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Abstract Neurodegenerative diseases (NDDs) affect more than 50 million people worldwide, posing a significant global health challenge as well as a high socioeconomic burden. With aging constituting one of the main risk factors for some NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD), this societal toll is expected to rise considering the predicted increase in the aging population as well as the limited progress in the development of effective therapeutics. To address the high failure rates in clinical trials, legislative changes permitting the use of alternatives to traditional pre‐clinical in vivo models are implemented. In this regard, microphysiological systems (MPS) such as organ‐on‐a‐chip (OoC) platforms constitute a promising tool, due to their ability to mimic complex and human‐specific tissue niches in vitro. This review summarizes the current progress in modeling NDDs using OoC technology and discusses five critical aspects still insufficiently addressed in OoC models to date. Taking these aspects into consideration in the future MPS will advance the modeling of NDDs in vitro and increase their translational value in the clinical setting.

Abstract Endothelial programmed death‐ligand 1 (PD‐L1) expression is higher in tumors than in normal tissues. Also, tumoral vasculatures tend to be leakier than normal vessels leading to a higher trans‐endothelial or transmural fluid flow. However, it is not clear whether such elevated transmural flow can control endothelial PD‐L1 expression. Here, a new microfluidic device is developed to investigate the relationship between transmural flow and PD‐L1 expression in microvascular networks (MVNs). After treating the MVNs with transmural flow for 24 h, the expression of PD‐L1 in endothelial cells is upregulated. Additionally, CD8 T cell activation by phytohemagglutinin (PHA) is suppressed when cultured in the MVNs pre‐conditioned with transmural flow. Moreover, transmural flow is able to further increase PD‐L1 expression in the vessels formed in the tumor microenvironment. Finally, by utilizing blocking antibodies and knock‐out assays, it is found that transmural flow‐driven PD‐L1 upregulation is controlled by integrin αVβ3. Overall, this study provides a new biophysical explanation for high PD‐L1 expression in tumoral vasculatures.

Abstract Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.

Abstract The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic‐based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on‐chip lymphatics with in vivo‐like morphology exhibit tissue‐scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three‐dimensional geometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on‐chip lymphatics enabled studies of lymphatic‐immune interactions that revealed inflammation‐driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on‐chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic‐based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.

Abstract Background There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. Methods Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. Results MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. Conclusion Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

Abstract Targeting DNA methyltransferase 1 (DNMT1) has immunomodulatory and anti-neoplastic activity, especially when paired with cancer immunotherapies. Here we explore the immunoregulatory functions of DNMT1 in the tumor vasculature of female mice. Dnmt1 deletion in endothelial cells (ECs) impairs tumor growth while priming expression of cytokine-driven cell adhesion molecules and chemokines important for CD8+ T-cell trafficking across the vasculature; consequently, the efficacy of immune checkpoint blockade (ICB) is enhanced. We find that the proangiogenic factor FGF2 promotes ERK-mediated DNMT1 phosphorylation and nuclear translocation to repress transcription of the chemokines Cxcl9/Cxcl10 in ECs. Targeting Dnmt1 in ECs reduces proliferation but augments Th1 chemokine production and extravasation of CD8+ T-cells, suggesting DNMT1 programs immunologically anergic tumor vasculature. Our study is in good accord with preclinical observations that pharmacologically disrupting DNMT1 enhances the activity of ICB but suggests an epigenetic pathway presumed to be targeted in cancer cells is also operative in the tumor vasculature.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer’s disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer’s disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer’s disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer’s disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer’s disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer’s disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer’s disease-specific microtissues displayed localized β-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

Abstract Three‐dimensional (3D) imaging techniques (e.g., confocal microscopy) are commonly used to visualize in vitro models, especially microvasculature on‐a‐chip. Conversely, 3D analysis is not the standard method to extract quantitative information from those models. We developed the μVES algorithm to analyze vascularized in vitro models leveraging 3D data. It computes morphological parameters (geometry, diameter, length, tortuosity, eccentricity) and intravascular flow velocity. μVES application to microfluidic vascularized in vitro models shows that they successfully replicate functional features of the microvasculature in vivo in terms of intravascular fluid flow velocity. However, wall shear stress is lower compared to in vivo references. The morphological analysis also highlights the model's physiological similarities (vessel length and tortuosity) and shortcomings (vessel radius and surface‐over‐volume ratio). The addition of the third dimension in our analysis produced significant differences in the metrics assessed compared to 2D estimations. It enabled the computation of new indices, such as vessel eccentricity. These μVES capabilities can find application in analyses of different in vitro vascular models, as well as in vivo and ex vivo microvasculature.

Gap formation in the vasculature underpins immune and tumour cell infiltration. Here the authors propose a chemo-mechanical model to analyse how feedback between mechanosensitive signalling, active cellular forces and adhesion governs the breakdown, recovery, and integrity of endothelial junctions.

Abstract Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

Emerging microfluidic disease models have amply demonstrated their value in many fields of cancer research. These in vitro technologies recapitulate key aspects of metastatic cancer, including the process of tumor cell arrest and extravasation at the site of the metastatic tumor. To date, extensive efforts have been made to capture key features of the microvasculature to reconstitute the pre-metastatic niche and investigate dynamic extravasation behaviors using microfluidic systems. In this mini-review, we highlight recent microfluidic vascular models of tumor cell extravasation and explore how this approach contributes to development of in vitro disease models to enhance understanding of metastasis in vivo.

Offeddu et al. employ a microfluidic vascular model to study the effects of selective removal of glycocalyx components from cancer cells. They find that cancer-associated hyaluronic acid and CD44 mediate firm adhesion to endothelial cells and extravasation during metastasis.

The lack of a conventional lymphatic system that permeates throughout the entire human brain has encouraged the identification and study of alternative clearance routes within the cerebrum. In 2012, the concept of the glymphatic system, a perivascular network that fluidically connects the cerebrospinal fluid to the lymphatic vessels within the meninges via the interstitium, emerged. Although its exact mode of action has not yet been fully characterized, the key underlying processes that govern solute transport and waste clearance have been identified. This review briefly describes the perivascular glial-dependent clearance system and elucidates its fundamental role in neurodegenerative diseases. The current knowledge of the glymphatic system is based almost exclusively on animal-based measurements, but these face certain limitations inherent to in vivo experiments. Recent advances in organ-on-a-chip technology are discussed to demonstrate the technology’s ability to provide alternative human-based in vitro research models. Herein, the specific focus is on how current microfluidic-based in vitro models of the neurovascular system and neurodegenerative diseases might be employed to (i) gain a deeper understanding of the role and function of the glymphatic system and (ii) to identify new opportunities for pharmacological intervention.

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell–cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the “black box” of living cells.

Cell therapies, including adoptive immune cell therapies and genetically engineered chimeric antigen receptor (CAR) T or NK cells, have shown promise in treating hematologic malignancies. Yet, immune cell infiltration and expansion has proven challenging in solid tumors due to immune cell exclusion and exhaustion and the presence of vascular barriers. Testing next-generation immune therapies remains challenging in animals, motivating sophisticated ex vivo models of human tumor biology and prognostic assays to predict treatment response in real-time while comprehensively recapitulating the human tumor immune microenvironment (TIME). This review examines current strategies for testing cell-based cancer immunotherapies using ex vivo microphysiological systems and microfluidic technologies. Insights into the multicellular interactions of the TIME will identify novel therapeutic strategies to help patients whose tumors are refractory or resistant to current immunotherapies. Altogether, these microphysiological systems (MPS) have the capability to predict therapeutic vulnerabilities and biological barriers while studying immune cell infiltration and killing in a more physiologically relevant context, thereby providing important insights into fundamental biologic mechanisms to expand our understanding of and treatments for currently incurable malignancies.

Biomechanical studies of eukaryotic cells have been limited due to low sensitivity and axial resolution in interferometric imaging. Here, the authors present depth-resolved confocal reflectance interferometric microscopy with high sensitivity and temporal resolution, which enables quantification of nucleic envelope and plasma membrane fluctuations.

Summary: This review describes recent research that has advanced our understanding of the role of immune cells in the tumor microenvironment (TME) using advanced 3D in vitro models and engineering approaches. The TME can hinder effective eradication of tumor cells by the immune system, but immunotherapy has been able to reverse this effect in some cases. However, patient-to-patient variability in response suggests that we require deeper understanding of the mechanistic interactions between immune and tumor cells to improve response and develop novel therapeutics. Reconstruction of the TME using engineered 3D models allows high-resolution observation of cell interactions while allowing control of conditions such as hypoxia, matrix stiffness, and flow. Moreover, patient-derived organotypic models are an emerging tool for prediction of drug efficacy. This review highlights the importance of modeling and understanding the immune TME and describes new tools for identifying new biological targets, drug testing, and strategies for personalized medicine.

Throughout the process of vascular growth and remodeling, the extracellular matrix (ECM) concurrently undergoes significant changes due to proteolytic activity—regulated by both endothelial and surrounding stromal cells. The role of matrix metalloproteinases has been well-studied in the context of vascular remodeling, but other proteases, such as cysteine cathepsins, could also facilitate ECM remodeling. To investigate cathepsin-mediated proteolysis in vascular ECM remodeling, and to understand the role of shear flow in this process, in vitro microvessels were cultured in previously designed microfluidic chips and assessed by immunostaining, zymography, and western blotting. Primary human vessels (HUVECs and fibroblasts) were conditioned by continuous fluid flow and/or small molecule inhibitors to probe cathepsin expression and activity. Luminal flow (in contrast to static culture) decreases the activity of cathepsins in microvessel systems, despite a total protein increase, due to a concurrent increase in the endogenous inhibitor cystatin C. Observations also demonstrate that cathepsins mostly co-localize with fibroblasts, and that fibrin (the hydrogel substrate) may stabilize cathepsin activity in the system. Inhibitor studies suggest that control over cathepsin-mediated ECM remodeling could contribute to improved maintenance of in vitro microvascular networks; however, further investigation is required. Understanding the role of cathepsin activity in in vitro microvessels and other engineered tissues will be important for future regenerative medicine applications.

Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular tissues and iBRB modeling. Recent BBB in vitro models using human cells of the neurovascular unit (NVU) showed physiological morphology and permeability values, and reproduced brain neurological disorder phenotypes that could be applicable to modeling the iBRB. Here, we describe similarities between iBRB and BBB properties, compare existing neurovascular barrier models, propose leverage of MPS-based strategies to develop new iBRB models, and explore potentials to personalize cellular inputs and improve pre-clinical testing.

Intratumoral recruitment of immune cells following innate immune activation is critical for anti-tumor immunity and involves cytosolic dsDNA sensing by the cGAS/STING pathway. We have previously shown that KRAS-LKB1 (KL) mutant lung cancer, which is resistant to PD-1 blockade, exhibits silencing of STING, impaired tumor cell production of immune chemoattractants, and T cell exclusion. Since the vasculature is also a critical gatekeeper of immune cell infiltration into tumors, we developed a novel microfluidic model to study KL tumor-vascular interactions. Notably, dsDNA priming of LKB1-reconstituted tumor cells activates the microvasculature, even when tumor cell STING is deleted. cGAS-driven extracellular export of 2′3′ cGAMP by cancer cells activates STING signaling in endothelial cells and cooperates with type 1 interferon to increase vascular permeability and expression of E selectin, VCAM-1, and ICAM-1 and T cell adhesion to the endothelium. Thus, tumor cell cGAS-STING signaling not only produces T cell chemoattractants, but also primes tumor vasculature for immune cell escape.

Cells in a tumor microenvironment are exposed to spatial and temporal variations in oxygen tension due to hyperproliferation and immature vascularization. Such spatiotemporal oxygen heterogeneity affects the behavior of cancer cells, leading to cancer growth and metastasis, and thus, it is essential to clarify the cellular responses of cancer cells to oxygen tension. Herein, we describe a new double-layer microfluidic device allowing the control of oxygen tension and the behavior of cancer cells under spatiotemporal oxygen heterogeneity. Two parallel gas channels were located above the media and gel channels to enhance gas exchange, and a gas-impermeable polycarbonate film was embedded in the device to prevent the diffusion of atmospheric oxygen. Variations in oxygen tension in the device with the experimental parameters and design variables were investigated computationally and validated by using oxygen-sensitive nanoparticles. The present device can generate a uniform hypoxic condition at oxygen levels down to 0.3% O2, as well as a linear oxygen gradient from 3% O2 to 17% O2 across the gel channel within 15 min. Moreover, human breast cancer cells suspended in type I collagen gel were introduced in the gel channel to observe their response under controlled oxygen tension. Hypoxic exposure activated the proliferation and motility of the cells, which showed a local maximum increase at 5% O2. Under the oxygen gradient condition, the increase in the cell number was relatively high in the central mild hypoxia region. These findings demonstrate the utility of the present device to study cellular responses in an oxygen-controlled microenvironment.

Abstract Placental vasculopathies are associated with a number of pregnancy‐related diseases, including pre‐eclampsia (PE)—a leading cause of maternal–fetal morbidity and mortality worldwide. Placental presentations of PE are associated with endothelial dysfunction, reduced vessel perfusion, white blood cell infiltration, and altered production of angiogenic factors within the placenta (a candidate mechanism). Despite maintaining vascular quiescence in other tissues, how pericytes contribute to vascular growth and signaling in the placenta remains unknown. Here, pericytes are hypothesized to play a detrimental role in the pathogenesis of placental vascular growth. A perfusable triculture model is developed, consisting of human endothelial cells, fibroblasts, and pericytes, capable of recapitulating growth and remodeling in a system that mimics inflamed placental microvessels. Placental pericytes are shown to contribute to growth restriction of microvessels over time, an effect that is strongly regulated by vascular endothelial growth factor and Angiopoietin/Tie2 signaling. Furthermore, this model is capable of recapitulating essential processes including tumor necrosis factor alpha (TNFα)‐mediated vascular leakage and leukocyte infiltration, both important aspects associated with placental PE. This placental vascular model highlights that an imbalance in endothelial–pericyte crosstalk can play a critical role in the development of vascular pathology and associated diseases.

Abstract Harmful materials in the blood are prevented from entering the healthy brain by a highly selective blood–brain barrier (BBB), and impairment of barrier function has been associated with a variety of neurological diseases. In Alzheimer's disease (AD), BBB breakdown has been shown to occur even before cognitive decline and brain pathology. To investigate the role of the cerebral vasculature in AD, a physiologically relevant 3D human neural cell culture microfluidic model is developed having a brain endothelial cell monolayer with a BBB‐like phenotype. This model is shown to recapitulate several key aspects of BBB dysfunction observed in AD patients: increased BBB permeability, decreased expression of claudin‐1, claudin‐5, and VE‐cadherin, increased expression of matrix‐metalloproteinase‐2 and reactive oxygen species, and deposition of β‐amyloid (Aβ) peptides at the vascular endothelium. Thus, it provides a well‐controlled platform for investigating BBB function as well as for screening of new drugs that need to pass the BBB to gain access to neural tissues.

Abstract The effectiveness of nanoparticles (NP) in nanomedicine depends on their ability to extravasate from vasculature towards the target tissue. This is determined by their permeability across the endothelial barrier. Unfortunately, a quantitative study of the diffusion permeability coefficients (Pd) of NPs is difficult with in vivo models. Here, we utilize a relevant model of vascular-tissue interface with tunable endothelial permeability in vitro based on microfluidics. Human umbilical vein endothelial cells (HUVECs) grown in microfluidic devices were treated with Angiopoietin 1 and cyclic adenosine monophosphate (cAMP) to vary the Pd of the HUVECs monolayer towards fluorescent polystyrene NPs (pNPs) of different sizes, which was determined from image analysis of their fluorescence intensity when diffusing across the monolayer. Using 70 kDa dextran as a probe, untreated HUVECs yielded a Pd that approximated tumor vasculature while HUVECs treated with 25 μg/mL cAMP had Pd that approximated healthy vasculature in vivo. As the size of pNPs increased, its Pd decreased in tumor vasculature, but remained largely unchanged in healthy vasculature, demonstrating a trend similar to tumor selectivity for smaller NPs. This microfluidic model of vascular-tissue interface can be used in any laboratory to perform quantitative assessment of the tumor selectivity of nanomedicine-based systems.

Hemodynamic and biochemical factors play important roles in critical steps of angiogenesis. In particular, interstitial flow has attracted attention as an important hemodynamic factor controlling the angiogenic process. Here, we applied a wide range of interstitial flow magnitudes to an in vitro three-dimensional (3D) angiogenesis model in a microfluidic device. This study aimed to investigate the effect of interstitial flow magnitude in combination with the vascular endothelial growth factor (VEGF) concentration on 3D microvascular network formation. Human umbilical vein endothelial cells (HUVECs) were cultured in a series of interstitial flow generated by 2, 8, and 25 mmH2O. Our findings indicated that interstitial flow significantly enhanced vascular sprout formation, network extension, and the development of branching networks in a magnitude-dependent manner. Furthermore, we demonstrated that the proangiogenic effect of interstitial flow application could not be substituted by the increased VEGF concentration. In addition, we found that HUVECs near vascular sprouts significantly elongated in >8 mmH2O conditions, while activation of Src was detected even in 2 mmH2O conditions. Our results suggest that the balance between the interstitial flow magnitude and the VEGF concentration plays an important role in the regulation of 3D microvascular network formation in vitro.

Angiogenesis is a crucial event for tumor progression and metastasis. It is the process through which new blood vessels are formed and has become a therapeutic target in many cancer therapies. However, current anti-angiogenic drugs such as Thalidomide still have detrimental teratogenic effects. This property could be caused by the presence of chiral carbons, intrinsic to such compounds. We synthesized four different phthalimide derivatives that lack chiral carbons in their chemical structure. We hypothesized that these achiral carbon compounds would retain similar levels of anti-angiogenic activity whilst reducing teratogenic effects. We tested for their anti-angiogenic functions using an in vitro 3D microfluidic assay with human endothelial cells. All four compounds caused a drastic inhibition of angiogenesis at lower effective concentrations compared to Thalidomide. Quantification of the blood vessel sprouting in each condition allowed us to classify compounds depending on their anti-angiogenic capabilities. The most effective identified compound (C4), was tested in vivo on a zebrafish embryo model. Blood vessel development was measured using number and lengths of the stalks visible in the fli1a:EGFP transgenic line. Potential teratogenic effects of C4 were monitored over zebrafish embryonic development. The in vivo results confirmed the increased potency of C4 compared to Thalidomide demonstrated by results in embryos exposed to concentrations as low as 0.02 μM. The teratogenic analysis further validated the advantages of using C4 over Thalidomide in zebrafish embryos. This study highlights how the use of in vitro 3D model can allow rapid screening and selection of new and safer drugs.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of “multi-cellular engineered living systems,” which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

The actin cytoskeleton is a complex network of filaments, cross-linking proteins and motors; although the components are recognised, the behaviour of the network is less understood. Here Mak et al.use a Brownian dynamics model that reveals actin turnover dynamics as a key regulatory mechanism controlling cytoskeletal states.

There is a demand for three-dimensional (3D) angiogenesis model including endothelial cells (ECs) and mesenchymal stem cells (MSCs), which are known to differentiate into pericytes, to construct stabilized and matured microvascular networks in vitro. However, it remains to be elucidated how MSCs affected on ECs in the process of 3D angiogenesis. In this study, we utilized a microfluidic device to develop a 3D coculture system including human umbilical vein ECs and human MSCs, which allowed us to investigate the effects of MSCs on ECs in the context of 3D angiogenesis. A series of EC:MSC ratio was tested in the EC-MSC coculture. First, we confirmed that MSCs differentiated into pericytes by direct EC-MSC contacts. Next, we found that MSCs attenuated vascular sprout formation of ECs regardless of EC:MSC ratio in the early stage of 3D angiogenesis as well as extension of microvascular networks in the later stage. ECs and MSCs were also cultured under interstitial flow to enhance angiogenesis. However, the stabilization effects of MSCs on the extension of capillary structures were dominant over the promotion effects of the interstitial flow. These results indicate the stabilization effect of MSCs on the formation of microvascular networks in vitro. Although some HMSCs differentiated into pericytes and located around microvascular networks, vascular structures became thick over time in coculture. The 3D EC-MSC coculture model described in this study is useful to further investigate culture microenvironments for constructing stabilized and matured microvascular networks with aligning pericytes.