Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS). In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared. Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated. For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = -0.791, p < 0.01). When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished. In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01). Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05). PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism. When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h. This toxic effect could be prevented by the inactivation of complement. When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line. However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin. We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.
Background: This study describes the pre‐clinical trials of an extracorporeal bioartificial liver support system (BALSS). It includes the biochemical changes which occur in the plasma and blood of pigs with devascularized livers when the plasma is treated in a BALSS, and the testing of the system for presence or absence of infective agents, pyrogens and for toxicity.
Methods: Hepatic cells were prepared from littermate juvenile white landrace pigs with a double‐step collagenase digest technique. The cell preparations were incubated with collagen‐coated dextran microspheres (CDM) for 3 h and the medium was tested to determine cellular metabolic activity. Incubation continued for a further 20 h during which the hepatic cells attach to the CDM. The CDM‐attached cells were inoculated into a hollow fibre bioreactor which was part of an extracorporeal liver support system.
Results: Hepatic cell content of the bioreactor was 6 × 109± 3 × 1018 cells, equivalent to those present in half a pig's liver. The system was tested in a controlled trial with the plasma of pigs with fulminant hepatic failure (FHF) due to devascularized livers. When plasma from FHF pigs was circulated through the device there was significantly less of an increase in the accumulation of ammonia, lactate and most amino acids when hepatic cells were included in the circuit compared with those in control experiments when they were excluded. Similar changes occurred in porcine blood. There were few infections diagnosed and an absence of pyrogens, endotoxins and toxicity in the bioreactor contents or in the terminating reservoir or animal blood samples.
Conclusions: We believe that the results, demonstrating function of the porcine hepatic cells in the circuit, together with low risks, justify a clinical trial of use of the BALSS in Australia.
The new 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) calorimetric assays for assessing hepatocyte density, viability and proliferation were evaluated and compared with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and tritiated thymidine (3HTdR) incorporation. OD values of MTS or XTT, which are metabolically reduced in viable cells to a watersoluble formazan product and can be read directly, had a good correlation coefficient with hepatocyte densities in a range of 2.5-40 x lo4 cells/ml (MTS r = 0.952; XTT r = 0.902) and with hepatocyte viability (MTS r = 0.974; XTT r = 0.975). At 0, 20, 40 and 60 hr cultures, the correlation coefficients with 3HTdR for assessing hepatocyte viability and proliferation (MTS r = 0.942-0.981; XTT r = 0.953-0.992) were excellent. In contrast with MTS and XTT, MTT OD values had a poor correlation coefficient with hepatocyte densities (r = 0.672), viability 0. = 0.622) and 3HTdR incorporation (r = 0.701-0.818 at 0, 20 and 40 hour cultures). This study shows that the MTS or XTT calorimetric assays for assessing hepatocyte density, viability and proliferation are more accurate, reliable and simpler than the widely used MTT assay. They avoid use of radioactive material as required for 3HTdR incorporation. Of the two, the MTS calorimetric assay is more sensitive than the XTT.
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