Air-liquid interface (ALI) systems have emerged as a physiologically relevant in vitro platform for evaluating the toxicological impact and potential health effects of airborne pollutants. When utilizing collected ultrafine particles (UFPs), application volume and liquid type are critical parameters. Using a smaller volume of liquid for the cell exposure results in a heterogeneous distribution of UFPs across the cell monolayer, whereas application of a sufficient volume optimises even UFP distribution. A buffered solution for UFP administration minimises potential side effects and unravels dose-dependent effects in toxicological endpoints. However, standardised exposure methodologies limit reproducibility and comparability across studies. Therefore, we propose a refined manual exposure technique of suspended airborne pollutants in an adequate exposure volume that bridges the gap between conventional submerged cultures and ALI systems. Our model uses cell culture inserts with A549 epithelial cells, THP-1 macrophages, and EA.hy926 endothelial cells to mimic the in vivo alveolar barrier within the lungs. This approach offers a balance of experimental reproducibility whilst addressing the current challenges of standardisation and feasibility in exposure studies with manual UFP exposure. In conjunction with existing aerosol ALI continuous flow exposure systems, our studies are advancing translational in vitro evaluations, aligning with the 3R principle.

BackgroundThe tumor microenvironment (TME) composition is among the critical events leading to the poor prognosis of head and neck cancers. TME includes immune cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAF), and other non-cancerous cells, which contribute to therapeutic outcome. The heterogeneity of the TME offers a multitude of potential targets for photodynamic therapy (PDT). Advanced 3D models that closely mimic the microenvironment are a promising tool to study tumor-stroma interactions, TAM plasticity and their impact on Foslip®-PDT outcome.ObjectiveThe aim of this study was to assess the effect of Foslip-PDT on photo-induced macrophage re-education in 3D tri-culture model composed of cancer cells, CAFs, and macrophages.MethodsA 3D model was established using FaDu cancer cells, MeWo fibroblasts, and PMA-differentiated U937 macrophages. The spheroids were characterized using immunochemistry, immunofluorescence, and qPCR. Foslip-based photoirradiation was applied to spheroids at different fluences to evaluate the photoinduced cell death. Macrophage phenotypes were assessed by flow cytometry.ResultsThe 3D tri-culture model displayed hallmarks of stromal–tumor interactions, including CAF clustering, macrophage infiltration (~30–40%), and epithelial–mesenchymal transition. Macrophages in the spheroids had prevailing M2 phenotype as deduced from overexpression of immunosuppressive markers (CD163, PDL-1, IL-10). The liposomal photosensitizer Foslip accumulated 2 to 3 times more in macrophage-enriched spheroids, however, PDT induced similar levels of cell death in all tested models. At the same time, Foslip-PDT produced immunomodulatory effect in tri-culture model characterized by the increase of CD80-M1 marker expression and the decrease in the expression of the CD206-M2 marker.ConclusionThe 3D tri-culture model integrated essential features of the HNSCC microenvironment. Foslip-PDT was effective in reprograming M2 macrophages to tumor-killing M1 macrophages. This study opens the way to combine direct tumor damage with TME modulation.

IntroductionNeurotoxicity is a critical liability for many environmental pollutants. Current in vitro neurotoxicity screens rely on direct exposure of cultured neurons to xenobiotics, often at exceeding physiologically relevant levels due to the restrictive nature of the blood -brain barrier (BBB). This limitation reduces the accuracy of central nervous system (CNS) exposure predictions.MethodsTo address this limitation, we have developed a novel human in vitro direct-contact triculture BBB model that more closely mimics the in vivo barrier. The triculture is formed by layering primary astrocytes, primary pericytes, and then brain microvessel endothelial cells (BMECs, HBEC-5i) in direct contact, increasing the restrictive nature of tight junctions and allowing cell -cell signaling that mimics the configuration found in the in vivo BBB. Using this model, we quantified the apparent bidirectional permeability (Papp) of more than 50 compounds, including environmental pollutants and CNS drugs, primarily by paracellular, passive transcellular and transporter-mediated pathways, to help develop a risk of exposure. In parallel with our in vitro BBB model, we are using the high-throughput toxicokinetics (HTTK) R library developed by the U.S. Environmental Protection Agency (EPA) as our model to predict brain exposure.ResultsThe triculture model demonstrated enhanced tight junction organization and increased efflux transporter expression compared with endothelial monocultures, indicating an improved barrier phenotype. Using our measured bidirectional Papp values, calculated efflux ratios, and EPA physiologically based pharmacokinetic (PBTK) reference data for compound parameters, we are developing predictions of toxicant accumulation in the brain parenchyma after chronic exposure in steady state.DiscussionIntegration of in vitro BBB permeability measurements with toxicokinetic modeling provides a physiologically relevant approach to predict CNS exposure and neurotoxicity risk. Through the development of this model, we postulate that future investigators could simply perform in vitro BBB permeability studies to determine the relative risk of potential brain accumulation and the risk of neurotoxicity.

In vitro models of the human airway are essential to study respiratory diseases and test potential therapeutics while reducing animal testing. Current models often use two-dimensional culture conditions rather than replicating the physiological 3D environment and do not allow direct cell-cell interactions between the diverse cell types found in the mucosa. Here, we provide a detailed step-by-step instruction for reproducibly generating a complex tri-culture model, which can be used to investigate the human airway environment in health and disease. The model is fabricated by preparing an epithelialized fibrin hydrogel with embedded endothelial and stromal cells. To assure complete differentiation into a mucociliary phenotype, samples are maintained at air-liquid interface (ALI) for 28 days. Afterwards, morphology and functionality can be validated using downstream analysis techniques such as immunohisto- and cytochemistry, electron microscopy, ciliary beating frequency analysis, measurement of mucociliary clearance and RNA isolation. After 4 weeks of maturation, a well-differentiated pseudostratified epithelium comprising basal, multiciliated and secretory cells is developed. We also observe a physiological ciliary beating frequency, mucus production and a functional particle clearance. Inside the hydrogel, endothelial cells form a three-dimensional network of vascular structures. These features make our model ideal for replicating human mucosal heterogeneity, especially compared to airway models using tumor-derived or immortalized cell lines, monocultures or rigid substrates. Hence, this protocol paves the way for fellow researchers to achieve robust airway in vitro modeling that can be performed in a standard cell culture lab without the need for extraordinary equipment or specialized expertise.

BackgroundOver the past decade, forward programming of human pluripotent stem cells (hPSCs) using various transcription factor (TF) combinations has been widely applied in neuroscience research. Ectopic NGN2 expression in hPSCs has been widely used for rapidly generating in vitro models of induced neurons (iNs) that are predominantly composed of excitatory glutamatergic neurons. Achieving a more balanced synaptic communication between excitatory and inhibitory neurons is essential for physiologically relevant in vitro models. Additionally, incorporating hPSC-derived astrocytes into models, rather than commonly used primary astrocytes, would more closely mimic in vivo disease phenotypes, especially for those associated with astrocyte dysfunction.MethodsInducible hPSC lines were generated by targeting the AAVS1 safe harbor site with TF transgene cassettes using CRISPR/Cas9. Forward programming was achieved through forced expression of NGN2 for glutamatergic neurons (iGlutNs), ASCL1/DLX2 for GABAergic neurons (iGABANs) and SOX9/NFIB for astrocytes (iAstros). Cell identity was validated by immunocytochemistry and bulk RNA sequencing. Functional properties were characterized on multielectrode arrays (MEAs).ResultsBulk RNA sequencing confirmed lineage-specific differentiation while revealing distinct transcriptomic profiles between iAstros and human primary astrocytes. Functional assays demonstrated robust inhibitory control of network dynamics in co-culture with iGABANs on MEA, with enhanced responses to GABAA receptor-targeting drugs including picrotoxin, bicuculline and clonazepam. Neurons co-cultured with iAstros showed reduced spontaneous activity compared to those cultured with primary astrocytes.ConclusionWe successfully generated hPSC-derived excitatory and inhibitory neurons to establish an appropriate E/I balance in vitro, supported by primary astrocytes. Although astrocyte identity was confirmed in our hPSC-derived astrocytes, further optimization is required to achieve full functional maturation. This approach to developing an isogenic co-culture system derived from a single hPSC line may more faithfully replicate native neural network dynamics, offering a physiologically relevant platform for studying neurological disorders and screening therapeutic compounds.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-026-04917-6.

3D-microcontact printing (3D-µCP) technique combines the advantages of microcontact printing and microthermoforming for the fabrication of functional biomaterials with complexity closer to real tissue. Despite its unmet advantages in terms of complexity and processability in a single step, this technique is limited to the front side of the substrate. Considering the advantage of inherent topography patterns on the reverse side of the substrate, an additional degree of patterning can be envisioned. However, selective patterning on the reverse side is challenging due to the fragility of the cell culture on the front side. Herein, a technically simple scraping technique is presented in combination with 3D-µCP to generate 3D cell patterns on both the front and the reverse side of a micro thermoformed substrate. The technical advancement of 3D-µCP with the scraping technique offers a complex, multi-layered co-culture system that can be used to study cell-to-cell crosstalk at the microlevel with topographies similar to real tissue. Direct seeding of the third cell type on the scaffold expands cell type complexity, resulting in a tri-culture model. As a proof of concept, a triculture of EA.Hy 926 endothelial cells, HepG2 hepatoblastoma cells, and NIH3T3 fibroblasts are generated, mimicking the tumor microenvironment in the liver.

Ethanol induces neuroimmune dysregulation and soluble TREM2 generation in a human iPSC neuron, astrocyte, microglia triculture model.

Bone is a primary site for metastasis in breast cancer, with up to 70 % of patients with metastatic breast cancer developing osteolytic bone lesions, wherein cancer cells drive osteoclast resorption of bone. However, progress in developing therapies is limited by the absence of predictive in vitro models. This study developed a unique organ-on-a-chip model to simulate osteolytic bone metastasis and utilised a multi-omics approach for characterisation/qualification and validation against in vivo data. Using the Emulate S1 platform, we co-cultured murine osteocytes and osteoclasts to recreate the bone microenvironment, alongside breast cancer cells in a separate channel separated by a porous membrane. Using RNA sequencing, cytokine profiling, and fluorescence staining, we demonstrated the importance of the complete tri-culture model in replicating key aspects of in vivo biology, and uncovered critical pathways involved in metastasis. A synergistic effect was observed in the tri-culture organ-chip model, leading to increased cancer cell migration and the upregulation of pro-metastatic and pro-inflammatory pathways that promote bone degradation and cancer progression. This study validates an organ-chip model of osteolytic breast cancer bone metastasis as a scalable alternative to traditional animal models. Furthermore, we show how multi-omics and bioinformatics techniques may be used for qualification and validation of organ-chip models; for unpicking the relative contribution of the different cell types; and to identify signalling pathways and therapeutic targets. STATEMENT OF SIGNIFICANCE: In this study, we develop a 3D organ-on-a-chip tri-culture model of the osteolytic metastatic niche, in which we verify expected bone and breast cancer cell behaviours. Importantly, we successfully validate our organ-chip against a dataset from the gold standard in vivo preclinical model of osteolytic breast metastases, using transcriptomics and proteomics to confirm strong alignment of gene expression profiles with in vivo mouse expression. Additionally, our multi-omics analysis sheds new light on both expected and novel molecular pathways for therapeutic targeting, demonstrating the utility of the organ-chip as a potential replacement for preclinical mouse models of breast cancer metastases in bone. Therefore, this study represents a key marker in the field of organ-chip research, demonstrating the importance of biomaterials technologies for preclinical science. Most importantly, our work demonstrates for biotech and pharma companies that qualified organ-chip devices can play a role as intermediate medium-throughput technologies for screening lead drug candidates.

Post-traumatic osteoarthritis (PTOA), a subtype of osteoarthritis initiated by joint trauma, is driven by unresolved early inflammation that ultimately leads to cartilage degeneration. Although animal models have advanced our understanding of disease progression, they offer limited resolution of the early molecular events following trauma. In this study, we developed a transwell-based in vitro triculture model mimicking the early joint environment post-injury, incorporating macrophages, fibroblast-like synoviocytes (FLSs), and human articular chondrocytes (HACs). In lieu of the commonly used macrophage activator, lipopolysaccharide (LPS), this study utilizes fibronectin fragments (Fnfs), which belong to the damage-associated molecules released upon trauma to cartilage, to activate macrophages and simulate post-traumatic inflammation. The triculture was maintained for 12 days while promoting paracrine-only communication between the cell types. The activation of macrophages by Fnfs led to a sustained expression of pNFκB in both HACs and FLSs, as shown by immunofluorescence, alongside increased gene expression of inflammatory mediators MMP3, MMP13, and TNF-α. Fnfs triggered catabolic signaling across all joint-resident cell types used in this model. To support the translational relevance of the in vitro findings, a complementary in vivo experiment in which Fnfs were injected intra-articularly showed increased MMP activity gene expression and reduced COL2A1 gene expression in joint cartilage. The cytokine and gene expression profiles observed in the triculture model closely mirrored those observed in early-stage in vivo PTOA models and in the patient-derived synovial fluid obtained in the early traumatic phase, underscoring the model’s physiological relevance. This triculture platform captures the key aspects of early PTOA processes driven by macrophage activation and offers a biologically relevant tool for mechanistic studies and therapeutic screening.

413P Single-cell transcriptomics uncovers cellular dynamics and potential anti-tumour macrophage repolarization in a tri-culture model of the tumour microenvironment

IntroductionNeuroinflammation triggered by viral infections is increasingly recognized as a driving force in neurodegenerative disease, promoting chronic neuronal injury and cognitive decline. A central mechanism in this process is impaired glutamate clearance due to downregulation of the astrocytic glutamate transporter GLT-1 (EAAT2/SLC1A2), which exacerbates excitotoxicity and neuronal death.MethodsIn this study, we assessed the neuroprotective effects of the β-lactam antibiotic ceftriaxone—a known upregulator of GLT-1—in an in vitro tri-culture model of neurons, microglia, and astrocytes challenged with the viral mimic polyinosinic:polycytidylic acid (Poly I:C).Results and discussionPoly I:C exposure elicited robust microglial and astrocytic activation and increased levels of TNF-α, IL-6, and IL-10. Concomitantly, we observed significant downregulation of GLT-1, synapse loss, impaired synaptic plasticity, and disrupted amino acid metabolism. A complementary Mendelian randomization analysis of GWAS data revealed that genetically determined alterations in plasma amino acid levels are significantly associated with the risk of five major neurodegenerative disorders, underscoring the role of metabolic dysregulation in disease pathogenesis. Treatment with ceftriaxone effectively reversed the Poly I:C–induced phenotypes: GLT-1 expression, dendritic spine density, and measures of synaptic plasticity were all restored, and abnormalities in amino acid and tricarboxylic acid cycle metabolites normalized. These findings highlight ceftriaxone’s multifaceted neuroprotective profile—modulating glutamate homeostasis, preserving synaptic integrity, and rebalancing metabolic pathways—and support its potential as a therapeutic agent to prevent neuronal degeneration in the context of virus-driven neuroinflammation.

SummaryHuman induced pluripotent stem cell (hiPSC) models enable disease modeling and drug screening, but standardized methods for multi-lineage co-culture remain limited. Here, we present a cryopreservation-compatible tri-culture system of neurons, astrocytes, and microglia. We describe steps for transducing induced pluripotent stem cells (iPSCs) with cell type-specific factors, generating intermediate cryopreserved stocks, differentiating each cell population, and assembling them into tri-culture. This protocol provides a reproducible platform to study dynamic interactions between human brain cells in a physiologically relevant environment.For complete details on the use and execution of this protocol, please refer to Lish et al.1,2

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. While in vitro patient-derived xenografts (PDX) lines are useful for studying GBM, they often exclude astrocytes and macrophages, which contribute significantly to tumor growth, invasion, and chemoradioresistance. Integrating these cells into tumor models is difficult due to their need for serum, which triggers GBM-PDX lines to lose their stem-like properties. The aim of this study was to develop a serum-free triculture model of GBM-PDX lines, normal human astrocytes (NHAs), and macrophages. Serum-free media alternatives were formulated for NHAs and identified for THP-1 macrophages, then combined with GBM PDX media to establish “PSX,” an experimental maintenance media. Cells were transitioned to serum-free media alternatives and functionally assessed through several parameters unique to each cell type. In addition to assessing GBM “stemness,” a custom 350-gene NanoString chip was used to assess differential gene expression in monocultured PDX cells versus PDX cells exposed to NHAs and macrophages. PSX maintained canonical function in astrocytes and macrophages while preserving the stem-like properties of GBM-PDX cells. Tri-culturing all three cells increased the expression of stemness-associated transcription factors and increased the expression of genes related to stemness and hypoxia in GBM cells. GBM PDX cells exposed to NHAs and macrophages in direct triculture exhibit increases in markers of stemness and hypoxia. These findings suggest that the serum-free triculture model presented herein may better recapitulate the tumoral heterogeneity of GBM in vitro, providing a novel model to utilize in current research.

Interactions among neurons, microglia, and endothelial cells (ECs) —the principal components of the neurovascular unit (NVU)—are vital for maintaining central nervous system (CNS) homeostasis and are implicated in numerous neurological disorders. However, mechanistic insights into their crosstalk remain limited due to the lack of physiologically relevant in vitro models. In this study, we present an improved 3D vascularized tri-culture model that integrates human-induced neural stem cells (hiNSCs), human vascular organoids (hVOs), and microglia within a geometrically engineered silk fibroin scaffold. This platform effectively recapitulates critical features of the native CNS microenvironment, including spatial neurovascular patterning and cell-type-specific interactions. Within this model, hVOs significantly promoted neuronal differentiation of hiNSCs, resulting in extended axonal networks and improved neurovascular alignment. Microglial effects were found to be phenotype-dependent: both resting (M0) and pro-inflammatory (M1) microglia inhibited hiNSCs differentiation and vascular development, with M1 cells exerting the strongest suppressive influence. In contrast, anti-inflammatory (M2) microglia displayed the least inhibitory effect and even modestly supported neurovascular maturation. Mechanistic studies revealed that M2 microglia cooperate with hVOs via the stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling axis to promote neuronal differentiation. To our knowledge, this represents the first demonstration of SDF-1/CXCR4-mediated immune-neurovascular interaction within a human tri-culture system. Thereafter, this 3D vascularized co-culture model provides a physiologically relevant in vitro platform to investigate neuroimmune and neurovascular interactions. It holds broad potential for mechanistic studies in neurodevelopment and neurodegeneration, drug evaluation, and the development of regenerative therapies.

Alcohol use disorders (AUDs) affect substantial populations worldwide and increase the risk of developing cognitive impairments and alcohol-associated dementia. While chronic inflammatory signaling likely plays an important role in alcohol-associated neurological sequalae, the precise mechanisms underlying alcohol-associated neuropathology remain enigmatic. We hypothesize that alcohol leads to neuroimmune dysregulation among neurons, astrocytes, and microglia; and is perpetuated by innate immune signaling pathways involving cell-cell signaling. To investigate how alcohol dysregulates neuroimmune interactions in a human context, we constructed a triculture model comprising neurons, astrocytes, and microglia derived from human induced pluripotent stem cells. After exposure to ethanol, we observed significant differential gene expression relating to innate immune pathways, inflammation, and microglial activation. Microglial activation was confirmed with morphological analysis and expression of CD68, a lysosomal-associated membrane protein and marker for phagocytic microglial activation. A striking finding in our study was the elevation of TREM2 expression and, specifically, TREM2 alternative splice variants that are predicted to give rise to soluble TREM2. TREM2 has been reported to be a risk factor for Alzheimer’s disease. These results suggest that ethanol exposure in the brain may lead to increased microglial activation and production of soluble isoform named TREM2219 through alternate splicing. Deciphering the molecular and cellular mechanisms underpinning ethanol-related neuroimmune dysregulation within a human context promises to shed light on the etiology of AUD-related disorders, potentially contributing to the development of effective therapeutic strategies.

Chronopharmacokinetics of the antidepressant paroxetine: An in vitro and in vivo approach.

Reconstructing Alzheimer's disease one cell type at a time using in vitro tricultures.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a growing health concern worldwide. Human cell-based in vitro culture models that retain disease-relevant phenotypic pathways and responses to assess the efficacy and liability of new therapeutics are needed. Obeticholic Acid (OCA), a Farnesoid X Receptor agonist, has been identified for MAFLD treatment, and clinically shown to have anti-inflammatory and anti-fibrotic effects. In this study, healthy and disease-origin primary human hepatocytes (PHHs) were cultured in TruVivo®, an all-human hepatic system for 14 days and treated with OCA to determine its' effects on lipogenic, inflammatory, and fibrogenic pathways. Decreases in lipogenesis and triglyceride levels were measured in OCA treated healthy and diseased PHHs. Significant decreases in CYP3A4 activity and gene expression were quantified. Macrophage marker expression, pro-inflammatory cytokines and fibrotic markers were lowered in OCA treated diseased PHHs. CYP7A1 gene expression decreased, while BSEP gene expression increased in OCA treated healthy and diseased PHHs. Overall, OCA treatment reduced lipogenic, inflammatory, and fibrogenic markers in diseased PHHs. Differences in the potency and efficacy of OCA against different disease-relevant pathways were observed in healthy and diseased PHHs indicating divergence of key regulatory mechanisms between healthy versus diseased phenotypes.

Background: The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) but also limits drug delivery. Insufficient knowledge of how the CNS promotes the onset and maintenance of peripheral neuropathic pain limits therapeutic methods for the treatment of persistent neuropathic pain. Thus, this study aimed to evaluate the ability of a novel combination of Palmitoylethanolamide (PEA) and Equisetum arvense L. (Equisetum A.L.) to cross the BBB and modulate nociceptive pathways. Methods: Using a humanised in vitro BBB tri-culture model, the permeability, cytotoxicity, and integrity of the barrier were assessed after exposure to two different PEA forms, PEA ultramicronized (PEA-um) and PEA80mesh, Equisetum A.L., and a combination of the last two samples. The samples exhibited no cytotoxicity, maintained tight junction integrity, and efficiently crossed the blood–brain barrier (BBB), with the combination displaying the highest permeability. The eluate from the BBB model was then used to stimulate the co-culture of CCF-STTG1 astrocytes and SH-SY5Y neurons pre-treated with H2O2 200 µM. Results: Treatment with the combination significantly increased cell viability (1.8-fold, p < 0.05), reduced oxidative stress (2.5-fold, p < 0.05), and decreased pro-inflammatory cytokines (TNFα, IL-1β) compared to single agents. Mechanistic analysis revealed modulation of key targets involved in pain pathways, including decreased FAAH and NAAA activity, increased levels of endocannabinoids (AEA and 2-AG), upregulation of CB2 receptor expression, enhanced PPARα activity, and reduced phosphorylation of PKA and TRPV1. Conclusions: These findings suggest that the combination of PEA and Equisetum A.L. effectively crosses the BBB and exerts combined anti-inflammatory and analgesic effects at the CNS level, suggesting a possible role in modulating neuroinflammatory and nociception responses.

Micro and nanoplastics (MNPs) are widespread environmental and food web contaminants that are absorbed by the intestine and distributed systemically, but the mechanisms of uptake are not well understood. In a triculture small intestinal epithelial model, we previously found that uptake of 26 nm polystyrene MNPs (PS26) occurred by both passive diffusion and active actin- and dynamin-dependent mechanisms. However, studies in a more physiologically relevant model are required to validate those results. Here, a microfluidic intestine-on-a-chip model was developed using primary human intestinal epithelial organoids from healthy and Crohn's disease donors, and used to evaluate the toxicity and mechanisms effectuating uptake of 25 nm polystyrene shell-gold core tracer MNPs (AuPS25). AuPS25 caused minimal toxicity after 24 h exposure in either healthy or Crohn's IOC models. RNAseq analysis of epithelial cells identified 9 genes dysregulated by AuPS25, including downregulation of IFI6 (interferon alpha-induced protein 6). Because IFI6 has important antiviral and immunosuppressive functions in the intestine, its downregulation suggests impairment of innate immune function, which could have important negative health consequences. Inhibitor studies revealed that AuPS25 uptake in the IOC occurred by both passive diffusion and active actin- and dynamin-dependent mechanisms, consistent with our previous findings in the triculture model.