In their normal in vivo matrix milieu, tissues assume complex well-organized three-dimensional architectures. Therefore, the primary aim in the tissue engineering design process is to fabricate an optimal analog of the in vivo scenario. This challenge can be addressed by applying emerging layered biofabrication approaches in which the precise configuration and composition of cells and bioactive matrix components can recapitulate the well-defined three-dimensional biomimetic microenvironments that promote cell-cell and cell-matrix interactions. Furthermore, the advent of and refinements in microfabricated systems can present physical and chemical cues to cells in a controllable and reproducible fashion unmatched with conventional cultures, resulting in the precise construction of engineered biomimetic microenvironments on the cellular length scale in geometries that are readily parallelized for high throughput in vitro models. As such, the convergence of layered solid freeform fabrication (SFF) technologies along with microfabrication techniques enables the creation of a three-dimensional micro-organ device to serve as an in vitro platform for cell culture, drug screening or to elicit further biological insights, particularly for NASA's interest in a flight-suitable high-fidelity microscale platform to study drug metabolism in space and planetary environments. The proposed model in this paper involves the combinatorial setup of an automated syringe-based, layered direct cell writing bioprinting process with micro-patterning techniques to fabricate a microscale in vitro device housing a chamber of bioprinted three-dimensional liver cell-encapsulated hydrogel-based tissue constructs in defined design patterns that biomimic the cell's natural microenvironment for enhanced biological functionality. In order to assess the structural formability and biological feasibility of such a micro-organ, reproducibly fabricated tissue constructs were biologically characterized for liver cell-specific function. Another key facet of the in vivo microenvironment that was recapitulated with the in vitro system included the necessary dynamic perfusion of the three-dimensional microscale liver analog with cells probed for their collective drug metabolic function and suitability as a drug metabolism model. This paper details the principles and methods that undergird the direct cell writing biofabrication process development and adaptation of microfluidic devices for the creation of a drug screening model, thereby establishing a novel drug metabolism study platform for NASA's interest to adopt a microfluidic microanalytical device with an embedded three-dimensional microscale liver tissue analog to assess drug pharmacokinetic profiles in planetary environments.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3- While important advances have been made toward understanding how Salmonella interacts with the intestinal epithelium to initiate disease (reviewed in references 6 and 44), investigations into the interaction of Salmonella with the human intestinal epithelium have been limited by the lack of in vitro and in vivo models which faithfully replicate the in vivo condition. In particular, it is well documented that important differences exist between the pathogenesis of Salmonella enterica serovar Typhimurium in human infections and that in widely used cell culture and animal models (34,40,47). In vitro assays using cultured mammalian epithelial cells have long been used as a model for investigating the interaction between Salmonella and the intestinal mucosa. However, there are inherent limitations associated with the use of these cultured cell lines (34), as they are not exact models of the conditions faced in vivo by Salmonella. Several characteristics of conventional tissue culture models have raised concerns regarding their overall efficacy as models for microbial infectivity in general (34) due to the dedifferentiation of these cells during conventional cell culture. Indeed, many of the physiological differences between cultured cells and their in vivo counterparts are believed to be the result of the dissociation of cells from their native three-dimensional (3-D) geometry in vivo to their propagation on a two-dimensional substrate in vitro (10). Likewise, many characteristics of animal models fail to mimic the human disease, and animal models present a complex system in which many variables cannot be controlled. A high-fidelity enteric cell culture model could provide new insights into studies of Salmonella infectivity by bridging the gap between the inherent limitations of cultured mammalian cells and intact animals.DFor humans, S. enterica serovar Typhimurium is among the most common Salmonella serotypes isolated from individuals suffering from infectious gastroenteritis and has long been recognized as a major public health problem (23). Gastroenteritis results from infection of the small intestine after ingestion with Salmonella. Indeed, the ability to colonize the intestinal epithelium is an essential feature in the pathogenicity of Salmonella infection. Moreover, the initial interactions between Salmonella and the host intestinal epithelium are believed to play a key role in mediating the intense inflammatory and secretory response which is a hallmark of serovar Typhimurium infections in humans (reviewed in reference 6). Studies with cultured intestinal epithelial cells have shown that,

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