Soluble Receptor for Advanced Glycation End Products (sRAGE) Is a Sensitive Biomarker in Human Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition with an unmet need for early diagnosis, better monitoring, and risk stratification. The receptor for advanced glycation end products (RAGE) is activated in response to hypoxia and vascular injury, and is associated with inflammation, cell proliferation and migration in PAH. For the adult cohort, we recruited 120 patients with PAH, 83 with idiopathic PAH (IPAH) and 37 with connective tissue disease-associated PAH (CTD-PAH), and 48 controls, and determined potential plasma biomarkers by enzyme-linked immunoassay. The established heart failure marker NTproBNP and IL-6 plasma levels were several-fold higher in both adult IPAH and CTD-PAH patients versus controls. Plasma soluble RAGE (sRAGE) was elevated in IPAH patients (3044 ± 215.2 pg/mL) and was even higher in CTD-PAH patients (3332 ± 321.6 pg/mL) versus controls (1766 ± 121.9 pg/mL; p < 0.01). All three markers were increased in WHO functional class II+III PAH versus controls (p < 0.001). Receiver-operating characteristic analysis revealed that sRAGE has diagnostic accuracy comparable to prognostic NTproBNP, and even outperforms NTproBNP in the distinction of PAH FC I from controls. Lung tissue RAGE expression was increased in IPAH versus controls (mRNA) and was located predominantly in the PA intima, media, and inflammatory cells in the perivascular space (immunohistochemistry). In the pediatric cohort, plasma sRAGE concentrations were higher than in adults, but were similar in PH (n = 10) and non-PH controls (n = 10). Taken together, in the largest adult sRAGE PAH study to date, we identify plasma sRAGE as a sensitive and accurate PAH biomarker with better performance than NTproBNP in the distinction of mild PAH from controls.

Rapid Prototyping of Multilayer Microphysiological Systems.

Microfluidic organs-on-chips aim to realize more biorelevant in vitro experiments compared to traditional two-dimensional (2D) static cell culture. Often such devices are fabricated via poly(dimethylsiloxane) (PDMS) soft lithography, which offers benefits (e.g., high feature resolution) along with drawbacks (e.g., prototyping time/costs). Here, we report benchtop fabrication of multilayer, PDMS-free, thermoplastic organs-on-chips via laser cut and assembly with double-sided adhesives that overcome some limitations of traditional PDMS lithography. Cut and assembled chips are economical to prototype ($2 per chip), can be fabricated in parallel within hours, and are Luer compatible. Biocompatibility was demonstrated with epithelial line Caco-2 cells and primary human small intestinal organoids. Comparable to control static Transwell cultures, Caco-2 and organoids cultured on chips formed confluent monolayers expressing tight junctions with low permeability. Caco-2 cells-on-chip differentiated ∼4 times faster, including increased mucus, compared to controls. To demonstrate the robustness of cut and assemble, we fabricated a dual membrane, trilayer chip integrating 2D and 3D compartments with accessible apical and basolateral flow chambers. As proof of concept, we cocultured a human, differentiated monolayer and intact 3D organoids within multilayered contacting compartments. The epithelium exhibited 3D tissue structure and organoids expanded close to the adjacent monolayer, retaining proliferative stem cells over 10 days. Taken together, cut and assemble offers the capability to rapidly and economically manufacture microfluidic devices, thereby presenting a compelling fabrication technique for developing organs-on-chips of various geometries to study multicellular tissues.

Cholinergic Activation of Primary Human Derived Intestinal Epithelium Does Not Ameliorate TNF-α Induced Injury.

INTRODUCTION: The intestinal epithelium contains specialized cells including enterocytes, goblet, Paneth, enteroendocrine, and stem cells. Impaired barrier integrity in Inflammatory Bowel Disease is characterized by elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Prior studies in immortalized lines such as Caco-2, without native epithelial heterogeneity, demonstrate the amelioration of TNF-α compromised barrier integrity via nicotinic (nAChR) or muscarinic (mAChR) acetylcholine receptor activation. METHODS: A tissue-engineered model of primary human small intestinal epithelium was derived from dissociated organoids cultured on collagen-coated Transwells. Differentiation was accomplished with serum-containing media and compared to Caco-2 and HT-29 regarding alkaline phosphatase expression, transepithelial electrical resistance (TEER), and IL-8 secretion. Inflammation was modeled via basal stimulation with TNF-α (25 ng/mL) with or without nicotine (nAChR agonist) or bethanechol (mAChR agonist). Apoptosis, density (cells/cm2), TEER, lucifer yellow permeability, 70 kDa dextran transport, cell morphology, and IL-8 secretion were characterized. RESULTS: Primary intestinal epithelium demonstrates significant functional differences compared to immortalized cells, including increased barrier integrity, IL-8 expression, mucus production, and the presence of absorptive and secretory cells. Exposure to TNF-α impaired barrier integrity, increased apoptosis, altered morphology, and increased secretion of IL-8. Stimulation of nAChR with nicotine did not ameliorate TNF-α induced permeability nor alter 70 kDa dextran transport. However, stimulation of mAChR with bethanechol decreased transport of 70 kDa dextran but did not ameliorate TNF-α induced paracellular permeability. CONCLUSIONS: A primary model of intestinal inflammation was evaluated, demonstrating nAChR or mAChR activation does not have the same protective effects compared to immortalized epithelium. Inclusion of other native stromal support cells are underway.

Effect of Monocyte Seeding Density on Dendritic Cell Generation in an Automated Perfusion-Based Culture System.

Dendritic cells (DCs) are increasingly important for research and clinical use but obtaining sufficient numbers of dendritic cells is a growing challenge. We systemically investigated the effect of monocyte (MO) seeding density on the generation of monocyte-derived immature DCs (iDCs) in MicroDEN, a perfusion-based culture system, as well as 6-well plates. Cell surface markers and the ability of the iDCs to induce proliferation of allogeneic T cells were examined. The data shows a strong relationship between iDC phenotype, specifically CD80/83/86 expression, and T cell proliferation. MicroDEN generated iDCs proved better than well plate generated iDCs at inducing T cell proliferation within the 200k-600k MO/cm2 seeding density range studied. We attribute this to perfusion in MicroDEN which supplies fresh differentiation medium continuously to the differentiating MOs while concurrently removing depleted medium and toxic byproducts of cellular respiration. MicroDEN generated fewer iDCs on a normalized basis than the well plates at lower MO seeding densities but generated equivalent numbers of iDCs at 600k MO seeding density. These results demonstrate that MicroDEN is capable of generating greater numbers of iDCs with less manual work than standard well plate culture and the MicroDEN generated iDCs have greater ability to induce T cell proliferation.

Automated generation of immature dendritic cells in a single-use system.

Dendritic cells (DCs) are an indispensable part of studying human responses that are important for protective immunity against cancer and infectious diseases as well as prevention of autoimmunity and transplant rejection. These cells are also key elements of personalized vaccines for cancer and infectious diseases. Despite the vital role of DCs in both clinical and basic research contexts, methods for obtaining these cells from individuals remains a comparatively under-developed and inefficient process. DCs are present in very low concentrations (<1%) in blood, thus they must be generated from monocytes and the current methodology in DC generation involves a laborious process of static culture and stimulation with cytokines contained in culture medium. Herein, we describe an automated fluidic system, MicroDEN, that allows for differentiation of monocytes into immature-DCs (iDCs) utilizing continuous perfusion of differentiation media. Manual steps associated with current ex vivo monocyte differentiation are vastly reduced and an aseptic environment is ensured by the use of an enclosed cartridge and tubing network. Benchmark phenotyping was performed on the generated iDCs along with allogeneic T-cell proliferation and syngeneic antigen-specific functional assays. MicroDEN generated iDCs were phenotypically and functionally similar to well plate generated iDCs, thereby demonstrating the feasibility of utilizing MicroDEN in the broad range of applications requiring DCs.

Electrically-Actuated Valves for Woven Fabric Lateral Flow Devices.

The integration of flow control elements into low-cost biosensors presents a significant engineering challenge. This Article describes the development and integration of active, chemical valves into lateral flow devices, using a scalable, single-step, weaving-based manufacturing approach. The valve was constructed from an electrically conductive polymer, polypyrrole. The polymer switches between wetting and nonwetting states when it is reduced and oxidized via the application of an electrochemical potential. In this work, yarns were first coated with polypyrrole and integrated into fabric lateral flow sensors. The coated yarns were stimulated in situ via integrated electrodes. Coated textiles were characterized for their response to variations in the applied electrical potential, the duration for which the potential is applied, and the chemical composition of the polymer. Among these tuning parameters, the concentration of iron (iii) chloride utilized to catalyze the synthesis of the polymer, was found to be a significant determinant in the wetting range of the polymer. Complete ON/OFF flow control was achieved at applied potentials of 20 V.cm-1, within 120 s of stimulation, using 0.1 M iron (iii) chloride, making the valve fairly easy to incorporate into point-of-care format. The practical utility of the valve was demonstrated by performing a Lowry protein assay in the device, wherein fluid flow was deactivated to allow individual reaction steps to go to completion prior to reactivation. Significant improvements in the sensitivity and linear range of the devices are reported in a simple straight-channel, lateral flow device, with the potential to develop more complex channel geometries via the weaving-based approach.

Enabling Microfluidics: from Clean Rooms to Makerspaces.

The traditional requirement for clean rooms and specialized skills has inhibited many biologists from pursuing new microfluidic innovations. Makerspaces provide a growing alternative to clean rooms: they provide low-cost access to fabrication equipment such as laser cutters, plotter cutters, and 3D printers; use commercially available materials; and attract a diverse community of product designers. This Opinion discusses the materials, tools, and building methodologies particularly suited for developing novel microfluidic devices in these spaces, with insight into biological applications and leveraging the maker community. The lower barrier to access of makerspaces ameliorates the otherwise poor accessibility and scalability of microfluidic prototyping.

Fluorescence-based lateral flow assays for rapid oral fluid roadside detection of cannabis use.

With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral-fluid testing for Δ9 -tetrahydrocannabinol (THC). This proof-of-concept assay uses a fluorescent-based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling-thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL.

One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices.

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.

Ultra-High-Throughput Sample Preparation System for Lymphocyte Immunophenotyping Point-of-Care Diagnostics.

Point-of-care (POC) microfluidic devices often lack the integration of common sample preparation steps, such as preconcentration, which can limit their utility in the field. In this technology brief, we describe a system that combines the necessary sample preparation methods to perform sample-to-result analysis of large-volume (20 mL) biopsy model samples with staining of captured cells. Our platform combines centrifugal-paper microfluidic filtration and an analysis system to process large, dilute biological samples. Utilizing commercialization-friendly manufacturing methods and materials, yielding a sample throughput of 20 mL/min, and allowing for on-chip staining and imaging bring together a practical, yet powerful approach to microfluidic diagnostics of large, dilute samples.

Galectin-3 and aldosterone as potential tandem biomarkers in pulmonary arterial hypertension.

BACKGROUND: Several studies have identified circulating biomarkers to be associated with the presence and severity of pulmonary arterial hypertension (PAH). Recent evidence supports a role for galectin-3 (Gal-3) and the mineralcorticoid aldosterone in left ventricular failure. However, studies on aldosterone together with Gal-3 in PAH are lacking. OBJECTIVE: We investigated a novel Aldosterone-galectin-3 (Gal-3) tandem and several other potential PAH biomarkers and their association with the disease severity. METHODS: A total of 57 patients, 41 with idiopathic PAH. (IPAH) and 16 with PAH associated with connective tissue disease (CTD), and 8 age-matched, non-relative controls were studied. Gal-3, aldosterone and other potential protein plasma concentrations were measured by single ELISA and multi-array MSD (Meso Scale Discovery) technology. RESULTS: Gal-3 values were increased in both patients with IPAH (12.2±0.6 ng/mL; p<0.05) and with PAH-CTD (14.1±1.6 ng/mL; p<0.05) versus control (8.5±0.9 ng/mL), while aldosterone was significantly elevated in IPAH only (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05). In addition, aldosterone, Gal-3, and N-terminal pro-brain natriuretic peptide (NT-proBNP) values were all higher in patients in WHO functional class II-III versus PAH functional class I or controls. The vascular injury marker intercellular adhesion molecule 1 (ICAM-1) was increased in IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively), whereas vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin-12 (IL-12) were elevated in PAH-CTD only (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively). CONCLUSIONS: Heightened Gal-3 and aldosterone plasma concentrations in PAH patients indicate a role for Gal-3 signalling in the pathobiology of IPAH and PAH-CTD, and may serve as biomarkers for functional status and progression of disease.

Accelerated adipogenic differentiation of hMSCs in a microfluidic shear stimulation platform.

The use of transplanted adipose tissue to repair crucial defects is clinically interesting for surgical reconstruction. Terminally differentiated adipocytes are utilized to promote the healthy regeneration of defective tissue. Use of differentiated mesenchymal stem cells, capable of differentiation into adipocytes, is advantageous because of their regenerative properties. Conventionally, the differentiation of hMSCs toward adipocytes occurs through chemical stimulation. We designed a microfluidic system, consisting of plastic tubing and a syringe pump, to create an environment of shear to accelerate this differentiation process. This system employed a flow rate equivalent to the accelerated flow rates found within the arterial system in order to promote and activate intracellular and extracellular proteins associated with the adipogenic lineage. Confirmation of sustained viability following shear exposure was obtained using a fluorescent live-dead assay. Visualization of intracellular lipid accumulation was achieved via Oil Red O staining. When placed into culture, shear stimulated hMSCs were further induced toward brown adipose tissue, as evidenced by a greater quantity of lipid triglycerides, relative to unstimulated hMSCs. qRT-PCR analysis validated the phenotypic changes observed when the hMSCs were later cultured in adipogenic differentiation media. Additionally, increased fold change for adipogenic markers such as LPL1, CFL1, and SSP1 were observed as a result of shear stimulation. The significance of this work lies in the demonstration that transient fluid shear exposure of hMSCs in suspension can influence differentiation into adipocytes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:440-446, 2016.

Suspension-based differentiation of adult mesenchymal stem cells toward chondrogenic lineage.

Human mesenchymal stem cells (hMSCs) are derived from bone marrow and have the ability to differentiate into cartilage and other mesenchymal cell types found throughout the body. Traditionally, the differentiation of hMSCs toward chondrocytes occurs through a combination of pelleted static cell culture and chemical stimuli. As an alternative to these protocols, we developed an in vitro flow through microfluidic method to induce the differentiation of hMSCs into chondrocytes. Suspensions of unattached hMSCs were exposed to a constant shear flow over a period of 20 minutes, which promoted phenotypic and gene expression changes toward the chondrogenic lineage. These internal and external changes of chondrogenic differentiation were then observed over 3 weeks later in culture, as confirmed through fluorescent immunocytochemical staining and real-time quantitative reverse transcriptase polymerase chain reaction. The increased concentration of Type II collagen on the surface of shear stimulated hMSCs with the upregulation of MAPK1 and SOX9 demonstrated the capabilities of our approach to induce sustained differentiation. In conclusion, our shear stimulation method, in combination with chemical stimuli, illustrates enhanced differentiation of hMSCs toward the chondrogenic lineage.

Effects of early adolescent environmental enrichment on cognitive dysfunction, prefrontal cortex development, and inflammatory cytokines after early life stress.

Early postnatal stress such as maternal separation causes cognitive dysfunction later in life, including working memory deficits that are largely mediated by the prefrontal cortex. Maternal separation in male rats also yields a loss of parvalbumin-containing prefrontal cortex interneurons in adolescence, which may occur via inflammatory or oxidative stress mechanisms. Environmental enrichment can prevent several effects of maternal separation; however, effects of enrichment on prefrontal cortex development are not well understood. Here, we report that enrichment prevented cognitive dysfunction in maternally separated males and females, and prevented elevated circulating pro-inflammatory cytokines that was evident in maternally separated males, but not females. However, enrichment did not prevent parvalbumin loss or adolescent measures of oxidative stress. Significant correlations indicated that adolescents with higher oxidative damage and less prefrontal cortex parvalbumin in adolescence committed more errors on the win-shift task; therefore, maternal separation may affect cognitive dysfunction via aberrant interneuron development. © 2015 Wiley Periodicals, Inc. Dev Psychobiol 58: 482-491, 2016.

Glycosyltransferase ST6GAL1 contributes to the regulation of pluripotency in human pluripotent stem cells.

Many studies have suggested the significance of glycosyltransferase-mediated macromolecule glycosylation in the regulation of pluripotent states in human pluripotent stem cells (hPSCs). Here, we observed that the sialyltransferase ST6GAL1 was preferentially expressed in undifferentiated hPSCs compared to non-pluripotent cells. A lectin which preferentially recognizes α-2,6 sialylated galactosides showed strong binding reactivity with undifferentiated hPSCs and their glycoproteins, and did so to a much lesser extent with differentiated cells. In addition, downregulation of ST6GAL1 in undifferentiated hPSCs led to a decrease in POU5F1 (also known as OCT4) protein and significantly altered the expression of many genes that orchestrate cell morphogenesis during differentiation. The induction of cellular pluripotency in somatic cells was substantially impeded by the shRNA-mediated suppression of ST6GAL1, partially through interference with the expression of endogenous POU5F1 and SOX2. Targeting ST6GAL1 activity with a sialyltransferase inhibitor during cell reprogramming resulted in a dose-dependent reduction in the generation of human induced pluripotent stem cells (hiPSCs). Collectively, our data indicate that ST6GAL1 plays an important role in the regulation of pluripotency and differentiation in hPSCs, and the pluripotent state in human cells can be modulated using pharmacological tools to target sialyltransferase activity.

Mass-spectrometry-based molecular characterization of extracellular vesicles: lipidomics and proteomics.

This review discusses extracellular vesicles (EVs), which are submicron-scale, anuclear, phospholipid bilayer membrane enclosed vesicles that contain lipids, metabolites, proteins, and RNA (micro and messenger). They are shed from many, if not all, cell types and are present in biological fluids and conditioned cell culture media. The term EV, as coined by the International Society of Extracellular Vesicles (ISEV), encompasses exosomes (30-100 nm in diameter), microparticles (100-1000 nm), apoptotic blebs, and other EV subsets. EVs have been implicated in cell-cell communication, coagulation, inflammation, immune response modulation, and disease progression. Multiple studies report that EV secretion from disease-affected cells contributes to disease progression, e.g., tumor niche formation and cancer metastasis. EVs are attractive sources of biomarkers due to their biological relevance and relatively noninvasive accessibility from a range of physiological fluids. This review is focused on the molecular profiling of the protein and lipid constituents of EVs, with emphasis on mass-spectrometry-based "omic" analytical techniques. The challenges in the purification and molecular characterization of EVs, including contamination of isolates and limitations in sample quantities, are discussed along with possible solutions. Finally, the review discusses the limited but growing investigation of post-translational modifications of EV proteins and potential strategies for future in-depth molecular characterization of EVs.

Cell chemotaxis on paper for diagnostics.

Microfluidic chemotaxis platforms have historically been utilized to probe phenomena such as neutrophil migration and are beginning to be developed for diagnostic applications; however, current microfluidic chemotaxis systems require specialized engineering equipment such as syringe pumps and long time frames (hours) to develop a chemokine gradient, and cell chemotaxis typically requires multiple additional hours. The paperfluidic device described in this work is a low-cost, sharp (2 mm wide), quasi-stable (at least 20 min) and rapidly generated (<1 s) chemokine gradient system capable of examining cell migration response over short time frames (20 min) that can be easily assembled. A proof-of-concept experiment on human pan-T cells showed significant (p ≪ 0.01) directed migration to the chemokine gradient over the control condition. This new technique for cell migration studies provides a foundational step in designing microfluidic chemotactic platforms for point-of-care diagnostics.

Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.