Perfusion of 3D encapsulated hepatocytes—A synergistic effect enhancing long‐term functionality in bioreactors

Long‐term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day⁻¹, for three operative weeks. Perfusion feeding led to a 10‐fold improvement in albumin synthesis in bioreactors containing non‐encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10⁻⁹ cm² s⁻¹ and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver‐specific functions of long‐term hepatocyte spheroid cultures. Biotechnol. Bioeng. 2011; 108:41–49. © 2010 Wiley Periodicals, Inc.     

Hydrogel‐based three‐dimensional cell culture for organ‐on‐a‐chip applications

Recent studies have reported that three‐dimensionally cultured cells have more physiologically relevant functions than two‐dimensionally cultured cells. Cells are three‐dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ‐on‐a‐chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation‐based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ‐on‐a‐chip. Last, we also discuss the challenges of hydrogel‐based three‐dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580–589, 2017     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

Disposable luciferase‐based microfluidic chip for rapid assay of water pollution

In the present study, we demonstrate the use of a disposable luciferase‐based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3‐dihydroxybenzene and 1,4‐benzoquinone for the proposed microfluidic biosensor measured 3 μM, 15 mM, and 2 μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.     

Quercetin and hydroxytyrosol as modulators of hepatic steatosis: A NAFLD‐on‐a‐chip study

Organs‐on‐chip (OoCs) are catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative. OoCs enable the creation of three‐dimensional (3D) tissue microenvironments with physiological and pathological relevance at unparalleled precision and complexity, offering new opportunities to model human diseases and to test the potential therapeutic effect of drugs, while overcoming the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver‐on‐a‐chip model to investigate the effects of two naturally occurring polyphenols, namely quercetin and hydroxytyrosol, on nonalcoholic fatty liver disease (NAFLD) using a high‐content analysis readout methodology. NAFLD is currently the most common form of chronic liver disease; however, its complex pathogenesis is still far from being elucidated, and no definitive treatment has been established so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid‐induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant and lipid‐lowering properties. In conclusion, the findings of the present work could guide novel strategies to contrast the onset and progression of NAFLD.     

Inhibition of miR‐103a‐3p suppresses lipopolysaccharide‐induced sepsis and liver injury by regulating FBXW7 expression

Inflammation, apoptosis, and oxidative stress are involved in septic liver dysfunction. Herein, the role of miR‐103a‐3p/FBXW7 axis in lipopolysaccharides (LPS)‐induced septic liver injury was investigated in mice. Hematoxylin‐eosin staining was used to evaluate LPS‐induced liver injury. Quantitative real‐time polymerase chain reaction was performed to determine the expression of microRNA (miR) and messenger RNA, and western blot analysis was conducted to examine the protein levels. Dual‐luciferase reporter assay was used to confirm the binding between miR‐103a‐3p and FBXW7. Both annexin V‐fluoresceine isothiocyanate/propidium iodide staining and caspase‐3 activity were employed to determine cell apoptosis. First, miR‐103a‐3p was upregulated in the septic serum of mice and patients with sepsis, and miR‐103a‐3p was elevated in the septic liver of LPS‐induced mice. Then, interfering miR‐103a‐3p significantly decreased apoptosis by suppressing Bax expression and upregulating Bcl‐2 levels in LPS‐induced AML12 and LO2 cells, and septic liver of mice. Furthermore, inhibition of miR‐103a‐3p repressed LPS‐induced inflammation by downregulating the expression of tumor necrosis factor, interleukin 1β, and interleukin 6 in vitro and in vivo. Meanwhile, interfering miR‐103a‐3p obviously attenuated LPS‐induced overactivation of oxidation via promoting expression of antioxidative enzymes, including catalase, superoxide dismutase, and glutathione in vitro and in vivo. Moreover, FBXW7 was a target of miR‐103a‐3p, and overexpression of FBXW7 significantly ameliorated LPS‐induced septic liver injury in mice. Finally, knockdown of FBXW7 markedly reversed anti‐miR‐103a‐3p‐mediated suppression of septic liver injury in mice. In conclusion, interfering miR‐103a‐3p or overexpression of FBXW7 improved LPS‐induced septic liver injury by suppressing apoptosis, inflammation, and oxidative reaction.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.