Advanced 3D Models of Human Brain Tissue Using Neural Cell Lines: State-of-the-Art and Future Prospects.

Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Functionalized Enzyme-Responsive Biomaterials to Model Tissue Stiffening in vitro.

The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix (ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5-4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D) in vitro models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).

Bioinspired liver scaffold design criteria.

Maintaining hepatic functional characteristics in-vitro is considered one of the main challenges in engineering liver tissue. As hepatocytes cultured ex-vivo are deprived of their native extracellular matrix (ECM) milieu, developing scaffolds that mimic the biomechanical and physicochemical properties of the native ECM is thought to be a promising approach for successful tissue engineering and regenerative medicine applications. On the basis that the decellularized liver matrix represents the ideal design template for engineering bioinspired hepatic scaffolds, to derive quantitative descriptors of liver ECM architecture, we characterised decellularised liver matrices in terms of their biochemical, viscoelastic and structural features along with porosity, permeability and wettability. Together, these data provide a unique set of quantitative design criteria which can be used to generate guidelines for fabricating biomaterial scaffolds for liver tissue engineering. As proof-of-concept, we investigated hepatic cell response to substrate viscoelasticity. On collagen hydrogels mimicking decellularised liver mechanics, cells showed superior morphology, higher viability and albumin secretion than on stiffer and less viscous substrates. Although scaffold properties are generally inspired by those of native tissues, our results indicate significant differences between the mechano-structural characteristics of untreated and decellularised hepatic tissue. Therefore, we suggest that design rules - such as mechanical properties and swelling behaviour - for engineering biomimetic scaffolds be re-examined through further studies on substrates matching the features of decellularized liver matrices.

Systemic and vascular inflammation in an in-vitro model of central obesity.

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.

Realisation and characterization of conductive hollow fibers for neuronal tissue engineering.

In this article, conductive hollow fibers have been fabricated using melt spinning technique. Multiwalled carbon nanotubes (MWNTs) and poly(3-hexylthiophene-2,5-diyl) (P3HT) have been used to fabricate conductive poly-caprolactone (PCL) composites. The hollow fibers have inner and outer diameter in the range of 300 µm and 500 µm, respectively. Critical parameters to tune the dimension of hollow fibers have been defined following two-dimensions mathematical model. Evaluation of the mechanical properties showed that the incorporation of 1-3 wt % MWNTs and 5-8 wt % P3HT increased Young Modulus of 10% and 20% respectively, compared with pure PCL. The electrical property assessment demonstrated that a minimum incorporation of 3 wt % MWNT and 8 wt % P3HT in PCL matrix transformed composite materials into conductive materials. In addition, SH-SY5Y human neuroblastoma cells were seeded on the fabricated samples an their adhesion, proliferation and neurite length growth were analysed. In particular we observed that these materials promoted cell activities and in particular on MWNT/PCL composites there was a significant increase of neurite growth.

A novel dual-flow bioreactor simulates increased fluorescein permeability in epithelial tissue barriers.

Permeability studies across epithelial barriers are of primary importance in drug delivery as well as in toxicology. However, traditional in vitro models do not adequately mimic the dynamic environment of physiological barriers. Here, we describe a novel two-chamber modular bioreactor for dynamic in vitro studies of epithelial cells. The fluid dynamic environment of the bioreactor was characterized using computational fluid dynamic models and measurements of pressure gradients for different combinations of flow rates in the apical and basal chambers. Cell culture experiments were then performed with fully differentiated Caco-2 cells as a model of the intestinal epithelium, comparing the effect of media flow applied in the bioreactor with traditional static transwells. The flow increases barrier integrity and tight junction expression of Caco-2 cells with respect to the static controls. Fluorescein permeability increased threefold in the dynamic system, indicating that the stimulus induced by flow increases transport across the barrier, closely mimicking the in vivo situation. The results are of interest for studying the influence of mechanical stimuli on cells, and underline the importance of developing more physiologically relevant in vitro tissue models. The bioreactor can be used to study drug delivery, chemical, or nanomaterial toxicity and to engineer barrier tissues.

The impact of fabrication parameters and substrate stiffness in direct writing of living constructs.

Biomolecules and living cells can be printed in high-resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods, we modeled the printing phase of two RP systems that use biomaterial inks containing living cells: a high-resolution inkjet system (BioJet) and a lower-resolution nozzle-based contact printing system (PAM(2)). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesize that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modeled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively, in BioJet and PAM(2). Results show that during inkjet printing impact energies of about 10(-8) J are transmitted to cells, whereas extrusion energies of the order of 10(-11) J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10(-9) J to avoid permanent cell damage. To obtain well-defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cells.

Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia.

Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system's metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system.

An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor.

The energy balance in vivo is maintained through inter-organ cross-talk involving several different tissues. As a first step towards recapitulating the metabolic circuitry, hepatocytes, endothelial cells and adipose tissue were connected in a multicompartmental modular bioreactor to reproduce salient aspects of glucose and lipid metabolism in vitro. We first examined how the two-way cellular interplay between adipose tissue and endothelial cells affects glucose and lipid metabolism. The hepatocyte cell line HepG2 was then added to the system, creating a three-way connected culture, to determine whether circulating metabolite concentrations were normalized, or whether metabolic shifts, which may arise when endothelial cells and adipose tissue are placed in connection, were corrected. The addition of hepatocytes to the system prevented the drop in the concentrations of glucose, L-alanine and lactate, and the rise in that of free fatty acids. There was no significant change in glycerol levels in either of the connected cultures. The results show that connected cultures recapitulate complex physiological systemic processes, such as glucose and lipid metabolism, and that the HepG2 hepatocytes normalize circulating metabolites in this in vitro environment in the presence of other cell types.

Sensing scaffolds to monitor cellular activity using impedance measurements.

Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density.

PAM2 (piston assisted microsyringe): a new rapid prototyping technique for biofabrication of cell incorporated scaffolds.

Rapid prototyping techniques are widely used to fabricate well-defined three-dimensional structures of tissue homologs. The piston-assisted microsyringe (PAM2) is a rapid prototyping technology specifically developed for low-shear stress extrusion of viscous hydrogel solutions containing cells. In this article the working parameters of the system were established to guarantee the realization of spatially controlled hydrogel scaffolds. Moreover the shear stresses acting on the cell membrane during extrusion was investigated through a computational fluid-dynamic analysis. The computational models show that the shear stress on the cells is of the order of 100 Pa during the extrusion process. HepG2 cells encapsulated in alginate were then extruded into spatially organized hepatic lobule-like architectures and their viability and function were evaluated. The results show that the metabolic fingerprint of the cells is preserved with respect to controls and the cells are uniformly distributed through the gel scaffold.

Cardiac progenitor cells: potency and control.

Stem cell-based regeneration of the heart has focused much scientific and public attention being cardiac diseases the major cause of disability and death in industrialized countries. Innumerable efforts have been taken to unveil the mechanisms undergoing stem cell proliferation and fate, but much remains to be endeavoured for their application in clinical practice. Nevertheless, the discovery of progenitor cells resident within the cardiac tissue has sparked off enthusiasm about the possibility of efficiently and safely engineering them to repair the injured myocardium. Indeed, the early applications of the cardiac progenitor cells, mostly based on simplistic concepts and techniques, have failed highlighting the prerequisite of expanding the knowledge about progenitor cell features and microenvironmental conditioning. In this review, recent information on resident cardiac progenitor cells has been systematically gathered in order to create a valuable instrument to support investigators in their efforts to establish an efficient cardiac cell therapy.

In vitro liver model using microfabricated scaffolds in a modular bioreactor.

Hepatocyte function on 3-D microfabricated polymer scaffolds realised with the pressure-activated microsyringe was tested under static and dynamic conditions. The dynamic cell culture was obtained using the multicompartment modular bioreactor system. Hepatocyte cell density, glucose consumption, and albumin secretion rate were measured daily over a week. Cells seeded on scaffolds showed an increase in cell density compared with monolayer controls. Moreover, in dynamic culture, cell metabolic function increased three times in comparison with static monolayer cultures. These results suggest that cell density and cell-cell interactions are mediated by the architecture of the substrate, while the endogenous biochemical functions are regulated by a sustainable supply of nutrients and interstitial-like flow. Thus, a combination of 3-D scaffolds and dynamic flow conditions are both important for the development of a hepatic tissue model for applications in drug testing and regenerative medicine.

Optimization of PAM scaffolds for neural tissue engineering: preliminary study on an SH-SY5Y cell line.

Engineering neural tissue is one of the most challenging goals of tissue engineering. Neural tissue is highly complex and possesses an organized three-dimensional (3D) distribution that is essential for tissue function. An optimal scaffold for tissue engineering has to provide this distribution until the cells are able to activate their normal functions and develop neural connections with the host tissue. Different strategies such as gene therapy and cell transplantation particularly in retinal tissue have been tested, but so far they have only induced retinal degeneration in animals. The objective of this work was to study neural cell assembly as a function of scaffold features and surface chemistry for application in retinal tissue engineering using microfabricated patterns with a well-defined geometry. Because retinal neurons are known to be arranged in hexagonal arrays, hexagonal scaffolds of poly(DL-lactide-co-glycolide) acid were fabricated using a pressure-assisted microsyringe (PAM) system. The behavior of a model cell, neuroblastoma originating from human retina (SH-SY5Y), was analyzed after seeding on the scaffolds, measuring cell density as a function of line width and length of the scaffold to identify the optimal hexagonal geometry. We also evaluated the influence of scaffold on cell metabolism using the methyl thiazolyl tetrazolium assay and on neurite extension. As far as two-dimensional scaffolds are concerned, the results show that although metabolic activity per cell remains constant, hexagons with sides of 500 microm and line widths of 20 +/- 5 microm are optimum for neural cell adhesion in terms of cell density. On 3D scaffolds, cell metabolism is about three times higher than controls, and the optimum number of layers in the scaffold is three or four.

Criticality of the biological and physical stimuli array inducing resident cardiac stem cell determination.

The replacement of injured cardiac contractile cells with stem cell-derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine-tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues. Disclosure of potential conflicts of interest is found at the end of this article.

PAM-microfabricated polyurethane scaffolds: in vivo and in vitro preliminary studies.

Polymeric scaffolds were realised with linear degradable PU in the form of square, hexagonal and octagonal grids. They were characterised in terms of their mechanical properties. Analysis shows that the mechanical properties of the scaffolds depend on their geometries which are easily modulated using PAM. In vitro biological assays showed that PU promotes the adhesion and proliferation of fibroblast cells and that cell activities are better on PU scaffolds than on PU films. In vivo implantation of PU and PLGA scaffolds and PU films demonstrated that the scaffolds are completely resorbed after three months with a slight inflammatory response, while the PU film was still present after six months with an intense granulomatous reaction.

Chitosan/gelatin blends for biomedical applications.

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40, 100/0 w/w) were produced as candidate materials for biomedical applications. Dehydro-thermal crosslinking was adopted to promote the formation of amide and ester bonds between the macromolecules ((CS/G)-t). The effect of composition and crosslinking on the physico-chemical properties of the samples was evaluated by scanning electron microscopy, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of (CS/G)-t samples were also determined through stress-strain and creep-recovery tests. The elastic moduli of dry blend samples showed a positive deviation from the additive law of the in-series model, because of interactions and/or chemical bonds between components. The comparison between the elastic moduli of wet samples and those of different human tissues showed that (CS/G)-t substrates can be suitable for soft-tissue reconstruction. (CS/G)-t two-dimensional scaffolds were fabricated by micro-molding, based on the use of a polydimethylsiloxane mould to create patterns with micro-scale resolution on cast films. Biocompatibility of (CS/G)-t samples was studied by means of cell tests using NIH-3T3 fibroblasts. Finally, the evaluation of the affinity of (CS/G)-t samples towards neuroblastoma cells adhesion and proliferation was performed, showing promising results for the blend containing 80 wt % gelatin.

Genipin-crosslinked chitosan/gelatin blends for biomedical applications.

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40 100/0 w/w) were produced, as candidate materials for biomedical applications. Different amounts of genipin (0.5 wt.% and 2.5 wt.%) were used to crosslink CS/G blends, promoting the formation of amide and tertiary amine bonds between the macromolecules and the crosslinker. The effects of composition and crosslinking on the physico-chemical properties of samples were evaluated by infrared analysis, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of crosslinked samples were also determined through stress-strain and creep tests: samples stiffness increased with increasing the crosslinker amount and the CS content. Blend composition affected mouse fibroblasts adhesion and proliferation on substrates, depending on the crosslinker amount. Finally, crosslinked CS/G blends containing 80 wt.% G were found to support neuroblastoma cells adhesion and proliferation which made them promising candidates for uses in the field of nerve regeneration.