caryophyllene (BCP) is a natural compound with established anti-oxidative and anti-inflammatory properties as shown in animal and cell culture studies. We examined whether BCP can impede LDL oxidation in an in vitro model. Methods: The anti-oxidative effect of BCP was evaluated in different concentrations (0, 25, 50, 100, and 150 µg/mL) with regard to scavenging reactive oxygen species (ROS) during LDL oxidation, which was initiated by the addition of copper chloride (CuCl₂) in a concentration of 10 µmol/L. Lipid hydroperoxides (LPO), malondialdehyde (MDA), dienes, cell viability, and reactions of BCP with ROS according to Gibbs free energies were applied to determine the oxidation state of LDL. Results: Our findings indicated that BCP is highly efficient in inhibiting LDL oxidation in a dose-dependent manner in this in vitro model. The lipid hydroperoxide content in oxLDL was significantly lower in the presence of 100 µg/mL BCP compared to oxLDL without BCP (p < 0.0001). This corresponds to the MDA levels, which were significantly lower in the presence of 100 µg/mL BCP compared to oxLDL without BCP (p = 0.0393). Furthermore, a dose-dependent inhibition of diene formation in the LDL particle was observed in the presence of ascending BCP concentrations which corresponds to a decrease in the cytotoxicity of oxLDL in EA.hy926 cells in the presence of increasing concentrations of BCP. Moreover, BCP’s anti-oxidant effectiveness exceeds that of the widely recognized anti-oxidant spermidine at equivalent concentrations. Our quantum chemical calculations showed that the reactions between BCP and hydroxyl radicals, hydroperoxyl radicals, or hydrogen peroxide are exergonic. We therefore conclude that BCP impedes the oxidation of LDL by its capability to scavenge (at least) these three reactive oxygen species. Conclusions: Our results indicate that BCP impedes the oxidation of LDL in vitro and therefore presumably has the potential to serve as an appropriate therapeutic agent to prevent atherogenesis and related (cardio)vascular diseases by balancing vascular oxidative stress. For this purpose, more prospective clinical studies in humans are required to assess the potential atheroprotective and health-promoting effects of BCP. Full article

The neurovascular unit (NVU) is composed of a diverse array of cells and an extracellular matrix (ECM). Neural cells and blood vessels are intricately interconnected, forming a cohesive whole. Specific cellular components and structures within the NVU play an indispensable role in maintaining homeostasis of the central nervous system (CNS). With the advancement and maturation of cell co-culture technology, various three-dimensional (3D) NVU models continue to emerge, offering a more objective and comprehensive perspective for in vitro studies of CNS diseases. Specifically, these 3D NVU models include Transwell Chamber models, gel-PDNS-based 3D models, self-assembled NVU models and microfluidic NVU models, which reconstruct the complex NVU architecture to varying degrees. This review systematically summarizes multiple 3D construction strategies for in vitro NVU to overcome the limitations of conventional cellular tests or animal experiments, highlights the critical roles of biomimetic gel in recapitulating native cell-gel crosstalk, comparatively analyzes four major 3D NVU technical routes in terms of cellular composition, vascular morphology, barrier performance, and reproducibility, categorizes application scenarios of 3D NVU platforms oriented to practical research demands, including oxygen-glucose deprivation/reoxygenation (OGD/R) injury modeling, blood-brain barrier (BBB) permeability assay, CNS drug penetration screening, neuroinflammation and neurotoxicity evaluation, proposes practical principles for model selection under different experimental purposes, and concludes with current bottlenecks, including imperfect vascular network maturation and lack of unified evaluation criteria, together with future perspectives for standardized 3D NVU in vitro. By comparing the advantages and limitations of these approaches, we aim to clarify their optimal applicability for investigating specific pathological mechanisms and screening potential therapeutics. Full article

Organoid and organ-on-chip technologies are rapidly evolving platforms for viral research that integrate stem cell biology, tissue engineering, and microfluidics to recapitulate key structural, mechanical, biochemical, and cellular features of human and animal physiology. By incorporating multicellular organoids into perfused microfluidic systems, these models can provide complex, dynamic, and physiologically relevant micro-environments for investigating virus–host interactions that are difficult to capture in conventional two-dimensional cultures and static organoids. Controlled flow, shear stress, extracellular matrix organization, tissue–tissue interfaces, and multicellular signaling enable mechanistic investigation of viral infectivity, dissemination, tissue injury and immune activation. Integration of real-time imaging and biosensors further permits longitudinal monitoring of viral replication, host responses, and tissue integrity, expanding the potential of these platforms for antiviral drug discovery. Recent organoid-on-chip studies using brain, skin, vaginal, respiratory, and intestinal models have demonstrated how tissue architecture, mechanical forces, glycocalyx dynamics, and immune–stromal interactions influence viral tropism and pathogenesis. In this review, we provide a mechanistic and translational overview of organoid and organ-on-chip technologies for studying viral infections, with particular emphasis on models of herpes simplex virus (HSV)-mediated disease. We further examine advances in immune integration, multi-organ systems, biosensing, and computational approaches that are expanding the complexity and predictive potential of these models. Importantly, patient-derived organoids and organ-on-chip platforms can capture interindividual differences in viral susceptibility, host responses, and therapeutic efficacy, providing pharmaceutical research with more precise, patient-relevant data to support drug prioritization and precision antiviral medicine. Finally, we discuss key barriers to broader adoption, including organoid maturation, biological and technical variability, reproducibility, scalability, biosafety, cost, standardization, and regulatory validation. Collectively, these advances position organoid and organ-on-chip technologies as powerful human-relevant models that bridge reductionist in vitro systems and human disease, while continued optimization, standardization, and validation will be essential to realize their full potential for mechanistically informed antiviral discovery, therapeutic development, and precision medicine. Full article

A standardized extract of cultured Lentinula edodes mycelia (ECLM), commercially known as AHCC^®, shows anti-inflammatory, immunomodulatory, and organ-protective properties. Experimental studies have indicated that ECLM modulates innate and adaptive immunity, including natural killer (NK) cell activity, antigen-presenting cell function, T-cell responses, and cytokine balance. These effects are particularly relevant in oncology because surgical stress and ischemia–reperfusion injury (IRI) generate a transient perioperative environment characterized by immune suppression, inflammation, and conditions favorable for metastatic progression. Recent animal studies have demonstrated the protective effects of ECLM in intestinal and hepatic IRI models, providing a potential mechanistic rationale for improving the perioperative host microenvironment. Clinical studies on hepatocellular carcinoma, pancreatic cancer, and gynecological malignancies suggest that ECLM may offer potential benefits in immune preservation, nutritional support, symptom management, and recurrence prevention, although most studies are small and hypothesis-generating. Emerging evidence from patient-derived xenograft and spontaneous carcinogenesis models further suggests that ECLM may influence tumor biology beyond host immune activation, although the underlying mechanisms require elucidation. This review summarizes the mechanistic, preclinical, translational, and clinical evidence supporting the use of ECLM as a candidate for perioperative immunonutritional strategies. We propose the hypothesis that ECLM may function as a perioperative microenvironmental stabilizer that integrates immune preservation and intestinal barrier protection. Full article

The problem: Johne’s disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), can be widespread in cattle herds. As a consequence of the slow progression of the infection and the delayed appearance of a humoral response, at any time few of the infected animals may be detected as positive with current serological tests. Many disease control initiatives have focused on removing infected animals by using serology to detect antibodies, principally to a purified MAP extract. However, the delayed humoral response to MAP extract, which can be preceded by shedding MAP in the faeces, limits the value of this control method. The approach: We used a longitudinal study to evaluate the timing and magnitude of antibody responses to lipid antigens. The method: Thirty breeding females from a beef cow herd with a history of Johne’s disease were recruited at 10–12 months old. Blood (serum) and faecal samples were collected at six-month intervals over five years. The serum samples were evaluated by enzyme-linked immunosorbent assay (ELISA) using six synthetic lipid antigens, each from a different class of chemical compound that are components of the mycobacterial cell wall. The responses were correlated with MAP infection status as determined by faecal culture, a commercial serum antibody ELISA (ID Screen^®), and interferon-γ release assays. Nineteen animals were positive using either culture or ID Screen^® at one or more time points; eleven animals were never positive in either assay. Results: In the lipid ELISA, the median responses for all animals were significantly higher at time point 5 (when the animals were three years old) than those at earlier time points. The timing of this response is compatible with, but does not prove, a shift in immune-response profile. For the antigens MOD171 and RT237F2, the responses for each individual animal were higher at time point 5 than at time point 4. Outcome: The increase in response to lipid antigens MOD171 and RT237F2 between the ages of two and four years may indicate infection with MAP at an earlier stage than is achieved with the established serology tests. Full article

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted primarily by Culex mosquitoes and causes severe neurological diseases in humans and animals. The main endemic areas are the Western Pacific and Southeast Asia, and its geographical distribution has expanded in recent years. The development of a diagnosis for orthoflavivirus infections is hampered by two main problems: the short duration of viremia, resulting in a narrow detection window, and severe cross-reactivity. NS1, a secreted nonstructural protein of orthoflavivirus, holds promise as a new target for overcoming these limitations. In this study, we established a highly specific and sensitive capture ELISA for the JEV NS1 protein. First, the JEV NS1 protein was successfully expressed in mammalian cells and purified using affinity chromatography. Seven mAbs recognizing JEV NS1 were generated, and the mAb 20B6 exhibited the strongest binding affinity. Based on 20B6, a capture ELISA was developed with an optimal coating concentration of 3 μg/mL and an optimal detection antibody working concentration of 0.432 μg/mL. No cross-reactivity was observed with other orthoflaviviruses (including WNV, KUNV, USUV, MVEV, SLEV, and ZIKV) or common porcine viruses. The method could effectively detect NS1 protein in cell culture medium, cell lysates, mouse tissues, and porcine serum samples from JEV-infected subjects. This study provides a solid foundation that may be further developed into an efficient and specific tool for epidemiological surveillance of Japanese encephalitis. Full article

Interest in sustainable protein sources is increasing because of environmental concerns related to animal agriculture and the growing burden of chronic non-communicable diseases. Plant-derived bioactive peptides (PDBAPs), amino acid sequences released from dietary proteins, are gaining attention because experimental studies have reported activities relevant to hypertension, type 2 diabetes, and cancer-associated processes. Although animal proteins have long been major sources of bioactive peptides, plant materials may offer advantages such as abundance, potentially lower production costs, and broad cultural acceptability; however, these benefits depend on the source, processing requirements, safety, and scale-up conditions. This review integrates plant sources, processing technologies, proposed mechanisms of action, and translational barriers. Current research covers traditional sources, including legumes and cereals, as well as agro-industrial by-products such as potato peels, spent coffee grounds, and broccoli stems. Modern processing strategies increasingly combine enzymatic hydrolysis or microbial fermentation with process-assisting technologies, including ultrasound treatment and subcritical water processing, to improve protein recovery or peptide release. Recent studies also examine proposed mechanisms of PDBAP activity, including Keap1/Nrf2-associated responses and inhibition of enzymes involved in metabolic disorders. Evidence is interpreted according to the stage of experimental validation, from computational prediction and cell-free assays to cellular, animal, and human studies. Key challenges remain, particularly digestive instability, uncertain systemic bioavailability, bitterness, safety standardization, and limited human clinical evidence. Future work should prioritize standardized extraction and analytical methods, optimized delivery systems, and robust clinical trials. Full article

Cardiac-related pain, including angina pectoris and pain accompanying myocardial infarction, remains clinically important, and existing treatments are inadequate for some patients. Animal and primary-tissue studies provide important mechanistic evidence, but species differences and limited access to patient-matched human neuro-cardiac tissue constrain translation. This review synthesizes the following separate advances relevant to future human iPSC-based cardiac-pain modeling: molecular mechanisms that may be reconstructed in vitro; platforms ranging from two-dimensional cultures to proposed sensory-innervated three-dimensional and microfluidic systems; and potential applications in target validation, patient-specific modeling, and analgesic screening. We distinguish direct human iPSC evidence from findings obtained in animal, primary-cell, cardiac-only, autonomic-neuron, or non-cardiac pain models. We also discuss maturation, standardization, and regulatory challenges. iPSC technologies may complement existing models and support mechanism-focused, patient-stratified research, but direct validation in human sensory–neuron–cardiac systems remains limited. Full article

Follicular-cell-derived thyroid cancers that progress from differentiated tumors to poorly differentiated or anaplastic states commonly lose thyroid lineage identity, radioiodine avidity, and favorable clinical behavior. Genetic and signaling alterations within tumor cells explain part of this transition, but they do not fully account for the coexistence of different differentiation states within the same molecular subtype or even within the same lesion. Increasing functional evidence indicates that dedifferentiation is maintained by reciprocal interactions between malignant cells and the immune, stromal, metabolic, and inflammatory microenvironment. Cancer-associated fibroblast glycolysis and lactate release, IL-6/CXCL8-driven inflammatory signaling, hypoxia, transforming growth factor-beta signaling, and tumor-associated macrophage feedback can suppress thyroid lineage programs while promoting plasticity, invasion, and treatment resistance. Here, we synthesize mechanistic studies supported by genetic perturbation, co-culture, pharmacologic blockade, iodine-uptake assays, animal models, or patient-level radioiodine endpoints. We distinguish functional dedifferentiation from epithelial–mesenchymal transition, stemness, and lymph-node metastasis, and discuss therapeutic strategies that combine tumor-cell redifferentiation with targeting of microenvironmental feedback to restore durable radioiodine sensitivity. Full article

Human cerebral organoids have emerged as a promising new therapeutic strategy for cell transplantation after brain injury. Researchers have explored the short-term survival and maturation of human cerebral organoids derived from human embryonic stem cells (hESCs) in animal models. However, the long-term survival and maturation of cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) have not been studied in depth. In this study, we generated cerebral organoids from hiPSCs in a feeder-free culture system. Then, the cerebral organoids were digested into small clusters and transplanted into the frontal cerebral cortex of postnatal day 0 (P0) SCID mice. The long-term survival and maturation of the grafted cerebral organoids were evaluated at 12 months post-transplantation. Our results indicate that grafted cerebral organoids survive well and maintain their forebrain identity in vivo over a long period. The transplanted cerebral organoids showed reduced proliferative capacity, indicating a low risk of tumor formation. The majority of transplanted cells differentiated into cortical neuronal subtypes in different cortical layers in anatomical lamination at 12 months post-transplantation. In addition, a small population of grafted cerebral organoids matured into GABAergic neurons and gliocytes, including astrocytes, microglia and oligodendrocytes. Furthermore, the grafts formed synapses with the host cells and achieved vascularization in the host brain. Our study demonstrates the long-term survival and maturation of cerebral organoids derived from hiPSCs in vivo and provides evidence for the feasibility of cerebral organoids derived from hiPSCs as a potential cell transplantation therapy. Full article

In drug-induced liver injury (DILI) prediction field, animal models and in vitro cell models are most commonly used. However, animal models require long experimental timelines and may exhibit species-specific differences compared with humans, whereas conventional two-dimensional (2D) cell culture models lack cell-to-cell and cell-to-extracellular matrix (ECM) interaction. Liver organoid models and liver organ-on-a-chip can better simulate the human liver microenvironment; however, the construction of liver organoids requires a long cycle and high costs, while liver organ-on-a-chip systems demand specialized equipment and professional technicians. Herein, we selected the human C3A cell line, characterized by its low cost and facile culture conditions to establish an in vitro three-dimensional (3D) liver model. Briefly, C3A cells were embedded in Matrigel and cultured for 7 days to allow model maturation. Compared with their 2D-cultured cell model, the established 3D model exhibited elevated mRNA expression levels of drug-metabolizing cytochrome P450 enzymes (CYPs). Moreover, the model displayed robust expression of key hepatic biomarkers, as well as bile duct biomarkers. To evaluate the model’s applicability for DILI prediction, we performed toxicity assessments using a panel of six well-characterized hepatotoxicants and three non-hepatotoxic compounds. Notably, the 3D C3A model achieved a sensitivity of 83.3%, specificity of 100%, and overall accuracy of 88.9%. Furthermore, treatment of this model with chlorpromazine, a well-characterized cholangiotoxic agent, resulted in suppressed expression of the bile duct biomarker cytokeratin 19 (CK19) and bile salt export pump (BSEP), accompanied by impaired bile acid transport capacity. Taken together, this study provided a simple, low-cost, easy to culture, and more readily scalable 3D hepatic model in comparison with conventional 2D primary human hepatocyte (PHHs) models and other advanced 3D liver models. Notably, the model displayed dual hepatic and biliary characteristics, supporting predictions of both DILI and drug-induced bile duct injury. It provided a promising in vitro platform for assessing drug-induced hepatobiliary toxicity, with potential to reduce reliance on animal experiments and accelerate early-stage screening of novel pharmaceutical candidates. Full article