Construction And Application Study Of Hepatic Tissue-like Bioreactor

Liver is one of the most important organs for maintaining normal physiological functions. Nowadays, the physiological and pathological studies on glucose, lipid metabolism in liver come into focus with the high incidence of diabetes and metabolic disorders. Hepatocytes in vitro culture models provide alternative platforms for less costive and high throughput screening studies. One of these models is hepatocytes in spheroid culture, which possesses the most in vivo-like structure and functions of liver. However, due to the lack of systemic studies of the mechanism of hepatocytes self-assembly, the functions and applications of hepatocytes spheroid are less evaluated. This paper firstly investigated the laws of cell self-assembly and mechanism of hepatocytes changes during spheroid culture. A hollow fiber based hepatic tissue-like bioreactor was constructed according to the observed mechansims. Finally, the glucose and lipid metabolism activities and its ability in reflecting effecacy of antihyperglycemic and antihyperlipidemic drugs were also investigated in this bioreactor.First of all, alginate and polyethylene glycol (PEG) hydrogel were taken as research platforms to investigate the effects of the lumen configuration, cell density and biomacromoleculars on hepatocytes self-assembly. It was found that the lumen greatly promoted cell aggregation. Lumen configuration/size and cell density can affect cell assembly by regulating "accumulated layer". And the spheroid achieves best morphology and functions at the fourth layer. At the same time, collagen addition can accelerate the aggregation of hepatocytes. The mechanism studies showed that cytoskeleton was engaged in hepatocytes spheroid formation. The expression of intergrin declined and E-cadherin increased with the compact of hepatocytes spheroid. Further more, collagen fibers could be rearranged during cell aggregation phase.Secondly, with the guidance of the laws obtained above, we constructed a hollow fiber membrane based hepatic tissue-like bioreactor. Hepatocytes can quickly assemble into spheroid within 18 hrs in the inner space of the hollow fiber lumen with the synergetic function of proper lumen, cell density and collagen addition. The cell viability, CYP450 activities and the liver-specific function of the bioreactor are superior to the same period of sandwich and monolayer culture models. More over, it can be used for human-derived liver cells in culture.Finally, glucose and lipid metabolism activities and the ability on reflecting drug efficacy were evaluated in "tissue-like" bioreactor. The results showed that hepatocytes displayed higher glucose and lipid metabolism activities within a longer culture time in the bioreactor than that of traditional culture models, which were closer to the in vivo data. Futher more, hepatocytes in bioreactor can be more sensitive in responding to hormonal stimulation and high glucose treatment. Meanwhile, our "tissue-like" bioreactor correctly reflected the efficacy of anti-hyperglycemic/anti-hyperlipidemic drugs (troglitazone, GW501516 and fenofibrate).In summary, this "tissue-like" bioreactor, which was constructed based on the guidance of cell-assembly mechanism study, behaved a more in vivo-like manner of liver in glucose and lipid metabolism and drug effecacy. Further more, this paper not only enriched the concept of the bioreactor design, but also laid the foundation for better application of the hepatic bioreactor in liver physiology and pathology studies.