Simona Salerno

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.

Improved functions of human hepatocytes on NH3 plasma-grafted PEEK-WC–PU membranes

PEEK-WC-PU membranes were modified with an NH(3) glow discharge process to graft N-containing functional groups at their surface in order to improve the maintenance of human hepatocytes. Native and modified membrane surfaces were characterized with XPS, ToF-SIMS and WCA measurements. We have investigated morphological behaviour and specific functions of primary human hepatocytes on native and modified PEEK-WC-PU membranes in a small-scale gas-permeable bioreactor. N-containing groups grafted at the surface of the membranes improved the initial steps of adhesion and the maintenance of phenotype and differentiated functions of cells. Confocal microscopy of cell morphology evidenced human hepatocytes exhibiting a polygonal shape and organizing a 3D structure. The presence of CK19 positive cells, a marker of biliary duct epithelium, was also found on native and modified membranes. Liver specific functions, investigated in terms of urea production, albumin synthesis and diazepam biotransformation, were maintained at high levels up to 19 days, particularly on surface-modified membranes.

Human hepatocytes and endothelial cells in organotypic membrane systems

The realization of organotypic liver model that exhibits stable phenotype is a major challenge in the field of liver tissue engineering. In this study we developed liver organotypic co-culture systems by using synthetic and biodegradable membranes with primary human hepatocytes and human umbilical vein endothelial cells (HUVEC). Synthetic membranes prepared by a polymeric blend constituted of modified polyetheretherketone (PEEK-WC) and polyurethane (PU) and biodegradable chitosan membranes were developed by phase inversion technique and used in homotypic and organotypic culture systems. The morphological and functional characteristics of cells in the organotypic co-culture membrane systems were evaluated in comparison with homotypic cultures and traditional systems. Hepatocytes in the organotypic co-culture systems exhibit compact polyhedral cells with round nuclei and well demarcated cell-cell borders like in vivo, as a result of heterotypic interaction with HUVECs. In addition HUVECs formed tube-like structures directly through the interactions with the membranes and hepatocytes and indirectly through the secretion of ECM proteins which secretion improved in the organotypic co-culture membrane systems. The heterotypic cell-cell contacts have beneficial effect on the hepatocyte albumin production, urea synthesis and drug biotransformation. The developed organotypic co-culture membrane systems elicit liver specific functions in vitro and could be applied for the realization of engineered liver tissues to be used in tissue engineering, drug metabolism studies and bioartificial liver devices.

Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites

In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly(l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1-3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 mum and ridges of 17 mum whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.

Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid–nanohydroxyapatite fiber scaffolds

The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

Biodegradable and synthetic membranes for the expansion and functional differentiation of rat embryonic liver cells

The insufficient availability of donor organs for orthotopic liver transplantation worldwide has urgently increased the requirement for new therapies for acute and chronic liver disease. The creation of an unlimited source of donor cells for hepatocyte transplantation therapy and pharmaceutical applications may be the isolation and expansion of liver progenitor cells or stem cells. Here we report the expansion and functional differentiation of rat embryonic liver cells on biodegradable and synthetic polymeric membranes in comparison with traditional substrates, such as collagen and polystyrene culture dishes. Membranes prepared from chitosan and modified polyetheretherketone were used for the culture of liver progenitor cells derived from rat embryonic liver. Cells proliferated, with a significant increase in their number within 8-11 days. The cells displayed functional differentiation showing urea synthesis, albumin production and diazepam biotransformation on all substrates investigated. In particular, on a chitosan membrane liver-specific functions were expressed at significantly higher levels for prolonged times compared with other synthetic membranes, utilizing traditional substrates (collagen and PSCD) as references. These results demonstrate that chitosan membranes offer cells favourable conditions to promote the expansion and functional differentiation of embryonic liver cells that could be effectively used in liver tissue engineering and in pharmaceutical applications.

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.

Neuronal growth and differentiation on biodegradable membranes

Semipermeable polymeric membranes with appropriate morphological, physicochemical and transport properties are relevant to inducing neural regeneration. We developed novel biodegradable membranes to support neuronal differentiation. In particular, we developed chitosan, polycaprolactone and polyurethane flat membranes and a biosynthetic blend between polycaprolactone and polyurethane by phase‐inversion techniques. The biodegradable membranes were characterized in order to evaluate their morphological, physicochemical, mechanical and degradation properties. We investigated the efficacy of these different membranes to promote the adhesion and differentiation of neuronal cells. We employed as model cell system the human neuroblastoma cell line SHSY5Y, which is a well‐established system for studying neuronal differentiation. The investigation of viability and specific neuronal marker expression allowed assessment that the correct neuronal differentiation and the formation of neuronal network had taken place in vitro in the cells seeded on different biodegradable membranes. Overall, this study provides evidence that neural cell responses depend on the nature of the biodegradable polymer used to form the membranes, as well as on the dissolution, hydrophilic and, above all, mechanical membrane properties. PCL–PU membranes exhibit mechanical properties that improve neurite outgrowth and the expression of specific neuronal markers. Copyright

3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells

In this study, a designed approach has been utilized for the development of a 3D liver system. This approach makes use of primary human sinusoidal endothelial cells, stellate cells and hepatocytes that are seeded sequentially on hollow fiber membranes (HF) in order to mimic the layers of cells found in vivo. To this purpose modified polyethersulfone (PES) HF membranes were used for the creation of a 3D human liver system in static and dynamic conditions. In order to verify the positive effect of non-parenchymal cells on the maintenance of hepatocyte viability and functions, homotypic cultures of hepatocytes alone on the HF membranes were further investigated. The membrane surface allowed the attachment and self-assembly of the cells, forming tissue-like structures around and between fibers. Sinusoidal cells formed tube-like structures that surrounded hepatocytes organized in cords within aggregates promoted by stellate cells. The co-culture of hepatocytes with sinusoidal endothelial and hepatic stellate cells preserved structural architecture of the construct and improved the liver-specific functions. Most importantly, cells co-cultured in a HF membrane bioreactor synthesized albumin and urea for 28 days. The liver membrane bioreactor also preserved the drug biotransformation activity with a continuous production of diazepam phase I metabolites for an extended period of time. Additionally, the cell oxygen uptake rates highlighted the maintenance of the actual oxygen concentration at a level compatible with their metabolic functions.

PAN hollow fiber membranes elicit functional hippocampal neuronal network.

This study focuses on the development of an advanced in vitro biohybrid culture model system based on the use of hollow fibre membranes (HFMs) and hippocampal neurons in order to promote the formation of a high density neuronal network. Polyacrylonitrile (PAN) and modified polyetheretherketone (PEEK-WC) membranes were prepared in hollow fibre configuration. The morphological and metabolic behaviour of hippocampal neurons cultured on PAN HF membranes were compared with those cultured on PEEK-WC HF. The differences of cell behaviour between HFMs were evidenced by the morphometric analysis in terms of axon length and also by the investigation of metabolic activity in terms of neurotrophin secretion. These findings suggested that PAN HFMs induced the in vitro reconstruction of very highly functional and complex neuronal networks. Thus, these biomaterials could potentially be used for the in vitro realization of a functional hippocampal tissue analogue for the study of neurobiological functions and/or neurodegenerative diseases.