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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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Emulsion templated scaffolds based on gelatin and glycosaminoglycans (Massimi: co-corresponding author)
No full textGelatin is one of the most commonly used biopolymer for creating cellular scaffolds due to its innocuous nature. To create stable gelatin scaffolds at physiological temperature (37 degrees C), chemical cross-linking is a necessary step. In a previous paper (Biomacromolecules 2006, 7, 3059-3068), cross-linking was carried out by either radical polymerization of the methacrylated derivative of gelatin (GMA) or through the formation of isopeptide bonds catalyzed by transglutaminase. The method of scaffold production was based on emulsion templating in which an organic phase is dispersed in the form of discrete droplets into a continuous aqueous solution of the biopolymer. Both kinds of scaffolds were tested as culture medium for hepatocytes. It turned out that the enzymatic cross-linked scaffold performed superiorily in this respect, even though it was mechanically less stable than the GMA scaffold. In the present paper, in an attempt to improve the biocompatibility of the GMA-based scaffold, biopolymers present in the extracellular matrix (ECM) were included in scaffold formulation, namely, chondroitin sulfate and hyaluronic acid. These biopolymers were derivatized with methacrylic moieties to undergo radical polymerization together with GMA. The morphology of the scaffolds was tuned to some extent by varying the volume fraction of the internal phase and to a larger extent by inducing a controlled destabilization of the precursor emulsion through the use of additives. In this way, scaffolds with 44% of the void volume attributable to voids with a diameter exceeding 60 microm and with 79% of the interconnect area attributable to interconnects with a diameter exceeding 20 microm in diameter could be successfully synthesized. To test whether the inclusion of ECM components into scaffold formulation resolves in an improvement of their biocompatibility with respect to GMA scaffolds, hepatocytes were seeded on both kinds of scaffolds and cell viability and function assays were carried out and compared
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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