- Email: [email protected]
In vitro optimization of alginate bead production for hepatocytes encapsulation in the fluidized bed bioartificial liver
$252
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
7206 Th, 11:30-11:45 (P41) Artificial liver support: New challenges for the application o f extracorporeal blood circuits J. Vienken. Fresenius Medical Care, BioSciences Department, Bad Homburg, Germany The application of extracorporeal blood circuits in liver failure therapy has its roots in the two functions of the liver as a detoxifying and synthetizing organ. Most recent developments in extracorporeal liver failure therapy have been targeted towards detoxification devices using specifically designed filters and adsorbers for the removal of toxins. This is mandatory, because in contrast to the hydrophilic uremic toxins, most of liver toxins are hydrophobic and bind preferentially to blood proteins. Consequently, hemodialysis is unable to remove the majority of these compounds. A current system uses albumin as a transport vehicle for hydrophobic compounds across a high flux membrane (e.g., Albumin-dialysis, MARS®-System, Gambro-Teraklin). In contrast to these devices, the Prometheus®-System of Fresenius Medical Care applies extremely permeable capillary membranes (molecular weight cut-off: >300,000). They allow for a direct filtration of most of the toxin-bearing proteins. In a secondary circuit these toxins are removed with the help of adsdorber columns. The proteinaceous toxin-free solution, then, returns back to the primary circuit. Such detoxification systems are currently assessed in clinical trials. Clinical results on safety parameters did prove that no considerable changes have been observed when albumin levels, cell counts and coagulation factors were assessed. Bilirubin-, bile acid- and plasma ammonia-levels were considerably reduced, as shown by these figures -21%, -43% and -40%, respectively. First successful therapeutical results have been seen on patients with drug abuse and during bridging to transplantation. Longterm applications of the Prometheus System, i.e. repeated use for more than 20 times, have proven that the system is safe and tolerable by patients on intensive care. Conclusion: Extracorporeal blood circuits are currently in clinical trials for the treatment of patients with liver failure. They mostly imply detoxification systems on the basis of filtering and adsorbing devices. Expected results of ongoing clinical trials will allow for the establishment of this approach as a routine therapy. 6941 Th, 11:45-12:00 (P41) In vitro optimization of alginate bead production for hepatocytes encapsulation in the fluidized bed bioartificial liver A. Gautier 1, A. Kinasiewicz 2, B. Carpentier 1, D. Lewiriska 2, P. Paullier 1, J. Bukowski 2, A. Weryr'lsky 2, C. Legallais 1. 1Universit6 de Technologie de Compiegne, CNRS UMR 6600, Biom#canique et G6nie Biom6dical, Compiegne, France, 2Institute of Biocybemetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland
Background: Bioartificial liver support needs metabolically active hepatocytes in high amounts. Cells cultured in 3D environment exhibit higher activity in comparison with monolayer culture. Fluidised bed bioreactors offer an environment of low shear stress and high mass transfer suitable for high density culture of encapsulated cells. They present an alternative to hollow fiber bioreactors previously used in bioartificial livers tested to date. Also their functionality has already been demonstrated in vitro with 1 mm diameter alginate bead, no study was led up to now to assess to optimize th bead diameter and the alginate composition. Methods: The aim of our study was to investigate two hepatocyte lines HepG2 and C3A in fluidized bed bioreactor, with different encapsulation protocols. HepG2 and C3A cells were cultured in DMEM Medium and encapsulated in alginate beads by two methods: air jet and in electrostatic field. The viability of cells was assessed by trypan blue exclusion test or by fluorescence staining with acridine orange and propidium iodide. After encapsulation capsule diameter and cell viability were measured. Then, capsules suspension was placed in fluidized bioreactor. Albumin and urea concentrations in culture medium were measured over time. Results: Diameter of microcapsules obtained by both methods ranged from 0.50 to 1.00mm. The electrostatic method was more efficient to produce homogeneous beads in the lower range of diameters, and resulted in a lower amount of residues. Cell viability after encapsulation was comparable in both methods, and ranged from 88 to 92%. Culture of cells in bioreactor was successfully maintained over 48 h under sterile conditions. They revealed significant increase in albumin production with time of culture. Conclusion: Both encapsulating methods seems suitable for hepatocyte entrapment before the perfusion in the fluidized bed bioartificial liver.
Oral Presentations 6608 Th, 12:00-12:15 (P41) Numerical modeling o f o x y g e n availability in the AMC bioartificial liver G. Mareels 1, EEC. Poyck 2, S. Eloot 1, R.A.EM. Chamuleau 2, ER. Verdonck 1. 1Cardiovascular Mechanics and Biofluid Dynamics research unit, Institute Biomedical Technology, Ghent University, Belgium, 2Departments of Experimental Surgery and Hepatology, Amsterdam Medical Center, University of Amsterdam, The Netherlands Oxygen (02) transfer to hepatocytes is considered the main limitation in the efficiency of bioartificial livers (BAL). A numerical model was developed and Computational Fluid Dynamics simulations were used to assess and possibly improve 02 availability in the AMC Bioartificial Liver (Academic Medical Center, Amsterdam, The Netherlands). The AMC-BAL is a cylindrical bioreactor with a non-woven mat containing functional active hepatocytes and gas capillaries to supply additional 02 to the hepatocytes. Two characteristic unit volumes (micro models) of the AMCBAL, each consisting of a piece of mat and capillaries either in an inline or triangular pattern, were identified and constructed in three-dimensional computer models. A 'theoretical' hepatocyte distribution (100% hepatocytes in mat) and a 'realistic' (50% hepatocytes in mat; 50% around capillaries) hepatocyte distribution were applied to each micro model. On these 4 cases, 02 transport and non-linear Michaelis-Menten 02 consumption were simulated under standard operating conditions to assess the 02 availability for the complete BAL. Oxygen availability was quantified by determining the percentage of hepatocytes which can consume 02 at a level of minimally 90% of maximal 02 uptake rate. Subsequently, operating conditions (culture medium flow rate and 02 level (pO2) and oxygenation gas pO2) and design parameters (number of capillaries) were altered to assess different strategies to improve 02 availability. Standard operating conditions only provided 16% of all hepatocytes in theoretical and 29% in realistic hepatocyte distribution with sufficient 02 in order to consume 02 at minimally 90% of maximal uptake rate. Capillary pattern had no significant influence. Optimal increase in 02 availability to 60% and 86% in theoretical and realistic hepatocyte distribution respectively, was achieved by combining double number of capillaries and double culture gas pO 2. Adaptation of the design may ultimately lead to a more efficient AMC-BAL. 7218 Th, 12:15-12:30 (P41) Development o f a standardised bioreactor with 3D capillary membrane structure U. Kertzscher 1, L. Goubergrits 1, K. Affeld 1, I. Guido 1, E Scharfschwert 1, J. Hengstler 2. 1Biofluidmechanics Laboratory, Char#& Berlin, Germany, 2 Universit#t Leipzig, Zentrum f#r Toxikologie, Leipzig, Germany All cells in bioreactors have to live without the natural network of capillaries, arteries and veins. In single layer cell cultures the mass flow of gases and nutrients can be achieved by diffusion alone. If, however, a higher cell volume requires a three dimensional structure, transport by diffusion alone will not suffice. One needs to introduce a convective transport. In nature, this is very effectively achieved by a network of capillaries. The next best we have in comparison to natural capillaries carrying blood are artificial capillaries carrying an appropriate gas or fluid. These artificial capillaries usually have pores, which permit the passage of the molecules needed by the cells. The aim of the project is the development of a standardized bioreactor with a 3D capillary structure which is easy to fabricate. In addition the bioreactor should be scalable to permit the culture of a larger or smaller cell mass and a window for microscopy is needed. For hepatocyte research we designed and fabricated four different sizes of bioreactors: 0.07, 4, 10, and 400ml. Purpose of the big cell chamber is an approach to a clinical useful size. The smallest bioreactor has superior transparency for microscopic use: It permits a 3D arrangement of cells; however the thickness still permits the microscopic observation. To enhance the transparency capillaries made of cellulose are used for the smallest bioreactor. In addition the mass transfer inside the bioreactors was calculated using methods of computational fluid dynamics and investigated by experimental methods. The experiments confirmed the numerical results. Future scientific aim is the possibility to manipulate different parameters in the bioreactor. With these manipulations it is possible to investigate the cell reaction to low oxygen supply, changing pH-value, not optimal temperature, oxidative stress caused by light and so on. 7744 Th, 14:00-14:30 (P44) Human bioartificial livers from hepatic stem cells W. Turner2,4, R. McClelland 1,4, L. Zhang 1, E. Schmelzer 1, E. Wauthier 1, A. Melhem 1, H. Yao 2, N. Cheng 1, J. Gerlach 3, L.M. Reid 1,2. 1Departments of Cell and Molecular Physiology and 2Biomedical Engineering, UNC School of Medicine, Chapel Hill, NC, USA, 3McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Sourcing of human liver cells has remained a hurdle obviating clinical trials of bioartificial livers. The expansion potential of human hepatic stem cells under
Journal o f Biomechanics 2006, Vol. 39 (Suppl 1)
7206 Th, 11:30-11:45 (P41) Artificial liver support: New challenges for the application o f extracorporeal blood circuits J. Vienken. Fresenius Medical Care, BioSciences Department, Bad Homburg, Germany The application of extracorporeal blood circuits in liver failure therapy has its roots in the two functions of the liver as a detoxifying and synthetizing organ. Most recent developments in extracorporeal liver failure therapy have been targeted towards detoxification devices using specifically designed filters and adsorbers for the removal of toxins. This is mandatory, because in contrast to the hydrophilic uremic toxins, most of liver toxins are hydrophobic and bind preferentially to blood proteins. Consequently, hemodialysis is unable to remove the majority of these compounds. A current system uses albumin as a transport vehicle for hydrophobic compounds across a high flux membrane (e.g., Albumin-dialysis, MARS®-System, Gambro-Teraklin). In contrast to these devices, the Prometheus®-System of Fresenius Medical Care applies extremely permeable capillary membranes (molecular weight cut-off: >300,000). They allow for a direct filtration of most of the toxin-bearing proteins. In a secondary circuit these toxins are removed with the help of adsdorber columns. The proteinaceous toxin-free solution, then, returns back to the primary circuit. Such detoxification systems are currently assessed in clinical trials. Clinical results on safety parameters did prove that no considerable changes have been observed when albumin levels, cell counts and coagulation factors were assessed. Bilirubin-, bile acid- and plasma ammonia-levels were considerably reduced, as shown by these figures -21%, -43% and -40%, respectively. First successful therapeutical results have been seen on patients with drug abuse and during bridging to transplantation. Longterm applications of the Prometheus System, i.e. repeated use for more than 20 times, have proven that the system is safe and tolerable by patients on intensive care. Conclusion: Extracorporeal blood circuits are currently in clinical trials for the treatment of patients with liver failure. They mostly imply detoxification systems on the basis of filtering and adsorbing devices. Expected results of ongoing clinical trials will allow for the establishment of this approach as a routine therapy. 6941 Th, 11:45-12:00 (P41) In vitro optimization of alginate bead production for hepatocytes encapsulation in the fluidized bed bioartificial liver A. Gautier 1, A. Kinasiewicz 2, B. Carpentier 1, D. Lewiriska 2, P. Paullier 1, J. Bukowski 2, A. Weryr'lsky 2, C. Legallais 1. 1Universit6 de Technologie de Compiegne, CNRS UMR 6600, Biom#canique et G6nie Biom6dical, Compiegne, France, 2Institute of Biocybemetics and Biomedical Engineering, Polish Academy of Sciences, Warsaw, Poland
Background: Bioartificial liver support needs metabolically active hepatocytes in high amounts. Cells cultured in 3D environment exhibit higher activity in comparison with monolayer culture. Fluidised bed bioreactors offer an environment of low shear stress and high mass transfer suitable for high density culture of encapsulated cells. They present an alternative to hollow fiber bioreactors previously used in bioartificial livers tested to date. Also their functionality has already been demonstrated in vitro with 1 mm diameter alginate bead, no study was led up to now to assess to optimize th bead diameter and the alginate composition. Methods: The aim of our study was to investigate two hepatocyte lines HepG2 and C3A in fluidized bed bioreactor, with different encapsulation protocols. HepG2 and C3A cells were cultured in DMEM Medium and encapsulated in alginate beads by two methods: air jet and in electrostatic field. The viability of cells was assessed by trypan blue exclusion test or by fluorescence staining with acridine orange and propidium iodide. After encapsulation capsule diameter and cell viability were measured. Then, capsules suspension was placed in fluidized bioreactor. Albumin and urea concentrations in culture medium were measured over time. Results: Diameter of microcapsules obtained by both methods ranged from 0.50 to 1.00mm. The electrostatic method was more efficient to produce homogeneous beads in the lower range of diameters, and resulted in a lower amount of residues. Cell viability after encapsulation was comparable in both methods, and ranged from 88 to 92%. Culture of cells in bioreactor was successfully maintained over 48 h under sterile conditions. They revealed significant increase in albumin production with time of culture. Conclusion: Both encapsulating methods seems suitable for hepatocyte entrapment before the perfusion in the fluidized bed bioartificial liver.
Oral Presentations 6608 Th, 12:00-12:15 (P41) Numerical modeling o f o x y g e n availability in the AMC bioartificial liver G. Mareels 1, EEC. Poyck 2, S. Eloot 1, R.A.EM. Chamuleau 2, ER. Verdonck 1. 1Cardiovascular Mechanics and Biofluid Dynamics research unit, Institute Biomedical Technology, Ghent University, Belgium, 2Departments of Experimental Surgery and Hepatology, Amsterdam Medical Center, University of Amsterdam, The Netherlands Oxygen (02) transfer to hepatocytes is considered the main limitation in the efficiency of bioartificial livers (BAL). A numerical model was developed and Computational Fluid Dynamics simulations were used to assess and possibly improve 02 availability in the AMC Bioartificial Liver (Academic Medical Center, Amsterdam, The Netherlands). The AMC-BAL is a cylindrical bioreactor with a non-woven mat containing functional active hepatocytes and gas capillaries to supply additional 02 to the hepatocytes. Two characteristic unit volumes (micro models) of the AMCBAL, each consisting of a piece of mat and capillaries either in an inline or triangular pattern, were identified and constructed in three-dimensional computer models. A 'theoretical' hepatocyte distribution (100% hepatocytes in mat) and a 'realistic' (50% hepatocytes in mat; 50% around capillaries) hepatocyte distribution were applied to each micro model. On these 4 cases, 02 transport and non-linear Michaelis-Menten 02 consumption were simulated under standard operating conditions to assess the 02 availability for the complete BAL. Oxygen availability was quantified by determining the percentage of hepatocytes which can consume 02 at a level of minimally 90% of maximal 02 uptake rate. Subsequently, operating conditions (culture medium flow rate and 02 level (pO2) and oxygenation gas pO2) and design parameters (number of capillaries) were altered to assess different strategies to improve 02 availability. Standard operating conditions only provided 16% of all hepatocytes in theoretical and 29% in realistic hepatocyte distribution with sufficient 02 in order to consume 02 at minimally 90% of maximal uptake rate. Capillary pattern had no significant influence. Optimal increase in 02 availability to 60% and 86% in theoretical and realistic hepatocyte distribution respectively, was achieved by combining double number of capillaries and double culture gas pO 2. Adaptation of the design may ultimately lead to a more efficient AMC-BAL. 7218 Th, 12:15-12:30 (P41) Development o f a standardised bioreactor with 3D capillary membrane structure U. Kertzscher 1, L. Goubergrits 1, K. Affeld 1, I. Guido 1, E Scharfschwert 1, J. Hengstler 2. 1Biofluidmechanics Laboratory, Char#& Berlin, Germany, 2 Universit#t Leipzig, Zentrum f#r Toxikologie, Leipzig, Germany All cells in bioreactors have to live without the natural network of capillaries, arteries and veins. In single layer cell cultures the mass flow of gases and nutrients can be achieved by diffusion alone. If, however, a higher cell volume requires a three dimensional structure, transport by diffusion alone will not suffice. One needs to introduce a convective transport. In nature, this is very effectively achieved by a network of capillaries. The next best we have in comparison to natural capillaries carrying blood are artificial capillaries carrying an appropriate gas or fluid. These artificial capillaries usually have pores, which permit the passage of the molecules needed by the cells. The aim of the project is the development of a standardized bioreactor with a 3D capillary structure which is easy to fabricate. In addition the bioreactor should be scalable to permit the culture of a larger or smaller cell mass and a window for microscopy is needed. For hepatocyte research we designed and fabricated four different sizes of bioreactors: 0.07, 4, 10, and 400ml. Purpose of the big cell chamber is an approach to a clinical useful size. The smallest bioreactor has superior transparency for microscopic use: It permits a 3D arrangement of cells; however the thickness still permits the microscopic observation. To enhance the transparency capillaries made of cellulose are used for the smallest bioreactor. In addition the mass transfer inside the bioreactors was calculated using methods of computational fluid dynamics and investigated by experimental methods. The experiments confirmed the numerical results. Future scientific aim is the possibility to manipulate different parameters in the bioreactor. With these manipulations it is possible to investigate the cell reaction to low oxygen supply, changing pH-value, not optimal temperature, oxidative stress caused by light and so on. 7744 Th, 14:00-14:30 (P44) Human bioartificial livers from hepatic stem cells W. Turner2,4, R. McClelland 1,4, L. Zhang 1, E. Schmelzer 1, E. Wauthier 1, A. Melhem 1, H. Yao 2, N. Cheng 1, J. Gerlach 3, L.M. Reid 1,2. 1Departments of Cell and Molecular Physiology and 2Biomedical Engineering, UNC School of Medicine, Chapel Hill, NC, USA, 3McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Sourcing of human liver cells has remained a hurdle obviating clinical trials of bioartificial livers. The expansion potential of human hepatic stem cells under