Search Results (257)

Male-factor infertility accounts for approxiamately half of all infertility cases globally, yet therapeutic options remain limited for individuals with no retrievable spermatozoa, such as those with non-obstructive azoospermia (NOA). In recent years, artificial gametogenesis has emerged as a promising avenue for fertility restoration, driven by advances in two complementary strategies: organotypic in vitro spermatogenesis (IVS), which aims to complete spermatogenesis ex vivo using native testicular tissue, and in vitro gametogenesis (IVG), which seeks to generate male gametes de novo from pluripotent or reprogrammed somatic stem cells. To evaluate the current landscape and future potential of these approaches, a narrative, semi-systematic literature search was conducted in PubMed and Scopus for the period January 2010 to February 2025. Additionally, landmark studies published prior to 2010 that contributed foundational knowledge in spermatogenesis and testicular tissue modeling were reviewed to provide historical context. This narrative review synthesizes multidisciplinary evidence from cell biology, tissue engineering, and translational medicine to benchmark IVS and IVG technologies against species-specific developmental milestones, ranging from rodent models to non-human primates and emerging human systems. Key challenges—such as the reconstitution of the blood–testis barrier, stage-specific endocrine signaling, and epigenetic reprogramming—are discussed alongside critical performance metrics of various platforms, including air–liquid interface slice cultures, three-dimensional organoids, microfluidic “testis-on-chip” devices, and stem cell-derived gametogenic protocols. Particular attention is given to clinical applicability in contexts such as NOA, oncofertility preservation in prepubertal patients, genetic syndromes, and reprocutive scenarios involving same-sex or unpartnered individuals. Safety, regulatory, and ethical considerations are critically appraised, and a translational framework is outlined that emphasizes biomimetic scaffold design, multi-omics-guided media optimization, and rigorous genomic and epigenomic quality control. While the generation of functionally mature sperm in vitro remains unachieved, converging progress in animal models and early human systems suggests that clinically revelant IVS and IVG applications are approaching feasibility, offering a paradigm shift in reproductive medicine. Full article

Light influence on shoot regeneration in Prunus salicina is a complex interaction that has been studied for the first time. Japanese plum plants were regenerated from calli and seeds of the scion cultivar ‘Victoria’. The effect of four different light spectra (white, blue, red, and mixed), along with three 6-benzyladenine (BA) concentrations (1, 1.5, and 2 mg L⁻¹), was studied in these two sources of explants. Organogenic calli were derived from the base of stem explants of the scion cultivar ‘Victoria’, whereas cotyledons and embryogenic axis slices were used as seed explants. Calli cultured with 2 mg L⁻¹ of BA and mixed light or 2.5 mg L⁻¹ of BA and control light showed the highest regeneration rates, with no significant differences compared to other treatments. Seed explants exposed to 2.5 mg L⁻¹ of BA and red light exhibited significantly higher organogenesis. In comparison, those in 1.5 mg L⁻¹ of BA with blue light or 2.5 mg L⁻¹ of BA with mixed/control light showed no regeneration. BA concentration did not have a significant effect in the induction of somatic shoots from any explant source. In contrast, a strong interaction between light and BA was noticed. This work presents a protocol that can be applied in transformation and editing research as light spectrum studies continue to advance. Full article

Living myocardial slices (LMSs) have shown great promise in cardiac research, allowing multicellular and complex interplay analyses with disease and patient specificity, yet their wider clinical use is limited by the large tissue sizes usually required. We therefore produced mini-LMSs (<10 mm²) from routine human cardiac surgery specimens and compared them with medium (10–30 mm²) and large (>30 mm²) slices. Size effects on biomechanical properties were examined with mathematical modeling, and viability, contraction profiles, and histological integrity were followed for 14 days. In total, 34 mini-, 25 medium, and 30 large LMS were maintained viable, the smallest measuring only 2 mm². Peak twitch force proved to be size-independent, whereas time-to-peak shortened as slice area decreased. Downsized LMSs displayed excellent contractile behavior for five to six days, after which a gradual functional decline and micro-architectural changes emerged. These findings confirm, for the first time, that mini-LMSs are feasible and viable, enabling short-term, patient-specific functional studies and pharmacological testing when tissue is scarce. Full article

Changes in hepatic lipoprotein metabolism are responsible for the majority of metabolic dysfunction-associated disorders, including familial hypercholesterolemia (FH), metabolic syndrome (MetS), metabolic dysfunction-associated fatty liver disease (MAFLD), and age-related diseases such as atherosclerosis, a major health burden in modern society. This review aims to advance the understanding of state-of-the-art mechanistic concepts in lipoprotein metabolism, with a particular focus on lipoprotein uptake and secretion and their dysregulation in disease, and to provide a comprehensive overview of experimental models used to study these processes. Human lipoprotein research faces several challenges. First, significant differences in lipoprotein metabolism between humans and other species hinder the reliability of non-human model systems. Additionally, ethical constraints often limit studies on human lipoprotein metabolism using tracers. Lastly, while 2D hepatocyte cell culture systems are widely used, they are commonly of cancerous origins, limiting their physiological relevance and necessitating the use of more physiologically representative models. In this review, we will elaborate on key findings in lipoprotein metabolism, as well as limitations and challenges of currently available study tools, highlighting mechanistic insights throughout discussion of these models. These include human tracer studies, animal studies, 2D tissue culture-based systems derived from cancerous tissue as well as from induced pluripotent stem cells (iPSCs)/embryonic stem cells (ESCs). Finally, we will discuss precision-cut liver slices, liver-on-a-chip models, and, particularly, improved 3D models: (i) spheroids generated from either hepatoma cancer cell lines or primary human hepatocytes and (ii) organoids generated from liver tissues or iPSCs/ESCs. In the last section, we will explore future perspectives on liver-in-a-dish models in studying mechanisms of liver diseases, treatment options, and their applicability in precision medicine approaches. By comparing traditional and advanced models, this review will highlight the future directions of lipoprotein metabolism research, with a focus on the growing potential of 3D liver organoid models. Full article

Consumer demand for natural, additive-free foods is increasing. Following the trend, this study evaluated the antifungal potential of lactic acid bacteria (LAB) against Penicillium species commonly found in cheese, using both LAB ferments and hydroxypropylmethylcellulose (HPMC) coatings. LAB strains were first screened with a dual-culture assay. Fermentations in Man–Rogosa–Sharpe (MRS) broth and milk whey were lyophilized and tested, with whey-based ferments showing greater antifungal activity. All whey ferments inhibited fungal growth, with KK₁₃, KB₂, KB₃, and KB₄ being the most effective based on MIC and MFC assays. KB₃-fermented whey had the highest levels of antifungal metabolites, such as phenyllactic acid. A coating containing 5% HPMC and 100 g/L of KB₃-fermented whey was applied to cheese slices, reducing the fungal counts of Penicillium commune by more than 1 Log₁₀ CFU per gram and extending shelf life by 12 days. In whole-cheese trials with natural contamination, this coating delayed visible fungal growth until day 60, extending shelf life by 45 days compared with uncoated samples and 33 days compared with coated controls. These findings support the use of LAB-fermented whey and HPMC coatings as natural preservation strategies, thereby contributing to the sustainable reuse of dairy by-products. Full article

Background/Objectives: Metastatic prostate cancer (PCa) remains a major clinical challenge with limited therapeutic options. The long non-coding RNA H19 has been implicated in regulating cell adhesion molecules and collective migration, key features of metastatic dissemination. This study investigates the role of the Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 in the H19-dependent transcriptional regulation of cell adhesion molecules. Currently, the major effects of BET inhibitors require androgen receptor (AR) expression. Methods: H19 was stably silenced in PC-3 (AR-null) and 22Rv1 (AR-positive) castration-resistant PCa cells. The cells were treated with the pan-BET inhibitors JQ1 and OTX015 or the BET degrader dBET6. In vivo, the effects of JQ1 were evaluated in xenograft mouse models. Chromatin immunoprecipitation (ChIP) and RNA-ChIP were used to assess BET protein recruitment and interaction with cell adhesion gene loci and H19. Organotypic slice cultures (OSCs) from fresh PCa surgical specimens were used as ex vivo models to validate transcriptional changes and BRD4 recruitment. Results: BET inhibition significantly reduced the expression of β4 integrin and E-cadherin and cell proliferation in both basal conditions, and following H19 knockdown in PC-3 and 22Rv1 cells. These effects were mirrored in JQ1-treated tumor xenografts, which showed marker downregulation and tumor regression. ChIP assays revealed that BRD4, more than BRD2/3, was enriched on β4 integrin and E-cadherin promoters, especially in regions marked by H3K27ac. H19 silencing markedly enhanced BRD4 promoter occupancy. RNA-ChIP confirmed a specific interaction between BRD4 and H19. These findings were validated in OSCs, reinforcing their clinical relevance. Conclusions: Our study demonstrates that BRD4 epigenetically regulates the H19-mediated transcriptional control of adhesion molecules involved in collective migration and metastatic dissemination. Importantly, these effects are independent of AR status, suggesting that targeting the H19/BRD4 axis may represent a promising therapeutic avenue for advanced PCa. Full article

Maternal depression negatively affects the neurodevelopment of offspring, but its pharmacological treatment during gestation remains controversial. This study reports the consequences of maternal depression and/or prenatal antidepressant treatment with mirtazapine on offspring early neurodevelopment via an animal model of maternal depression induced by pregestational chronic unpredictable stress (CUS). Offspring from four groups were studied: nonstressed vehicle-treated dams, nonstressed mirtazapine-treated dams, stressed vehicle-treated dams, and stressed mirtazapine-treated dams. The hippocampal excitability of offspring was examined in primary hippocampal cultures established on the first postnatal day, reflecting mostly prenatal development, and in hippocampal slices prepared on postnatal days 11–13, reflecting an early postnatal development. The pregestational CUS modeling of maternal depression moderately suppressed offspring hippocampal excitability in primary cultures but facilitated it in slices. Mirtazapine administered to CUS-exposed dams partly rectified the changes observed in primary cultures of pups from untreated dams and, more prominently, in slices. Mirtazapine itself negatively affected the hippocampal excitability of nonstressed dam offspring in primary culture, and this effect was diminished in slices. Since altered hippocampal neurotransmission might be responsible, at least in part, for the neuropsychopathologies frequently observed in the offspring of depressed mothers, and mirtazapine was able to partly relieve such changes, this treatment may be also beneficial during the prenatal and perinatal periods. Full article

The cultivation of excitable cells typically profits from continuous electrical stimulation, but electrochemical consequences are mostly harmful and must be minimized. The properties of the electrode materials and stimulation impulses are key. Here, we developed an easy method to analyze the electrochemical impact of biphasic, current-controlled impulses, applied via graphite electrodes, using phenol red as the redox indicator. We also tested the stimulation conditions for the long-term cultivation of myocardial tissue. The colorimetric assay was able to detect ±0.2% deviations in typical positive and negative pulse charges. Phenol red was best preserved (20% degradation over 24 h) by impulses of equivalent positive and negative charges (full charge balance), generated with either manual calibration, capacitive electrode coupling, or feedback regulation of electrode polarization. Feedback regulation established full charge balance at pre-pulse voltages of about 300 mV, but also provided the option to selectively compensate irreversible electrode reactions. Modifications to shape and timing did not affect the electrochemical effects of symmetric impulses. Charge-balanced stimulation maintained more than 80% of the contractility of porcine left ventricular myocardium after 10 days of culture, whereas disbalances of 2–4% provoked weakening and discoloration of the tissues. Active polarization regulation, in contrast to capacitive electrode coupling, reproduced the biological advantages of full charge balance. Full article

The patch clamp technique has become the gold standard for neuron electrophysiology research in brain science. Brain slices have been widely utilized as the targets of the patch clamp technique due to their higher optical transparency compared to a live brain and their intercellular connectivity in comparison to cultured single neurons. However, the narrow working space, small scope, and depth of the field of view make the positioning of the operation’s micropipette to the target neuron a time-consuming task reliant on a high level of experience, significantly slowing down operation of the patch clamp technique in brain slices. Further, the current poor controllability in gigaseal formation, which is the key to electrophysiology signal recording, significantly lowers the patch clamp success rate. In this paper, a stepwise navigation of the micropipette is conducted to accelerate the positioning process of the micropipette tip to the target neuron in the brain slice. Then, a fuzzy proportional–integral–derivative controller is designed to control the gigaseal formation process along a designed resistance curve. The experimental results demonstrate an almost doubled patch clamp technique speed, with a 25% improvement in the success rate compared to the conventional manual method. The above advantages may promote the application of our method in brain science research based on brain slice platforms. Full article

Proof-of-concept to determine the direct biomechanical effects of cardiac contractility modulation (CCM) on living myocardial slices (LMS) from patients with end-stage heart failure (HF). Left ventricular LMS from patients with end-stage HF were produced and cultured in a biomimetic system with mechanical loading and electrical stimulation. CCM stimulation (80 mA, 40 ms delay, 21 ms duration) enhanced maximum contractile force (CCM: 1229 µN (587–2658) vs. baseline: 1066 µN (529–2128), p = 0.05) and area under the contractile curve (CCM: 297 (151–562) vs. baseline: 243 (129–464), p = 0.05) but did not significantly impact contractile duration, time to peak, or time to relaxation. Increasing CCM stimulation delay, duration, and amplitude resulted in a higher fraction of LMS with a positive inotropic response. Furthermore, CCM attenuated the negative force-frequency relationship in HF-LMS. CCM stimulation enhanced contractile force in HF-LMS. The fraction of LMS exerting a positive inotropic response to CCM increased with increasing delay, duration, and amplitude settings, suggesting that personalizing stimulation parameters could optimize the beneficial effects of CCM. CCM is a novel device-based therapy that may improve contractile function, ejection fraction, functional outcomes, and quality of life in patients with heart failure. However, continuous efforts are needed to identify true responders to CCM therapy, understand the exact mechanisms, and optimize the contractile response to CCM stimulation. The present study revealed that CCM enhanced the contractile force of HF-LMS in a stimulation setting-dependent manner, reaching a larger fraction of the myocardium while increasing delay, duration, and amplitude. This understanding may contribute to the individualization of CCM stimulation settings. Full article

Intracerebral hemorrhage leads to immediate brain injury due to local mechanical damage, on which current treatment approaches are focused, but it also induces secondary brain injury. The purpose of this study is to characterize blood components, degradation products and their effects in secondary brain injury. Immunocyto- and immunohistochemistry, Fluorescence-Activated Cell Sorting, WST-1 assays and RNA sequencing were applied using human cell cultures and human ex vivo brain tissue slices. Brain tissue was immediately collected, cooled and sliced during neurosurgical operations to perform experiments on living tissue slices of the human brain. Among the blood degradation products, free iron (Fe²⁺ and Fe³⁺), but not hemoglobin, induced detrimental effects on pericyte function and survival (78.5% vs. 94.3%; p-value < 0.001). RNA sequencing revealed ferroptosis as the underlining cellular mechanism, mediated via GPX-4 (log2 fold change > 1.0, p-value < 1.08 × 10⁻³⁰) in pathway analysis and eventually resulting in oxidative cell death. Pericytes located at cerebral capillary branching sites were specifically affected by ferroptosis, leading to capillary disruption and vasoconstriction, which were partially prevented by ferrostatin-1. Free iron induces the pericyte-dependent disruption of cerebral capillary function and represents a therapeutic target to attenuate secondary injury after intracerebral hemorrhage. Full article

Particulate matter (PM) is a major component of ambient air pollution. PM exposure is linked to numerous adverse health effects, including chronic lung diseases. Air quality guidelines designed to regulate levels of ambient PM are currently based on the mass concentration of different particle sizes, independent of their origin and chemical composition. The objective of this study was to assess the relative hazardous effects of carbonaceous particles (soot), ammonium nitrate, ammonium sulfate, and copper oxide (CuO), which are standard components of ambient air, reflecting contributions from primary combustion, secondary inorganic constituents, and non-exhaust emissions (NEE) from vehicular traffic. Human epithelial cells representing bronchial (BEAS-2B) and alveolar locations (H441 and A549) in the airways, human lung fibroblasts (HFL-1), and rat precision-cut lung slices (PCLS) were exposed in submerged cultures to different concentrations of particles for 5–72 h. Following exposure, cell viability, metabolic activity, reactive oxygen species (ROS) formation, and inflammatory responses were analyzed. CuO and, to a lesser extent, soot reduced cell viability in a dose-dependent manner, increased ROS formation, and induced inflammatory responses. Ammonium nitrate and ammonium sulfate did not elicit any significant cytotoxic responses but induced immunomodulatory alterations at very high concentrations. Our findings demonstrate that secondary inorganic components of PM have a lower hazard cytotoxicity compared with combustion-derived and indicative NEE components, and alveolar epithelial cells are more sensitive to PM exposure. This information should help to inform which sources of PM to target and feed into improved, targeted air quality guidelines. Full article

Asthma is a major non-communicable disease whose pathogenesis is still not fully elucidated. One of the asthma research models is precision-cut lung slices (PCLSs), and among the therapeutic options, miRNA molecules are of great interest. The aim of our study was to investigate whether inhibition of miR-223-3p and miR328a-3p affects the inflammatory response in PCLSs derived from a rat with HDM-induced allergic inflammation and a control rat. We generated rat PCLSs and transfected them with miR-223-3p and miR-328a-3p inhibitors. RNA was isolated from PCLSs and analyzed by qPCR. We also examined the proteins in the culture medium using the Magnetic Luminex Assay. The comparison between miRNA-transfected PCLSs and non-transfected controls showed significant differences in the expression of several genes associated with allergic inflammation, including Il-33, Ccl5, Prg2 and Tslp, in both the rat with allergic inflammation and the control rat. In the culture medium, we found no significant differences in protein levels between rat with allergic inflammation and the control. Our study highlighted some important issues: the need to extend the model by including more biological replicates, the need to standardize culture conditions, and the need to consider co-transfection with several miRNA inhibitors when modifying miRNAs expression in the PCLS model. Full article

Ischemic heart disease is the leading cause of death worldwide. Reduced oxygen supply and myocardial hypoxia lead to tissue damage and impairment of the heart function. To the best of our knowledge, the primary functional effects of hypoxia in the multicellular model of living myocardial slices (LMSs) have not been investigated so far. In this study, we analyzed force generation, ultrastructure, gene expression, and proteome changes in rat LMS after 24 h of ex vivo culture in normal and reduced levels of oxygen (O₂). We observed a significant reduction in absolute force and a slowdown of force kinetics as well as an increase in cardiomyocyte apoptosis and myofibrillar and mitochondrial damage, as well as transcriptomic changes. Proteome analysis revealed the deregulation of proteins involved in metabolic processes, hypoxic response, and neutralizing of reactive oxygen species. Our results indicate that hypoxia induces substantial primary changes in heart tissue, which are independent of perfusion and immune responses. Our new LMS model could serve as a screening system for drug development and new mechanistic insights. Full article

Tissue engineering research for neurological applications has demonstrated that biomaterial-based structural bridges present a promising approach for promoting regeneration. This is particularly relevant for penetrating traumatic brain injuries, where the clinical prognosis is typically poor, with no available regeneration-enhancing therapies. Specifically, repurposing clinically approved biomaterials offers many advantages (reduced approval time and achieving commercial scaleup for clinical applications), highlighting the need for detailed screening of potential neuromaterials. A major challenge in experimental testing is the limited availability of neuromimetic, technically accessible, cost-effective, and humane models of neurological injury for efficient biomaterial testing in injury-simulated environments. Three dimensional (3D) organotypic brain slices bridge the gap between live animal models and simplified co-cultures and are a versatile tool for studies on neural development, neurodegenerative disease and in drug testing. Despite this, their utility for investigation of neural cell responses to biomaterial implantation is poorly investigated. We demonstrate that murine brain organotypic slices can be used to develop a model of penetrating traumatic brain injury, wherein a surgical-grade biomaterial scaffold can be implanted into the lesion cavity. Critically, the model allowed for examination of key cellular responses involved in CNS injury pathology/biomaterial handling: astrogliosis, microglial activation and axonal sprouting. The approach offers a technically simple and versatile methodology to study biomaterial interventions as a regenerative therapy for neurological injuries. Full article