Bartlettpurcell4341

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.Tissue engineering is an elegant tool to create organs in vitro, that can help obviate the lack of organ donors in transplantation medicine and provide the opportunity of studying complex biological systems in vitro, thereby reducing the need for animal experiments. Artificial intestine models are at the core of Fish-AI, an EU FET-Open research project dedicated to the development of a 3D in vitro platform that is intended to enable the aquaculture feed industry to predict the nutritional and health value of alternative feed sources accurately and efficiently.At present, it is impossible to infer the health and nutrition value through the chemical characterization of any given feed. Therefore, each new feed must be tested through in vivo growth trials. The procedure is lengthy, expensive and requires the use of many animals. Furthermore, although this process allows for a precise evaluation of the final effect of each feed, it does not improve our basic knowledge of the cellular and molecular mechanisms determining such end-results. In turn, this lack of mechanistic knowledge severely limits the capacity to understand and predict the biological value of a single raw material and of their different combinations.The protocol described herein allows to develop the two main components essential to produce a functional platform for the efficient and reliable screening of feeds that the feed industry is currently developing for improving their health and nutritional value. It is here applied to the Rainbow Trout, but it can be fruitfully used to many other fish species.Oviduct and uterus are key female reproductive organs lined by ciliated simple columnar epithelia, which are the first line of maternal contact with gametes and the developing embryo during reproduction and which warrant the optimal developmental environment for the conceptus. Selleck CAY10683 A major challenge for modeling these epithelia in vitro is the preservation of apical-basal polarization and cilia formation. The air-liquid interface (ALI) culture approach is a technology originally invented for modeling epidermal and airway epithelia. It has recently been shown that it also allows the establishment of highly differentiated in vitro models of epithelia that do not have access to ambient air in vivo. In this chapter, we present a comprehensive ALI procedure to model female reproductive tract (FRT) epithelia of different mammalian species in vitro over extended time periods. As a working example, the protocol focuses on primary oviductal epithelial cells (OEC) isolated from domestic pig. Hints on protocol variations for the culture of OEC from other species are provided in the Subheading 4.Various approaches have been evaluated for developing three-dimensional (3D) scaffolds for modeling or engineering of the bone tissue. However, most of such attempts have come up short in mimicking the natural bone tissue extracellular matrix (ECM) microenvironment, especially its natural bioactive content. Here we describe the methodology for the preparation of a natural ECM-based multichannel construct as a biomimetic 3D bone tissue model. We elucidate the construction of the composite scaffold incorporating decellularized small intestinal submucosa ECM, synthetic hydroxyapatite and poly(ε-caprolactone), and the mechanical stimulation of the cell-seeded construct under bioreactor culture.Intercellular communication can be carried out by circulating systemic and/or locally released extracellular vesicles (EVs), produced by nearly every cell type and tissue, and are involved in physiological and pathological processes. In recent years, EVs have been identified in reproductive tissues, such as oviduct and uterus, and have been shown to be related to several events important for reproductive success. The understanding of their functions in reproduction has important implications for assisted reproductive technologies, for the treatment of infertility in humans and improvement of reproduction efficiency in animals. To study such EVs, it is necessary to isolate and concentrate them from fluid samples, which in the case of reproductive tissues, are usually of limited volume. Several methods for EV isolation are available such as chromatography, ultracentrifugation, polymer-based precipitation, and immunoaffinity.Outcomes can be variable in terms of the amount and quality of isolated EVs, due to the type of isolation method. The choice of method, or a different combination of methods, may depend on the type of sample and scientific question to be addressed in a given study. In this chapter, we describe a method for isolation of EVs from bovine oviductal and uterine fluids for use in functional studies. The method combines size exclusion chromatography and ultracentrifugation. We also describe the different protocols for characterization of isolated EVs (transmission electron microscopy, nanoparticle tracking analysis, and western blot), as well as the isolation of RNA content in EVs, and their miRNAs profiling for functional studies.