Effects of simulated microgravity on expression profile of microRNA in human lymphoblastoid cells.

This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison with static 1 × g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a high aspect ratio vessel (bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNAs was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22, miR-141, miR-618, and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using quantitative RT-PCR. Expression of several transcription factors including EGR2, ETS1, and c-REL was altered in simulated microgravity conditions. Taken together, the results reported here indicate that simulated microgravity alters the expression of miRNAs and genes in TK6 cells. To our knowledge, this study is the first to report the effects of simulated microgravity on the expression of miRNA and related genes.

Microprinting of liver micro-organ for drug metabolism study.

In their normal in vivo matrix milieu, tissues assume complex well-organized 3D architectures. Therefore, a primary aim in the tissue engineering design process is to fabricate an optimal analog of the in vivo scenario, in which the precise configuration and composition of cells and bioactive matrix components can establish the well-defined biomimetic microenvironments that promote cell-cell and cell-matrix interactions. With the advent and refinements in microfabricated systems which can present physical and chemical cues to cells in a controllable and reproducible fashion unrealizable with conventional tissue culture, high-fidelity, high-throughput in vitro models are achieved. The convergence of solid freeform fabrication (SFF) technologies, namely microprinting, along with microfabrication techniques, a 3D microprinted micro-organ, can serve as an in vitro platform for cell culture, drug screening, or to elicit further biological insights. This chapter firstly details the principles, methods, and applications that undergird the fabrication process development and adaptation of microfluidic devices for the creation of a drug screening model. This model involves the combinatorial setup of an automated syringe-based, layered direct cell writing microprinting process with soft lithographic micropatterning techniques to fabricate a microscale in vitro device housing a chamber of microprinted 3D micro-organ that biomimics the cell's natural microenvironment for enhanced performance and functionality. In order to assess the structural formability and biological feasibility of such a micro-organ, 3D cell-encapsulated hydrogel-based tissue constructs are microprinted reproducibly in defined design patterns and biologically characterized for both viability and cell-specific function. Another key facet of the in vivo microenvironment that is recapitulated with the in vitro system is the necessary dynamic perfusion of the 3D microscale liver analog with cells probed for their collective drug metabolic function and suitability as a drug metabolism model.

Biofabrication of a three-dimensional liver micro-organ as an in vitro drug metabolism model.

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.

Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. These results reveal that many genes play previously unrecognized roles in multiple DNA repair responses, all of which are required for successful repair of IR-induced damage.

Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis.

In vitro cell culture models used to study how Salmonella initiates disease at the intestinal epithelium would benefit from the recognition that organs and tissues function in a three-dimensional (3-D) environment and that this spatial context is necessary for development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3-D models of human colonic epithelium and apply them to study the early stages of enteric salmonellosis. The human colonic cell line HT-29 was cultured in 3-D and characterized by immunohistochemistry, histology, and scanning electron microscopy. Wild-type Salmonella typhimurium and an isogenic SPI-1 type three secretion system (TTSS) mutant derivative (invA) were used to compare the interactions with 3-D cells and monolayers in adherence/invasion, tissue pathology, and cytokine expression studies. The results showed that 3-D culture enhanced many characteristics normally associated with fully differentiated, functional intestinal epithelia in vivo, including better organization of junctional, extracellular matrix, and brush-border proteins, and highly localized mucin production. Wild-type Salmonella demonstrated increased adherence, but significantly lower invasion for 3-D cells. Interestingly, the SPI-I TTSS mutant showed wild-type ability to invade into the 3-D cells but did not cause significant structural changes to these cells. Moreover, 3-D cells produced less interleukin-8 before and after Salmonella infection. These results suggest that 3-D cultures of human colonic epithelium provide valuable alternative models to study human enteric salmonellosis with potential for novel insight into Salmonella pathogenesis.