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In vivo studies clearly demonstrate the participation and subsequent death of non-parenchymal liver cells (NPCs) with corresponding hepatocyte effects. This results in a critical need to investigate how major liver cell types function cohesively during hepatotoxicity. However, virtually no studies replicate these phenomena in vitro. We report the design of multi-cellular three-dimensional (3D) organotypic liver models of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). LSECs and KCs were separated from hepatocytes by a detachable membrane that emulates the physical and chemical properties of the Space of Disse. Acetaminophen (APAP)-induced changes to cellular function and phenotype were investigated. LSECs exhibited approximately 40% cell death at 20mM APAP. KCs exhibited decreased interleukin-10 and increased tumor necrosis factor-alpha and interferon-gamma secretion. The secretion of these proteins altered hepatocyte function and signaling. Both LSECs and KCs maintained phenotypic markers. At 20mM APAP, the 3D models exhibited aspartate aminotransferase to alanine aminotransferase ratios from 2.1-2.5 and 45% glutathione depletion, corresponding to what is seen in vivo. At 10 and 20mM APAP, the 3D models exhibited cell death, primarily through necrosis. Therefore, the 3D cultures described in this report demonstrate significant potential as realistic models for hepatotoxicity studies.

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UNLABELLED: The design of antimicrobial membranes and thin films are critical for the design of biomaterials that can combat bacterial contamination. Since the long-term use of conventional antibiotics can result in bacterial resistance, there is a critical need to incorporate natural antimicrobial peptides (AMPs) that not only prevent a wide range of pathogens from causing infections but can also promote many beneficial outcomes in wounded tissues. We report the design and antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a naturally occurring human AMP. LL-37 was physically adsorbed and chemically immobilized on the surface of PEMs. The antimicrobial and cytotoxic properties of PEMs were tested with Gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. The ability to prevent bacterial adhesion and to neutralize an E. coli layer was investigated as a function of LL-37 concentration. An interesting trend was that even unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, bacterial adhesion was significantly lower on the surface of the films as well as in the surrounding broth. Immobilizing LL-37 resulted in less than 3% bacterial adhesion on the surface due to the presence of the peptide. LL-37 modified PEMs did not result in any cytotoxicity up to input concentrations of 16µM. More importantly, urea and albumin secretion by hepatocytes were unaffected even at high LL-37 concentrations. The COL/HA PEMs can serve as antimicrobial coatings, biological membranes and as in vitro platforms to investigate pathogen-tissue interactions. STATEMENT OF SIGNIFICANCE: Antimicrobial peptides (AMPs) are emerging as an alternative to conventional antibiotics. We report the antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a human AMP. The antimicrobial and cytotoxic properties were tested with gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. Unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, the sustained release of the active peptide killed planktonic bacteria. Immobilizing LL-37 resulted in less than 3% bacterial adhesion. LL-37 modified PEMs did not result in cytotoxicity up to input concentrations of 16µM. The COL/HA PEMs can serve as antimicrobial coatings and to investigate pathogen-cell interactions.

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BACKGROUND: Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. METHODS: To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. RESULTS: All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based ß-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. CONCLUSIONS: Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.