Microfluidic Conditions Research Articles

NX210c drug candidate peptide strengthens mouse and human blood-brain barriers

BackgroundAlterations of blood-brain barrier (BBB) and blood-spinal cord barrier have been documented in various animal models of neurodegenerative diseases and in patients. Correlations of these alterations with functional deficits suggest that repairing barriers integrity may represent a disease-modifying approach to prevent neuroinflammation and neurodegeneration induced by the extravasation of blood components into the parenchyma. Here, we screened the effect of a subcommissural organ-spondin-derived peptide (NX210c), known to promote functional recovery in several models of neurological disorders, on BBB integrity in vitro and in vivo.MethodsIn vitro, bEnd.3 endothelial cell (EC) monolayers and two different primary human BBB models containing EC, astrocytes and pericytes, in static and microfluidic conditions, were treated with NX210c (1-100 µM), or its vehicle, for 4 h and up to 5 days. Tight junction (TJ) protein levels, permeability to dextrans and transendothelial electrical resistance (TEER) were evaluated. In vivo, young and old mice (3- and 21-month-old, respectively) were treated daily intraperitoneally with NX210c at 10 mg/kg or its vehicle for 5 days and their brains collected at day 6 to measure TJ protein levels by immunohistochemistry.ResultsNX210c induced an increase in claudin-5 protein expression after 24-h and 72-h treatments in mouse EC. Occludin level was also increased after a 24-h treatment. Accordingly, NX210c decreased by half the permeability of EC to a 40-kDa FITC-dextran and increased TEER. In the human static BBB model, NX210c increased by ∼ 25% the TEER from 3 to 5 days. NX210c also increased TEER in the human 3D dynamic BBB model after 4 h, which was associated with a reduced permeability to a 4-kDa FITC-dextran. In line with in vitro results, after only 5 days of daily treatments in mice, NX210c restored aging-induced reduction of claudin-5 and occludin levels in the hippocampus, and also in the cortex for occludin.ConclusionsIn summary, we have gathered preclinical data showing the capacity of NX210c to strengthen BBB integrity. Through this property, NX210c holds great promises of being a disease-modifying treatment for several neurological disorders with high unmet medical needs.

Microfluidics and Chip Development

During the course of the summer, I had the privilege to work with Dr. Carlos Escobedo and his team of researchers on the topics of microfluidics and Lab-On-Chip (LOC) technology. Microfluidics is a branch of fluid dynamics which utilizes small volumes of fluid to perform tests under ideal conditions, and LOC is the process of using microfluidics for experiments in small-scale environments. I participated in two LOC projects, the first for bacterial analysis and the second for microplastic separation. The goal of the first project was to observe if Vibrio Cholerae bacteria was attracted to a variety of chemo attractants. To observe this interaction under microfluidic conditions, we developed a chip which when flooded with chemoattractant trapped a portion in the chip. We could then remove the non-entrapped chemoattractant from the sides and refill the chip with bacteria. Observations of the bacteria’s movement were then taken using a microscope attached to a digital camera. The second project focused on microplastics, which are plastic particulates under 5mm in diameter, our smallest sample being 10μm. Due to their size standard filtering techniques were ineffective at separating them, which led to us using Deterministic Lateral Displacement (DLD). DLD utilizes an array of pillars to create spots of changing pressure for passing particles. The array is designed so larger particles are unaffected while smaller particulates will be redirected and collected separately. To arrange the plastics into their types, a centrifugal design was used. This design functions similarly to a centrifuge, which by rapidly spinning a substance separates out materials based on density. The chip works under a similar theory, but instead uses a spiral channel to force the particles in a repeated circular spinning motion. The particles then exit with differing directionalities based on where they were in the channel, allowing for collection.

Probing Phoretic Transport of Oxidative Enzyme-Bound Zn(II)-Metallomicelle in Adenosine Triphosphate Gradient via a Spatially Relocated Biocatalytic Zone.

Although cellular transport machinery is mostly ATP-driven and ATPase-dependent, there has been a recent surge in understanding colloidal transport processes relying on a nonspecific physical interaction with biologically significant small molecules. Herein, we probe the phoretic behavior of a biocolloid [composed of a Zn(II)-coordinated metallomicelle and enzymes horseradish peroxidase (HRP) and glucose oxidase (GOx)] when exposed to a concentration gradient of ATP under microfluidic conditions. Simultaneously, we demonstrate that an ATP-independent oxidative biocatalytic product formation zone can be modulated in the presence of a (glucose + ATP) gradient. We report that both directionality and extent of transport can be tuned by changing the concentration of the ATP gradient. This diffusiophoretic mobility of a submicrometer biocolloidal object for the spatial transposition of a biocatalytic zone signifies the ATP-mediated functional transportation without the involvement of ATPase. Additionally, the ability to analyze colloidal transport in microfluidic channels using an enzymatic fluorescent product-forming reaction could be a new nanobiotechnological tool for understanding transport and spatial catalytic patterning processes. We believe that this result will inspire further studies for the realization of elusive biological transport processes and target-specific delivery vehicles, considering the omnipresence of the ATP-gradient across the cell.

Simultaneous effects of capillary number, viscosity ratio, and contraction ratio on droplet dynamics in contraction microchannel

Abstract In droplet-based microfluidic systems, fluid properties, flow conditions, and channel geometry play crucial roles in the movement and manipulation of droplets. This study employs a three-dimensional numerical simulation method to investigate droplet dynamics in a contraction microchannel under the simultaneous influence of different factors, such as capillary number (Ca), viscosity ratio (λ), and contraction ratio (C). Specifically, a theoretical model is introduced for predicting the transition of droplets from trap to squeeze, and it is observed to be 〖Ca〗_I = f(C,λ). Meanwhile, droplet dynamics are investigated for the transition from squeeze to breakup for finding critical capillary number value (CaII). In addition, the critical viscosity ratio for the process from squeeze to breakup has been figured out by numerical results for a wide range of contraction ratios. Eventually, droplet dynamics including deformation, velocity ratio, and breakup during contraction microchannel are also quantitatively studied under the factors above.

Linear and nonlinear interfacial rheology of responsive microgels at the oil-water interface

Microgelation can significantly enhance the interfacial activity of polysaccharide and the ability to stabilize emulsion, but the interfacial behaviour of microgels and the intrinsic mechanism of stabilizing emulsion is not fully understood. In this study, chitosan microgels (h-CSMs) with uniform size were successfully prepared by electrostatic crosslinking hydrophobically modified chitosan (h-CS) with sodium phytate (SP) using microfluidic technique. The microfluidic conditions for fabricating of h-CSM were optimized: the mass ratio of h-CS: SP = 5: 1 with h-CS concentration of 0.5 mg/ml, and the pressure ratio of Ph-CS (Pa):PSP (Pa) = 1600:1000. The prepared h-CSMs were pH-responsive, with a swollen state at pH 5 and a shrunken state at pH 3 and 7. Further, the interfacial adsorption kinetics, and the interfacial rheology particularly the nonlinear interfacial rheology were systematically studied. The instantaneous Si-factor was proposed to describe the nonlinear responses of the microgel interface during an individual dilatational oscillation cycle. The results showed that microgelation significantly improved the interfacial ability of chitosan molecules at the oil-water interface. The h-CSM interface exhibited strain-softening response during extension and strain-hardening response during compression. The interface formed by h-CSMs under the swollen state had higher viscoelastic modulus, lower degree of nonlinearity and higher emulsifying capacity compared to that formed under the shrinking state. Low ionic strength significantly increased the viscoelastic modulus and reduced the nonlinearity and enhanced emulsifying capacity of the h-CSM interface, whereas high ionic strength disrupted the interaction between the h-CSMs and increased the nonlinear of the h-CSM interface due to too strong electrostatic screening effect.

Capillary effects of submicron heterogeneities in aqueous solutions of sugars. The additional mechanism of microfluidics

In this paper, we study submicron heterogeneities (SMH) that spontaneously form in aqueous solutions of sugars: glucose, fructose and sucrose, with a concentration of 0.5 mol%. These solutions were passed through glass capillaries with a diameter of about 6 and 13 µm, which led to a decrease in the concentration of SMH without significantly changing their size. For capillaries of smaller diameter, this effect is more pronounced. The decrease in the concentration of SMH is not associated with their mechanical destruction or adsorption on the surface of capillaries. This phenomenon is explained by the destruction of SMH as a result of their interaction with the hydrophilic surface of the capillary. The fundamental differences between the presence of SMH in microfluidic conditions and bulk reservoirs are discussed. This phenomenon should be taken into account in microfluidics.

Diffusion-based culture and real-time impedance monitoring of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions

Tumor spheroids are widely studied for in vitro modeling of tumor growth and responses to anticancer drugs. However, current methods are mostly limited to static and perfusion-based cultures, which can be improved by more accurately mimicking pathological conditions. Here, we developed a diffusion-based dynamic culture system for tumor spheroids studies using a thin membrane of hydrogel microwells and a microfluidic device. This allows for effective exchange of nutrients and metabolites between the tumors and the culture medium flowing underneath, resulting in uniform tumor spheroids. To monitor the growth and drug response of the spheroids in real-time, we performed spectroscopic analyses of the system's impedance, demonstrating a close correlation between the tumor size and the resistance and capacitance of the system. Our results also indicate an enhanced drug effect on the tumor spheroids in the presence of a low AC electric field, suggesting a weakening mechanism of the spheroids induced by external perturbation.

A Real-Time LSPR-Based Study of Metal-Organic Framework (MOF) Growth.

MOFs are known for their absorption properties and widely used for accumulation, filtering, sensorics, photothermal, catalytical and other applications. Their combination with plasmonic metal nanoparticles leads to hybrid structures that profit from the stabilizing effect and high porosity of the MOF as well as the optical and electronic properties of the nanoparticles. The growth of MOFs on plasmonic nanoparticles can be monitored in-situ using LSPR spectroscopy, simultaneously applying microfluidic reaction conditions for the fabrication of NP@MOF structures. Here, a systematic study is conducted using LSPR spectroscopy for the monitoring of the Layer-by-Layer deposition of twelve different MOFs, determining the suitability of LSPR spectroscopy for this purpose. In addition to some well-investigated materials like HKUST-1, other MOFs such as MIL-53, MIL-88 A and Cu-BDC are deposited successfully. For some MOFs such as Zn-Fum, the LSPR experiment indicates that no deposition had taken place. The results are confirmed with AFM, SEM and XPS measurements. This work shows that LSPR spectroscopy is suitable for the in-situ monitoring of LbL MOF growth and the microfluidic setup is a very promising method for the controlled manufacturing of NP@MOF hybrid structures. Further studies may include the optimization of the synthesis process or the transfer to other materials.

Chitosan-Coated Fosetyl-Al Nanocrystals' Efficacy on Nicotiana tabacum Colonized by Xylella fastidiosa.

Xylella fastidiosa (Xf) is a quarantine plant pathogen capable of colonizing the xylem of a wide range of hosts. Currently, there is no cure able to eliminate the pathogen from a diseased plant, but several integrated strategies have been implemented for containing the spread of Xf. Nanotechnology represents an innovative strategy based on the possibility of maximizing the potential antibacterial activity by increasing the surface-to-volume ratio of nanoscale formulations. Nanoparticles based on chitosan and/or fosetyl-Al have shown different in vitro antibacterial efficacy against Xf subsp. fastidiosa (Xff) and pauca (Xfp). This work demonstrated the uptake of chitosan-coated fosetyl-Al nanocrystals (CH-nanoFos) by roots and their localization in the stems and leaves of Olea europaea plants. Additionally, the antibacterial activity of fosetyl-Al, nano-fosetyl, nano-chitosan, and CH-nanoFos was tested on Nicotiana tabacum cultivar SR1 (Petite Havana) inoculated with Xff, Xfp, or Xf subsp. multiplex (Xfm). The bacterial load was evaluated with qPCR, and the results showed that CH-nanoFos was the only treatment able to reduce the colonization of Xff, Xfm, and Xfp in tobacco plants. Additionally, the area under the disease progress curve, used to assess symptom development in tobacco plants inoculated with Xff, Xfm, and Xfp and treated with CH-nanoFos, showed a reduction in symptom development. Furthermore, the twitching assay and bacterial growth under microfluidic conditions confirmed the antibacterial activity of CH-nanoFos.

Machine learning-driven optimization of mRNA-lipid nanoparticle vaccine quality with XGBoost/Bayesian method and ensemble model approaches

To enhance the efficiency of vaccine manufacturing, this study focuses on optimizing the microfluidic conditions and lipid mix ratios of messenger RNA-lipid nanoparticles (mRNA-LNP). Different mRNA-LNP formulations (n = 24) were developed using an I-optimal design, where machine learning tools (XGBoost/Bayesian optimization and self-validated ensemble (SVEM)) were used to optimize the process and predict lipid mix ratio. The investigation included material attributes, their respective ratios, and process attributes. The critical responses like particle size (PS), polydispersity index (PDI), Zeta potential, pKa, heat trend cycle, encapsulation efficiency (EE), recovery ratio, and encapsulated mRNA were evaluated. Overall prediction of SVEM (> 97%) was comparably better than that of XGBoost/Bayesian optimization (> 94%). Moreover, in actual experimental outcomes, SVEM prediction is close to the actual data as confirmed by the experimental PS (94∼96 nm) is close to the predicted one (95∼97 nm). The other parameters including PDI and EE were also close to the actual experimental data.

Liver-on-chip model and application in predictive genotoxicity and mutagenicity of drugs

Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.

Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Microfluidic production of silver nanoparticles demonstrates ability for on demand synthesis of a wide size distribution of particles

Silver nanoparticles (AgNP) can help prevent infection of virus and bacteria. The size and morphology of AgNP can be crucial to function, with smaller nanoparticles (< 20 nm) able to penetrate the cell wall. This is significant as oxidative stress and genotoxicity are associated with some sizes and coatings of AgNP, contraindicating the use of AgNP to reduce infection. We present evidence that a microfluidic chip can synthesize larger sizes and distributions of AgNP from the nano-to-micro size range. We show results from a microfluidic mixing chip that can produce a wide range of nano-to-micro size (~ 24–400 nm) AgNP. Synthesis is based on a modified Turkevich method, using a single-step AgNP synthesis on the microfluidic chip using two chemical components, trisodium citrate (NaCit) and AgNO3. To make AgNP more accessible, we describe the microfluidic chip and conditions capable of synthesis. We also describe how modification of flow rate and chemical reagent concentration change particle diameter during production. In our experiments, we found that AgNP production created a visible adsorption line in the microfluidic device, possibly owing to AgNP surface interaction at the polydimethylsiloxane (PDMS) interface. We characterize these particles with dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Based on optical light microscopy, we hypothesize that AgNP formation primarily occurs at the interface between the two chemical reagent streams. We also conclude that AgNP size increases could be due to interaction with the PDMS surface, which is known to be porous. Future work will help to understand how surface interaction may influence the formation of larger particles.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

Electron microscopy and X-ray techniques correlative in situ studies in microfluidic conditions on hybrid perovskites

Electron microscopy and X-ray techniques correlative in situ studies in microfluidic conditions on hybrid perovskites

Rocking- and diffusion-based culture of tumor spheroids-on-a-chip.

Tumor spheroids are now intensively investigated toward preclinical and clinical applications, necessitating the establishment of accessible and cost-effective methods for routine operations. Without losing the advantage of organ-chip technologies, we developed a rocking system for facile formation and culture of tumor spheroids in hydrogel microwells of a suspended membrane under microfluidic conditions. While the rocking is controlled with a step motor, the microfluidic device is made of two plastic plates, allowing plugging directly syringe tubes with Luer connectors. Upon injection of the culture medium into the tubes and subsequent rocking of the chip, the medium flows back and forth in the channel underneath the membrane, ensuring a diffusion-based culture. Our results showed that such a rocking- and diffusion-based culture method significantly improved the quality of the tumor spheroids when compared to the static culture, particularly in terms of growth rate, roundness, junction formation and compactness of the spheroids. Notably, dynamically cultured tumor spheroids showed increased drug resistance, suggesting alternative assay conditions. Overall, the present method is pumpless, connectionless, and user-friendly, thereby facilitating the advancement of tumor-spheroid-based applications.

Shrimp oil nanoemulsions prepared by microfluidization and ultrasonication: characteristics and stability.

Shrimp oil (SO) nanoemulsions stabilized by fish myofibrillar protein, considered as functional foods, were prepared via microfluidization and ultrasonication. The study explored varying microfluidization (pressure and cycles) and ultrasonication (amplitude and sonication time) conditions that influenced emulsion properties and stability. Ultrasonicated emulsions exhibited superior emulsifying properties, adsorbed protein content, thermal stability, and centrifugal stability than microfluidized emulsions (p < 0.05). Microfluidization at 6.89 and 13.79 MPa with 2 or 4 cycles yielded larger droplets (536 to 638 nm) (p < 0.05), while ultrasonication at 40% and 50% amplitude for 5, 10 and 15 min produced smaller droplets (426 to 494 nm) (p < 0.05). Optimal conditions were obtained for microfluidization (13.79 MPa, 2 cycles) and ultrasonication (50% amplitude, 10 min). Ultrasonicated emulsions had generally smaller d32 and d43, lower polydispersity and higher ζ-potential than their microfluidized counterparts. Microstructural analysis and CLSM images confirmed their superior stability during storage. SO nanoemulsions could be applied as functional food.

"On-The-Fly" Synthesis of Self-Supported LDH Hollow Structures Through Controlled Microfluidic Reaction-Diffusion Conditions.

Layered double hydroxides (LDHs) are a class of functional materials that exhibit exceptional properties for diverse applications in areas such as heterogeneous catalysis, energy storage and conversion, and bio-medical applications, among others. Efforts have been devoted to produce millimeter-scale LDH structures for direct integration into functional devices. However, the controlled synthesis of self-supported continuous LDH materials with hierarchical structuring up to the millimeter scale through a straightforward one-pot reaction method remains unaddressed. Herein, it is shown that millimeter-scale self-supported LDH structures can be produced by means of a continuous flow microfluidic device in a rapid and reproducible one-pot process. Additionally, the microfluidic approach not only allows for an "on-the-fly" formation of unprecedented LDH composite structures, but also for the seamless integration of millimeter-scale LDH structures into functional devices. This method holds the potential to unlock the integrability of these materials, maintaining their performance and functionality, while diverging from conventional techniques like pelletization and densification that often compromise these aspects. This strategy will enable exciting advancements in LDH performance and functionality.

Green chemistry approach for stereoselective aldol condensation catalyzed by amino acids under microflow conditions

Over the recent years, the pharmaceutical field has been under significant pressure to adapt to the principles of green chemistry. The industry is facing the challenge of environmental protection and securing people's health while sustaining its fundamental goals of efficiency and selectivity optimization. In this work, the reaction processes optimization has been acheived through the microfluidic approach to the widely used aldol condensation reaction. In accordance with the green chemistry principles, we have found that the best conditions include aqueous ethanol solvent and easily available amino acids used as catalysts. Asymmetric aldol condensation of para-nitrobenzaldehyde with cyclohexanone catalyzed by L-tert-Leu under microfluidic conditions provided the product with high yield and diastereoselectivity (anti/syn = 28:1) and 98% enantiomeric excess. The microfluidic reactor process, compared to the classical batch method (in a flask), provides comparable product yield, while the reaction time was reduced by an order of magnitude. It should be noted that this approach allows to carry out the reactions at low (below zero) temperatures with greater enantioselectivity.

PODO/TERT256 – A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 μM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 μM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.