Integrated-gut-liver-on-a-chip platform as an in vitro human model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) afflicts a significant percentage of the population; however, no effective treatments have yet been established because of the unsuitability of in vitro assays and animal experimental models. Here, we present an integrated-gut-liver-on-a-chip (iGLC) platform as an in vitro human model of the gut-liver axis (GLA) by co-culturing human gut and liver cell lines interconnected via microfluidics in a closed circulation loop, for the initiation and progression of NAFLD by treatment with free fatty acids (FFAs) for 1 and 7 days, respectively. Co-cultured Caco-2 gut-mimicking cells and HepG2 hepatocyte-like cells demonstrate the protective effects from apoptosis against FFAs treatment, whereas mono-cultured cells exhibit induced apoptosis. Phenotype and gene expression analyses reveal that the FFAs-treated gut and liver cells accumulated intracellular lipid droplets and show an increase in gene expression associated with a cellular response to copper ions and endoplasmic reticulum stress. As an in vitro human GLA model, the iGLC platform may serve as an alternative to animal experiments for investigating the mechanisms of NAFLD.

Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems.

A microphysiological system (MPS) holds great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform faces several challenges in terms of the materials used (e.g. polydimethylsiloxane; PDMS). For instance, absorption of drug candidates and fluorescent dyes into PDMS, as well as the effect elicited by materials on cultured cells, can cause inaccurate or misleading results in cell assays. The use of PDMS also poses challenges for mass production and long-term storage of fabricated MPSs. Hence, to circumvent these issues, herein we describe the development of a cyclo olefin polymer (COP)-based MPS using photobonding processes and vacuum ultraviolet (VUV), designated as COP-VUV-MPS. COP is an amorphous polymer with chemical/physical stability, high purity and optical clarity. Due to the thermostability and high modulus of COP, the metal molding processes was applied for mass production of MPSs without deformation of microstructures and with quick fabrication cycle time (approx. 10 min/cycle). Moreover, VUV photobonding process with an excimer light at a 172nm wavelength allowed assembling COP materials without the use of additional solvents and tapes, which might cause cell damages. In comparison with the conventional MPS made of PDMS (PDMS-MPS), COP-VUV-MPS showed improved chemical resistance without causing molecule absorption. Moreover, COP-VUV-MPS maintained the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes or gene expression. These results suggest that COP-VUV-MPS may be broadly applicable for the advancement of MPS and applications in drug development, as well asin vitrotoxicological testing.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.