Empirical and Computational Evaluation of Hemolysis in a Microfluidic Extracorporeal Membrane Oxygenator Prototype.

Microfluidic devices promise to overcome the limitations of conventional hemodialysis and oxygenation technologies by incorporating novel membranes with ultra-high 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 been performed to empirically and computationally study hemolysis in medical devices, such as valves and blood pumps, little is known about blood damage in microfluidic devices. In this study, four variants of a representative microfluidic membrane-based oxygenator and two controls (positive and negative) are introduced, and computational models are used to predict hemolysis. The simulations were performed in ANSYS Fluent for nine shear stress-based parameter sets for the power law hemolysis model. We found that three of the nine tested parameters overpredict (5 to 10×) hemolysis compared to empirical experiments. However, three parameter sets demonstrated higher predictive accuracy for hemolysis values in devices characterized by low shear conditions, while another three parameter sets exhibited better performance for devices operating under higher shear conditions. Empirical testing of the devices in a recirculating loop revealed levels of hemolysis significantly lower (<2 ppm) than the hemolysis ranges observed in conventional oxygenators (>10 ppm). Evaluating the model's ability to predict hemolysis across diverse shearing conditions, both through empirical experiments and computational validation, will provide valuable insights for future micro ECMO device development by directly relating geometric and shear stress with hemolysis levels. We propose that, with an informed selection of hemolysis parameters based on the shear ranges of the test device, computational modeling can complement empirical testing in the development of novel high-flow blood-contacting microfluidic devices, allowing for a more efficient iterative design process. Furthermore, the low device-induced hemolysis measured in our study at physiologically relevant flow rates is promising for the future development of microfluidic oxygenators and dialyzers.

Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production.

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC-to-iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.

Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier.

The microSiM (µSiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the µSiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between 'blood' and 'brain' chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the µSiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

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 antibiotic's mechanism of action and its effect on EV release, but these results underline the importance of antibiotic choice when treating sepsis patients.

Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Optimization of Parylene C and Parylene N thin films for use in cellular co-culture and tissue barrier models.

Parylene has been used widely used as a coating on medical devices. It has also been used to fabricate thin films and porous membranes upon which to grow cells. Porous membranes are integral components of in vitro tissue barrier and co-culture models, and their interaction with cells and tissues affects the performance and physiological relevance of these model systems. Parylene C and Parylene N are two biocompatible Parylene variants with potential for use in these models, but their effect on cellular behavior is not as well understood as more commonly used cell culture substrates, such as tissue culture treated polystyrene and glass. 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 physical and chemical changes in material surface properties. Our results support and build on previous findings of positive effects of plasma treatment on Parylene biocompatibility while showing a more pronounced improvement for Parylene C compared to Parylene N. We measured relatively minor changes in surface roughness following plasma treatments, but significant changes in oxygen concentration at the surface persisted for 7 days and was likely the dominant factor in improving cellular behavior. Overall, this study offers facile and relatively low-cost plasma treatment protocols that provide persistent improvements in cell-substrate interactions on Parylene that match and exceed tissue culture polystyrene.

Exosomes, microvesicles, and other extracellular vesicles-a Keystone Symposia report.

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs.

Outer membrane vesicles as molecular biomarkers for Gram-negative sepsis: Taking advantage of nature's perfect packages.

Sepsis is an often life-threatening response to infection, occurring when host proinflammatory immune responses become abnormally elevated and dysregulated. To diagnose sepsis, the patient must have a confirmed or predicted infection, as well as other symptoms associated with the pathophysiology of sepsis. However, a recent study found that a specific causal organism could not be determined in the majority (70.1%) of sepsis cases, likely due to aggressive antibiotics or localized infections. The timing of a patient's sepsis diagnosis is often predictive of their clinical outcome, underlining the need for a more definitive molecular diagnostic test. Here, we outline the advantages and challenges to using bacterial outer membrane vesicles (OMVs), nanoscale spherical buds derived from the outer membrane of Gram-negative bacteria, as a diagnostic biomarker for Gram-negative sepsis. Advantages include OMV abundance, their robustness in the presence of antibiotics, and their unique features derived from their parent cell that could allow for differentiation between bacterial species. Challenges include the rigorous purification methods required to isolate OMVs from complex biofluids and the additional need to separate OMVs from similarly sized extracellular vesicles, which can share physical properties with OMVs.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological 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 experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

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

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix 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 (<50µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130µm). 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 platform's 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.

Engineering cell-substrate interactions on porous membranes for microphysiological systems.

Microphysiological systems are now widely used to recapitulate physiological and pathological microenvironments in order to study and understand a variety of cellular processes as well as drug delivery and stem cell differentiation. Central to many of these systems are porous membranes that enable tissue barrier formation as well as compartmentalization while still facilitating small molecule diffusion, cellular transmigration and cell-cell communication. The role or impact of porous membranes on the cells cultured upon them has not been widely studied or reviewed. Although many chemical and physical substrate characteristics have been shown to be effective in controlling and directing cellular behavior, the influence of pore characteristics and the ability to engineer porous membranes to influence these responses is not fully understood. In this mini-review, we show that many studies point to a multiphasic cell-substrate response, where increasing pore sizes and pore-pore spacing generally leads to improved cell-substrate interactions. However, the smallest pores in the nano-scale sometimes promote the strongest cell-substrate interactions, while the very largest micron-scale pores hinder cell-substrate interactions. This synopsis provides an insight into the importance of membrane pores in controlling cellular responses, and may help with the design and utilization of porous membranes for induction of desired cell processes in the development of biomimetic platforms.

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes.

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. 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.

Microengineered 3D Collagen Gels with Independently Tunable Fiber Anisotropy and Directionality.

Cellular processes, including differentiation, proliferation, and migration, have been linked to the alignment (anisotropy) and orientation (directionality) of collagen fibers in the native extracellular matrix (ECM). Given the critical role that biophysical cell-matrix interactions play in regulating biological functions, several microfluidic-based methods have been used to establish 3D collagen gels with defined fiber properties; these gels have helped to establish quantitative relationships between structural ECM cues and observed cell responses. Although existing microfluidic fabrication methods provide excellent definition over collagen fiber anisotropy, they have not demonstrated the independent control over fiber anisotropy and directionality necessary to replicate in vivo collagen architecture. Therefore, to advance collagen microengineering capabilities, we present a user-friendly technology platform that uses controlled fluid flows within a non-uniform microfluidic channel network to create collagen landscapes that can be tuned as a function of extensional strain rate. Herein, we demonstrate capabilities to i) control the degree of fiber anisotropy, ii) create spatial gradients in fiber anisotropy, iii) independently define fiber directionality, and iv) generate multi-material interfaces within a 3D environment. We then address the practical issue of integrating cells into microfluidic systems by using a peel-off template technique to provide direct access to microengineered collagen gels, and demonstrate that cells respond to the defined properties of the landscape. Finally, the platform's modular capability is highlighted by integrating a sub-micrometer thick porous parylene membrane onto the microengineered collagen as a method to define cell-substrate interactions.

Engineering fiber anisotropy within natural collagen hydrogels.

It is well known that biophysical properties of the extracellular matrix (ECM), including stiffness, porosity, composition, and fiber alignment (anisotropy), play a crucial role in controlling cell behavior in vivo. Type I collagen (collagen I) is a ubiquitous structural component in the ECM and has become a popular hydrogel material that can be tuned to replicate the mechanical properties found in vivo. In this review article, we describe popular methods to create 2-D and 3-D collagen I hydrogels with anisotropic fiber architectures. We focus on methods that can be readily translated from engineering and materials science laboratories to the life-science community with the overall goal of helping to increase the physiological relevance of cell culture assays.

Trilayer Interlinked Graphene Oxide Membrane for Wearable Hemodialyzer.

2D nanomaterials have long been considered for development of high permeability membranes. However, current processes have yet to yield a viable membrane for practical use due to the lack of scalability and substantial performance improvements over existing membranes. Herein, an ultrathin graphene oxide (GO) membrane with a permeability of 1562 mL h-1 mmHg-1 m-2, two orders of magnitude higher than the existing nanofiltration membranes, and a tight molecular weight cut-off is presented. To build such a membrane, a new process involving self-assembly and optimization of GO nanoplatelet physicochemical properties is developed. The process produces a highly organized mosaic of nanoplatelets enabling ultra-high permeability and selectivity. An adjustable molecular interlinker between the layers enables absolute nanometer-scale size cut-offs. These characteristics promise significant improvements to many nanoparticle and biological separation applications. In this work, the performance of the membrane in blood dialysis scenarios is evaluated. Urea and cytochrome-c sieving coefficients of 0.5 and 0.4 are achieved while retaining 99% of albumin. Hemolysis, complement activation, and coagulation studies exhibit a performance on par or superior to the existing dialysis membrane materials.

Endothelial cell apicobasal polarity coordinates distinct responses to luminally versus abluminally delivered TNF-α in a microvascular mimetic.

Endothelial cells (ECs) are an active component of the immune system and interact directly with inflammatory cytokines. While ECs are known to be polarized cells, the potential role of apicobasal polarity in response to inflammatory mediators has been scarcely studied. Acute inflammation is vital in maintaining healthy tissue in response to infection; however, chronic inflammation can lead to the production of systemic inflammatory cytokines and deregulated leukocyte trafficking, even in the absence of a local infection. Elevated levels of cytokines in circulation underlie the pathogenesis of sepsis, the leading cause of intensive care death. Because ECs constitute a key barrier between circulation (luminal interface) and tissue (abluminal interface), we hypothesize that ECs respond differentially to inflammatory challenge originating in the tissue versus circulation as in local and systemic inflammation, respectively. To begin this investigation, we stimulated ECs abluminally and luminally with the inflammatory cytokine tumor necrosis factor alpha (TNF-α) to mimic a key feature of local and systemic inflammation, respectively, in a microvascular mimetic (µSiM-MVM). Polarized IL-8 secretion and polymorphonuclear neutrophil (PMN) transmigration were quantified to characterize the EC response to luminal versus abluminal TNF-α. We observed that ECs uniformly secrete IL-8 in response to abluminal TNF-α and is followed by PMN transmigration. The response to abluminal treatment was coupled with the formation of ICAM-1-rich membrane ruffles on the apical surface of ECs. In contrast, luminally stimulated ECs secreted five times more IL-8 into the luminal compartment than the abluminal compartment and sequestered PMNs on the apical EC surface. Our results identify clear differences in the response of ECs to TNF-α originating from the abluminal versus luminal side of a monolayer for the first time and may provide novel insight into future inflammatory disease intervention strategies.

Free Standing, Large-Area Silicon Nitride Membranes for High Toxin Clearance in Blood Surrogate for Small-Format Hemodialysis.

Developing highly-efficient membranes for toxin clearance in small-format hemodialysis presents a fabrication challenge. The miniaturization of fluidics and controls has been the focus of current work on hemodialysis (HD) devices. This approach has not addressed the membrane efficiency needed for toxin clearance in small-format hemodialysis devices. Dr. Willem Kolff built the first dialyzer in 1943 and many changes have been made to HD technology since then. However, conventional HD still uses large instruments with bulky dialysis cartridges made of ~2 m2 of 10 micron thick, tortuous-path membrane material. Portable, wearable, and implantable HD systems may improve clinical outcomes for patients with end-stage renal disease by increasing the frequency of dialysis. The ability of ultrathin silicon-based sheet membranes to clear toxins is tested along with an analytical model predicting long-term multi-pass experiments from single-pass clearance experiments. Advanced fabrication methods are introduced that produce a new type of nanoporous silicon nitride sheet membrane that features the pore sizes needed for middle-weight toxin removal. Benchtop clearance results with sheet membranes (~3 cm2) match a theoretical model and indicate that sheet membranes can reduce (by orders of magnitude) the amount of membrane material required for hemodialysis. This provides the performance needed for small-format hemodialysis.

Micropatterned Poly(ethylene glycol) Islands Disrupt Endothelial Cell-Substrate Interactions Differently from Microporous Membranes.

Porous membranes are ubiquitous in cell co-culture and tissue-on-a-chip studies. These materials are predominantly chosen for their semi-permeable and size exclusion properties to restrict or permit transmigration and cell-cell communication. However, previous studies have shown pore size, spacing and orientation affect cell behavior including extracellular matrix production and migration. The mechanism behind this behavior is not fully understood. In this study, we fabricated micropatterned non-fouling polyethylene glycol (PEG) islands to mimic pore openings in order to decouple the effect of surface discontinuity from potential grip on the vertical contact area provided by pore wall edges. Similar to previous findings on porous membranes, we found that the PEG islands hindered fibronectin fibrillogenesis with cells on patterned substrates producing shorter fibrils. Additionally, cell migration speed over micropatterned PEG islands was greater than unpatterned controls, suggesting that disruption of cell-substrate interactions by PEG islands promoted a more dynamic and migratory behavior, similarly to enhanced cell migration on microporous membranes. Preferred cellular directionality during migration was nearly indistinguishable between substrates with identically patterned PEG islands and previously reported behavior over micropores of the same geometry, further confirming disruption of cell-substrate interactions as a common mechanism behind the cellular responses on these substrates. Interestingly, compared to respective controls, there were differences in cell spreading and a lower increase in migration speed over PEG islands compared prior results on micropores with identical feature size and spacing. This suggests that membrane pores not only disrupt cell-substrate interactions, but also provide additional physical factors that affect cellular response.

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis.

Extracellular vesicles (EVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.