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Materials and methods
All media and chemicals used in the cell culturing, transport and dissolution of the liver are as described by Nieuwoudt et al (2005)[87]. Perfluorocarbon-lecithin emulsions were prepared according to the method of Moolman et al (2004) [10,94].
The emulsions were mixed with double concentrated MEM and sterile deionised water, to achieve a 20% v/v PFC emulsion-MEM mixture. The mixture was pH adjusted and used as a culturing medium. For liver perfusion and hepatocyte isolation the method of Nieuwoudt et al (2005)[87] was used throughout. Cell count and viability was assessed with Trypan-blue. The suspension was then seeded into a sealed recirculating system in an incubator, representing a simplified dynamic model of the BALSS. Briefly, each system (figure 3.2.1) was composed of LS 25 Masterflex tubing that connected a 500 ml glass reservoir, a 100 ml internal volume bioreactor, with a sampling port, and the reservoir medium was oxygenated using an aquarium bubbler. All was housed in a non-gas incubator at 37 oC (Scientific 2000, Instrulab, South Africa). To facilitate homogenous seeding throughout the matrix, the bioreactor was designed for even flow using computational fluid dynamics (CFD), see section 3 above. A total of eight, 7-day long metabolic trials were conducted on the dynamic and static systems. Five studies employed ordinary MEM, (PFC(-)), while 3 used the supplemented PFC-MEM emulsion (PFC(+)). Cell culturing and metabolic evaluation All methods are as defined by Nieuwoudt et al [88] and presented in Appendix A3. Briefly, daily samples were taken for LD, AST, glucose, lactate and pyruvate. Blood gas samples for pH, pO2, pCO2 were taken and the oxygen uptake rate (OUR) was calculated.
Thoughts and recommendations
The above in vitro cell biology and bioreactor studies presented a large scale, sterile primary cell isolation procedure, an investigation of hepatocyte metabolism over 7-days in a direct plasma contact hepatocyte bioreactor and the novel use of PET to discriminate between O2 challenged PFC versus non-PFC flow optimized bioreactors. From a simple statistical perspective the primary cell isolation method has been very successful: In 40 procedures over a 3 year period, a mean of 2.14 x 1010 + 8.60 x 108 hepatocytes and 2.43 x 109 + 1.82 x 108 stellate cells were isolated from the livers of 30 kg pigs with liver masses of approximately 1.5 kg each. Bacterial or fungal contaminations were not found in any of these, which verified the sterility of the procedures.
Of interest, assuming an adult liver has between 1.0 and 1.5 x 1011 hepatocytes, 2.14 x 1010 equates to approximately 15 – 20 % of the total amount, or assuming the liver is composed of 70 % hepatocytes and has a mass of 1.5 kg, the isolated hepatocyte mass weighs 158 – 210 g. These values are within the amount often quoted to be sufficient for an effective bioartificial liver support device [70,83,92]. Having said that, it is ideal to have as large a cell mass as possible, assuming the conditions in the bioreactor are sufficient to maintain them. The disposable BRAT bowls used in the above method were of a fixed volume (either 250 or 165 ml) which limited the total amount of cells that could be isolated. There are consequently efforts underway to use other larger volume apparatus which will increase the isolatable quantity. The second study successfully demonstrated that there was metabolic activity in both PFC and non-PFC bioreactors over a 7 day period, but presumably due to the employed gas mix was unable to show a difference between them. During the course of this study and in unpublished efforts subsequently, some of the difficulties involved in successfully demonstrating the efficacy of such bioreactors was further highlighted. For example, the mentioned lack of consensus regarding reporting methods, the great variations in reported results and the difficulty of measuring in situ cell functions due to a lack of non invasive methods enabling direct access to them. In specifically the latter, we found that the PFC and fragments of the PUF matrix were difficult to remove from cell aggregate samples taken from dismantled bioreactors after termination. Gene expression and flow cytometry experiments were consequently unreliable.
The subsequent novel use of PET was an attempt to solve the above problems and to conclusively demonstrate that the PFC facilitated cell function under hypoxic conditions, as may be found in the treatment of an acute liver failure patient. They were also performed on the same duration following a primary cell isolation procedure that would occur if such cells were employed in the BAL device. The success of the experiments was partly owing to the use of radio-transparent bioreactor material and attention to maintaining exactly the same experimental conditions.
1 INTRODUCTION
1.1 Background
1.2 Defining Models
1.2 Problem statement
1.3 Thesis structure
1.4 Copyright and authorship issues
2 The Clinical And Biological Background Of Acute Liver Failure and Liver Support Technology
2.1 Introduction
2.2 Defining ALF
2.3 Epidemiology and etiology
2.4 Pathogenesis and the clinical syndrome
2.5 Prognostic scoring systems
2.6 Orthotopic Liver Transplantation
2.7 Liver support systems
3 The design of the UP-CSIR BALSS
3.1 Bioreactor optimization
3.2 Circulation system optimization
4 In Vitro Cell biology studies overview
4.1 A large scale automated method for hepatocyte isolation
4.2 A study to determine hepatocyte function in the UP-CSIR radial-flow bioreactor using a perfluorocarbon oxygen carrier
4.3 Imaging glucose metabolism in hepatocyte-stellate co-culture bioreactors using positron emission tomography
4.4 Thoughts and recommendations
5 IN VIVO ANIMAL STUDIES overview
5.1 Non-toxicity of IV injected perfluorocarbon oxygen carrier in an animal model of liver regeneration following surgical injury
5.2 Standardization criteria for an ischemic surgical model of acute hepatic failure in pigs
5.3 Thoughts and recommendations
6 Mathematical MODELING STUDIES overview
6.1 A pharmacokinetic compartment model of the UP-CSIR BALSS
6.2 Developing an on-line predictive clinical monitoring system for acute liver failure patients
6.3 Thoughts and recommendations
7 SUMMARY AND CONCLUSION
8 References
9 AppendiCES
Appendix A: In vitro study methods
Appendix B: In vivo study methods
Appendix C: The derivation of the compartmental model equations
Appendix D: On line model sensitivity and verification
