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Human hepatocytes in three-dimensional culture on Innovative biopolymeric scaffolds as a useful system for in vitro toxicology tests
No full textP 1.6
HUMAN HEPATOCY
TES IN THREE-
DIMENSIONAL CULTURE ON INNOVATIVE
BIOPOLYMERIC SCAFFOLDS
AS AN USEFUL SYSTEM
FOR
IN VITRO
TOXICOLOGY TESTS
Stampella A. (a), Massimi M. (b), Barbetta A.
(c), Rizzitelli G. (c), Dentini M. (c), Conti
Devirgiliis L. (a)
(a) Department of Biology and Biotechnology Charles Darwin, Sapienza University of
Rome, Rome, Italy
(b) Department of Basic and Applied Biology, University of L’Aquila, L’Aquila, Italy
(c) Department of Chemistry, Sapienza University of Rome, Rome, Italy
Many innovative biomaterials have recently be developed as scaffolds to replace
physiological matrix components and their im
provement has led to significant advances in
culture techniques in terms of cell survival, quantitative expansion, maintenance of
differentiated phenotype and specific cell functions. A key point in achieving these goals
has been to maintain a three-dimensional culture and the typical cyto-architecture of the
tissue by improving the extracellular matrix geometry and by promoting cell-cell contacts
and reciprocal adhesions. These bio-artificial
systems represent a real hope as functional
substitutes for damaged organs and tissues and have provoked a great interest in the field of
regenerative medicine. Concerning hepatocyte cultures, since the liver is the main organ
involved in detoxification processes and in the defence of organisms against harmful
molecules, in addition to their biomedical applications, these systems can be utilized as
invaluable tool for toxicology tests for analyzing the effects on metabolism of new drugs,
or for screening potentially toxic substances. The aim of our research was to identify the
most suitable biomaterial for the technological applications with hepatocytes. Since the
possibility to improve the performance of thes
e systems depends strongly on the methods
used to create the scaffolds, here we analyzed
porous matrices made of gelatin or blends of
gelatin and glycosaminoglycans, obtained with different methods for the culture of the C3A
cell line, considered a
good model of human hepatocytes. Scaffolds were obtained using
either a concentrated emulsi
on-templating technique known as High Internal Phase
Emulsion (HIPE) or a gas foaming technique; the latter method uses an inert gas instead of
the internal liquid phase toluene, avoiding the use of organic solvent and allowing the
creation of scaffolds with la
rger pores and interconnections. Cell viability was analysed
using MTS and LDH assays; ultrastructural morphology and three-dimensional cell
organization into the scaffold were assessed by SEM; albumin and urea secretion, as the
main metabolic markers of hepatocyte functions, were monitored using, respectively, an
ELISA kit and a colorimetric assay. Finally
Cytochrome P450-3A4 activity was quantified
by a luminescent method. Values of activity of this important enzyme of the detoxification
system, obtained in the absence or in the pr
esence of specific inducing molecules, were
compared between the different culture conditions
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A three-dimensional hepatocyte-alginate culture system as a tool for in vitro pharmacological tests
No full textDielectric characterization of Hepatocytes in suspension and embedded into two different polymeric scaffolds
No full textThe dielectric and conductometric properties of hepatocytes in two different environments (in aqueous suspension and embedded into polymeric scaffolds) have been investigated in the frequency range from 10 kHz to 2 GHz, where the interfacial electrical polarization gives rise to marked dielectric relaxation effects. We analyzed the dielectric behavior of hepatocytes in complete medium aqueous suspensions in the light of effective medium approximation for heterogeneous systems and hepatocytes cultured into two different highly porous and interconnected polymeric structures. In the former case, we have evaluated the passive electrical parameters associated with both the plasmatic and nuclear membrane, finding a general agreement with the values reported elsewhere, based on a partially different analysis of the experimental spectra. In the latter case, we have evaluated the cell growth into two different polymeric scaffolds made of alginate and gelatin with a similar pore distribution and similar inter-connectivity. Based on a qualitative analysis of the dielectric spectra, we were able to provide evidence that alginate scaffolds allow an overall survival of cells better than gelatin scaffold can do. These indications, confirmed by biological tests on cell viability, suggest that hepatocytes embedded in alginate scaffolds are able to perform liver specific functions even over on extended period of time
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Satisfactory performance of hepatocytes in three-dimensional culture on innovative biopolymeric scaffolds
No full textThe introduction in recent years of innovative biomaterials as scaffolds to replace physiological matrix
components has led to significant advances in culture techniques, in terms of cell survival, quantitative
expansion, maintenance of differentiated phenotype and specific functions of cells. ese bio-artificial
tissues can be used as functional substitutes for damaged organs, as models to study complex biological
processes, or for tissue-specific toxicology tests. e aim of our research was to identify the most suitable
biomaterial for technological applications with hepatocytes, e.g. tissue therapy, cellular transplantation,
metabolic analysis of new drugs or screening for potentially toxic substances. Since the possibility to
improve the performance of these systems depends strongly on the methods used to create the scaffolds,
here we analyzed porous matrices made of gelatin or blends of gelatin and glycosaminoglycans, obtained
with different methods for the culture of the C3A cell line, considered a good model of human hepatocytes.
Scaffolds were obtained using either a concentrated emulsion-templating technique known as high internal
phase emulsion (HIPE) or a gas foaming technique; the latter method uses an inert gas instead of the
internal liquid phase toluene, avoiding the use of organic solvent and allowing the creation of scaffolds
with larger pores and interconnections. By analyzing cell adhesion, viability, ultrastructural morphology,
production of albumin and urea and induction of cytochrome P450-3A4 as the main metabolic markers of
hepatocyte functions, it was possible to compare the performances of matrices prepared with the different
methods. Taken together our results suggest that the morphology of the scaffolds (surface porosity, void/
interconnection size) is less crucial than the utilization of potentially harmful molecules during their
creation
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