Bio-artificial liver from cultured human foetal hepatocytes: feasibility and prospects.
BACKGROUND
Conventional therapy for acute liver failure has not been able to improve survival beyond 40%. Apart from liver transplantation, the most promising development in this field is the utilization of cultured hepatocytes to make 'bio-artificial liver support systems' as a 'bridge to transplantation' or ideally as a 'bridge to total recovery'. This study examines the feasibility of culturing foetal hepatocytes without the use of growth factors and formulating a bio-artificial liver support device in our set-up.


METHODS
Foetal hepatocytes were harvested from the liver obtained from mid trimester abortions at Armed Forces Medical College and Command Hospital (SC), Pune. The liver was perfused with Phosphate Buffered Saline (PBS) and collagenase type IV and was cut with a pair of sterile scissors into tiny pieces. Cells so separated, were washed with PBS plus foetal calf serum and stirred to disperse the cell aggregates. Filtered cell suspensions were inoculated in polystyrene flasks containing hepatocyte culture medium (MEM E: 75%, M199: 25%, BSA: 0.1%, Bovine Insulin 5 micrograms/ml, FCS: 10%, Penicillin: 10 i.u., Streptomycin 50 micrograms/ml, Hydrocortisone 5 micrograms/ml and incubated at 37 degrees C. The functional capabilities of the cultured hepatocytes were analyzed by studying production of albumin and a foetoprotein. Structural integrity of hepatocytes was assessed by light and electron microscopy.


RESULTS
The hepatocyte yield varied from 2 to 60 x 10(6) cells/L with an average of 38 x 10(6) cells/L in the eight consecutive experiments. Initial hepatocyte viability varied from 25% to 90% with an average of 61%. The yield and the viability of hepatocytes were adversely affected by the condition of foetus at birth and use of intra-amniotic injections for inducing abortions. Hepatocyte monolayers and colonies formed in 75% experiments. The cultures could be maintained in incubation without the use of epidermal or hepatocyte growth factors for 2-25 days with a mean survival of 8.9 days. The cells in culture were found to be structurally normal and functionally active and could be cryo-preserved. These hepatocytes were inoculated into a hollow fiber module to formulate bio-artificial liver support device. The cultures ultimately developed either cellular disintegration or bacterial infections despite use of antibiotics in the culture medium.


CONCLUSIONS
We conclude that it is feasible to maintain foetal hepatocyte cultures without the use of expensive growth factors for over 8 days. Bio-artificial liver formulated with cultured foetal hepatocytes is now a step closer to clinical trials.
Anand
A C
AC
Department of Gastroenterology, Command Hospital (WC), Chandimandir, India.
Bhonde
R R
RR
Kurup
S
S
Kochhar
S P
SP
D001187:Artificial Organs; D017690:Cell Transplantation; D002478:Cells, Cultured; D005240:Feasibility Studies; D005333:Fetus / Q000166:cytology*; D006801:Humans; D008099:Liver / Q000166:cytology*; D008854:Microscopy, Electron; D013997:Time Factors