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When cultured endothelial cells (ECs) are exposed to shear flow, initially cobblestone-like ECs spontaneously elongate and align along the flow direction, acquiring a similar architecture to that of native endothelium in blood vessels. Though previous works have revealed how individual cells sense and respond to shear flow, little is known about th...
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... In summary, the high temporal and spatial resolution achieved in our experimental setup allowed us to describe the ordering kinetics of endothelial cell layers within the framework of active nematic liquid crystals, highlighting the governing role of topological excitation and explaining endothelial cell alignment as a dynamic transition with cellular misalignment as an intermediate stage. The introduced unifying framework reconciles previous contradictory observations reporting perpendicular or parallel endothelial cell alignment depending on experimental parameters [25][26][27][40][41][42]. The importance of the transient regime in endothelial cell alignment reveals the need for precise temporal resolution in detecting changes in protein expression, exemplified by the previously observed transient upregulation of JNK2 in bovine aortic endothelial cells [43]. ...
Many physiological processes, such as the shear flow alignment of endothelial cells in the vasculature, depend on the transition of cell layers between disordered and ordered phases. Here, we demonstrate that such a transition is driven by the non-monotonic evolution of nematic topological defects and the emergence of topological strings that bind the defects together, unveiling an intermediate phase of ordering kinetics in biological matter. We used time-resolved large-scale imaging and physical modeling to resolve the nature of the non-monotonic decrease in the number of defect pairs. The interaction of the intrinsic cell layer activity and the alignment field determines the occurrence of defect domains, which defines the nature of the transition. Defect pair annihilation is mediated by topological strings spanning multicellular scales within the cell layer. We propose that these long-range interactions in the intermediate ordering phase have significant implications for a wide range of biological phenomena in morphogenesis, tissue remodeling, and disease progression.
... On the basis of our finding that the in vitro vessels cultured under bidirectional oscillatory flow and unidirectional laminar flow showed no difference in the helical handedness, and the fact that such biased cell alignment was also seen in previous in vitro studies under static conditions (26), we conclude that the flow profiles or even the presence of flow are not the determinant factors for the directionality of chiral vascular morphogenesis. As for the influence of other specific flow profiles, such as pulsatile flow, on the extent of chiral biases, we will leave it for future more detailed studies to evaluate the role of cell chirality in vascular disorders (61)(62)(63)(64)(65). ...
The cellular helical structure is well known for its crucial role in development and disease. Nevertheless, the underlying mechanism governing this phenomenon remains largely unexplored, particularly in recapitulating it in well-controlled engineering systems. Leveraging advanced microfluidics, we present compelling evidence of the spontaneous emergence of helical endothelial tubes exhibiting robust right-handedness governed by inherent cell chirality. To strengthen our findings, we identify a consistent bias toward the same chirality in mouse vascular tissues. Manipulating endothelial cell chirality using small-molecule drugs produces a dose-dependent reversal of the handedness in engineered vessels, accompanied by non-monotonic changes in vascular permeability. Moreover, our three-dimensional cell vertex model provides biomechanical insights into the chiral morphogenesis process, highlighting the role of cellular torque and tissue fluidity in its regulation. Our study unravels an intriguing mechanism underlying vascular chiral morphogenesis, shedding light on the broader implications and distinctive perspectives of tubulogenesis within biological systems.
... Furthermore, the high ODECs or HUVECs seeding density that was used may also have impeded the flow induced alignment, as was observed previously. [55] ECs are known to regulate phenotype switching of VSMCs. [56,57] In our experiments a significant upregulation Adv. ...
3D‐scaffold based in vitro human tissue models accelerate disease studies and screening of pharmaceutics while improving the clinical translation of findings. Here is reported the use of human induced pluripotent stem cell (hiPSC)‐derived vascular organoid cells as a new cell source for the creation of an electrospun polycaprolactone‐bisurea (PCL‐BU) 3D‐scaffold‐based, perfused human macrovessel model. A separation protocol is developed to obtain monocultures of organoid‐derived endothelial cells (ODECs) and mural cells (ODMCs) from hiPSC vascular organoids. Shear stress responses of ODECs versus HUVECs and barrier function (by trans endothelial electrical resistance) are measured. PCL‐BU scaffolds are seeded with ODECs and ODMCs, and tissue organization and flow adaptation are evaluated in a perfused bioreactor system. ODECs and ODMCs harvested from vascular organoids can be cryopreserved and expanded without loss of cell purity and proliferative capacity. ODECs are shear stress responsive and establish a functional barrier that self‐restores after the thrombin challenge. Static bioreactor culture of ODECs/ODMCs seeded scaffolds results in a biomimetic vascular bi‐layer hierarchy, which is preserved under laminar flow similar to scaffolds seeded with primary vascular cells. HiPSC‐derived vascular organoids can be used as a source of functional, flow‐adaptive vascular cells for the creation of 3D‐scaffold based human macrovascular models.
... Changes in cell morphology in response to various mechanical stimuli such as shear stress, [33] tensile stress, [34] and pressure, [35] often lead to a physiological and pathological mechanism of cells. [36,37] Among various types of cells, the morphology of endothelial cells (ECs) is subjected to shear stresses derived from blood flow. ...
In recent years, microfluidic systems have been extensively utilized for biological analysis. The integration of pumps in microfluidic systems requires precise control of liquids and effort‐intensive set‐ups for multiplexed experiments. In this study, a 3D‐printed centrifugal pump driven by magnetic force is presented for microfluidics and biological analysis. The permanent magnets implemented in the centrifugal pump synchronized the rotation of the driving and operating parts. Precise control of the flow rate and a wide range and variety of flow profiles are achieved by controlling the rotational speed of the motor in the driving part. The compact size and contactless driving part allow simple set‐ups within commercially available culture dishes and tubes. It is demonstrated that the fabricated 3D‐printed centrifugal pump can induce laminar flow in a microfluidic device, perfusion culture of in vitro tissues, and alignment of cells under shear stress. This device has a high potential for applications in microfluidic devices and perfusion culture of cells.
... Although EC responses to flow have been investigated at both the single cell and monolayer levels, very few studies have specifically tackled the influence of cell density on EC flow responses. One study reported that only confluent ECs aligned in response to a 2 Pa shear stress 188 . Cell density also influences EC migration: while low density ECs migrate in the direction of the flow, ECs in dense monolayers move against the flow direction 136 . ...
Endothelial cells (ECs) lining all blood vessels are subjected to large mechanical stresses that regulate their structure and function in health and disease. Here, we review EC responses to substrate-derived biophysical cues, namely topography, curvature, and stiffness, as well as to flow-derived stresses, notably shear stress, pressure, and tensile stresses. Because these mechanical cues in vivo are coupled and are exerted simultaneously on ECs, we also review the effects of multiple cues and describe burgeoning in vitro approaches for elucidating how ECs integrate and interpret various mechanical stimuli. We conclude by highlighting key open questions and upcoming challenges in the field of EC mechanobiology.
... (1) In tissues, the application of stretch induces elongation of cells in the strain direction [160], in contrast to the transverse alignment found in single cells. Indeed, because adherens junctions enable the propagation of tension from one cell to another [188], the strategy of perpendicular orientation does not reduce cellular tension. Instead, by elongating in the strain direction, cells redistribute the available material and reduce the tension [160]). ...
... Indeed, my system is the first one to allow the direct application of tension on a confluent endothelium, thanks to flow actuation. Because the monolayer is grown on a soft hydrogel, it can be viewed as "a thin stiff film bound to a soft elastic substrate", as perfectly described by Harris et al. for an epithelium on a hydrogel [188]. The collagen hydrogel in my chip has a Young's modulus of around 1 kPa, while the Young's modulus of a monolayer (of epithelial cells) has been reported to be around 20 kPa [187]. ...
... Although EC responses to flow have been investigated at both the single cell and monolayer levels, very few studies have specifically tackled the influence of cell density on EC flow responses. One study reported that only confluent ECs aligned in response to a 2 Pa shear stress 188 . Cell density also influences EC migration: while low density ECs migrate in the direction of the flow, ECs in dense monolayers move against the flow direction 136 . ...
Endothelial cells (ECs) lining the inner surfaces of blood vessels sense and respond to the numerous mechanical forces present in the microvascular environment. Although the influence of wall shear stress, stiffness and curvature has been thoroughly investigated, the role of strain and wall tension remains less clear, most notably in the case of confluent monolayers. In vitro platforms such as organ-on-chips are ideal systems to investigate EC mechanobiology as they offer a controllable and well defined set of mechanical stimuli.I started by developing a hydrogel-based microvessel-on-chip that encompasses both shear stress and circumferential strain in a pulsatile manner based on luminal flow actuation. The concept is the following: by imposing a given flow rate inside the channel, the luminal pressure is increased due to the channel's hydraulic resistance, which dilates the vessel. I then performed an extensive characterization of the mechanical behavior of the system and demonstrated that the shear and strain span the physiological ranges. Because both stresses derive from the luminal pressure, shear and strain are tightly coupled. To enable independent control of each stress, I explored three strategies: (1) adding a hydraulic resistance of variable length at the channel output, (2) changing the width of the hydrogel, and (3) tuning the hydrogel concentration. I also demonstrated the endothelial monolayer has an effect of flow shielding which explains the higher deformation obtained in endothelialized channels relative to cell-free channels.The luminal flow actuation in the microvessel-on-chip can also be viewed as a hydraulic compression assay. The second part of my Ph.D. was devoted to the study of the dynamics of the poroelastic gel in this novel assay. Three major responses were investigated: the dynamics of the channel dilation after a pressure step, the strain distribution in the hydrogel and wave propagation. Each of these responses leads to complex dynamic behavior that can be exploited to derive the important poroelastic parameters of the hydrogel, most notably the Young's modulus and the permeability.The final part of my Ph.D. was the investigation of the response of EC monolayers to static tensile stresses where I discovered a new behavior. ECs showed a strong reorganization, aligning their overall shape, cytoskeleton and nuclei in the stretch direction, and the effect was magnitude dependent. Associated with this overall reorientation, adherens junctions (AJs) remodeled into focal AJs (FAJs), a specialized structure that forms under tension. The cortical network of actin filaments also remodeled into thick bundles of central stress fibers. The association of the stress fibers and FAJs enabled the formation of circumferential transendothelial actin cables. In parallel, I proposed a hypothetical framework for the long term monolayer mechanics observed in this assay.In summary, during my Ph.D. I developed a microvessel-on-chip that can subject cells to wall tension, characterized its mechanical behavior in detail, and investigated the response of the cells to the tensile stresses in this system, revealing a novel collective behavior.
... For all the experiments shown here, ECs were plated on coverslips functionalized with fibronectin (20 mg mL À1 ) and grown to near confluency in RPMI-1640 medium at 37 C humidified atmosphere and in the presence of 5% CO 2 . We chose near rather than full confluency because EC elongation along the flow in an in vitro setting is either observed on fully confluent cells after prolonged shear-flow stimulation (typically 24 h) or at shorter times when seeding the cells slightly below full confluency (19,20,32,33). We opted for the latter to reduce the experimental times and minimize the occurrence of bubbles inside the chamber. ...
The leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well-established, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow-chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces caused actin re-arrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1 engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited shorter-lived interactions with ECs than when adhered to non-mechanically stimulated ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shear-forces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscores the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.
... This overall decrease of the intercellular stress and junctional tension could retain the integrity of cell-cell junctions and maintain EC barrier function. Further, the crucial effects of the cell collectivity in driving large-scale EC-alignment under flow is most clearly seen in experiments showing that isolated ECs elongate their cell body in response to flow, but do not align with their neighbors in contrast to those in a confluent monolayer83 . ...
Both biological and engineering approaches have contributed significantly to the recent advance in the field of mechanobiology. Collaborating with biologists, bio-engineers and materials scientists have employed the techniques stemming from the conventional semiconductor industry to rebuild cellular milieus that mimic critical aspects of in vivo conditions and elicit cell/tissue responses in vitro . Such reductionist approaches have help to unveil important mechanosensing mechanism in both cellular and tissue level, including stem cell differentiation and proliferation, tissue expansion, wound healing, and cancer metastasis. In this mini-review, we discuss various microfabrication methods that have been applied to generate specific properties and functions of designer substrates/devices, which disclose cell-microenvironment interactions and the underlying biological mechanisms. In brief, we emphasize on the studies of cell/tissue mechanical responses to substrate adhesiveness, stiffness, topography, and shear flow. Moreover, we comment on the new concepts of measurement and paradigms for investigations of biological mechanotransductions that are yet to emerge due to on-going interdisciplinary efforts in the fields of mechanobiology and microengineering.
... Consistent with reports by others [17,44], the ECs appeared more elongated under perfusion while their F-actin remodeled from cortical stress fibers located at the cell periphery to dorsal stress fibers throughout the cytoplasm. However, we did not observe a predominant global cell alignment, which could indicate either that a certain threshold of shear stress was not reached with the applied flow protocol [45], that reorientation was hindered by the high density of the cells [46], or that adhesion of the monolayer to the underlying basement membrane was not sufficiently strong [47]. ...
... Indeed, we used a high cell seeding density to ensure the formation of a tightly connected network of ECs. However, it has been previously shown that overconfluence can impede the alignment of cells in the monolayer in the direction of flow within the observation period of 48 h [46]. Regulation of cell morphology and F-actin organization in response to shear stress has been additionally shown to require a firm anchorage to the basement membrane via focal adhesionassociated proteins, such as vinculin [48,49]. ...
Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e., endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFβ, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.
... In addition, perfusion culture causes cell alignment of VECs and LECs in the microchannels (Fig. 4). It is well known that VECs typically align with the flow direction as a consequence of shear stress in order to strength the cell-cell junctions and stabilize vessel structures [48,49]. Lymphatics exhibit similar cell alignment behavior, but over a different range of shear stresses [50]. ...
In this study, we modeled lymphangiogenesis and vascular angiogenesis in a microdevice using a tissue-engineering approach. Lymphatic vessels (LV) and blood vessels (BV) were fabricated by sacrificial molding with seeding human lymphatic endothelial cells and human umbilical vein endothelial cells into molded microchannels (600-μm diameter). During subsequent perfusion culture, lymphangiogenesis and vascular angiogenesis were induced by addition of phorbol 12-myristate 13-acetate (PMA) and VEGF-C or VEGF-A and characterized by podoplanin and Prox-1 expression. Our results showed that while blood capillaries consistently exhibited zipper-like junctions, lymphatic capillaries formed button-like junctions when treated with dexamethasone. To test the potential for screening anti-angiogenic (vascular and lymphatic) factors, antagonists of VEGF were introduced. We found that an inhibitor of VEGF-R3 did not completely suppress lymphatic angiogenesis with BVs present, although lymphatic angiogenesis was selectively prevented by addition of a VEGF-R3 inhibitor without BVs. To probe the mechanism of action, we focus on matrix metalloproteinase (MMP) secretion by vascular endothelial cells and lymphatic endothelial cells under monoculture or co-culture conditions. We found that vascular angiogenesis facilitated lymphangiogenesis via remodeling of the local microenvironment by the increased secretion of MMP, mainly by vascular endothelial cells. Applications of this model include a drug-screening assay for corneal disease and models for tumorigenesis including lymphatic angiogenesis and vascular angiogenesis.
