Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model.

OBJECTIVE: The liver acts as an innate immunity-dominant organ and natural killer (NK) cells, are the main lymphocyte population in the human liver. NK cells are in close interaction with other immune cells, acting as the first line of defense against pathogens, infections, and injury. A previously developed, three-dimensional, perfused liver-on-a-chip comprised of human cells was used to integrate NK cells, representing pivotal immune cells during liver injury and regeneration. The objective of this study was to integrate functional NK cells in an in vitro model of the human liver and assess utilization of the model for NK cell-dependent studies of liver inflammation. RESULTS: NK cells from human blood and liver specimen were isolated by Percoll separation with subsequent magnetic cell separation (MACS), yielding highly purified blood and liver derived NK cells. After stimulation with toll-like-receptor (TLR) agonists (lipopolysaccharides, Pam3CSK4), isolated NK cells showed increased interferon (IFN)-gamma secretion. To study the role of NK cells in a complex hepatic environment, these cells were integrated in the vascular compartment of a microfluidically supported liver-on-a-chip model in close interaction with endothelial and resident macrophages. Successful, functional integration of NK cells was verified by immunofluorescence staining (NKp46), flow cytometry analysis and TLR agonist-dependent secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. Lastly, we observed that inflammatory activation of NK cells in the liver-on-a-chip led to a loss of vascular barrier integrity. Overall, our data shows the first successful, functional integration of NK cells in a liver-on-a-chip model that can be utilized to investigate NK cell-dependent effects on liver inflammation in vitro.

Human iPSC-Derived Proinflammatory Macrophages cause Insulin Resistance in an Isogenic White Adipose Tissue Microphysiological System.

Chronic white adipose tissue (WAT) inflammation has been recognized as a critical early event in the pathogenesis of obesity-related disorders. This process is characterized by the increased residency of proinflammatory M1 macrophages in WAT. However, the lack of an isogenic human macrophage-adipocyte model has limited biological studies and drug discovery efforts, highlighting the need for human stem cell-based approaches. Here, human induced pluripotent stem cell (iPSC) derived macrophages (iMACs) and adipocytes (iADIPOs) are cocultured in a microphysiological system (MPS). iMACs migrate toward and infiltrate into the 3D iADIPOs cluster to form crown-like structures (CLSs)-like morphology around damaged iADIPOs, recreating classic histological features of WAT inflammation seen in obesity. Significantly more CLS-like morphologies formed in aged and palmitic acid-treated iMAC-iADIPO-MPS, showing the ability to mimic inflammatory severity. Importantly, M1 (proinflammatory) but not M2 (tissue repair) iMACs induced insulin resistance and dysregulated lipolysis in iADIPOs. Both RNAseq and cytokines analyses revealed a reciprocal proinflammatory loop in the interactions of M1 iMACs and iADIPOs. This iMAC-iADIPO-MPS thus successfully recreates pathological conditions of chronically inflamed human WAT, opening a door to study the dynamic inflammatory progression and identify clinically relevant therapies.

Human macrophage polarization determines bacterial persistence of Staphylococcus aureus in a liver-on-chip-based infection model.

Infections with Staphylococcus aureus (S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis. Although considered an extracellular pathogen, S. aureus can invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate macrophages is considered a critical step to evade host antimicrobial reactions. We leveraged a recently established human liver-on-chip model to demonstrate that S. aureus specifically targets macrophages as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs). In vitro, M2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about macrophage activation in the liver and its impact on bacterial persistence and dissemination in the course of infection.

Keeping Candida commensal: how lactobacilli antagonize pathogenicity of Candida albicans in an in vitro gut model.

The intestine is the primary reservoir of Candida albicans that can cause systemic infections in immunocompromised patients. In this reservoir, the fungus exists as a harmless commensal. However, antibiotic treatment can disturb the bacterial microbiota, facilitating fungal overgrowth and favoring pathogenicity. The current in vitro gut models that are used to study the pathogenesis of C. albicans investigate the state in which C. albicans behaves as a pathogen rather than as a commensal. We present a novel in vitro gut model in which the fungal pathogenicity is reduced to a minimum by increasing the biological complexity. In this model, enterocytes represent the epithelial barrier and goblet cells limit C. albicans adhesion and invasion. Significant protection against C. albicans-induced necrotic damage was achieved by the introduction of a microbiota of antagonistic lactobacilli. We demonstrated a time-, dose- and species-dependent protective effect against C. albicans-induced cytotoxicity. This required bacterial growth, which relied on the presence of host cells, but was not dependent on the competition for adhesion sites. Lactobacillus rhamnosus reduced hyphal elongation, a key virulence attribute. Furthermore, bacterial-driven shedding of hyphae from the epithelial surface, associated with apoptotic epithelial cells, was identified as a main and novel mechanism of damage protection. However, host cell apoptosis was not the driving mechanism behind shedding. Collectively, we established an in vitro gut model that can be used to experimentally dissect commensal-like interactions of C. albicans with a bacterial microbiota and the host epithelial barrier. We also discovered fungal shedding as a novel mechanism by which bacteria contribute to the protection of epithelial surfaces.This article has an associated First Person interview with the joint first authors of the paper.

A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.

CAAP48, a New Sepsis Biomarker, Induces Hepatic Dysfunction in an in vitro Liver-on-Chip Model.

Sepsis is a leading cause of mortality in the critically ill, characterized by life-threatening organ dysfunctions due to dysregulation of the host response to infection. Using mass spectrometry, we identified a C-terminal fragment of alpha-1-antitrypsin, designated CAAP48, as a new sepsis biomarker that actively participates in the pathophysiology of sepsis. It is well-known that liver dysfunction is an early event in sepsis-associated multi-organ failure, thus we analyzed the pathophysiological function of CAAP48 in a microfluidic-supported in vitro liver-on-chip model. Hepatocytes were stimulated with synthetic CAAP48 and several control peptides. CAAP48-treatment resulted in an accumulation of the hepatocyte-specific intracellular enzymes aspartate- and alanine-transaminase and impaired the activity of the hepatic multidrug resistant-associated protein 2 and cytochrome P450 3A4. Moreover, CAAP48 reduced hepatic expression of the multidrug resistant-associated protein 2 and disrupted the endothelial structural integrity as demonstrated by reduced expression of VE-cadherin, F-actin and alteration of the tight junction protein zonula occludens-1, which resulted in a loss of the endothelial barrier function. Furthermore, CAAP48 induced the release of adhesion molecules and pro- and anti-inflammatory cytokines. Our results show that CAAP48 triggers inflammation-related endothelial barrier disruption as well as hepatocellular dysfunction in a liver-on-chip model emulating the pathophysiological conditions of inflammation. Besides its function as new sepsis biomarker, CAAP48 thus might play an important role in the development of liver dysfunction as a consequence of the dysregulated host immune-inflammatory response in sepsis.

Corrigendum: Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model.

This corrects the article DOI: 10.1038/srep21868.

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research.

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy.

Near infrared spectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of near infrared spectroscopy from biochips are the basis for a novel method of fast, continuous, and contact-free analysis of cell viability and real-time monitoring of cell growth. The technique thus paves the way for a robust and reliable high-throughput analysis of biochip-embedded cell cultures.

Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model.

Liver dysfunction is an early event in sepsis-related multi-organ failure. We here report the establishment and characterization of a microfluidically supported in vitro organoid model of the human liver sinusoid. The liver organoid is composed of vascular and hepatocyte cell layers integrating non-parenchymal cells closely reflecting tissue architecture and enables physiological cross-communication in a bio-inspired fashion. Inflammation-associated liver dysfunction was mimicked by stimulation with various agonists of toll-like receptors. TLR-stimulation induced the release of pro- and anti-inflammatory cytokines and diminished expression of endothelial VE-cadherin, hepatic MRP-2 transporter and apolipoprotein B (ApoB), resulting in an inflammation-related endothelial barrier disruption and hepatocellular dysfunction in the liver organoid. However, interaction of the liver organoid with human monocytes attenuated inflammation-related cell responses and restored MRP-2 transporter activity, ApoB expression and albumin/urea production. The cellular events observed in the liver organoid closely resembled pathophysiological responses in the well-established sepsis model of peritoneal contamination and infection (PCI) in mice and clinical observations in human sepsis. We therefore conclude that this human liver organoid model is a valuable tool to investigate sepsis-related liver dysfunction and subsequent immune cell-related tissue repair/remodeling processes.

A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo.

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability.

A microfluidically perfused three dimensional human liver model.

Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.