A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

In this study, we demonstrate the ability of a three-dimensional co-culture model to preserve some key aspects of differentiated hepatocyte function in vitro. Freshly isolated rat hepatocytes in co-culture with activated stellate cells rapidly aggregate to form well-defined viable spheroids. After 5 days in culture, the spheroids have a complex extracellular matrix support and hepatic ultrastructure including bile canaliculi, tight junctions, desmosomes and lipid storage. Co-culture spheroids have superior cytochrome P₄₅₀ (CYP₄₅₀) 3A and 2B function, and increased inducibility of 2B function, relative to a range of hepatocyte monoculture techniques (high-performance liquid chromatography of testosterone metabolites). Increased function in co-culture is supported by greater expression of CYP₄₅₀ 3A23, 1A2, and 2E1 mRNA relative to monoculture (reverse transcriptase quantitative polymerase chain reaction). Also, high hepatocyte growth factor mRNA expression in co-culture suggests a post-traumatic, or possibly regenerative, environment. A preliminary study of human hepatocytes co-cultured with rat stellate cells demonstrated prolonged function of CYP₄₅₀ 3A4, 2C19 and 2C9. This study shows that stellate cells facilitate spheroid formation, influence spheroid architecture, and are an effective method of preserving some aspects of hepatocyte function in the early stage of culture.

One of the greatest challenges in the attempt to create functional liver tissue in vitro is the maintenance of hepatocyte-specific functions. The pharmaceutical industry has long awaited the development of engineered liver tissue, which could represent a long-term, inducible, high-fidelity model for high-throughput screening of new drug compounds. It is also anticipated that such engineered models could one day be used in liver transplants, where replacement is limited by chronic donor shortages. As isolated hepatocytes dedifferentiate rapidly in culture the use of hepatocytes in long-term studies has proved to be a difficult challenge. Here we report a system of rat hepatocytes cocultured with primary rat hepatic stellate cells on a biodegradable poly(DL-lactic acid) substratum. These coculture conditions were found to encourage the rapid self-organization of three-dimensional spheroids. The spheroids formed exhibit hepatocyte-specific functionality (CYP-450 activity and albumin secretion) after almost 2 months in static culture.

The advancement of elastomeric patterning techniques in recent years has significantly enhanced our ability to spatially control biomaterial surface chemistry at the micrometre level. The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors, combinatorial library screening and the formation of tissue engineering templates. In this paper, we describe the direct patterning of protein by microcontact printing. An important consideration for the fabrication of protein micropatterns intended for these applications is the nature of the protein immobilization to a substrate. To date, the patterning of proteins by direct microcontact printing (microCP) has relied on the non-covalent adsorption to a substrate. Ideally, the proteins need to be firmly anchored onto a surface without adversely effecting their activity. Here, the high affinity avidin-biotin receptor-ligand interaction has been exploited to form arrays of avidin molecules onto a polymeric substrate expressing biotin moieties. This has created a generic technique by which any biotinylated species can be subsequently immobilized into defined patterns. Utilizing atomic force microscopy (AFM), the patterned surfaces have been characterized to molecular resolution. The micropatterned sample supported cell adhesion when biotin-(G)11-GRGDS was bound to the avidin bearing arrays.

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