Hypothermic Preservation Of Miniaturized Bioartificial Liver And Its Mass Transfer Study

Hypothermic preservation has long been appreciated in storage and transportation of bioartificial liver (BAL) by down-regulating the cell metabolism and prolonging cell survival. But the deterioration of cell activity induced by the process of hypothermic preservation (hypothermia and re warming) limits the clinical use of BAL. In this paper, the hollow fiber based BAL was miniaturized as a hypothermic preservation model for proposing a new strategy to prevent hypothermia/rewarming-induced cell injury.Through screening of protective agents for hypothermic preservation, it was found that diammonium glycyrrhizinate, vitamine E, trehalose and matrine obviously alleviated the hypothermia/rewarming-induced cell injury. The combined use of these four protective agents improved 38% of cell viability and 32% of intracellular GSH content during rewarming, compared with the cells treated without protective agents.The hypothermic/rewarming-induced cell injury was addressed by clarifying the protective stage and the corresponding protective mechanism of different protective agents. It was found that the antioxidants exerted protection against cellular damage only during pre-incubation and rewarming, the membrane stabilizers and calcium inhibitors only exerted their protections at hypothermia and rewarming respectively, while the inhibitor of mitochondrial permeability transition exerted its protection during hypothermia and rewarming. The independence between the increased intracellular ATP content by adding 1,6-fructose diphosphate and cell viability presented that the decline of ATP during hypothermia did not induce the cell injury. Besides, the addition of di-rhamnolipid at hypothermic stage could improve the cell activity via enhancing the trehalose absorption, while Matrine could alleviate cell injury through inhibiting the accumulation of cytosolic free calcium during rewarming. The results mentioned above indicated that the intracellular redox balance, cytoskeleton and mitochondrial membrane potential were damaged during hypothermia and further deteriorated during rewarming. Along with the accumulation of intracellular free calcium during rewarming, the damage of cells was exacerbated and finally induced cell death.Then, the efficacy of protective agents was enhanced by optimize the addition time of different protective agents. Through establishing a microelectrode-based method, the diffusion coefficients of protective agents were assayed in the bioreactor and the time requested for reaching equilibrium in the bioreactor was determined. Based on these results, the addition time of protective agents was adjusted as below:trehalose was added into culture medium 0.75 h before the end of pre-incubation, while vitamine E, Matrine and diammonium glycyrrhizinate were added separately into culture medium 4.58 h,1.38 h and 0.88 h before the end of hypothermia. After optimizing the addition time of protective agents, both of cell viability and intracellular GSH content were improved during rewarming. Besides, the addition of di-rhamnolipid at hypothermic stage could also improve 20% of cell viability.Based on the optimized strategy of hypothermic preservation, the miniaturized bioartificial liver post to 36 h of hypothermia and 144 h of rewarming maintained the 87% of cell viability, 80-92% of intracellular GSH content and urea/albumin secretion, and 80-86% of CYP 450 activity, compared to hepatocytes without hypothermia. Our preservation performed better than current available reports, and could meet the requirement of BAL. The extended use of these protective agents in cryopreserved hepatocytes also successfully alleviated cell injury and sustained cell activity after rewarming, which also validated their protective effects.In conclusion, by screening protective agents, studying on the mechanism of protection and cell injury, and detecting the mass transfer on bioreactor to improve the addition of protective agents, the new strategy of hypothermic preservation can sustain well the cell viability and functions. This thesis could offer a potential application of this new strategy in hypothermic preservation of bioartificial liver.