A Experimental Study On Development Of Bioartificial Liver And Its Treatment Of Acute Liver Failure

The treatment of acute liver failure (ALF) is extremely difficult in the current medicine. With a routine treatment, the mortality of ALF is high about 80%. Although liver transplantation (LT) can reduce that to 30-50%, LT has the shortcomings such as the shortage of donor liver so not to be able to meet the urgent need of treating ALF with emergency LT, complex operation, the life-long immunosuppressant treatment on sufferer and high expenses. As another way of liver-substituting therapy, the study on artificial liver has made great achievements in the past forty years. Nowadays the animal experiments and primary clinical applications with bioartificial liver (BAL) based on cultured hepatocytes and artificial assistant device at abroad have shown that BAL can not only be possible to compenste the functions of detoxification and biosynthesis of liver, stabilization of inner condition and interdiction of vicious circle, but also provide conditions and time for regeneration of hepatocytes which possess great ability to regenerate. With gradually maturation and perfection BAL can be promise to bring the lightest hope for the treatment of ALF just as the revolutionary change of the treatment of kidney failure with kidney dialysis instead of only being a bridging therapy for waiting LT. Combining with the prophase work on this subject, we cultured the normal primary porcine hepatocytes on microcarriers suspendingly as biomaterial to develop BAL to experiment on treating ALF pigs with aiming to provide evidences for BAL to enter phase I clinical examination earlier. ?? Part I Seperation and culture on microcarriers suspendingly of porcine hepatocytes Aim To establish a technique of harvesting hepatocytes of abundant sources. Methods Hepatocytes were from the donors of native hybrid pigs and isolated on the way of two-step orthotopic collagenase circulating perfusing method as described by Seglen. The yield and viability were assessed by trypan blue exclusion test. Microcarriers Cytodex-3 were added at the concentration of Smg/L into the STUART stirring culture vessel siliconized by 5% methyl silicon resin ethyl acetate. Porcine hepatocytes were entirely inoculated into the vessel at the concentration of 5 X 106/mL. Together with the supplemental factors, the culture matrix was added in according to one third of the final volume. After being stirred for 6 hours the culture matrix and supplemental factors were added to the 200mL final volume and from then on stirring were kept on. Cell growth was observed on light and electron microscopes. Concentrations of albumin and urea were examined dynamically in the supemation. Results Each liver provided (4.26?.75) X 1010 hepatocytes averagely and their vivid rate was (91.22 ?0.83)%. Most hepatocytes tended to conglomerate obviously after being inoculated and 48-72 hours later typical multicellular aggregate spheroids could be observed. Their morphological characteristics and abilities to synthesize albumin and urea could be maintained for about 4 weeks. Conclusion large-amount and high-activity normal primary porcine hepatocytes could be isolated on the way of two-step orthotopic tyep IV collagenase circulating perfusing method. When being cultured on microcarriers they could be achieved to the objects of high...