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G
ENERAL
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NTRODUCTION
In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous controlled in vivo experiments (when it is possible to do these).
In a search for an alternative to in vivo experiments, our work aims at developing a special culture-connection device in which different cell types are located in a different environments but connected by a closed loop media circulation. The signals exchanged between cells from various organs are, thus reproducing the in vivo inter-tissue communications. With this perspective, we evaluated a new biomimetic system as an in vitro simulator of Human metabolism. (As a point of reference the aim is to study salient aspect of glucose metabolism). This new system is the Multi-Compartmental modular Bioreactor (McmB) designed and realized at the Interdepartmental Research Center “E. Piaggio”. The biomimetic system is conceived to represent at least four different human cell types (from human tissues or as lineages: pancreatic, hepatic, adipose and endothelial), in different cell culture chambers connected only by flow. The new concept of “connect cultures” seeks to mimic the physiological milieu in which cells from different tissue or organs are exposed to soluble ligands though blood, lymph or other fluids in the extra cellular matrix. In this way the whole organism is able to maintain the physiological homeostasis. Homeostasis refers to the ability of on organism to maintain constant vital parameters, such as blood pressure, blood composition, energy balance and hytratation level. It requires that each organ and within it, each cell adjusts its program according to the information it gains from the systemic environment; that is, mainly from the blood, which carries informative molecules. Understating how a tissue or a cell modifies its functional status in repose to given stimulus is well known in a classical cell culture system, which combines ease of manipulation with cell- specific response. However, few models are available at present for understanding regulatory networks at the molecular level. Primary cultures in monolayer are simple and amenable to molecular studies but cannot be considered for crosstalk analysis. In vivo models of metabolic studies have provided a wealth of information, and the generation of mice that are functionally deleted for one gene has renewed the interest for such studies. However, the interpretation of mice phenotype at the molecular level, in basal condition and upon challenges, remains complex and often hypothetical. The need for in vitro tools has prompted the development of coculture methods. In one type of application, cell-cell interactions are direct and provide a structural organization, reminiscent of that of an organ, in which different cell types create a matrix that participants in organ-specific function maintenance. Closer to our interest are the connected cultures that allow free diffusion but no direct physical contact between different cell types.
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The multi-compartimental modular bioreactor (McmB) allows:
free diffusion and access to the various cell types of any secreted molecules that may act as a signal;
the possibility of retrieving each cell type without contamination by the others, in order to analyze the cell-specific change in gene transcription program and ease of manipulation and of production of the corresponding materials and cells experimental reliability.
The development of this connection culture system combining several cell types could also be used to understand intracellular communication mechanisms in normal and pathological conditions and could also be a substitute for numerous preliminary experiments conducted in animals. The present feasibility study was based on a 4 compartment bioreactor containg hepatocytes, adipose tissue, endothelial cells and virtually pancreatic isles (through the use of insulin).
Hepatocytes were fundamental for orchestrating glucose metabolism; in fact in the liver little of glucose taken up in human eating a mixed diet is oxidized completely to CO2 and H2O to produce high-energy bonds. The
bulk is converted instead to glycogen to a maximum storage capacity of 65g glycogen per kg liver tissue. During fasting, hepatic stores of glycogen are used to maintain a physiologic concentration of glucose in the blood.
The liver is also the major site for synthesis of fatty acids from excess sugar in the fed state but does not store an abundance of fatty acids. The function of fatty acids storage pertains to adipose tissue. Fatty acids are mobilized from storage in adipocytes during fasting.
Vascular tissue mediates all plasmatic exchange processes. Thus the presence of endothelial cells in our experiments is as a conditioning tissue. Morover vasculalar tissue represents in this study not only a spectator organ but a target one during the simulation of Hyperglycemia. In fact chronic elevation of blood glucose level leads to damage of blood vessels (angiopathy). The endothelial cells lining the blood vessels take in more glucose than normal, since they don't depend on insulin. They then form more surface glycoproteins than normal, and cause the basement membrane to grow thicker and weaker. For example, hypertension is common in diabetes associated with a diminished production of NO by endothelial cells.
