Assessment of resin perfusion in hepatic failure in vitro and in vivo.

AIM: To observe the adsorbent effect of resin on endotoxin, cytokine, bilirubin in plasma of patients with hepatic failure and to determine the resin perfusion as an artificial liver support system in the treatment of hepatic failure. METHODS: One thousand milliliters of discarded plasma was collected from each of 6 severe hepatitis patients treated with plasma exchange. The plasma was passed through a resin perfusion equipment for 1-2 h via extracorporeal circulation, and then absorbent indicators of transaminase, bilirubin, blood ammonia, endotoxin and cytokines were examined. In the meantime, study of in vivo resin plasma perfusion was performed on 7 severe hepatitis patients to compare the changes of endotoxin and cytokines in blood before and after perfusion. RESULTS: The levels of total bilirubin, endotoxin, interleukin 1beta and TNF-alpha in plasma were significantly decreased after in vitro resin plasma perfusion. The levels of interleukin 1beta, TNF-alpha and endotoxin in blood were also evidently declined after in vivo resin plasma perfusion. Nevertheless, no obvious changes in IL-6, creatinine (Cr) and urea nitrogen (UN), blood ammonia and electrolytes were found both in vitro and in vivo. CONCLUSION: Bilirubin, endotoxin and cytokines in plasma of patients with hepatic failure can be effectively adsorbed by resin in vitro. Most cytokines and endotoxin in plasma can also be effectively removed by resin in vivo. It demonstrates that resin perfusion may have good treatment efficacy on hepatic failure and can be expected to slow down the progression of hepatic failure.

Primary hepatocyte culture in collagen gel mixture and collagen sandwich.

AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes. METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined. RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration. Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phase-contrast microscope. There was little LDH-release during the culture period. CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivo environment, and are helpful for maintaining specific hepatic functions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the study of bioartificial livers.

[Preliminary evaluation on the effects of a hybrid bioartificial liver support system in the treatment of hepatic failure].

OBJECTIVES: To construct a novel hybrid artificial liver support system and evaluate its clinical efficacy in the treatment of hepatic failure. METHODS: A hybrid bioartificial liver support system consisting of plasma exchange device, charcoal perfusion column, and bioreactor cultured human or porcine hepatocytes was developed. 30 patients with hepatic failure were treated using this hybrid system. RESULTS: Both the excellent rate and effectual rate of the artificial liver support system were 43.3% (13/30). The total effectual rate was 86.7%. Finally, eleven out of 30 patients recovered completely. Six patients were bridged to liver transplantation. Six patients (20%) died of hepatic failure and seven patients (23.3%) discharged due to worsening of disease. CONCLUSIONS: The hybrid artificial liver support system has prominent liver support effects for hepatic failure, which can be regarded as an efficient measure for the treatment of severe hepatitis.

[Preparation of hollow fiber bioreactor for culturing pig hepatocytes].

OBJECTIVE: To study the method of preparing the hollow fiber bioreactor for culturing pig hepatocytes. METHODS: Hepatocytes were isolated from experimental suckling minipigs by two-step perfusion with collagenase, and seeded onto hollow fiber bioreactor, then cultured with an artificial capillary cell culture system. The albumin-excretion, lidocaine-transforming rate, lactate dehydrogenase (LDH) release and the cell viability in bioreactors were examined. RESULTS: The porcine albumin could be detected by SDS/PAGE on the 2nd, 4th, 6th day. The rates of lidocaine-transforming ranged from 89.6% to 96.1%. The release of LDH into the culture medium increased from (23.7+/-4.6) U/L to (127.8+/-17.4) U/L (F=39.582, P<0.01) during the experiments, and the viability of pig hepatocytes in hollow fiber bioreactor reduced from 95.8%+/-0.3% to 83.8%+/-4.7% (t=5.135, P<0.01). CONCLUSION: The hollow fiber bioreactor for culturing pig hepatocytes can be prepared by artificial capillary cell culture system, which provides a certain liver-specific function in 1 week.