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ONCLUSION
This PhD thesis describes the development of a 3D multi-compartmental modular bioreactor model for studying metabolism in vitro. In particular, the focus was on establishing the presence of cell-cell cross-talk through the investigation of glucose metabolism and its associated by- products and intermediates. The starting point for the system was the liver, which is the key orchestrator of metabolism in the body. In vitro liver cultures of HepG2 were first studied in 2D polymeric membranes and scaffolds. The results of this first stage demonstrated that our 3D scaffolds promote higher cell proliferation, such that final cell densities are about 4 to 5 times higher than on 2D surfaces. Other metabolic parameters were unchanged, indicative that the baseline cell function was unaltered. In the second phase of experiments the scaffolds were placed in the first compartment of the bioreactor and culture medium was pumped through the system in a closed-loop. Neither cell proliferation nor glucose consumption increased significantly with respect to the scaffolds in static conditions, but protein synthesis increased dramatically about 6 to 10 fold. These 2 experiments established then that 3D HepG2 cultures in a dynamic environment are metabolically more efficient than classic hepatocyte cultures.
In the next phase, having established a common medium between hepatocytes, HUVEC and adipose tissue, the metabolic activity of the 3 cells were compared first singly and them in a combination of 2 (adipose tissue and HUVEC) and all 3. It was observed that overall HepG2 is capable of conditioning and controlling cell-cross-talk by regulating glycemia, as vell as triglyceride synthesis and transport.
Cross-talk in the system was therefore confirmed, and the connected culture was shown to present the basic elements of glucose metabolism. In the final stage of the research, the 3 cell connected culture was exposed to different concentration of glucose and insulin to simulate a sort-of post-prandial stimulus. It was possible to replicate some features of the physiological response although the timescales were not consistent. The model used is necessarily extremely simple and only combines 3 specific elements of the very complex in-vivo environment. Nevertheless, this work is the first experimental demonstration of a 3 cell connected culture system in which tissues are shown to be capable of establishing communication through soluble ligands, and reciprocally influencing metabolic states.
