Construction And Evaluation Of A New BAL System Based On A Choanoid Fluidized Bed Bioreactor

Background:Bioartificial liver support system which incorporating isolated hepatocytes in a bioreactor has been proposed as a promising treatment method for liver failure by replacing liver function of detoxification and synthesis and simultaneously bridging patients for liver transplantation or by assisting in regeneration of patients' diseased livers.Many institutes in the world have been dedicated in this field and developed several kinds of bioartificial liver devices successively. At present, most of them have completed the animal experiments or even the clinical trails step by step. However, the progress of bioartificial liver in china is slowly and the key technology for example the construction of bioreactor is still depended on foreign countries. Therefore, it is necessitous for us to construct a new bioartificial liver system based on ourselves patents.We have been dedicated in this field for many years and acquired improvement in researching of both cell resources and design of bioreactors. As to the cell resource, we have established humanity immortalized hepatocytes lines including HepLL and HepLi4. In addition, we have created a choanoid fluidized bed bioreactor and got the certification of national invention patents.Here, we try to construct a new bioartificial liver support system based on the choanoid fluidized bed bioreactor. Evaluation of the efficiency and safety for this device was carried out through the fulminant liver failure pigs' model caused by D-galactosamine. Part?construction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactorObjective:Construction of a new bioartificial liver support system based on a choanoid fluidized bed bioreactor (CFBB) and to evaluate the feasibility of this device through healthy pig model.Methods:The BAL system consisted of two parallel circuits; the first one was a plasma separation circuit which included an arterial pump, a heparin pump, a separating pump, a returning pump, and a membrane plasma separator (OP-02 W, Asahi-Kasei, Japan) to provide plasma to the second circuit and send the plasma which has been deeply contacted with hepatocytes back to the body. The second circuit consisted of a reservoir, a roller pump, a membrane oxygenator. a bioreactor and an oxygen electrode, the speed of this circuit is faster than the first one which can provide multi-circulation for the plasma to the bioreactor. All the equipment of this circuit were placed in an incubator maintaining an internal temperature of 37??After incorporated with 300ml empty encapsulations in bioreactor, the feasibility of this new BAL system was evaluated through 8 hours operation in healthy Chinese experiment mini pig models.Results:This new BAL system was stable during the course of 8 hours' operation. and no obvious clotting or hemolytic complications were observed. All animals that received BAL treatment tolerated the procedure well and no allergic reactions or other significant side effects occurred. Conclusion:We provide a new BAL support system based on choanoid fluidized bed bioreactor and this new system is feasibility and safety for further animal experiment.Part?Evaluation of a new bioartificial liver support system through fulminant hepatic failure pig modelsObjective:We provided a fulminant hepatic failure in Chinese experiment miniature pigs without the use of anesthetic. The efficiency and safety of this new BAL system was evaluated by incorporating encapsualted primary porcine hepatocytes in bioreactor.Methods:FHF was induced with intravenous administration of D-galactosamine (1.5g/Kg). Primary porcine hepatocytes were isolated with four-step collagenase perfusion method and single-stage procedure was adopted to produce alginate-chitosan encapsulation. Thirty FHF pigs were divided into three groups:(1) an FHF group which was only given intensive care; (2) a sham BAL group which was treated with the BAL system with empty encapsulation and (3) a BAL group which was treated with the BAL system containing encapsulated freshly isolated primary porcine hepatocytes. The survival times and biochemical parameters of these animals were measured, also, properties of the encapsulations and hepatocytes before and after perfusion were evaluated.Results:Symptom such as oanorexia, restlessness was occurred after the administration of D-galactosamine. Compared to the two control groups, the BAL treated group had prolonged the survival time for 18.1 and 18.3 hours respectively, and decreased the blood lactate levels, blood glucose and amino acids remained stable. No obvious ruptured beads or statistical decline in viability or function of encapsulated hepatocytes were observed.Conclusion:Administration of D-galactosamine can establish a fulminant hepatic failure animal model successfully. This new fluidized bed bioartificial liver system is safe and efficient. It may represent a feasible alternative in the treatment of liver failure.