Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

Application of perfluoropolyether elastomers in microfluidic drug metabolism assays.

Recently, increasing attention has been paid to liver-on-a-chip models for both pharmacokinetics and toxicity (ADMET) screenings. Although polydimethylsiloxane (PDMS) is the most popular material for the fabrication of microfluidic devices, its extensive sorption of hydrophobic drugs limits its applications. Therefore, we investigated a chemically repellent material, perfluoropolyether (PFPE) elastomer, as an alternative to PDMS. Primary rat hepatocytes cultured in the PFPE microfluidic device were polygonal or cuboidal in shape and had one or two prominent nuclei, as when cultured in 96-well plates. When hepatocytes were cultured in the PFPE microfluidic device and exposed to dynamic flow, the production of albumin and urea increased 3.94- and 1.72-fold, respectively, compared with no dynamic flow. Exposure to dynamic flow did not result in obvious changes in the expression of cytochrome P450, but increased the metabolic activity of hepatocytes compared to under static conditions. PFPE devices did not absorb midazolam, which was extensively absorbed by PDMS devices. However, the sorption of bufuralol could not be avoided even with PFPE devices. Solvent swelling experiments highlighted much better chemical repellency with PFPE than with PDMS. Hansen solubility parameters and sphere radius were estimated from the solvent swelling experiments. The relative energy distance (RED) of bufuralol to PFPE was much smaller than that of other three drugs tested, reasonably explaining the high sorption of bufuralol to PFPE. Although sorption into PFPE cannot be completely avoided, PFPE microfluidic devices may provide a better performance in ADMET evaluation than PDMS.

SARS-CoV-2 disrupts respiratory vascular barriers by suppressing Claudin-5 expression.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Generation of Tetrafluoroethylene-Propylene Elastomer-Based Microfluidic Devices for Drug Toxicity and Metabolism Studies.

Polydimethylsiloxane (PDMS) is widely used to fabricate microfluidic organs-on-chips. Using these devices (PDMS-based devices), the mechanical microenvironment of living tissues, such as pulmonary respiration and intestinal peristalsis, can be reproduced in vitro. However, the use of PDMS-based devices in drug discovery research is limited because of their extensive absorption of drugs. In this study, we investigated the feasibility of the tetrafluoroethylene-propylene (FEPM) elastomer to fabricate a hepatocyte-on-a-chip (FEPM-based hepatocyte chip) with lower drug absorption. The FEPM-based hepatocyte chip expressed drug-metabolizing enzymes, drug-conjugating enzymes, and drug transporters. Also, it could produce human albumin. Although the metabolites of midazolam and bufuralol were hardly detected in the PDMS-based hepatocyte chip, they were detected abundantly in the FEPM-based hepatocyte chip. Finally, coumarin-induced hepatocyte cytotoxicity was less severe in the PDMS-based hepatocyte chip than in the FEPM-based hepatocyte chip, reflecting the different drug absorptions of the two chips. In conclusion, the FEPM-based hepatocyte chip could be a useful tool in drug discovery research, including drug metabolism and toxicity studies.

Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology.

Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multiciliated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.

Usability of Polydimethylsiloxane-Based Microfluidic Devices in Pharmaceutical Research Using Human Hepatocytes.

A liver-on-a-chip (liver-chip) is a microfluidic device carrying liver cells such as human hepatocytes. It is used to reproduce a part of liver function. Many microfluidic devices are composed of polydimethylsiloxane (PDMS), which is a type of silicone elastomer. PDMS is easy to process and suitable for cell observation, but its high hydrophobicity carries the risk of drug absorption. In this study, we evaluated drug absorption to the PDMS device and investigated the drug responsiveness of human hepatocytes cultured in the PDMS device (hepatocyte-chips). First, the absorption rates of 12 compounds to the PDMS device were measured. The absorption rates of midazolam, bufuralol, cyclosporine A, and verapamil were 92.9, 71.7, 71.4, and 99.6%, respectively, but the other compounds were poorly absorbed. Importantly, the absorption rate of the compounds was correlated with their octanol/water distribution coefficient (log D) values (R2 = 0.76). Next, hepatocyte-chips were used to examine the response to drugs, which are typically used to evaluate hepatic functions. Using the hepatocyte-chips, we could confirm the responsiveness of drugs including cytochrome P450 (CYP) inducers and farnesoid X receptor (FXR) ligands. We believe that our findings will contribute to drug discovery research using PDMS-based liver-chips.

Biomimetic aorta-gonad-Mesonephros-on-a-Chip to study human developmental hematopoiesis.

A fundamental limitation in the derivation of hematopoietic stem and progenitor cells is the imprecise understanding of human developmental hematopoiesis. Herein we established a multilayer microfluidic Aorta-Gonad-Mesonephros (AGM)-on-a-chip to emulate developmental hematopoiesis from pluripotent stem cells. The device consists of two layers of microchannels separated by a semipermeable membrane, which allows the co-culture of human hemogenic endothelial (HE) cells and stromal cells in a physiological relevant spatial arrangement to replicate the structure of the AGM. HE cells derived from human induced pluripotent stem cells (hiPSCs) were cultured on a layer of mesenchymal stromal cells in the top channel while vascular endothelial cells were co-cultured on the bottom side of the membrane within the microfluidic device. We show that this AGM-on-a-chip efficiently derives endothelial-to-hematopoietic transition (EHT) from hiPSCs compared with regular suspension culture. The presence of mesenchymal stroma and endothelial cells renders functional HPCs in vitro. We propose that the AGM-on-a-chip could serve as a platform to dissect the cellular and molecular mechanisms of human developmental hematopoiesis.

Editorial for the Special Issue on Organs-on-Chips.

Recent advances in microsystems technology and cell culture techniques have led to the development of organ-on-chip microdevices to model functional units of organs [...].

Tetrafluoroethylene-Propylene Elastomer for Fabrication of Microfluidic Organs-on-Chips Resistant to Drug Absorption.

Organs-on-chips are microfluidic devices typically fabricated from polydimethylsiloxane (PDMS). Since PDMS has many attractive properties including high optical clarity and compliance, PDMS is very useful for cell culture applications; however, PDMS possesses a significant drawback in that small hydrophobic molecules are strongly absorbed. This drawback hinders widespread use of PDMS-based devices for drug discovery and development. Here, we describe a microfluidic cell culture system made of a tetrafluoroethylene-propylene (FEPM) elastomer. We demonstrated that FEPM does not absorb small hydrophobic compounds including rhodamine B and three types of drugs, nifedipine, coumarin, and Bay K8644, whereas PDMS absorbs them strongly. The device consists of two FEPM layers of microchannels separated by a thin collagen vitrigel membrane. Since FEPM is flexible and biocompatible, this microfluidic device can be used to culture cells while applying mechanical strain. When human umbilical vein endothelial cells (HUVECs) were subjected to cyclic strain (~10%) for 4 h in this device, HUVECs reoriented and aligned perpendicularly in response to the cyclic stretch. Moreover, we demonstrated that this device can be used to replicate the epithelial-endothelial interface as well as to provide physiological mechanical strain and fluid flow. This method offers a robust platform to produce organs-on-chips for drug discovery and development.

Effects of age-dependent changes in cell size on endothelial cell proliferation and senescence through YAP1.

Angiogenesis - the growth of new blood capillaries- is impaired in aging animals. Biophysical factors such as changes in cell size control endothelial cell (EC) proliferation and differentiation. However, the effects of aging on EC size and the mechanism by which changes in cell size control age-dependent decline in EC proliferation are largely unknown. Here, we have demonstrated that aged ECs are larger than young ECs and that age-dependent increases in EC size control EC proliferation and senescence through CDC42-Yes-associated protein (YAP1) signaling. Reduction of aged EC size by culturing on single-cell sized fibronectin-coated smaller islands decreases CDC42 activity, stimulates YAP1 nuclear translocation and attenuates EC senescence. Stimulation of YAP1 or inhibition of CDC42 activity in aged ECs also restores blood vessel formation. Age-dependent changes in EC size and/or CDC42 and YAP1 activity may be the key control point of age-related decline in angiogenesis.

Engineering of vascularized 3D cell constructs to model cellular interactions through a vascular network.

Current in vitro 3D culture models lack a vascular system to transport oxygen and nutrients, as well as cells, which is essential to maintain cellular viability and functions. Here, we describe a microfluidic method to generate a perfusable vascular network that can form inside 3D multicellular spheroids and functionally connect to microchannels. Multicellular spheroids containing endothelial cells and lung fibroblasts were embedded within a hydrogel inside a microchannel, and then, endothelial cells were seeded into both sides of the hydrogel so that angiogenic sprouts from the cell spheroids and the microchannels were anastomosed to form a 3D vascular network. Solution containing cells and reagents can be perfused inside the cell spheroids through the vascular network by injecting it into a microchannel. This method can be used to study cancer cell migration towards 3D co-culture spheroids through a vascular network. We recapitulated a bone-like microenvironment by culturing multicellular spheroids containing osteo-differentiated mesenchymal stem cells (MSCs), as well as endothelial cells, and fibroblasts in the device. After the formation of vascularized spheroids, breast cancer cells were injected into a microchannel connected to a vascular network and cultured for 7 days on-chip to monitor cellular migration. We demonstrated that migration rates of the breast cancer cells towards multicellular spheroids via blood vessels were significantly higher in the bone-like microenvironment compared with the microenvironment formed by undifferentiated MSCs. These findings demonstrate the potential value of the 3D vascularized spheroids-on-a-chip for modeling in vivo-like cellular microenvironments, drug delivery through blood vessels, and cellular interactions through a vascular network.

Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device.

Creating vascular networks in tissues is crucial for tissue engineering. Although recent studies have demonstrated the formation of vessel-like structures in a tissue model, long-term culture is still challenging due to the lack of active perfusion in vascular networks. Here, we present a method to create a three-dimensional cellular spheroid with a perfusable vascular network in a microfluidic device. By the definition of the cellular interaction between human lung fibroblasts (hLFs) in a spheroid and human umbilical vein endothelial cells (HUVECs) in microchannels, angiogenic sprouts were induced from microchannels toward the spheroid; the sprouts reached the vessel-like structures in a spheroid to form a continuous lumen. We demonstrated that the vascular network could administer biological substances to the interior of the spheroid. As cell density in the spheroid is similar to that of a tissue, the perfusable vasculature model opens up new possibilities for a long-term tissue culture in vitro.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

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

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

Engineered in vitro disease models.

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro.

Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

Microengineered physiological biomimicry: organs-on-chips.

Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

Mechanochemical control of mesenchymal condensation and embryonic tooth organ formation.

Mesenchymal condensation is critical for organogenesis, yet little is known about how this process is controlled. Here we show that Fgf8 and Sema3f, produced by early dental epithelium, respectively, attract and repulse mesenchymal cells, which cause them to pack tightly together during mouse tooth development. Resulting mechanical compaction-induced changes in cell shape induce odontogenic transcription factors (Pax9, Msx1) and a chemical cue (BMP4), and mechanical compression of mesenchyme is sufficient to induce tooth-specific cell fate switching. The inductive effects of cell compaction are mediated by suppression of the mechanical signaling molecule RhoA, and its overexpression prevents odontogenic induction. Thus, the mesenchymal condensation that drives tooth formation is induced by antagonistic epithelial morphogens that manifest their pattern-generating actions mechanically via changes in mesenchymal cell shape and altered mechanotransduction.

High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array.

Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.

Transwells with microstamped membranes produce micropatterned two-dimensional and three-dimensional co-cultures.

This article describes a simple and rapid cell patterning method to form co-culture microarrays in commercially available Transwells. A thin poly(dimethylsiloxane) (PDMS) layer is printed on the underside of a Transwell using a PDMS stamp. Arbitrary cellular patterns are generated according to the geometric features of the thin PDMS layer through hydrodynamic forces that guide cells onto the membrane only over the PDMS-uncoated regions. Micropatterns of surface-adhered cells (we refer to this as two-dimensional) or non-surface-adhered clusters of cells (we refer to this as three-dimensional) can be generated depending on the surface treatment of the filter membrane. Additionally, co-cultures can be established by introducing different types of cells on the membrane or in the bottom chamber of the Transwell. We show that this co-culture method can evaluate mouse embryonic stem (mES) cell differentiation based on heterogeneous cell-cell interactions. Co-culture of mES cells and HepG2 cells decreased SOX17 expression of mES cells, and direct cell-cell contact further decreased SOX17 expression, indicating that co-culture with HepG2 cells inhibits endoderm differentiation through soluble factors and cell-cell contact. This method is simple and user-friendly and should be broadly useful to study cell shapes and cell-cell interactions.