Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen.

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patient's sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

Evaluation of primary human liver cells in bioreactor cultures for extracorporeal liver support on the basis of urea production.

Primary human liver cells from donor organs unsuitable for transplantation were cultivated in bioreactors developed for extracorporeal liver support. Because each system contains cells originating from an individual organ, each bioreactor culture must be individually characterized. The objective of this study was to identify suitable decisive parameters for the evaluation of cell culture performance. We analyzed the data from 47 bioreactor cultures containing 437 +/- 110 g of cells. Choosing urea production as the decisive parameter, the bioreactor cultures were divided into high-performance (daily urea production > or = 110 mg per bioreactor between culture days 3 and 14) and low-performance cultures. Comparing the mean courses of the groups revealed a significant distinction in most other investigated biochemical parameters. In conclusion, urea production seems to be an appropriate parameter for evaluating the performance of liver cell cultures in bioreactors because it corresponds to all other evaluated parameters of cell function.

The SlideReactor--a simple hollow fiber based bioreactor suitable for light microscopy.

Most bioartificial liver support systems are based on hollow fiber capillaries within modified dialysis cartridges or more sophisticated bioreactor constructions. Due to their design microscopic follow-up of reorganization and growth of tissue between the hollow fibers is not possible. The SlideReactor is a simple hollow fiber based bioreactor construction suitable for light microscopy and time-lapse video observation. The SlideReactor offers a cell compartment separated from a medium inflow and outflow compartment. Cell compartment access ports enable easy filling of the cell compartment with cell suspension, as well as fixation of the tissue. For more complex procedures or full access to all the cells, the bioreactor can be opened easily by cutting the silicone seal with a scalpel. Due to its simple design and the utilization of standard materials, it could serve as a suitable, cost-efficient tool to evaluate the behavior of cells cultured between hollow fiber capillaries. The paper describes the production process: similar to open source projects in software engineering, we would like to propose the concept as an open platform to anyone interested in hollow fiber based cell culture.

In vitro evaluation of the transportability of viable primary human liver cells originating from discarded donor organs in bioreactors.

BACKGROUND: The use of primary human liver cells obtained from discarded donor organs is increasingly favored for cell-based extracorporeal liver support systems. However, as cryopreservation of primary human hepatocytes causes a significant loss of metabolic activity, the transport of bioreactors with viable liver cells is required. The aim of this study was to evaluate the impact of two major potential threats to viable cells during transport: temperature changes and mechanical stress. METHODS: In each experiment three hollow fiber-based bioreactors were charged with primary human liver cells originating from the same discarded donor organ and were simultaneously kept under culture conditions for 8 days. In total, 18 bioreactors were evaluated. On the fifth day the bioreactors were exposed to hypothermia (4 degrees C, n = 3), to hyperthermia (42 degrees C, n = 3), or served as normothermic controls (37 degrees C, n = 3). In a second test series bioreactors were exposed to vibration (21 Hz for 20 min, thereafter 7 Hz for 160 min, n = 3), or were operated as control cultures (n = 6). The release of hepatocyte-specific enzymes was determined as an indicator for cell damage. RESULTS: Hypothermic stress resulted in a significant release of transaminases and led to disturbances of the histological integrity, all indicating a high degree of cell damage. When compared with the control cultures, hyperthermia and mechanical stress in terms of vibration had no significant effect on the cells. CONCLUSION: The transport of hollow fiber bioreactors charged with viable primary human liver cells appears to be feasible in transport monitors for perfusion and temperature control.

In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD).

The detoxification capacities of single-pass albumin dialysis (SPAD), the molecular adsorbents recirculation system, (MARS) and continuous veno-venous hemodiafiltration (CVVHDF) were compared in vitro. In each experiment 4,100 mL of toxin-loaded human plasma was processed for 6.5 hours. MARS treatment (n = 6) was undertaken in combination with CVVHDF. For SPAD (n = 6) and CVVHDF (n = 6) a high-flux hollow fiber hemodiafilter (identical to the MARS filter) was used. Levels of ammonia, urea, creatinine, bilirubin, and bile acids were determined. Concentrations before and after application of detoxification procedures were expressed as differences and were compared using the Kruskal-Wallis test. Post hoc comparisons for pairs of groups were adjusted according to Bonferroni-Holm. Time, group, and interaction effects were tested using the nonparametric ANOVA model for repeated measurements. SPAD and CVVHDF induced a significantly greater reduction of ammonia levels than MARS. No significant differences were found among SPAD, MARS, and CVVHDF with respect to other water-soluble substances. SPAD induced a significantly greater reduction in bilirubin levels than MARS. Reductions in bile acid levels were similar for SPAD and MARS. When operating MARS in continuous veno-venous hemodialysis mode, as recommended by the manufacturer, no significant differences in the removal of bilirubin, bile acids, urea, and creatinine were found. However, MARS in continuous veno-venous hemodialysis mode was significantly less efficient in removing ammonia than MARS in CVVHDF mode. In conclusion, the detoxification capacity of SPAD is similar to or even greater than that of MARS.