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Problem background
Acute liver failure is a devastating condition with high mortality rates (higher than 80% – Dixit and Gitnick, 1996:101). Orthotopic liver transplantation is currently the only available treatment. The high mortality rate i due to a number of reasons, including shortage of suitable donors, short time window between onset of acute liver failure and death (a few weeks) and the availability of medical centers able and equipped to perform the procedure.
The high mortality rate of patients suffering from acute liver failure could be reduced if a therapy was available that could support the patient’s liver functions, either until patient recovery (due to the liver’s well-known regeneration ability) or until liver transplantation.
Such a therapy or treatment system would have to meet a number of requirements in order to bridge the patient until transplantation or recovery:
It must perform 20 – 40% of normal adult human liver function (Tsiaoussis et al., 2001:5; Olson, Bradley and Mate, 1999; Sussman and Kelly, 1997:S67). There is still uncertainty as to exactly which of the large number of liver functions (some of which are still not understood) are essential for patient survival (Allen, Hassanein and Bhatia, 2001:447; Brems et al., 2001; Park, Iwata and Ikada, 2001:296). Some authors think that as little as 3-5% of total liver mass could make a difference (Kamohara, Rozga and Demetriou, 1998:278).
There must be a sufficient rate of exchange / circulation of blood between patient and system to have an impact on levels of relevant molecules in patient’s blood (Iwata, Park and Ikada, 1998:235).
Immune reactions & risk of zoonosis (incl. cell debris) must be minimized / properly managed The system must be as easy to use as possible (e.g. must fit on a bedside trolley; require minimal human intervention)
The system must be cost-effective and costs should compare favourably with normal intensive care treatment for patients suffering from acute liver failure. Extracorporeal support for liver failure patients has been researched for over 40 years (Allen, Hassanein and Bhatia, 2001:447), but no commercial system is available that has had clinical success.
Please note that all raw experimental data is contained in spreadsheets (Microsoft Excel 2000) included on a CD attached to the inside back cover of this dissertation.
Due to the complexity of the liver, a simple filtration system (analogous to the kidney dialysis machine) is not sufficient for liver support. The liver performs a large number of functions, including metabolic, regulatory and secretory functions. This complexity necessitates the inclusion of live liver cells (hepatocytes) in any system that has to support or perform liver functions.
Systems containing hepatocytes in a bioreactor for the purpose of liver support are generally referred to as Bioartificial Liver Support Systems (BALSS). The inclusion of hepatocytes in the system adds additional requirements to the BALSS:
A suitable environment / surface for cell adhesion and proliferation (hepatocytes are anchoragedependent – Busse and Gerlach, 1999:328);
Oxygen and nutrient supply for sustaining cell growth and function – hepatocytes have very high oxygen demands compared to most other mammalian cells (Hay, Veitch and Gaylor, 2001:119);
Regulation of carbon dioxide levels for optimal cell functioning – carbon dioxide levels in blood plasma influence the pH of the blood plasma due to the dissociation of dissolved carbon dioxide (H2CO3) into bicarbonate (HCO3 – ) and hydrogen (H+ ) ions. Proper control of the pH in the circulation system is of critical importance to maintain optimal cell function. Acidosis could occur if carbon dioxide generated by the cells is not removed. The pH of blood plasma is directly related to the carbon dioxide partial pressure and can be calculated from the Henderson-Hasselbalch equation (Ludwig, 2002):
Immune protection for both patient and hepatocyte bioreactor;
Effective mass transfer between hepatocytes and exchange fluid (either whole blood or blood plasma) from patient (Flendrig, Te Velde and Chamuleau, 1997:1177).
The above set of requirements contains two conflicting requirements: effective mass transfer vs. immune protection. While a barrier such as a membrane could provide immune protection, it simultaneously introduces but introduces an additional mass transfer resistance. The solution to this problem is currently approached in one of two ways:
1. Whole blood – indirect contact: A barrier or membrane is provided, with main purpose to protect the hepatocytes from leukocytes (white blood cells) while still allowing exchange of smaller molecules. Whole blood is perfused on one side of the membrane, while hepatocytes are grown on the other side. The membrane unfortunately also introduces an additional resistance to mass transfer. Hepatocytes further away from the membrane are starved of oxygen, while also functioning sub-optimally due to decreased mass transfer of metabolites, toxins, etc. (because of
increased diffusional distance).
2. Blood plasma – direct contact: Blood plasma is separated from whole blood through filtration, and only the blood plasma is contacted with the hepatocytes. Most molecules related to liver function are dissolved in the blood plasma, allowing liver support through treatment of blood plasma alone (Kamohara, Rozga and Demetriou, 1998:279). As the white blood cells or leukocytes are removed, the risk of immune response activation is reduced, allowing direct perfusion of the blood plasma over the hepatocytes. While this allows higher mass transfer rates between cells and blood plasma compared to membrane-type BALSS, another problem is created.
1. Introduction
1.1. Problem background
1.2. Problem statement
1.3. Project background
2. Liver support systems
2.1. The human liver
2.1.1. Normal liver function
2.1.2. Liver failure and epidemiology
2.2. History of liver support systems
2.2.1. Artificial systems
2.2.2. Biological approaches
2.2.3. Bio-artificial systems
2.2.4. Current development status of liver support systems
2.3. UP-CSIR bio-artificial liver support system
3. Oxygen carrier development
3.1. Overview of oxygen carriers
3.1.1. Hemoglobin-based oxygen carriers
3.1.2. Perfluorocarbon-based oxygen carriers
3.1.3. Oxygen carrier selection for the UP-CSIR bio-artificial liver support system
3.2. Emulsion preparation & stability
3.2.1. Emulsion formulation
3.2.2. Emulsion manufacturing procedure
3.2.3. Emulsion sterilization
3.2.4. Emulsion droplet size distributions
3.2.5. Emulsion stability
3.3. Emulsion rheology
3.3.1. Viscosity of the continuous phase
3.3.2. Volume fraction of the dispersed phase
3.3.3. Droplet size and distribution
3.3.4. Interfacial interactions
3.4. Emulsion concentration
3.4.1. Centrifugal separation
3.4.2. Ultrafiltration
3.5. Perfluorooctyl bromide recovery from emulsion
4. Mass transfer considerations
4.1. Modelling aspects
4.2. Mass transfer model: gas-sparged oxygenator
4.2.1. Basic approach
4.2.2. Mole balances
4.2.3. Specific surface areas
4.2.4. Mass transfer coefficients
4.2.5. Other gas-sparger parameters
4.3. Mass transfer model: membrane oxygenator
4.3.1. Basic approach
4.3.2. Mole balances
4.3.3. Specific surface areas
4.3.4. Mass transfer coefficient
4.3.4. Other membrane oxygenator parameters
4.4. Model and experimental results
4.4.1. Experimental setup
4.4.2. Determination of experimental mass transfer coefficients
4.4.3. Model results and discussion
4.4.4. Conclusions
5. Influence of emulsion on cell growth kinetics and cell function
5.1. Background
5.1.1. Perfluorocarbons and cell culture
5.1.2. Modelling of UP-CSIR BALSS
5.2. Experimental setup
5.3. Results & discussion
6. Conclusions
6.1. Bio-artificial liver support systems
6.2. Perfluorocarbon emulsions
6.3. Mass transfer modelling
6.4. Perfluorocarbons in cell culture
6.5. Recommendations
APPENDIX A: Analytical solution of simultaneous differential equations
for gas-sparger
Appendix B: Derivation of fractional gas-PFOB interfacial specific surface
area
Appendix C: Calculation procedures for membrane oxygenator mass
transfer coefficients
Appendix D: Basic bioreactor design equations
References
