Emergent mechanical control of vascular morphogenesis.

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.

Fibroblast activation in response to TGFß1 is modulated by co-culture with endothelial cells in a vascular organ-on-chip platform.

Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFß1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFß1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFß1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.

A platform to reproducibly evaluate human colon permeability and damage.

The intestinal epithelium comprises diverse cell types and executes many specialized functions as the primary interface between luminal contents and internal organs. A key function provided by the epithelium is maintenance of a barrier that protects the individual from pathogens, irritating luminal contents, and the microbiota. Disruption of this barrier can lead to inflammatory disease within the intestinal mucosa, and, in more severe cases, to sepsis. Animal models to study intestinal permeability are costly and not entirely predictive of human biology. Here we present a model of human colon barrier function that integrates primary human colon stem cells into Draper's PREDICT96 microfluidic organ-on-chip platform to yield a high-throughput system appropriate to predict damage and healing of the human colon epithelial barrier. We have demonstrated pharmacologically induced barrier damage measured by both a high throughput molecular permeability assay and transepithelial resistance. Using these assays, we developed an Inflammatory Bowel Disease-relevant model through cytokine induced damage that can support studies of disease mechanisms and putative therapeutics.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery.

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

Measurement of oxygen consumption rates of human renal proximal tubule cells in an array of organ-on-chip devices to monitor drug-induced metabolic shifts.

Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.

Evaluation of rapid transepithelial electrical resistance (TEER) measurement as a metric of kidney toxicity in a high-throughput microfluidic culture system.

Rapid non-invasive kidney-specific readouts are essential to maximizing the potential of microfluidic tissue culture platforms for drug-induced nephrotoxicity screening. Transepithelial electrical resistance (TEER) is a well-established technique, but it has yet to be evaluated as a metric of toxicity in a kidney proximal tubule (PT) model that recapitulates the high permeability of the native tissue and is also suitable for high-throughput screening. We utilized the PREDICT96 high-throughput microfluidic platform, which has rapid TEER measurement capability and multi-flow control, to evaluate the utility of TEER sensing for detecting cisplatin-induced toxicity in a human primary PT model under both mono- and co-culture conditions as well as two levels of fluid shear stress (FSS). Changes in TEER of PT-microvascular co-cultures followed a dose-dependent trend similar to that demonstrated by lactate dehydrogenase (LDH) cytotoxicity assays and were well-correlated with tight junction coverage after cisplatin exposure. Additionally, cisplatin-induced changes in TEER were detectable prior to increases in cell death in co-cultures. PT mono-cultures had a less differentiated phenotype and were not conducive to toxicity monitoring with TEER. The results of this study demonstrate that TEER has potential as a rapid, early, and label-free indicator of toxicity in microfluidic PT-microvascular co-culture models.

A high-throughput microfluidic bilayer co-culture platform to study endothelial-pericyte interactions.

Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.

Correction: A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions.

Correction for 'A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions' by Kelly Tan et al., Lab Chip, 2019, 19, 1556-1566, DOI: .

A thermoplastic microfluidic microphysiological system to recapitulate hepatic function and multicellular interactions.

Hepatic in vitro platforms ranging from multi-well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next-generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high-throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue-culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media-to-cell ratio in comparison with multi-well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte-Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic-based high-throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi-cellular interactions and therapeutic responses.

Interstitial flow promotes macrophage polarization toward an M2 phenotype.

Tumor tissues are characterized by an elevated interstitial fluid flow from the tumor to the surrounding stroma. Macrophages in the tumor microenvironment are key contributors to tumor progression. While it is well established that chemical stimuli within the tumor tissues can alter macrophage behaviors, the effects of mechanical stimuli, especially the flow of interstitial fluid in the tumor microenvironment, on macrophage phenotypes have not been explored. Here, we used three-dimensional biomimetic models to reveal that macrophages can sense and respond to pathophysiological levels of interstitial fluid flow reported in tumors (∼3 µm/s). Specifically, interstitial flow (IF) polarizes macrophages toward an M2-like phenotype via integrin/Src-mediated mechanotransduction pathways involving STAT3/6. Consistent with this flow-induced M2 polarization, macrophages treated with IF migrate faster and have an enhanced ability to promote cancer cell migration. Moreover, IF directs macrophages to migrate against the flow. Since IF emanates from the tumor to the surrounding stromal tissues, our results suggest that IF could not only induce M2 polarization of macrophages but also recruit these M2 macrophages toward the tumor masses, contributing to cancer cell invasion and tumor progression. Collectively, our study reveals that IF could be a critical regulator of tumor immune environment.

Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation.

Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell-cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil-endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC-PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN-tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor-PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site.