With the progression of late-onset Alzheimer disease (LOAD), there is a dysregulation and then a breakdown of the blood-brain barrier (BBB). An important pathological feature in the brains of patients is the accumulation of amyloid beta (Aβ) peptides. Their aggregation leads to the formation of particularly harmful Aβ oligomers (Aβ-O). Unfortunately, our understanding of changes in the blood-brain barrier, particularly with regard to the effects of Aβ-O, is still very limited. This study investigated a LOAD-specific and induced pluripotent stem cell (hiPSC)-based in vitro model of the BBB for disease mechanisms and validated the findings in two independent laboratories. This study also investigated Aβ transport across the BBB. Furthermore, obtained in vitro findings were confirmed in the cerebrospinal fluid proteome of a LOAD patient cohort. Control and LOAD hiPSCs exhibited comparable efficiency in forming brain capillary endothelial-like cells (BCECs). Although transendothelial electrical resistance (TEER) assessments indicated no significant differences in barrier tightness between LOAD and control BCECs, high-throughput multiplex qPCR analysis revealed subtle alterations in barrier integrity. This included changes in various barrier markers, such as mucins (MUC1, MUC20), aquaporins (AQP5, AQP10), junctional transcripts (CLDNs, TJP1, OCLN), and receptors (LRP1, INSR, LSR), which were confirmed in LOAD patients. High-content imaging and flow cytometry indicated reduced cadherin 5 (CDH5) levels in LOAD BCECs. Importantly, the results also highlighted a difference in the transport of Aβ-O across the BBB. This model demonstrates a LOAD-relevant phenotype with decreased Aβ transport and alterations in key transcripts and could thus serve for future translational studies to rescue pathogenic phenotypes.

Using human induced pluripotent stem cell (hiPSC)-based in vitro blood-brain barrier (BBB) models holds a great promise for disease modeling and testing therapeutic approaches while reducing limitations of animal models due to interspecies differences in preclinical research. Especially in the case of rare diseases, the development of these models is still ongoing. In this study, an in vitro, patient-derived BBB model of SYNGAP1 disorder was established by using a hiPSC line carrying the Leu323Arg missense mutation, two isogenic controls corrected by CRISPR/Cas9 gene editing, and a sham isogenic control (treated also in the CRISPR/Cas9 gene editing process but without being corrected). The brain capillary endothelial-like cell (BCELC) differentiation protocol was adapted for each line individually, which resulted in functional barrier formation (transendothelial electrical resistance = TEER > 1000 Ω*cm2) and BBB-relevant marker expression (Claudin5, ZO-1, Occludin, Cadherin-5) in all models. Unexpectedly, the established BBB models showed position-dependent TEER in 24-well multiwell plates, having higher values in the middle compared to outer positions, pointing out the importance of experimental layout for reproducibility. Adaptation steps also included the incorporation of a pre-enrichment approach into the BCELC differentiation protocol. This helped to reduce cell population heterogeneity, confirmed by a reduction in Nanog expression and an increase in Cadherin-5 expression in differentiated samples. All in all, this lessons-learned study highlights the challenges and the possible solutions for protocol translation to a patient-derived, hiPSC-based BBB model of the SYNGAP1 disorder.

Background/Objectives: Systemic inflammation and circulating proinflammatory cytokines can impair blood–brain barrier (BBB) integrity. Many in vitro BBB models lack the complexity to fully recapitulate systemic inflammation and its long-term effects on the BBB. This study aimed to develop a hollow-fiber flow-based dynamic in vitro (DIV) BBB model for investigating prolonged proinflammatory responses under physiological flow conditions. Methods: Culture conditions for hCMEC/D3 in a DIV model (Flocel) were optimized by varying serum concentrations over seven weeks. Barrier integrity (transendothelial electrical resistance (TEER), permeability studies), proliferation, metabolism (NMR spectroscopy) and molecular changes (high-throughput qPCR) were assessed. Optimized triple-cultures with hCMEC/D3, human primary astrocytes and pericytes were established. After four weeks of barrier establishment, the triple-cultures were exposed to TNF-α, IL-1β and IFN-γ (0.1 ng/mL or 10 ng/mL each) for two weeks. The inflammatory response was assessed with a multiplex cytokine array. Results: Reduced serum concentration (0.25% FBS) decreased proliferation, promoted aerobic respiration, and altered tight junction and transporter gene expression, accompanied by moderately improved barrier integrity compared with 1% or 5% FBS. In optimized triple-cultures, cytokine exposure induced concentration-dependent secretion of IL-6, IL-8, and MCP-1 and changes in mRNA levels, with minor effects on barrier integrity. Sustained cytokine release over two weeks demonstrated stable induction of inflammatory responses at the BBB. Conclusions: An organotypic DIV model of the human BBB, incorporating hCMEC/D3, human primary astrocytes and pericytes was successfully established. By enabling long-term exposure to physiologically relevant cytokine concentrations under flow conditions, this model may provide a platform to investigate functional and molecular BBB responses in inflammation-driven disease progression.

Endocrine disruptors (EDs) are implicated in adverse developmental and reproductive outcomes, yet their identification remains a major challenge in chemical safety assessment. Current testing strategies rely heavily on animal models, which are constrained by ethical concerns, interspecies differences, and limited mechanistic resolution but justified by the complexity of the endocrine system and its physiology. Capturing the complex biology of intact organisms and incorporating toxicokinetic properties in alternative test methods is challenging. To address this, the European Partnership for the Assessment of Risks from Chemicals (PARC) is advancing the development and regulatory integration of new approach methodologies (NAMs). This project specifically contributes by developing and validating human-relevant NAMs to identify key aspects of endocrine disruption relevant to developmental and reproductive toxicity (DART). Key innovative activities include predictive modeling, refinement of zebrafish and amphibian embryo assays, and establishment of advanced in vitro systems for assessing toxicity in the oocyte, testis, placenta, and brain. By combining mechanistic insights with multi-modality and high throughput testing strategies, this work aims to improve the predictive power and regulatory utility of NAMs for ED identification within the One Health paradigm.

Ischemic stroke disrupts blood-brain barrier (BBB) integrity and alters small extracellular vesicle (sEV) signaling, yet the mechanisms underlying sEV transport across compromised barriers remain poorly understood. This study investigated BBB-sEV interactions under normal and stroke-mimicking oxygen/glucose deprivation (OGD) conditions using an in vitro human BBB co-culture model consisting of brain capillary endothelial (BCECs, hCMEC/D3) and astrocytoma cells (1321N1). Model characterization revealed that co-culture with 1321N1 cells enhanced BBB vulnerability to OGD compared to hCMEC/D3 mono-culture. OGD exposure (5 h and 24 h) progressively decreased transendothelial electrical resistance (TEER) and increased FITC-dextran 4 (FD4) permeability, with more severe impairment in co-cultures (e.g. TEER - after 5 h: 0.85-fold, after 24 h: 0.55-fold for co-culture related to mono-culture). Following 19 h oxygen and glucose restoration ('recovery') after 5 h OGD, barrier integrity loss was halted but not reversed. Transcriptomic analysis revealed adaptive cellular responses including upregulated glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF), alongside temporal changes in tight junction protein expression (CLDN5, CDLN6). sEV secretion kinetics in apical and basolateral compartments demonstrated that both cell types released particles in response to OGD in a time-dependent manner, with co-cultures showing enhanced secretion compared to mono-cultures. sEV uptake and permeation studies using eight cancer cell line-derived sEVs revealed cell-origin dependent internalization patterns by BCECs, with the highest uptake for HEK293T and SH-SY5Y sEVs. These internalized sEVs were predominantly targeted to lysosomes. Despite severe barrier disruption due to OGD transcellular permeation of single sEV particles was not detectable.

This article highlights the benefits of preregistration in improving the reliability and transparency of in vitro studies. By preregistering their studies, researchers can make their findings more reproducible, paving the way for medical progress, and accelerating the replacement of animal experiments.

Abstract Background The composition of tight junctions (TJs) in epithelia is well understood, whereas that one in endothelial cells at the blood-brain barrier (BBB) is controversial. Although freeze fracture EM remains the gold standard for assessing TJ nano-structures, advances in fluorescence microscopy, such as stimulated emission depletion (STED) together with image analysis tools, allow immunostaining-based quantitative analysis of the protein composition and the nano-organization of TJ strands. Methods Here, we present two approaches for quantitative analysis of the BBB TJ meshwork utilizing confocal and STED imaging together with open access image analysis tools CellProfiler, iLastik and ImageJ. For the first use case, a BBB model based on brain capillary endothelial-like cells (BCELCs) differentiated from human induced pluripotent stem cells (hiPSC) was employed. For the second one, monolayers of human primary brain microvascular endothelial cells (pBMVECs) were used. Results STED analysis of BCELC monolayers revealed that here claudin-5 co-polymerizes with claudin-4 and − 6 into continuous TJ strand meshworks. Treatments with established TJ openers (1.4 M mannitol or C. perfringens enterotoxin-based claudin binders) strongly decreased transendothelial resistance (TEER) accompanied by a reduction in the nanoscopic co-localization of claudin-5/-4, -5/-6 and amount of junctional claudin-5. These findings suggested that in hiPSC-based BBB models multiple claudins together constitute TJs, leading to a tight paracellular barrier against solutes. Using STED imaging of pBMVECs immunostained for Cldn5, extensive meshworks of continuous TJ strands were detected. In contrast to the hiPSC-based model, other claudins were not detected. TEER and morphometric analyses of Cldn5-positive strands revealed that the barrier defect caused by claudin-5 binders is due to impaired TJ structural integrity. Conclusions The findings suggest that Cldn5 is sufficient to form the tight paracellular barrier at the BBB. The presence of claudin-4, -5 and − 6 in hiPSC-derived cells reflects a mixed phenotype depending on their differentiation process. In sum, we demonstrated that, unlike confocal imaging, STED enables monitoring the composition and structural integrity of BBB TJ strands.

Abstract New Approach Methodologies (NAMs) are increasingly promoted for animal-free nanosafety assessment, yet their regulatory use remains constrained by fragmented regulatory criteria and nanomaterial-specific testing complexities. This review comprehensively analyzes the convergence of advanced cell biology, nanotechnology, and information technology in reshaping safety evaluations, with a focus on regulatory translation across global frameworks. We first summarize enabling technologies, including advanced in vitro and ex vivo systems, organoids, microphysiological systems, high-dimensional single-cell multi-omics and label-free hyperspectral imaging, AutoML, generative AI, physiologically based kinetic modeling, and in vitro-to-in vivo extrapolation. We then analyze the regulatory landscape, emphasizing OECD harmonization efforts, the Mutual Acceptance of Data system, and regional developments across Europe, Africa, the Americas, and Asia–Pacific. A central theme is that regulatory acceptance of NAMs for nanomaterials requires not only biological relevance, reproducibility, and context-of-use definition, but also robust physicochemical characterization, exposure control, dosimetry, and data interoperability. Industrial implementation in pharmaceuticals, cosmetics, agrochemicals, antimicrobials, and environmental protection is discussed as a measure of regulatory readiness and scalability. Finally, we outline future directions involving Smart NAMs, digital twins, and Safe and Sustainable by Design (SSbD) frameworks. By integrating technological, regulatory, and implementation perspectives, this review identifies pathways toward harmonized, mechanistic, and human- or target species-relevant next-generation risk assessment (NGRA) for nanotechnology. Graphical abstract

The increasing production and accumulation of plastic waste, coupled with insufficient recycling practices, contribute to the growing presence of plastic in the environment. Nanoplastic particles are of particular concern, as they pose greater (health and environmental) risks and exhibit wider dispersion compared to macroplastics. The blood–brain barrier may be exposed to nanoplastics present in the blood, which could affect its functionality or even pass through and damage the central nervous system. This study examined the effects of polystyrene (PS) nanoparticles with different chemical surface modifications (pristine, carboxylated, aminated) and sizes (50 nm and 100 nm) on cells of the neurovascular unit (NVU): human brain endothelial cells, astrocytes, and pericytes. Results indicated that only high concentrations of nanoparticles (100 μg/mL and 300 μg/mL) applied for 48 h decreased cell viability and barrier integrity significantly. Specifically, 50 nm carboxylated PS particles reduced barrier integrity and altered tight junction gene expression substantially. Fluorescent labelling of the investigated particles enabled to confirm their uptake by all tested cell types of the NVU, but also highlighted that the labelling changes the particles’ properties. Furthermore, cell culture medium-dependent particle agglomeration and increase of size were inversely correlated with cellular internalisation, which has to be considered for future risk assessments.

Human-derived biomaterials offer several advantages over animal-derived or synthetic alternatives, including improved biocompatibility, ethical acceptability, sustainability, and clinical translatability. Here we present new applications of human placenta-derived materials - specifically HUMAN PLACENTA substrate, collagen type-I, and Laminin-111 - as 2D coating materials and 3D matrices for the cultivation of spheroids and adherent cells. Collagen type-I coatings supported colorectal cancer spheroid formation without the need for growth-factor supplementation. Lm-111 significantly enhanced NIH3T3 fibroblast adhesion compared with poly-L-lysine and rat-tail collagen type-I, performing comparably to bovine fibronectin. In a transwell blood-brain barrier model, HUMAN PLACENTA substrate coatings enabled confluent endothelial monolayers with transendothelial electrical resistance values not significantly different from the conventional human collagen type-IV/bovine fibronectin mixture. Across these in vitro models, placenta-derived materials performed comparably or better than conventional animal-derived and synthetic coatings, supporting robust cell viability, adhesion, and barrier formation. Due to their human origin, these biomaterials exhibit reduced biological complexity while enhancing biocompatibility and translational relevance. Therefore, they provide a sustainable, ethically acceptable alternative for advanced cell culture systems.

Bitter taste disorders are common chemotherapy-induced side effects that compromise nutrition and quality of life. Since no treatment exists for conditions such as bitter taste hypersensitivity and phantogeusia, we hypothesized that a homoeriodictyol sodium (Na-HED) mouthwash reduces chemotherapy-induced bitterness sensitivity caused by salivary platinum (Pt) species derived from intravenously administered carboplatin. In a clinical pilot study involving patients with gynecological cancer (n = 8) receiving carboplatin-based chemotherapy, dietary intake, salivary Pt concentrations, and changes in taste perception were investigated. Patients exhibited decreased energy and protein intake, and salivary Pt. Using an in vitro blood-saliva barrier model, the transfer of Pt-based agents, specifically carboplatin and cisplatin, was demonstrated. Standardized sensory testing in patients revealed increased bitter taste sensitivity compared with the chemo-naïve baseline. A rinse-and-spit solution containing Na-HED, an approved flavoring compound and antagonist of bitter taste receptors (TAS2Rs), reduced the perceived bitterness of caffeine in patients receiving carboplatin (n = 15). As various anticancer agents are predicted to bind to TAS2Rs, a prerequisite for bitter taste sensation, this bitter-masking strategy may have broader applicability. Our preliminary findings show that the Na-HED mouthwash reduces bitter taste sensitivity in patients, supporting its potential to alleviate chemotherapy-induced bitter taste hypersensitivity and phantogeusia.

The involvement of non-scientific staff in discussions about animal welfare and scientific quality is essential for biomedical research progress. In this study, we developed a survey to collect the self-perception of animal care staff (ACS) and laboratory technicians about their involvement in scientific planning and conduct. Participants were contacted to complete an anonymous online questionnaire. We obtained 850 responses, mainly from Europe: 564 from ACS and 286 from laboratory technicians. Job satisfaction was assessed as positive by ACS and laboratory technicians despite the low frequency of culture of care activities and mental health meetings. Both groups expressed their desire to be trained in research planning and conduct; however, regular training was not reported. In addition, the inability to act on animal welfare concerns owing to experimental reasons was reported by both groups. Over half of the participants felt valued and appreciated by the lead scientists or animal facility manager; however, it is not clear how they are acknowledged, as their names on the authors list or in the manuscript acknowledgments are barely included. Our results indicated that involvement of ACS and laboratory technicians in planning and conducting studies would improve their understanding of how experiments are done, and therefore communication processes, work satisfaction, animal welfare, and scientific quality. Finally, we provided recommendations to improve the engagement of ACS and laboratory technicians in discussions about animal research planning and conduct.

Extensively studied blood-brain barrier (BBB) in-vitro models are established on 2D cell culture inserts. However, they do not accurately represent 3D in-vivo microenvironments due to lack of direct neurovascular unit cellular contacts. Here, the establishment and characterization of a self-assembled 3D BBB spheroid model using human-induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (iBCECs) in combination with primary human astrocytes (ACs) and pericytes (PCs) are reported. This investigation compares 3D spheroids with 2D mono-cultured iBCECs derived from two different hiPSC lines and two differentiation strategies. It is observed that spheroid properties vary depending on the differentiation strategy or type of hiPSC line applied for model generation. However, spheroids demonstrate in-vivo like tight junction ultrastructure and, in comparison to 2D models, higher transcript expression of BBB specific genes. Furthermore, they possess characteristic barrier integrity, barrier functionality, and protein expression. It is inferred that hiPSC-derived BBB spheroids hold a strong potential as a reliable future BBB in-vitro test system.

S100B is a brain protein, produced mainly by astrocytes, that indicates neurological injury by leaking into the bloodstream, cerebrospinal fluid (CSF), and urine. Elevated levels of S100B in blood and CSF serve as a marker for acute neural injury such as traumatic brain injury (TBI) and stroke. The extent of S100B elevation can help predict clinical outcomes after brain injury and monitor the effectiveness of treatment. Measuring S100B levels over time, or using a trajectory analysis, can provide more reliable information about injury progression and help predict secondary injuries. In order to predict clinical outcomes after brain injury, as well as to provide a basis for appropriate treatment and indicate treatment success, it is imperative to have appropriate analytical tools at hand. In this review, we focus on the research progress of S100B as an "alert" signalling molecule in the connection of brain injuries and critically assess current diagnostic assays for S100B, including Enzyme-Linked Immunosorbent Assay (ELISA) kits, biosensors, and point-of-care (PoC) devices.

Tumor-derived small extracellular vesicles (sEVs) have been implicated in changes of the blood–brain barrier (BBB) during pre-metastatic niche formation. Although it was postulated that sEVs can traverse the highly restrictive BBB via transcytosis—data mostly based on the indirect detection of transported cargo—direct evidence for sEV transport across the BBB remains elusive due to challenges in sEV labelling, detection limits, and inherent limitations of existing in vitro BBB models. This study investigated the interaction and effects of sEVs derived from low (LNCaP) and moderately metastatic (DU145) prostate cancer (PCa) cell lines with the human brain endothelial cell line hCMEC/D3. Systematic optimization of the cell culture membrane insert set-up for sEV transport studies was accomplished with inserts with different pore sizes, varied coating procedures and medium compositions. Particle size distribution, quantification and zeta-potential was measured with nanoparticle tracking analysis. Uptake of fluorescent labelled sEVs by hCMEC/D3 cell layers was determined by flow cytometry, barrier integrity was measured by transendothelial electrical resistance (TEER). Effects of inflammatory cytokines and PCa lines-derived sEVs on hCMEC/D3 at the transcriptomic level were investigated by means of high-throughput qPCR based on Fluidigm Biomark® platform. Improved conditions for sEV transport studies included the application of membrane inserts with 1 µm pore size and of 1

Traditional in vivo methodologies have long formed the foundation of chemical and material safety assessment, yet they are increasingly inadequate to meet modern regulatory, ethical, and sustainability demands. These conventional approaches are resource-intensive, ethically questionable, and often fail to accurately predict human or environmental toxicity, particularly for emerging pollutants such as PFAS, (nano-) pesticides, and 2D materials. In response, the EU has launched initiatives like the Chemical Strategy for Sustainability and the Zero Pollution Action Plan under the European Green Deal to promote innovation in safer, and more sustainable chemicals. Central to this transformation is the Safe and Sustainable by Design (SSbD) framework, developed by the European Commission’s Joint Research Center, which provides structured methodologies and metrics to integrate safety and sustainability into material innovation from the earliest stages of design.Building on this vision, the CHIASMA project aims to advance Next generation Safety Assessment (NGSA) by developing innovative New Approach Methodologies (NAMs) that combine experimental, computational, and Life Cycle Assessment (LCA) tools. Focusing on key biological systems and exposure routes, CHIASMA integrates Artificial Intelligence (AI), Machine Learning (ML), and Knowledge Graph (KG) technologies to enhance data interoperability and predictive accuracy. By embedding FAIR data principles and aligning with Good Laboratory Practice (GLP) standards, CHIASMA promotes transparency and regulatory acceptance. Fully aligned with SSbD principles, CHIASMA establishes a digital, interoperable infrastructure for predictive safety evaluation, that leverage on state-of-art experimental New Approach Methodologies (NAMs) bridging critical data gaps and supporting the transition towards sustainable, science-driven, and ethically responsible chemical and material innovation in Europe and beyond.

Sex steroids shape human brain development, yet the cellular and molecular consequences of androgen addition need further exploration. Here, we used a human neural organoid model - brain microphysiological system (bMPS), derived from nine induced pluripotent stem cell (iPSC) lines to model the impact of dihydrotestosterone (DHT), a potent non-aromatizable androgen. All lines differentiated reproducibly into electrically active, neuron-glia-oligodendrocyte organoids expressing robust androgen-signaling components. DHT was bioavailable, elicited nuclear androgen receptor (AR) translocation and increased organoid size in most cell lines, consistent with androgen-responsive mTOR and metabolic pathway activation. Bulk RNA-seq across nine lines revealed that transcriptional responses varied across donor backgrounds, but DHT-responsive genes converged on mitochondrial energetics, lysosomal function, glycoprotein processing, apoptosis, and mTOR signaling. Cell-type expression profiling showed an androgen-driven shift primarily in male lines toward astrocytic profiles with reductions in oligodendrocyte, oligodendrocyte progenitor (OPC), excitatory, and inhibitory neuronal signatures, supported by immunohistochemistry and AR enrichment in astrocytes and OPCs. DHT also altered neurodevelopmental pathways, increasing variation in synaptic pruning and decreasing variation in neuronal migration, with autism spectrum disorder (ASD) diagnosis and seizure status of donors moderating these effects more strongly than sex. Baseline transcriptional differences distinguished iPSC lines which responded more strongly to DHT from weak responders: responders displayed enhanced synaptic maturity and reduced ECM gene expression. Using isogenic XX/XY lines, we found that differences in sex-chromosome expression exceeded DHT-induced changes and that DHT decreased expression of inhibitory neuron genes in males and increased it in females. Finally, DHT induced extensive DNA methylation changes, targeting HOX genes, patterning, and synaptic genes. Collectively, these findings reveal that androgen signaling shapes transcription, cell populations, and epigenetic landscapes in a genetic background-dependent manner. This work contributes to understanding how androgens influence human brain development and highlights how in vitro models can contribute to representing inter-individual variability in neurodevelopment and neurodevelopmental disorders.

This study investigates the effects of N‑PEP‑12, a neuroprotective dietary supplement, on cognitive function, neuroplasticity, and neurogenesis in aged 129S1/SvImJ (S1) mice, which are known for age‑associated cognitive impairments. The primary objective was to determine whether N‑PEP‑12 could improve memory retention and enhance neural health by modulating hippocampal plasticity and neurogenesis. S1 mice were chronically treated with N‑PEP‑12 or a Vehicle to assess its impact on cognitive performance using cued fear conditioning (CFC) and object location memory (OLM) tests. Additionally, in vitro studies examined the effects of N‑PEP‑12 on neuroplasticity markers such as Neurofilament Light Chain (NF‑L) expression and vasopressin (AVP) promoter methylation, to elucidate the molecular mechanisms underlying cognitive enhancements. N‑PEP‑12 treatment significantly improved associative and contextual memory in the CFC and OLM tests respectively. In vitro assays revealed that N‑PEP‑12 increased NF‑L expression and decreased AVP promoter methylation, indicating enhanced neuroplasticity and neurogenesis. Furthermore, N‑PEP‑12 preserved blood‑brain barrier integrity under oxidative stress conditions, suggesting a protective role against vascular‑related cognitive decline. The findings suggest that N‑PEP‑12 promotes hippocampal health by enhancing neurogenesis and neuroplasticity, potentially mitigating age‑related cognitive decline. These results highlight N‑PEP‑12 as a promising agent for supporting healthy cognitive function in aging populations through the modulation of neurobiological pathways associated with learning and memory.

This study presents first polymeric hollow fibers (HFs) with sufficient optical transparency for real-time and non-invasive monitoring of biological systems, one of the most important hurdles accounted in HF bioreactor technology. Biocompatible polymers, including polyvinylidene fluoride (PVDF), polycaprolactone, and polyacrylonitrile, were processed via dry-jet wet spinning. A comprehensive analysis of the morphological, structural, and transport properties of the HFs provided valuable insights into their optical performance. Our findings revealed the critical interplay of HF wall thickness, porosity, polymer crystal size, and intrinsic refractive index in achieving transparency. These findings may serve as a useful guidance to produce transparent polymeric HFs with similar or different polymeric systems. Remarkably, PVDF HFs achieved over 83% transmittance, exceeding standards for in vitro cell observation, and was achieved without the presence of modifiers. As a proof of concept, labeled human cerebral microvascular endothelial cells (hCMEC/D3) were cultured within the HFs and successfully monitored in real time under both static and dynamic conditions. These transparent HFs demonstrate transformative potential, enabling real-time decision making while significantly reducing cost and time. This cutting-edge membrane material paves the way for advanced applications in microfluidics, mass transport studies, and biomedical research, establishing these transparent HFs as key enabling materials for strategic biomedical technologies.