Protocol for fabricating and characterizing microvessel-on-a-chip for human umbilical vein endothelial cells.

Organ-on-a-chip technologies enable the fabrication of endothelial tissues, so-called microvessels (MVs), which emulate the endothelial barrier function in healthy or disease conditions. In this protocol, we describe the fabrication of perfusable open-chamber style MVs embedded in collagen gels. We then report a simple technology to characterize the MV barrier properties in static or under pressure based on fluorescence confocal imaging. Finally, we provide quantification techniques that enable us to infer the structure of MV paracellular pores. For complete details on the use and execution of this protocol, please refer to Cacheux et al.1.

CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment.

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-ß (TGF-ß) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-ß signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-ß-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-ß-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-ß-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.

Asymmetry of tensile versus compressive elasticity and permeability contributes to the regulation of exchanges in collagen gels.

The Starling principle describes exchanges between blood and tissues based on the balance of hydrostatic and osmotic flows. However, the permeation properties of the main constituent of tissues, namely, collagen, in response to the stress exerted by blood pressure remain poorly characterized. Here, we develop an instrument to determine the elasticity and permeability of collagen gels under tensile and compressive stress based on measuring the temporal change in pressure in an air cavity sealed at the outlet of a collagen slab. Data analysis with an analytical model reveals a drop in the permeability and enhanced strain stiffening of native collagen gels under compression versus tension, both effects being essentially lost after chemical cross-linking. Furthermore, we report the control of the permeability of native collagen gels using sinusoidal fluid injection, an effect explained by the asymmetric response in tension and compression. We lastly suggest that blood-associated pulsations could contribute to exchanges within tissues.

Endothelial tissue remodeling induced by intraluminal pressure enhances paracellular solute transport.

The endothelial layers of the microvasculature regulate the transport of solutes to the surrounding tissues. It remains unclear how this barrier function is affected by blood flow-induced intraluminal pressure. Using a 3D microvessel model, we compare the transport of macromolecules through endothelial tissues at mechanical rest or with intraluminal pressure, and correlate these data with electron microscopy of endothelial junctions. On application of an intraluminal pressure of 100 Pa, we demonstrate that the flow through the tissue increases by 2.35 times. This increase is associated with a 25% expansion of microvessel diameter, which leads to tissue remodeling and thinning of the paracellular junctions. We recapitulate these data with the deformable monopore model, in which the increase in paracellular transport is explained by the augmentation of the diffusion rate across thinned junctions under mechanical stress. We therefore suggest that the deformation of microvasculatures contributes to regulate their barrier function.

Solute transport in the brain tissue: what are the key biophysical parameters tying in vivo and in vitro studies together?

The mechanisms of solute transport in brain tissues are still under debate. The medical relevance of this topic has put the blood-brain barrier and the mechanisms of solute transport through the brain parenchyma in the spotlight, notably in the context of brain clearance. In the last decade, the classical view of pure diffusive flow across the brain parenchyma was tested against the recent proposal of an active, convectional fluid flow model known as the glymphatic model. Experimental studies of brain transport on living humans and animals have temporal and spatial limitations to validate any of these models. Therefore, detailed microscopic observations, mostly ex vivo tissue and simplified in vitro brain models with the support from computational models, are necessary to understand transport mechanisms in brain tissues. However, standardization is lacking between these experimental approaches, which tends to limit the generality of conclusions. In this review, we provide an overview of the output and limitations of modern brain solute transport studies to search for key parameters comparable across experimental setups. We emphasize that in vitro models relying on physiological material and reproducing the biophysical setting of the brain, as well as computational/mathematical models constitute powerful solutions to understand the solute transport phenomena inside of the brain tissue. Finally, we suggest the blood-brain barrier permeability and the apparent diffusion coefficient through the brain parenchyma to be robust biophysical parameters for the extraction of cross-model conclusion.

Phenotypic and transcriptional characterization of oligodendrocyte precursor cells in a 3D culture.

Remyelination of the central nervous system (CNS) is a regenerative response that depends on the development of oligodendrocyte precursor cells (OPCs), which are generated from neural stem cells in developmental stages and exist as tissue stem cells in the adult CNS. Three-dimensional (3D) culture systems that recapitulate the complexity of the in vivo microenvironment are important for understanding the behavior of OPCs in remyelination and for exploring effective therapeutic approaches. In general, functional analysis of OPCs has mainly used two-dimensional (2D) culture systems; however, the differences between the properties of OPCs cultured in 2D and 3D have not been fully elucidated despite cellular functions being affected by the scaffold. In this study, we analyzed the phenotypic and transcriptomic differences in OPCs from 2D and collagen gel-based 3D cultures. In the 3D culture, the OPCs exhibited less than half ratio of proliferation and almost half ratio of differentiation to mature oligodendrocytes, compared to the 2D culture in the same culturing period. RNA-seq data showed robust changes in the expression level of genes associated with oligodendrocyte differentiation, and there were more up-regulated genes than down-regulated genes in 3D cultures compared to 2D cultures. In addition, the OPCs cultured in collagen gel scaffolds at lower collagen fiber densities showed higher proliferation activity compared with those cultured in collagen gel with higher collagen fiber densities. Our findings have identified the effect of culture dimension as well as the complexity of the scaffold on OPC responses at the cellular and molecular levels.

Enhanced podocyte differentiation and changing drug toxicity sensitivity through pressure-controlled mechanical filtration stress on a glomerulus-on-a-chip.

Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.

Image-based crosstalk analysis of cell-cell interactions during sprouting angiogenesis using blood-vessel-on-a-chip.

BACKGROUND: Sprouting angiogenesis is an important mechanism for morphogenetic phenomena, including organ development, wound healing, and tissue regeneration. In regenerative medicine, therapeutic angiogenesis is a clinical solution for recovery from ischemic diseases. Mesenchymal stem cells (MSCs) have been clinically used given their pro-angiogenic effects. MSCs are reported to promote angiogenesis by differentiating into pericytes or other vascular cells or through cell-cell communication using multiple protein-protein interactions. However, how MSCs physically contact and move around ECs to keep the sprouting angiogenesis active remains unknown. METHODS: We proposed a novel framework of EC-MSC crosstalk analysis using human umbilical vein endothelial cells (HUVECs) and MSCs obtained from mice subcutaneous adipose tissue on a 3D in vitro model, microvessel-on-a-chip, which allows cell-to-tissue level study. The microvessels were fabricated and cultured for 10 days in a collagen matrix where MSCs were embedded. RESULTS: Immunofluorescence imaging using a confocal laser microscope showed that MSCs smoothed the surface of the microvessel and elongated the angiogenic sprouts by binding to the microvessel's specific microstructures. Additionally, three-dimensional modeling of HUVEC-MSC intersections revealed that MSCs were selectively located around protrusions or roots of angiogenic sprouts, whose surface curvature was excessively low or high, respectively. CONCLUSIONS: The combination of our microvessel-on-a-chip system for 3D co-culture and image-based crosstalk analysis demonstrated that MSCs are selectively localized to concave-convex surfaces on scaffold structures and that they are responsible for the activation and stabilization of capillary vessels.

Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.

De novo Fc-based receptor dimerizers differentially modulate PlexinB1 function.

Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.

Nailfold capillary patterns correlate with age, gender, lifestyle habits, and fingertip temperature.

Nailfold capillaroscopy is a simple and noninvasive imaging tool to visualize the pattern of capillaries. Microvascular abnormalities have been previously observed in autoimmune disease such as systemic sclerosis and diabetes. Thus, early detection of microvascular dysfunction or changes has promising way for the one of the disease preventions. In this study, for routine health checkups, we evaluated the relationship between the structure of nailfold capillaries and lifestyle habits in healthy participants. First, we analyzed the correlation of structural parameters of nailfold capillaries with values of responses to questions on their lifestyle habits in 224 participants. The results suggested that an unhealthy lifestyle, including poor sleeping habits, smoking, intense exercise, and drinking alcohol, causes a change in the pattern of nailfold capillaries. We then investigated whether the pattern of nailfold capillaries changed after a conscious improvement in lifestyle habits. One to two weeks after the self-improvement of lifestyle habits, the hairpin loops sharpened or straightened. In conclusion, this study is the first report indicating a correlation between the structure of nailfold capillaries and lifestyle habits in a non-clinical population. The simple, inexpensive, and noninvasive method using nailfold microscopy can be employed for routine health checkups everywhere even at a bedside.

Glomerulus-on-a-Chip: Current Insights and Future Potential Towards Recapitulating Selectively Permeable Filtration Systems.

Glomerulopathy, characterized by a dysfunctional glomerular capillary wall, results in proteinuria, leading to end-stage renal failure and poor clinical outcomes, including renal death and increased overall mortality. Conventional glomerulopathy research, including drug discovery, has mostly relied on animal experiments because in-vitro glomerulus models, capable of evaluating functional selective permeability, was unavailable in conventional in-vitro cell culture systems. However, animal experiments have limitations, including time- and cost-consuming, multi-organ effects, unstable reproducibility, inter-species reliability, and the social situation in the EU and US, where animal experiments have been discouraged. Glomerulus-on-a-chip, a new in-vitro organ model, has recently been developed in the field of organ-on-a-chip research based on microfluidic device technology. In the glomerulus-on-a-chip, the podocytes and endothelial cells are co-cultured in a microfluidic device with physical stimuli that mimic the physiological environment to enhance cell function to construct a functional filtration barrier, which can be assessed by permeability assays using fluorescently labeled molecules including inulin and albumin. A combination of this glomerulus-on-a chip technology with the culture technology to induce podocytes and endothelial cells from the human pluripotent stem cells could provide an alternative organ model and solve the issue of animal experiments. Additionally, previous experiments have verified the difference in the leakage of albumin using differentiated podocytes derived from patients with Alport syndrome, such that it could be applied to intractable hereditary glomerulopathy models. In this review, we provide an overview of the features of the existing glomerulus-on-a-chip systems, focusing on how they can address selective permeability verification tests, and the challenges they involved. We finally discuss the future approaches that should be developed for solving those challenges and allow further improvement of glomerulus-on-a-chip technologies.

Stratum corneum levels of calprotectin proteins S100A8/A9 correlate with disease activity in psoriasis patients.

Psoriasis is an intractable inflammatory skin disorder characterized by scaly erythema and plaques. The Psoriasis Area and Severity Index (PASI) is widely used to score disease severity, but evaluation is subjective, and an objective biomarker would be useful. The stratum corneum (SC), which can be non-invasively harvested, may reflect psoriasis-associated changes in epidermal keratinocytes, such as the upregulation of the calprotectin proteins S100A8 and S100A9. The aim of this study was to examine the availability of S100A8/A9 protein levels in SC as a biomarker of psoriasis disease activity. Fifty-three patients with psoriasis, 30 with psoriasis vulgaris (PsV), and 23 with psoriatic arthritis (PsA) participated. SC cells from lesional and non-lesional skin were collected by tape-stripping. S100A8/A9 levels in serum and in SC were quantified by enzyme-linked immunosorbent assay and compared with PASI score before and after treatment initiation or switching. Atopic dermatitis (AD) patients and disease-free individuals were used as controls. Expression of S100A8/A9 in SC of lesional skin of psoriasis patients was significantly higher than in non-lesional skin or AD skin. There was no significant difference of SC S100A8/A9 levels between PsV and PsA patients. The S100A8/A9 levels in SC of psoriasis patients were significantly positively correlated with the PASI score. When patients' skin lesions cleared (PASI clear) in response to treatment, expression of S100A8/A9 in SC was no longer detectable. S100A8/A9 protein levels in SC may be available as an objective, non-invasive biomarker of psoriasis activity to complement PASI scoring.

Extracellular matrix components and elasticity regulate mouse vaginal epithelial differentiation induced by mesenchymal cells†.

Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM's components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.

Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.

Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.

A 3D tissue model-on-a-chip for studying the effects of human senescent fibroblasts on blood vessels.

All human tissues experience aging that eventually causes organ dysfunction and disease. Cellular senescence was discovered in fibroblasts cultured in vitro. In adults, it is a primary defense mechanism against cancer, but also a major contributor to lifespan limits and disorders associated with aging. To assess how human blood vessels change in an aged environment, we developed an elementary tissue model-on-a-chip that comprises an in vitro three-dimensional model of a blood vessel embedded in a collagen gel with young or senescent skin fibroblasts. We found that senescent fibroblasts mechanically altered the surrounding extracellular matrix by exerting excessive traction stress. We then found that senescent fibroblasts induced sprouting angiogenesis of a microvessel via their senescence-associated secretory phenotype (SASP). Finally, we gathered evidence that the mechanical changes of the microenvironment play a role in sustaining SASP-induced angiogenesis. The model proved useful in monitoring morphological changes in blood vessels induced by senescent fibroblasts while controlling the proportion of senescent cells, and enabled the study of SASP inhibitors, a class of drugs useful in aging and cancer research.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation.

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

A simple three-dimensional gut model constructed in a restricted ductal microspace induces intestinal epithelial cell integrity and facilitates absorption assays.

The intestine acts as a center for nutrient and water absorption at the epithelium and plays an important role in immunity. Considering the complexity of its function and roles in living systems, a physiologically relevant gut in vitro model is desirable in both basic biology and the analysis of effects of some substances on functions of the gut; these analyses include the screening of drug and food candidates with regard to intestinal disorder at an early stage of medical development. In the present study, we constructed a three-dimensional (3D) gut model using human absorptive enterocytes (CACO-2 cells) by reconstitution of the gut epithelial sheet restricted on a high-reproducible ductal scaffold of collagen gel. Moreover, using the 3D gut model, we evaluated the morphology at the cellular and tissue levels and conducted a phenotypic analysis of the intestinal physiological functions, which involved a permeability assay mimicking barrier disruption inducing inflammation and an absorption assay reflecting ingestive effects. The ductal structure, in vivo-like 3D epithelial structures, epithelial barrier, and effective absorptive function characterized the 3D gut model. The epithelial cells formed a villus-like buckling epithelium, vertical microvilli of increased density on the cell surface, and a crypt-like localized cell proliferating region. The mature shape of the epithelium may contribute to mimicking barrier function and effective absorption compared with that in the 2D gut model. Furthermore, we successfully mimicked the dextran sodium sulfate-induced epithelial barrier dysfunction as a trigger phenomenon of gut inflammation in the 3D gut model. The integrity of the epithelium and phenotypic analysis of the intestinal physiological functions in the simple and reproducible 3D gut model will allow for a drug screening system for assessing the effects on the functions of the gut epithelium from the lumen side.

TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals.

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.