Liver Sinusoid on a Chip: Long-Term Layered Co-Culture of Primary Rat Hepatocytes and Endothelial Cells in Microfluidic Platforms

We describe the generation of microfluidic platforms for the co-culture of primary hepatocytes and endothelial cells; these platforms mimic the architecture of a liver sinusoid. This paper describes a progressional study of creating such a liver sinusoid on a chip system. Primary rat hepatocytes (PRHs) were co-cultured with primary or established endothelial cells in layers in single and dual microchannel configurations with or without continuous perfusion. Cell viability and maintenance of hepatocyte functions were monitored and compared for diverse experimental conditions. When primary rat hepatocytes were co-cultured with immortalized bovine aortic endothelial cells (BAECs) in a dual microchannel with continuous perfusion, hepatocytes maintained their normal morphology and continued to produce urea for at least 30 days. In order to demonstrate the utility of our microfluidic liver sinusoid platform, we also performed an analysis of viral replication for the hepatotropic hepatitis B virus (HBV). HBV replication, as measured by the presence of cell-secreted HBV DNA, was successfully detected. We believe that our liver model closely mimics the in vivo liver sinusoid and supports long-term primary liver cell culture. This liver model could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, cancer research, and analysis of pathological effects and replication strategies of various hepatotropic infectious agents

Metabolic Patterning on a Chip: Towards in vitro Liver Zonation of Primary Rat and Human Hepatocytes

An important number of healthy and diseased tissues shows spatial variations in their metabolic capacities across the tissue. The liver is a prime example of such heterogeneity where the gradual changes in various metabolic activities across the liver sinusoid is termed as “zonation” of the liver. Here, we introduce the Metabolic Patterning on a Chip (MPOC) platform capable of dynamically creating metabolic patterns across the length of a microchamber of liver tissue via actively enforced gradients of various metabolic modulators such as hormones and inducers. Using this platform, we were able to create continuous liver tissues of both rat and human origin with gradually changing metabolic activities. The gradients we have created in nitrogen, carbohydrate and xenobiotic metabolisms recapitulated an in vivo like zonation and zonal toxic response. Beyond its application in recapitulation of liver zonation in vitro as we demonstrate here, the MPOC platform can be used and expanded for a variety of purposes including better understanding of heterogeneity in many different tissues during developmental and adult stages

Progressive Hypoxia-on-a-chip: An In Vitro Oxygen Gradient Model for Capturing the Effects of Hypoxia on Primary Hepatocytes in Health and Disease

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia‐inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia‐on‐a‐chip platform can be used to study the role of oxygen and hypoxia‐associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen‐limited conditions

Freeform Bioprinting of Liver Encapsulated in Alginate Hydrogels Tissue Constructs for Pharmacokinetic Study

An in vitro model that can be realistically and inexpensively used to predict human response to various drug administration and toxic chemical exposure is needed. By fabricating a microscale 3D physiological tissue construct consisting of an array of channels and tissue-embedded chambers, one can selectively develop various biomimicking mammalian tissues for a number of pharmaceutical applications, for example, experimental pharmaceutical screening for drug efficacy and toxicity along with apprehending the disposition and metabolic profile of a candidate drug. This paper addresses issues relating to the development and implementation of a bioprinting process for freeform fabrication of a 3D cell-encapsulated hydrogel-based tissue construct, the direct integration onto a microfluidic device for pharmacokinetic study, and the underlying engineering science for the fabrication of a 3D microscale tissue chamber as well as its application in pharmacokinetic study. To this end, a prototype 3D microfluidic tissue chamber embedded with liver cells encapsulated within a hydrogel matrix construct is bioprinted as a physiological in vitro model for pharmacokinetic study. The developed fabrication processes are further validated and parameters optimized by assessing cell viability and liver cell phenotype, in which metabolic and synthetic liver functions are quantitated.Mechanical Engineerin

Bioreactor technologies to support liver function in vitro

Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drives efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.National Institutes of Health (U.S.) (R01 EB010246)National Institutes of Health (U.S.) (P50-GM068762-08)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109)5UH2TR000496-02National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (CBET-0939511)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039

DESIGN AND DEVELOPMENT OF A MICROFLUIDIC DEVICE FOR THE ASSESSMENT OF FIRST-PASS METABOLISM

The aim of the thesis is to develop a microfluidic platform in order to mimic the first pass metabolism of oral ingested compounds. In the first part of the thesis, there is an introduction about the in vivo mechanism involved in the in process of first pass metabolism. First pass metabolism is strictly correlated to oral bioavailability of new developed drugs. The prediction of the dose of drug that reaches the blood flow and the target is fundamental. The organ involved in the first pass metabolism are principally the intestine, where a first metabolic process takes place, and the liver where the quote of drugs is metabolized again. New bioengineered in vitro model to assess first pass metabolism are explained, with a particular attention on 3D intestine and liver model. Furthermore, the first chapter is focused on the recent studies on organ-on-chip device that can recapitulate the in vivo physiology and microenvironment, with the relative steps of fabrications. To achieve the reproduction of the first pass metabolism on chip, we first focussed on the production of an innovative hepatic three dimensional tissues and then on the development of a organotypic intestinal tissues. In the chapter 2 it is presented the comparison of two kind of hepatic 3D model: spheroids and microtissues. The 3D-hepatic model chosen, was cultured into the new developed liver-on-chip device in order to have a perfusion culture. The chapter 3 is focused on the fabrication of an organotypic intestinal 3D tissues cultured in both in static and dynamic conditions. In particular a gut-on-chip microfluidic device was fabricated in order to obtain an air-liquid interface culture. The combination of the two hepatic and intestine model on chip, is addressed in chapter 4. In this last chapter a microfluidic biochip, can accommodate both hepatic microtissues and 3D human intestinal equivalent. By the selective communication of the two tissues recreated into the biochip, it is possible to simulate in vitro the mechanism of orally ingested drugs

Advanced 3D cell culture techniques in micro-bioreactors, Part II: Systems and applications

In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems

Microfabricated Physiological Models for In Vitro Drug Screening Applications

Microfluidics and microfabrication have recently been established as promising tools for developing a new generation of in vitro cell culture microdevices. The reduced amounts of reagents employed within cell culture microdevices make them particularly appealing to drug screening processes. In addition, latest advancements in recreating physiologically relevant cell culture conditions within microfabricated devices encourage the idea of using such advanced biological models in improving the screening of drug candidates prior to in vivo testing. In this review, we discuss microfluidics-based models employed for chemical/drug screening and the strategies to mimic various physiological conditions: fine control of 3D extra-cellular matrix environment, physical and chemical cues provided to cells and organization of co-cultures. We also envision future directions for achieving multi-organ microfluidic devices

Cell Culture on MEMS Platforms: A Review

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bioincompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bioincompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.Institute of Bioengineering and Nanotechnology (Singapore)Singapore. Biomedical Research CouncilSingapore. Agency for Science, Technology and ResearchSingapore. Agency for Science, Technology and Research (R-185-001-045-305)Singapore. Ministry of EducationSingapore. Ministry of Education (Grant R-185- 000-135-112)Singapore. National Medical Research CouncilSingapore. National Medical Research Council (Grant R-185-000-099-213)Jassen Cilag (Firm)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Global Enterprise for Micro-Mechanics and Molecular Medicin

Liver ‘organ on a chip’

© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systemsNational Institutes of Health (U.S.) (Grant UH3TR000496)National Institutes of Health (U.S.) (Grant UH3TR000503