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Novel bioactive polymeric membranes to elicit specific human hepatocyte responses
Desalination 199 (2006) 261–262
Novel bioactive polymeric membranes to elicit specific human hepatocyte responses Loredana De Bartoloa*, Sabrina Morellib, Simona Salernoa, Carla Campanaa, Antonella Piscioneria, Maria Rendea, Augustinus Baderb, Enrico Driolia a
Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, Via P. Bucci, cubo 17/C, Rende (CS), Italy b Biomedical-Biotechnological Center, BBZ, University of Liepzig, Germany email: [email protected] Received 26 October 2005; accepted 2 March 2006
Keywords: Polymeric membranes; Human hepatocytes; Liver specific functions; Culture
1. Introduction The aim of the paper was the design and development of new bioactive polymeric membranes to be used for the in vitro reconstruction of liver tissue-model [1–2]. Significant efforts have also been made to develop new polymeric materials able to favour the liver reconstruction and to modulate liver cell behaviour. We developed membranes from a polymeric blend of modified polyether-etherketone (PEEK-WC) and polyurethane (PU) by inverse phase technique by using the direct immersionprecipitation method (PEEK-WC-PU) [3]. The morphological and physico-chemical properties of the PEEK-WC-PU membranes were established before their use in the cell culture system. *Corresponding author.
Preliminarily a screening of this new material was performed investigating the specific liver functions in terms of albumin and urea synthesis using primary human hepatocytes (Cambrex Bio Science) cultured on PEEK-WC-PU membranes in comparison with collagen film. The biotransformation functions of human liver cells were evaluated using diclofenac (DIC), a nonsteroidal anti-inflammatory drug that is widely used in painful and inflammatory rheumatic and nonrheumatic conditions. Then the performance of the novel PEEK-WC-PU membranes was validated by using the biohybrid system constituted by human hepatocytes and PEEK-WC-PU membrane in a oxygen permeable membrane bioreactor. Liver specific functions in terms of urea synthesis and total protein secretion was evaluated in the time. Drug biotransformation was also assessed using Diazepam, an anti anxiety agent (benzodiazepines), as model drug.
Presented at EUROMEMBRANE 2006, 24–28 September 2006, Giardini Naxos, Italy. 0011-9164/06/$– See front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2006.03.063
262
L. De Bartolo et al. / Desalination 199 (2006) 261–262
2. Results and discussion The morphology of PEEK-WC-PU membrane was characterized by scanning electron microscopy (SEM) that showed a surface with small pores regularly distributed over the surface with an average pore size of 0.16 mm, a porosity of 62 ± 9% and a thickness of 46 ± 4 mm. The physico-chemical properties of the membranes were characterized by means of water contact angle (WCA) measurements. This analysis demonstrated that the PEEK-WC-PU membrane surface has moderate wettability with a water contact angle of about 74° ± 3, which was constant in the time. The liver specific functions of the human hepatocytes on membranes were investigated by estimating the rate of albumin production and urea synthesis in culture time up to 24 days. In both case the metabolic activity was best expressed when hepatocytes were cultured on the novel membranes with respect the collagen film, for all culture time. Proteins secreted by cells in the medium were identified by gel electrophoresis that evidenced a band corresponding to molecules with MW of 66 KD, like albumin, which is more marked on polymeric membrane than on collagen. Also the biotransformation functions were maintained for all culture period. In particular, the DIC elimination rate reached a peak on day 2, followed by a decrease and, thereafter, the values remained constant during the culture time. The performance of PEEK-WC-PU membranes was also evaluated on the basis of human hepatocyte culture on this new material in a bioreactor. The modified membrane was deposed at the bottom of the chamber delimited by flat-sheet gas-permeable polymeric membranes that ensured the diffusion of O2 and CO2. The obtained results confirmed the ability of novel PEEK-WC-PU membranes to support the specific liver metabolic functions. In particular urea and
protein synthesis were maintained at good level during the culture time up to 17 days. Human hepatocytes cultured in this PEEK-WC-PU membrane bioreactor were also able to eliminate diazepam at the concentration of 10 mg/mL. 3. Conclusions Novel PEEK-WC-PU membrane results an interesting material which is able to support the long-term maintenance and differentiation of human hepatocytes. These results evidenced a good performance of PEEK-WC-PU membranes in terms of albumin and urea synthesis. Moreover the experiments with the bioreactor demonstrated that the biohybrid system constituted by human hepatocytes and PEEK-WC-PU membrane also elicit the specific metabolic activity and the biotransformation function of the human hepatocytes. Acknowledgments This work was supported by grants from European Commission through the Livebiomat project, Contract No. NMP3-CT-2005-013653. References [1]
[2]
[3]
L. De Bartolo, S. Morelli, A. Bader and E. Drioli, Evaluation of cell behaviour related to physicochemical properties of polymeric membranes to be used in bioartificial organs, Biomaterials, 23 (2002) 2485–2497. L. De Bartolo, S. Morelli, M. Rende, A. Gordano and E. Drioli, New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes, Biomaterials, 25 (2004) 3621–3629. L. De Bartolo, S. Morelli, M.C. Gallo, C. Campana, G. Statti, M. Rende, S. Salerno and E. Drioli, Effect of isoliquiritigenin on viability and differentiated functions of human hepatocytes on PEEK-WC-polyurethane membranes, Biomaterials, 25 (2005) 6625–6634.
Novel bioactive polymeric membranes to elicit specific human hepatocyte responses Loredana De Bartoloa*, Sabrina Morellib, Simona Salernoa, Carla Campanaa, Antonella Piscioneria, Maria Rendea, Augustinus Baderb, Enrico Driolia a
Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, Via P. Bucci, cubo 17/C, Rende (CS), Italy b Biomedical-Biotechnological Center, BBZ, University of Liepzig, Germany email: [email protected] Received 26 October 2005; accepted 2 March 2006
Keywords: Polymeric membranes; Human hepatocytes; Liver specific functions; Culture
1. Introduction The aim of the paper was the design and development of new bioactive polymeric membranes to be used for the in vitro reconstruction of liver tissue-model [1–2]. Significant efforts have also been made to develop new polymeric materials able to favour the liver reconstruction and to modulate liver cell behaviour. We developed membranes from a polymeric blend of modified polyether-etherketone (PEEK-WC) and polyurethane (PU) by inverse phase technique by using the direct immersionprecipitation method (PEEK-WC-PU) [3]. The morphological and physico-chemical properties of the PEEK-WC-PU membranes were established before their use in the cell culture system. *Corresponding author.
Preliminarily a screening of this new material was performed investigating the specific liver functions in terms of albumin and urea synthesis using primary human hepatocytes (Cambrex Bio Science) cultured on PEEK-WC-PU membranes in comparison with collagen film. The biotransformation functions of human liver cells were evaluated using diclofenac (DIC), a nonsteroidal anti-inflammatory drug that is widely used in painful and inflammatory rheumatic and nonrheumatic conditions. Then the performance of the novel PEEK-WC-PU membranes was validated by using the biohybrid system constituted by human hepatocytes and PEEK-WC-PU membrane in a oxygen permeable membrane bioreactor. Liver specific functions in terms of urea synthesis and total protein secretion was evaluated in the time. Drug biotransformation was also assessed using Diazepam, an anti anxiety agent (benzodiazepines), as model drug.
Presented at EUROMEMBRANE 2006, 24–28 September 2006, Giardini Naxos, Italy. 0011-9164/06/$– See front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2006.03.063
262
L. De Bartolo et al. / Desalination 199 (2006) 261–262
2. Results and discussion The morphology of PEEK-WC-PU membrane was characterized by scanning electron microscopy (SEM) that showed a surface with small pores regularly distributed over the surface with an average pore size of 0.16 mm, a porosity of 62 ± 9% and a thickness of 46 ± 4 mm. The physico-chemical properties of the membranes were characterized by means of water contact angle (WCA) measurements. This analysis demonstrated that the PEEK-WC-PU membrane surface has moderate wettability with a water contact angle of about 74° ± 3, which was constant in the time. The liver specific functions of the human hepatocytes on membranes were investigated by estimating the rate of albumin production and urea synthesis in culture time up to 24 days. In both case the metabolic activity was best expressed when hepatocytes were cultured on the novel membranes with respect the collagen film, for all culture time. Proteins secreted by cells in the medium were identified by gel electrophoresis that evidenced a band corresponding to molecules with MW of 66 KD, like albumin, which is more marked on polymeric membrane than on collagen. Also the biotransformation functions were maintained for all culture period. In particular, the DIC elimination rate reached a peak on day 2, followed by a decrease and, thereafter, the values remained constant during the culture time. The performance of PEEK-WC-PU membranes was also evaluated on the basis of human hepatocyte culture on this new material in a bioreactor. The modified membrane was deposed at the bottom of the chamber delimited by flat-sheet gas-permeable polymeric membranes that ensured the diffusion of O2 and CO2. The obtained results confirmed the ability of novel PEEK-WC-PU membranes to support the specific liver metabolic functions. In particular urea and
protein synthesis were maintained at good level during the culture time up to 17 days. Human hepatocytes cultured in this PEEK-WC-PU membrane bioreactor were also able to eliminate diazepam at the concentration of 10 mg/mL. 3. Conclusions Novel PEEK-WC-PU membrane results an interesting material which is able to support the long-term maintenance and differentiation of human hepatocytes. These results evidenced a good performance of PEEK-WC-PU membranes in terms of albumin and urea synthesis. Moreover the experiments with the bioreactor demonstrated that the biohybrid system constituted by human hepatocytes and PEEK-WC-PU membrane also elicit the specific metabolic activity and the biotransformation function of the human hepatocytes. Acknowledgments This work was supported by grants from European Commission through the Livebiomat project, Contract No. NMP3-CT-2005-013653. References [1]
[2]
[3]
L. De Bartolo, S. Morelli, A. Bader and E. Drioli, Evaluation of cell behaviour related to physicochemical properties of polymeric membranes to be used in bioartificial organs, Biomaterials, 23 (2002) 2485–2497. L. De Bartolo, S. Morelli, M. Rende, A. Gordano and E. Drioli, New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes, Biomaterials, 25 (2004) 3621–3629. L. De Bartolo, S. Morelli, M.C. Gallo, C. Campana, G. Statti, M. Rende, S. Salerno and E. Drioli, Effect of isoliquiritigenin on viability and differentiated functions of human hepatocytes on PEEK-WC-polyurethane membranes, Biomaterials, 25 (2005) 6625–6634.