Biology subjects

Gaborski, T. R.

Publications and source records attributed to Gaborski, T. R..

17 recordsLinked to original sources

bioRxiv2026

Bacterial extracellular vesicles indirectly destabilize a human stem cell-derived blood-brain barrier on-chip through pro-inflammatory stimulation of immune cells

Pathogenic bacterial extracellular vesicles (BEVs) can disrupt the blood-brain barrier (BBB), leading to neuroinflammation. Prior in vitro studies of this process were performed in simple models that may have lacked important physiological factors. We sought to determine if treatment with Escherichia coli-derived BEVs could directly compromise the integrity of a BBB lab-on-chip model or if an immune component was required. Our device featured isogenic human induced pluripotent stem cell-derived brain microvascular endothelial-like cells (BMECs) and pericytes separated by an ultrathin, porous silicon nitride membrane. BEVs and free lipopolysaccharide (LPS) were capable of causing upregulation of intercellular adhesion molecule-1 on the BMEC surfaces, which is important for immune cell recruitment. However, neither BEVs nor LPS at physiological doses caused pronounced loss of BMEC tight junction proteins, nor did they increase barrier permeability to small dye molecules. In contrast, stimulating THP-1 macrophages with BEVs led to increased production of pro-inflammatory cytokines, and conditioned media from the stimulated macrophages disrupted BMEC tight junctions and increased barrier permeability. Our work demonstrates the importance of incorporating an immune component in studies of BEV-mediated disruption of BBB models.

cell biology↗

bioRxiv2025

Pericytes Repair Engineered Defects in the Basement Membrane to Restore Barrier Integrity in an in vitro Model of the Blood-Brain Barrier.

Pericytes play a key role in the brain where they support the blood-brain barrier (BBB). Their loss has been reported in response to systemic inflammation and neurodegenerative disease. We recently demonstrated that iPSC-derived brain pericyte-like cells (BPLCs) and brain microvascular endothelial cell (BMEC)-like cells collaboratively form a nascent, 3D basement membrane when cultured across a nanoporous membrane1. Building on this, we aimed to engineer defects in the basement membrane to investigate whether pericytes could facilitate its repair. In BMEC monocultures, we observed that micropore patterns in nanomembranes created discontinuities in laminin, which destabilized barrier function. Remarkably, the addition of pericytes to the basal side of the membrane restored both laminin integrity and barrier function. Our results align with the role of pericytes as support cells for microvasculature and encourage the use of our tissue barrier platform (the {micro}SiM) to model neurological disorders involving pericyte dysfunction and/or disruption of basement membrane.

bioengineering↗

bioRxiv2025

Targeting Fibrotic Scars with Extracellular Vesicles Extracted from Mature Aloe vera: Enhanced Antioxidant, Anti-inflammatory, and Antifibrotic Activity through M2 Macrophage Polarization and Myofibroblasts Inhibition

BackgroundAbnormal scarring and fibrotic skin disorders arise from dysregulated wound healing processes. While Aloe vera is widely recognized for its therapeutic properties, the potential of its extracellular vesicles (Av-EVs) remains underexplored. ObjectiveThis study aimed to isolate and characterize Av-EVs and evaluate their antioxidant, anti-inflammatory, and antifibrotic properties in vitro, focusing on the impact of extraction method and plant maturity. MethodsAv-EVs were isolated from mature and young Aloe vera leaves using manual (NB) or blender-based (B) homogenization. Vesicles were characterized by nanoparticle tracking analysis, transmission electron microscopy, and protein quantification. Antioxidant and cytotoxicity assays (DPPH, alamarBlue) were followed by functional anti-inflammatory and antifibrotic analyses respectively in LPS-stimulated THP-1 macrophages and TGF-{beta}1/Vitamin C-activated human dermal fibroblasts (RT-qPCR, immunofluorescence, proteomics). ResultsNB-derived EVs from mature leaves exhibited the most potent activity across all assays, showing superior antioxidant capacity, greater suppression of pro-inflammatory cytokines, enhanced M2 macrophage polarization, and significant downregulation of COL1A1 and -SMA. In contrast, B-derived and young leaf-derived EVs showed reduced bioactivity, with young EVs failing to inhibit fibrotic markers. ConclusionManually extracted Av-EVs from mature leaves demonstrate superior multifunctional bioactivity, highlighting their potential as plant-derived nanotherapeutics for fibrotic scar modulation.

cell biology↗

bioRxiv2025

Development of a PEGylated Parylene Nanopocket Membrane for the Capture and Release of Lipid Vesicles

Membranes are commonly used for the separation and purification of a variety of biological species. In this study, we developed a nanopocket membrane that can capture nanoparticles and extracellular vesicles in tangential flow filtration by pulling the species of interest into a nanopocket, while tangential flow washes away particles too large to be captured. We developed the pores using a four-step lithography process. By optimizing photolithography parameters, particularly exposure dose, we achieved well-defined pore shapes with the desired wall tilt, forming nanopocket geometries. We validated membrane performance using bead mixtures of varying diameters (50 nm -10 {micro}m), demonstrating its ability to pass small particles, capture mid-sized particles, and exclude larger ones. Surface modification with poly(l-lysine)- grafted poly(ethylene glycol) enhanced this performance by reducing pore size and improving particle release. Additionally, we demonstrated selective capture and release of liposomes as well as EVs in the presence of fluorescently labeled BSA (f-BSA) protein. During this process, EVs were retained and later released, while f-BSA passed through, confirming selective capture and reduced protein contamination. These PEGylated nanopocket membranes constitute a low-pressure platform for gentle, size-selective isolation of nanoscale bioparticles, laying the groundwork for improved extracellular-vesicle analytics and point-of-care diagnostic applications.

bioengineering↗

bioRxiv2025

Antibiotic treatment modulates Escherichia coli-derived bacterial extracellular vesicle (BEV) production and their capacity to upregulate ICAM-1 in human endothelial cells

Antibiotic treatment is often necessary to eliminate life-threatening bacterial infections. However, these treatments can alter production of bacterial extracellular vesicles (BEVs), which often contain pro-inflammatory biomolecules. In this study, we examined how the clinically-relevant antibiotics meropenem, tobramycin, and ciprofloxacin impacted BEV production from a urinary tract infection-associated Escherichia coli strain (CFT073 [WAM2267]) and a meningitis-associated strain (K1 RS218). BEVs from both strains caused a dose-dependent increase in expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells, priming the endothelium for interactions with immune cells. Blockade of toll-like receptor 4 revealed that this receptor was responsible for BEV-endothelial interactions. Treatment with meropenem, a {beta}-lactam antibiotic, increased production of BEVs from strain K1 RS218. Furthermore, meropenem treatment caused strain CFT073 [WAM2267] to produce BEVs with heightened stimulatory capacity, possibly by amplifying the content of lipoprotein Lpp in these BEVs as measured by mass spectrometry. To our knowledge, this is the first study examining the interplay between antibiotic treatment and the effects of the resulting BEVs on endothelial ICAM-1 expression. Our results indicate treatment risks of certain antibiotics against specific strains of E. coli and could help identify therapeutic targets to reduce BEV-mediated endothelial stimulation during infection.

cell biology↗

bioRxiv2023

The effect of clinically relevant beta-lactam, aminoglycoside, and quinolone antibiotics on bacterial extracellular vesicle release from E. coli

Sepsis, a leading cause of death in hospitals, can be defined as a dysregulated host inflammatory response to infection, which can lead to tissue damage, organ failure, and cardiovascular complications. Although there is no cure for sepsis, the condition is typically managed with broad spectrum antibiotics to eliminate any potential bacterial source of infection. However, a potential side-effect of antibiotic treatment is the enhanced release of bacterial extracellular vesicles (BEVs). BEVs are membrane-bound nanoparticles produced by a variety of mechanisms, one of which includes the pinching-off of the outer membrane (in Gram-negative bacteria) to enclose proteins and other biological molecules for transport and intercellular communication. Some of the Gram-negative EV cargo, including Peptidoglycan associated lipoprotein (Pal) and Outer membrane protein A (OmpA), have been shown to induce both acute and chronic inflammation in host tissue. We hypothesize that antibiotic concentration and its mechanism of action can have an effect on the amount of released BEVs, which could potentially exacerbate the host inflammatory response. In this study, we evaluated nine clinically relevant antibiotics for their effect on EV release from Escherichia coli. EVs were characterized using immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. Several beta-lactam antibiotics caused significantly more EV release, while quinolone and aminoglycosides caused relatively less vesiculation. Further study is warranted to corroborate the correlation between an antibiotics mechanism of action and its effect on EV release, but these results underline the importance of antibiotic choice when treating sepsis patients.

microbiology↗

bioRxiv2022

Effects of Oxygen Plasma Treatment on Parylene C and Parylene N Membrane Biocompatibility for Tissue Barrier Models

Porous membranes are integral components of in vitro tissue barrier and co-culture models and their interaction with cells and tissues directly affects the performance and credibility of these models. Plasma-treated Parylene C and Parylene N are two biocompatible Parylene variants with perceived potential for use in these models but their biocompatibility and biological interactions at their interface with cells are not well understood. Here, we use a simple approach for benchtop oxygen plasma treatment and investigate the changes in cell spreading and extracellular matrix deposition as well as the changes in material surface properties. Our results support the previous findings on the persistent effects of plasma treatment on Parylene biocompatibility while showing a more pronounced improvement for Parylene C over Parylene N. It is observed that although both increased surface roughness and persistent increases in oxygen species govern the plasma-driven improvement, the changes in oxygen concentration at the interface are the dominant factor. Overall, the results of this study provide a clear picture of potential mechanisms of plasma-induced changes in synthetic polymers which have implications for their use in in vitro model systems and other BioMEMS applications.

bioengineering↗

bioRxiv2022

The Modular μSiM Reconfigured: Integration of Microfluidic Capabilities to Study in vitro Barrier Tissue Models under Flow

Microfluidic approaches to study tissue barriers have emerged to address the lack of fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional, tried-and-true experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the advantages of both conventional open-well and microfluidic systems. Here, we develop a plug-and-play flow module to add on-demand microfluidic capabilities to a modular microfluidic system featuring a silicon membrane "m-SiM" as an open-well device with live-cell imaging capabilities. The magnetic latching assembly of our design enables bi-directional reconfiguration between open-well and fluidic modes. This design feature allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. Our work also provides an experimentally-validated flow model to help select desired flow conditions based on the experimental needs. As a proof-of-concept, we demonstrate flow-induced alignment of endothelial cells and visualize different phases of neutrophil transmigration across an endothelial monolayer under flow. We anticipate that our reconfigurable design will be adopted by both engineering and bioscience laboratories due to the compatibility with standard open-well protocols and the simple flow addition capabilities.

bioengineering↗

bioRxiv2022

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix (ECM) are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50m in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130m). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platforms capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

bioengineering↗

bioRxiv2021

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization through Diminished Cell Spreading and Enhance Adipogenesis

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular co-culture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and sub-micron scale pores affect YAP localization and differentiation of adipose-derived stem cells (ADSCs). We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis, while decreasing osteogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/429012v3_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@5fa0daorg.highwire.dtl.DTLVardef@3981d9org.highwire.dtl.DTLVardef@17bd8f5org.highwire.dtl.DTLVardef@1e0c9e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Numerical Model to Predict Hemolysis and Transport in a Membrane-Based Microfluidic Device

Microfluidic devices promise to overcome the limitations of conventional hemodialysis and oxygenation technologies by incorporating novel membranes with ultrahigh permeability into portable devices with low blood volume. However, the characteristically small dimensions of these devices contribute to both non-physiologic shear that could damage blood components and laminar flow that inhibits transport. While many studies have applied empirical and computational models of hemolysis to medical devices, such as valves and blood pumps, little is known about blood damage in the microfluidic flow regime. In this study, four design variants of a microfluidic membrane-based oxygenator and two controls (positive and negative) are introduced and modeled using a Computational Fluid Dynamics (CFD) model to predict hemolysis. The simulations were performed in ANSYS Fluent for nine shear stress-based Power Law hemolysis model variants. Empirical testing of the devices in a recirculating loop revealed levels of hemolysis significantly lower (3 ppm hemolysis for pump, tube, and device combined) than the hemolysis ranges (>10 ppm) observed in conventional oxygenators. We found that most of the nine tested hemolysis models overpredict (5x to 10x) hemolysis compared to empirical experiments. However, two models demonstrated higher predictive accuracy for hemolysis values in devices characterized by low shear conditions, while another set of three models exhibited better performance for devices operating under higher shear conditions. Our study highlights the limitations of combining hemolysis models with computational fluid dynamics models for a priori in silico device-induced hemolysis. Nevertheless, with a judicious selection of hemolysis models based on the shear ranges of the test device, we propose that computational modeling can complement empirical testing in the development of novel micro-dialyzers or oxygenators, allowing for a more efficient iterative design process. Furthermore, the low device-induced hemolysis (< 2 ppm) measured in our study at physiologically relevant flow rates is promising for the future development of microfluidic dialyzers and oxygenators.

bioengineering↗

bioRxiv2020

Microengineered three-dimensional collagen landscapes with independently tunable fiber anisotropy and directionality

Fibrillar collagens are structural proteins in the extracellular matrix (ECM), and cellular processes, including differentiation, proliferation, and migration, have been linked to the orientation (directionality) and alignment (anisotropy) of collagen fibers. Given the importance of cell-substrate interactions in driving biological functions, several microfluidic approaches have demonstrated three-dimensional (3D) collagen gels with defined fiber properties that enable quantitative correlations between structural cues and observed cell responses. Although existing methods provide excellent definition over collagen fiber anisotropy, independent control over both anisotropy and directionality (that we collectively refer to as the collagen landscape) has not been demonstrated. Therefore, to advance collagen microengineering capabilities, we present a user-friendly approach that uses controlled fluid flows within a non-uniform microfluidic channel network to create well-defined collagen landscapes. We demonstrate capabilities including i) control over fiber anisotropy, ii) spatial gradients in fiber anisotropy, iii) defined fiber directionality, and iv) multi-material interfaces. We then show that cells respond to the microengineered topographic cues by aligning along the anisotropy domains and following fiber directionality. Finally, this platforms modular capability is demonstrated by integrating an ultrathin porous parylene (UPP) membrane on the microengineered collagen as a mask to control cell-substrate interactions.

bioengineering↗

bioRxiv2020

Bilayer graphene oxide membranes for a wearable hemodialyzer

2D nanomaterials have long been considered for development of ultra-high throughput membranes, due to their atomically thin nature and high mechanical strength. However, current processes have yet to yield a viable membrane for practical applications due to the lack of scalability and substantially improved performance over existing membranes. Herein, a graphene oxide (GO) bilayer membrane with a permeability of 1562 mL/hr.mmHg.m2, two orders of magnitude higher than existing nanofiltration membranes, and a tight molecular weight cut-off (MWCO) is presented. To build such a membrane, we have developed a new process involving self-assembly and optimization of GO nanoplatelets physicochemical properties. The process produced a highly organized mosaic of nanoplatelets enabling ultra-high permeability and selectivity with only three layers of GO. Performance of the membrane has been evaluated in a simulated hemodialysis application, where it presents a great value proposition. The membrane has a precise molecular cut-off size of 5 nm, adjusted using a molecular interlinker, designed to prevent loss of critical blood proteins. Urea, cytochrome-c, and albumin are used as representative test molecules. Urea and cytochrome-c sieving coefficients of 0.5 and 0.4 were achieved under physiological pressure conditions, while retaining 99% of albumin. Hemolysis, complement activation, and coagulation studies exhibit a performance on par or superior to the existing hemodialyzer materials.

bioengineering↗

bioRxiv2020

Robust and Gradient Thickness Porous Membranes for In Vitro Modeling of Physiological Barriers

Porous membranes are fundamental elements for tissue-chip barrier and co-culture models. However, the exaggerated thickness of commonly available membranes impedes an accurate in vitro reproduction of the biological multi-cellular continuum as it occurs in vivo. Existing techniques to fabricate membranes such as solvent cast, spin-coating, sputtering and PE-CVD result in uniform thickness films. To understand critical separation distances for various barrier and co-culture models, a gradient thickness membrane is needed. Here, we developed a robust method to generate ultrathin porous parylene C (UPP) membranes not just with precise thicknesses down to 300 nm, but with variable gradients in thicknesses, while at the same time having porosities up to 25%. We also show surface etching and increased roughness lead to improved cell attachment. Next, we examined the mechanical properties of UPP membranes with varying porosity and thickness and fit our data to previously published models, which can help determine practical upper limits of porosity and lower limits of thickness. Lastly, we validate a straightforward approach allowing the successful integration of the UPP membranes into a prototyped 3D-printed scaffold enabling in vitro barrier modeling and investigation of cell-cell interplay over variable distances using thickness gradients.

bioengineering↗

bioRxiv2019

Nanopatterned Thermoresponsive Functionalization of Substrates via Nanosphere Lithography

1Self-assembled monolayers (SAMs) have been widely utilized as a way of tailoring surface chemistry through the adsorption of organic molecules to different materials. SAMs are easy to prepare and offer a wide variety of organic molecules that afford additional or improved properties to the coated material. Spatial control of SAM placement has been achieved over many length-scales, even at the nanoscale. However, nanopatterned SAMs are usually prepared through serial processes utilizing atomic scanning probes or soft-lithography utilizing elastomeric masters. These techniques are expensive or not repeatable. Here we present the use of nanospheres for the creation of nanopatterned Au:Cu films which spatially control the grafting of a thermoresponsive SAM made from poly(N-isopropyl acrylamide) (PNIPAM). Chemical characterization validates the presence of PNIPAM and environmental atomic force microscopy showed its response to temperature which was evidenced by a change in stiffness. Our approach represents an affordable large area methodology for repeatable spatial control of SAMs at the nanoscale.

bioengineering↗

bioRxiv2019

Use of Nanosphere Self-Assembly to Pattern Nanoporous Membranes for the Study of Extracellular Vesicles

Nanoscale biocomponents naturally released by cells, such as extracellular vesicles (EVs), have recently gained interest due to their therapeutic and diagnostic potential. Membrane based isolation and co-culture systems have been utilized in an effort to study EVs and their effects. Nevertheless, improved platforms for the study of small EVs are still needed. Suitable membranes, for isolation and co-culture systems, require pore sizes to reach into the nanoscale. These pore sizes cannot be achieved through traditional lithographic techniques and conventional thick nanoporous membranes commonly exhibit low permeability. Here we utilized nanospheres, similar in size and shape to the targeted small EVs, as patterning features for the fabrication of freestanding SiN membranes (120 nm thick) released in minutes through a sacrificial ZnO layer. We evaluated the feasibility of separating subpopulation of EVs based on size using these membranes. The membrane used here showed an effective size cut-off of 300 nm with the majority of the EVs [≤]200 nm. This work provides a convenient platform with great potential for studying subpopulations of EVs.

bioengineering↗

bioRxiv2019

Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Exosomes are membrane vesicles secreted by cells and distributed widely in all biofluids. Exosomes can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of exosomes is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for exosome labelling. One concern with the use of lipophilic dyes is an increase in the exosome size due to membrane fusion or intercalation. This size shift alone may undermine the validity of exosomes tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled exosomes was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled exosomes were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled exosomes was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for exosomes tracking.

biochemistry↗