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Metabolic dysfunction‐associated steatotic liver disease (MASLD) is hallmarked by hepatic steatosis, cell injury, inflammation, and fibrosis. This study elaborates on a multicellular biochip‐based liver sinusoid model to mimic MASLD pathomechanisms and investigate the therapeutic effects of drug candidates lanifibranor and resmetirom. Mouse liver primary hepatocytes, hepatic stellate cells, Kupffer cells, and endothelial cells are seeded in a dual‐chamber biocompatible liver‐on‐a‐chip (LoC). The LoC is then perfused with circulating immune cells (CICs). Acetaminophen (APAP) and free fatty acids (FFAs) treatment recapitulate acute drug‐induced liver injury and MASLD, respectively. As a benchmark for the LoC, multiplex immunofluorescence on livers from APAP‐injected and dietary MASLD‐induced mice reveals characteristic changes on parenchymal and immune cell populations. APAP exposure induces cell death in the LoC, and increased inflammatory cytokine levels in the circulating perfusate. Under FFA stimulation, lipid accumulation, cellular damage, inflammatory secretome, and fibrogenesis are increased in the LoC, reflecting MASLD. Both injury conditions potentiate CIC migration from the perfusate to the LoC cellular layers. Lanifibranor prevents the onset of inflammation, while resmetirom decreases lipid accumulation in hepatocytes and increases the generation of FFA metabolites in the LoC. This study demonstrates the LoC potential for functional and molecular evaluation of liver disease drug candidates. [ABSTRACT FROM AUTHOR]

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells. [ABSTRACT FROM AUTHOR]

The gut is a tubular organ responsible for nutrient absorption and harbors our intestinal microbiome. This organ is composed of a multitude of specialized cell types arranged in complex barrier-forming crypts and villi covered by a mucosal layer controlling nutrient passage and protecting from invading pathogens. The development and self-renewal of the intestinal epithelium are guided by niche signals controlling the differentiation of specific cell types along the crypt-villus axis in the epithelium. The emergence of microphysiological systems, or organ-on-chips, has paved the way to study the intestinal epithelium within a dynamic and controlled environment. In this review, we describe the use of organ-on-chip technology to control and guide these differentiation processes in vitro. We further discuss current applications and forthcoming strategies to investigate the mechanical processes of intestinal stem cell differentiation, tissue formation, and the interaction of the intestine with the microbiota in the context of gastrointestinal diseases. [ABSTRACT FROM AUTHOR]

Fungal infections (mycoses) affect over a billion people per year. Approximately, two million of these infections are life-threatening, especially for patients with a compromised immune system. Fungi of the genera Aspergillus , Candida , Histoplasma and Cryptococcus are opportunistic pathogens that contribute to a substantial number of mycoses. To optimize the diagnosis and treatment of mycoses, we need to understand the complex fungal–host interplay during pathogenesis, the fungal attributes causing virulence and how the host resists infection via immunological defenses. In vitro models can be used to mimic fungal infections of various tissues and organs and the corresponding immune responses at near-physiological conditions. Furthermore, models can include fungal interactions with the host–microbiota to mimic the in vivo situation on skin and mucosal surfaces. This article reviews currently used in vitro models of fungal infections ranging from cell monolayers to microfluidic 3D organ-on-chip (OOC) platforms. We also discuss how OOC models can expand the toolbox for investigating interactions of fungi and their human hosts in the future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: In this work, Raman spectroscopic cell imaging approaches and a discrimination between HepG2, nondifferentiated hepatic stem cell line HepaRG, and differentiated hepatocyte‐like HepaRG cells are presented. Raman spectroscopic imaging was used to visualize the cell nuclei by means of false color imaging using a marker band, and a cell segmentation was performed by means of clustering. Furthermore, a 3‐class‐classification model based on the mean Raman spectra of individual cells was established for a classification between different cell types. A high average sensitivity of 96% was achieved by the applied classification model. Based on the results of clustering and classification, the main spectral contributions to different cell types and cell segments were analyzed in detail. Thereby, HepG2, nondifferentiated hepatic stem cell line HepaRG, and differentiated hepatocyte‐like HepaRG cells were Raman spectroscopically characterized and proven to be significantly different. [ABSTRACT FROM AUTHOR]

(1) Background: Epithelial ovarian cancer (EOC) is the most lethal cancer of the female reproductive system. In an earlier study, we identified multiple genes as hypermethylated in tumors of patients with poor prognosis. The most promising combination of markers to predict a patient's outcome was CaMKIINa and RUNX3. Aim of this study was to functionally validate the importance of both genes. (2) Methods: IC50 measurements, cell cycle distribution-, proliferation, and migration experiments were conducted after transgene overexpression in two EOC cell lines. (3) Results: We showed that CaMKIINa has tumor suppressive functions in vitro and reduces proliferation, migration, and colony formation. However, it had no effect on the reversion of the resistance to cisplatin. RUNX3 exhibited dualistic functions related to cisplatin sensitivity and migration capacity, depending on the respective transcript variant (TV). A2780 cells expressing RUNX3 TV2--the promoter of which harbors a CpG (50-C-phosphate-G-30) island and is potentially inactivated by hypermethylation--exhibited increased cisplatin sensitivity and reduced migration properties. However, RUNX3 TV1, not affected by CpG island methylation could be characterized as mediating resistance and enhancing migration in A2780. The higher resistance of RUNX3 TV1 transfected cells correlates with a reduction of cell proliferation. Moreover, RUNX3 TV1 expressing cells exhibit a reduced cell cycle arrest at the gap-2 or mitosis phase (G2/M) under cisplatin treatment comparable to resistant A2780 subcultures. (4) Conclusion: It appears that CaMKIINα and RUNX3 TV2 can reduce the malignant potential of EOC cells. [ABSTRACT FROM AUTHOR]

Background: Taurolidine has been used for peritonitis, oncological and catheter-lock treatment because of its anti-inflammatory properties. It has been suggested that taurolidine has no severe side-effects, but after long-term use morphological and functional changes of the liver were reported. The aim of this study was to investigate the effect of short-term use of taurolidine on the liver. Methods: In HepaRG cell cultures and on a novel liver biochip dose-dependent effects of taurolidine treatment on hepatocyte adherence and cell viability was investigated. Furthermore, liver enzymes and interleukin- (IL-) 6 were measured in supernatants. Male rats were treated with low- or high-dose taurolidine, respectively, and compared to controls with physiological saline solution administration regarding blood serum parameters and histology. Results: In HepaRG cell cultures, hepatocyte adherence was significantly decreased, cell death and cleaved caspase-3 were significantly increased after administration of taurolidine in a dose-dependent manner. High-dose application of taurolidine led to elevated liver enzymes and IL-6 secretion in hepatic organoid. After 24 h a significant increase of serum GLDH and ASAT was observed in rats treated with high-dose taurolidine treatment. Conclusions: Our results suggest that taurolidine caused liver injury after short-term use in in vitro and in vivo models probably due to direct toxic effects on hepatocytes. Therefore, the taurolidine dose should be titrated in further investigations regarding liver injury and inflammation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The development of therapeutic substances to treat diseases of the central nervous system is hampered by the tightness and selectivity of the blood-brain barrier. Moreover, testing of potential drugs is time-consuming and cost-intensive. Here, we established a new microfluidically supported, biochip-based model of the brain endothelial barrier in combination with brain cortical spheroids suitable to detect effects of neuroinflammation upon disruption of the endothelial layer in response to inflammatory signals. Unilateral perfusion of the endothelial cell layer with a cytokine mix comprising tumor necrosis factor, IL-1β, IFNγ, and lipopolysaccharide resulted in a loss of endothelial von Willebrand factor and VE-cadherin expression accompanied with an increased leakage of the endothelial layer and diminished endothelial cell viability. In addition, cytokine treatment caused a loss of neocortex differentiation markers Tbr1, Tbr2, and Pax6 in the cortical spheroids concomitant with reduced cell viability and spheroid integrity. From these observations, we conclude that our endothelial barrier/cortex model is suitable to specifically reflect cytokine-induced effects on barrier integrity and to uncover damage and impairment of cortical tissue development and viability. With all its limitations, the model represents a novel tool to study cross-communication between the brain endothelial barrier and underlying cortical tissue that can be utilized for toxicity and drug screening studies focusing on inflammation and neocortex formation. [ABSTRACT FROM AUTHOR]

Background: Francisella tularensis, a gram-negative bacterium replicates intracellularly within macrophages and efficiently evades the innate immune response. It is able to infect and replicate within Kupffer cells, specialized tissue macrophages of the liver, and to modulate the immune response upon infection to its own advantage. Studies on Francisella tularensis liver infection were mostly performed in animal models and difficult to extrapolate to the human situation, since human infections and clinical observations are rare. Results: Using a human co-culture model of macrophages and hepatocytes we investigated the course of infection of three Francisella tularensis strains (subspecies holarctica – wildtype and live vaccine strain, and mediasiatica - wildtype) and analyzed the immune response triggered upon infection. We observed that hepatocytes support the intracellular replication of Franciscella species in macrophages accompanied by a specific immune response inducing TNFα, IL-1β, IL-6 and fractalkine (CX3CL1) secretion and the induction of apoptosis. Conclusions: We could demonstrate that this human macrophage / hepatocyte co-culture model reflects strain-specific virulence of Francisella tularensis. We developed a suitable tool for more detailed in vitro studies on the immune response upon liver cell infection by F. tularensis. [ABSTRACT FROM AUTHOR]

Simple Summary: Epithelial ovarian cancer treatment is limited by missing predictive markers, frequent chemotherapy resistance and an incomplete understanding of the biology of tumors. Earlier work proved that hypermethylation of the gene RUNX3 coding for a transcription factor has prognostic value, and RUNX3 transcript variant overexpression, regulated by this epigenetic mechanism, influences cisplatin sensitivity and malignant properties of cells contrary. The present data validate RUNX3 transcript variant-specific effects for high-grade serous ovarian cancer and identify RUNX3-regulated genes and processes. Specifically, DNA damage repair and angiogenesis are influenced by RUNX3, and transcript variant 1 mediates stronger carcinogenic properties. The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis. [ABSTRACT FROM AUTHOR]

Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20–30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32–48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) is activated by vascular endothelial growth factor (VEGF) in endothelial cells and it is significantly involved in VEGF-induced angiogenesis. This study investigates whether the VEGF/AMPK pathway regulates autophagy in endothelial cells and whether this is linked to its pro-angiogenic role. We show that VEGF leads to AMPKα1-dependent phosphorylation of Unc-51-like kinase 1 (ULK1) at its serine residue 556 and to the subsequent phosphorylation of the ULK1 substrate ATG14. This triggers initiation of autophagy as shown by phosphorylation of ATG16L1 and conjugation of the microtubule-associated protein light chain 3B, which indicates autophagosome formation; this is followed by increased autophagic flux measured in the presence of bafilomycin A1 and by reduced expression of the autophagy substrate p62. VEGF-induced autophagy is transient and probably terminated by mechanistic target of rapamycin (mTOR), which is activated by VEGF in a delayed manner. We show that functional autophagy is required for VEGF-induced angiogenesis and may have specific functions in addition to maintaining homeostasis. In line with this, inhibition of autophagy impaired VEGF-mediated formation of the Notch intracellular domain, a critical regulator of angiogenesis. Our study characterizes autophagy induction as a pro-angiogenic function of the VEGF/AMPK pathway and suggests that timely activation of autophagy-initiating pathways may help to initiate angiogenesis. [ABSTRACT FROM AUTHOR]

This corrects the article DOI: 10.1038/srep21868 [ABSTRACT FROM AUTHOR]

Harvesting cultivated macrophages for tissue engineering purposes by enzymatic digestion of cell adhesion molecules can potentially result in unintended activation, altered function, or behavior of these cells. Thermo-responsive polymer is a promising tool that allows for gentle macrophage detachment without artificial activation prior to subculture within engineered tissue constructs. We therefore characterized different species of thermo-responsive polymers for their suitability as cell substrate and to mediate gentle macrophage detachment by temperature shift. Primary human monocyte- and THP-1-derived macrophages were cultured on thermo-responsive polymers and characterized for phagocytosis and cytokine secretion in response to lipopolysaccharide stimulation. We found that both cell types differentially respond in dependence of culture and stimulation on thermo-responsive polymers. In contrast to THP-1 macrophages, primary monocyte–derived macrophages showed no signs of impaired viability, artificial activation, or altered functionality due to culture on thermo-responsive polymers compared to conventional cell culture. Our study demonstrates that along with commercially available UpCell carriers, two other thermo-responsive polymers based on poly(vinyl methyl ether) blends are attractive candidates for differentiation and gentle detachment of primary monocyte–derived macrophages. In summary, we observed similar functionality and viability of primary monocyte–derived macrophages cultured on thermo-responsive polymers compared to standard cell culture surfaces. While this first generation of custom-made thermo-responsive polymers does not yet outperform standard culture approaches, our results are very promising and provide the basis for exploiting the unique advantages offered by custom-made thermo-responsive polymers to further improve macrophage culture and recovery in the future, including the covalent binding of signaling molecules and the reduction of centrifugation and washing steps. Optimizing these and other benefits of thermo-responsive polymers could greatly improve the culture of macrophages for tissue engineering applications. [ABSTRACT FROM AUTHOR]

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells. [ABSTRACT FROM AUTHOR]