Maria Rende

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor.

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 microg/h 10(6) cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

Distinct alpha subunits of the GABAA receptor are responsible for early hippocampal silent neuron-related activities.

The modulatory actions of GABAA receptor subunits are crucial for morphological and transcriptional neuronal activities. In this study, growth of hamster hippocampal neurons on biohybrid membrane substrates allowed us to show for the first time that the two major GABAA α receptor subunits (α2,5) are capable of early neuronal shaping plus expression differences of some of the main neuronal cytoskeletal factors (GAP‐43, the neurotrophin––BDNF) and of Gluergic subtypes. In a first case the inverse α5 agonist (RY‐080) seemed to account for the reduction of dendritic length at DIV7, very likely via lower BDNF levels. Conversely, the effects of the preferentially specific agonist for hippocampal α2 subunit (flunitrazepam) were, instead, directed at the formation of growth cones at DIV3 in the presence of greatly (P < 0.01) diminished GAP‐43 levels as displayed by strongly reduced axonal sprouting. It is interesting to note that concomitantly to these morphological variations, the transcription of some Gluergic receptor subtypes resulted to be altered. In particular, flunitrazepam was responsible for a distinctly rising expression of axonal NR1 mRNA levels from DIV3 (P < 0.01) until DIV7 (P < 0.001), whereas RY‐080 evoked a very great (P < 0.001) downregulation of dendritic GluR2 at only DIV7. Together, our results demonstrate that GABAA α2,5 receptor‐containing subunits by regulating the precise synchronization of cytoskeletal factors are considered key modulating neuronal elements of hippocampal morphological growth features. Moreover, the notable NR1 and GluR2 transcription differences promoted by these GABAA α subunits tend to favorably corroborate the early role of α2 + α5 on hippocampal neuronal networks in hibernating rodents through the recruitment and activation of silent neurons, and this may provide useful insights regarding molecular neurodegenerative events.

Flat and tubular membrane systems for the reconstruction of hippocampal neuronal network.

The selection of appropriate biomaterials that promote cellular adhesion and growth is particularly important for the in vitro reconstruction of neuronal network. This study focused on the development of new polymeric membranes in flat and tubular (hollow‐fibre) configurations as novel biomaterials for neuronal outgrowth. Two membrane systems constituted by modified polyetheretherketone (PEEK‐WC) and polyacrylonitrile (PAN) membranes were developed and used for the culture of hamster hippocampal neurons. We demonstrated that all investigated membranes supported the adhesion and growth of hippocampal neurons enhancing neuronal differentiation and neurite alignment. The differences in cell behaviours between cells cultured on flat and hollow‐fibre (HF) membranes were highlighted by the quantitative analysis of neuronal marker fluorescence intensity, morphometric analysis, RT–PCR analysis and also by metabolic activity measurements. In particular, the PAN HF membranes showed ideal growth culture conditions, guaranteeing adequate levels of metabolic features. Primary hippocampal cells cultured on PAN HF membranes were able to recreate in vitro a 3D neural tissue‐like structure that, mimicking the hippocampal tissue, could be used as a tool for the study of natural and pathological neurobiological events. Copyright

Membrane Approaches for Liver and Neuronal Tissue Engineering

Many advances have been made in the last century to replace and restore damaged organs and tissues due to the increase of the aging population and the shortage of suitable organs for transplantation. Tissue engineering approaches based on the combination of smart biomaterials with advanced cell therapy should closely mimic and replicate the physical and biochemical milieu of the natural tissues and organs. Many efforts have been made to improve the design of natural and synthetic, resorbable and nonbiodegradable biomaterials in supporting cell adhesion, differentiation, and proliferation toward the formation of a new tissue. Micro- and nanostructured membranes have a great potential to create a biomimetic environment for cells providing them the biochemical, physical, and mechanical cues, which are necessary for the tissue formation. Tailor-made membranes are designed according to well-defined engineering criteria, taking into account the chemical composition and the physicochemical, morphological, and transport properties. These systems on the basis of their characteristics compartmentalize cells in a well-controlled microenvironment at molecular level providing a selective mass transfer of nutrients and metabolites to and from cells ensuring the system homeostasis. In this article, advances achieved in the development of liver and neuronal membrane systems as bioartificial organs and tissues were reviewed focusing on their evaluation in both the preclinical and clinical systems and their validation as in vitro platforms.