The Study Of Microcapsulated Immortalized Hepatocytes After Cryopreservation For Bioartificial Liver System

BackgroundThe number of deaths caused by liver diseases, especially late stage liver failure, continues to an increasing trend annually. Artificial liver becomes a research hotspot in recent years, which is designed to "bridge" patients to liver transplantation or spontaneous recovery. Many non-bioartificial liver support systems have been investigated over the past30years, and recent developments appear promising, but there are still gaps compared with developed countries for bioartificial liver system research.AimThis study is to choose the immortalized hepatocytes with best functions, encapsulate them and culture these microspheres with patients’plasma in our bioartificial support system after cryopreservation in large scale.Methods1. The functional evaluation of immortalized human hepatocyte. Cell proliferation was detected by MTT and functions were extamined by Real-time quantitative PCR. immunofluoresence, Western blot, drug metabolizing capacity and ELISA.2. Fluidized microencapsulated hepatocyte cell lines were compared with the two-dimensional cultured hepatocytes and microencapsulated cells on the changes of gene and protein expression of phase I enzymes, II phase enzyme and specific nuclear receptors. Cryopreservation and recovery conditions of microencapsulated hepatocytes and the effection of large scale cryopreservation were optimized by the synthesis of ALB and urea.3. The evaluation of effectiveness of bioartificial liver. The patient’s plasma was collected. Microencapsulated hepatocyte cells were recovered after crypopreservation and added in the optimized bioreactor to be cultured with the plasma for24h. The effect of plasma on activity and functions of cells and the changes of small molecules in the plasma were determined.Results1. HepLi3cells were better according to their better proliferation, available to large-scale cultivation and liver functions.2. The microencapsulated hepatocytes maintained high activity during the fluidization culture. And this condition could improve the expression of liver specific genes and protein, which may enhance cells’functions through suppressing kinases.3.2hours incubation before and after the cryopreservation were determined, during which the number of recovered alive cells was more than50%. Forethemore, the cryopreservation volume expansion did not affect the liver functions.4. HepLi3cells as seeded cells after cryopreservation were used for treating plasma from liver failure patients. Microencapsulated cells could stable the environment of patients which may improve their conditions. Conclusion1. HepLi3was the better seeded cells for BAL.2. Optimized the production of microcapsules, liver cell cryopreservation and recovery conditions, protein kinases surpression may be the mechnism of promoting liver cell functions in microcapsulated culture.3. The microencapsulated hepatocytes maintained high activity after large scale cryopreservation. They could be offered as seeded cells resource for BAL system.4. BAL was proved to remove toxic substances, effectively improve the environment of patients by using microencapsulated HepLi3.