Luminal endothelialization of small caliber silk tubular graft for vascular constructs engineering

: The constantly increasing incidence of coronary artery disease worldwide makes necessary to set advanced therapies and tools such as tissue engineered vessel grafts (TEVGs) to surpass the autologous grafts [(i.e., mammary and internal thoracic arteries, saphenous vein (SV)] currently employed in coronary artery and vascular surgery. To this aim, in vitro cellularization of artificial tubular scaffolds still holds a good potential to overcome the unresolved problem of vessel conduits availability and the issues resulting from thrombosis, intima hyperplasia and matrix remodeling, occurring in autologous grafts especially with small caliber (<6 mm). The employment of silk-based tubular scaffolds has been proposed as a promising approach to engineer small caliber cellularized vascular constructs. The advantage of the silk material is the excellent manufacturability and the easiness of fiber deposition, mechanical properties, low immunogenicity and the extremely high in vivo biocompatibility. In the present work, we propose a method to optimize coverage of the luminal surface of silk electrospun tubular scaffold with endothelial cells. Our strategy is based on seeding endothelial cells (ECs) on the luminal surface of the scaffolds using a low-speed rolling. We show that this procedure allows the formation of a nearly complete EC monolayer suitable for flow-dependent studies and vascular maturation, as a step toward derivation of complete vascular constructs for transplantation and disease modeling

Bioreactors as physiologicallike in vitro models

Bioreactors are powerful tools for in vitro development of engineered substitutes through controlled biological, physical, and mechanical culture conditions: bioreactor technology allows a closer in vitro replication of native tissues. One of bioreactors applications is the design of in vitro 3D tissue models as a bridge between 2D and in vivo models, allowing the application of 3R (replacement, reduction, refinement) principle. To this aim, bioreactors can be used to culture cells seeded on engineered scaffolds under in vivo-like conditions. Another key use of bioreactors is for perfusion decellularization of tissues and organs to be used as scaffolds. This contribution describes a dynamic stretching. bioreactor, imposing a mechanical stretching to the cultured constructs, allowing the development of skeletal muscle engineered constructs, and a decellularization bioreactor, designed for decellularization of blood vessels

A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc(+)HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc(+)HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models

PhotoMEA: development of optical tools for the study of the functional properties of neuronal networks

A large number of studies on neuronal physiology and plasticity have provided a detailed picture of the molecular machinery underlying the modulation of neuronal activity. On the contrary, the mechanisms controlling properties of complex neuronal networks remain poorly understood. At present, the neuronal functional properties are investigated either by a large-scale approach (i.e. MicroElectrode Array devices, MEAs) that enables the study of the general activity of a complex neuronal network, or alternatively by a micro-scale approach (i.e. intracellular or patch electrodes) suitable for the detailed analysis of the molecular mechanisms that actively contribute to the generation and modulation of the single neuron activity. Systems based on electrodes have yielded important results in neurophysiology, but now they start to show some severe limits, such as the possibility of inducing cellular damage in the case of intracellular electrodes and the poor spatial resolution in the case of MEAs. Optical methods for neuronal stimulation, e.g. using caged compounds, and for neuronal activity recording, e.g. using Voltage-Sensitive fluorescent Dyes (VSDs), can be useful tools to overcome these limits. Local light stimulations are obtained activating caged glutamate by UV light pulses. Single neurons or selected parts of them can be stimulated using optical fibres micro-positioned in the neuronal culture or optical waveguides micro-structured in the glass coverslip, on which the neurons are cultured, to precisely drive UV light. Optical recordings of the electrical activity from the entire network are performed using di-4-ANEPPS Voltage-Sensitive Dye and a standard epi-fluorescence microscope equipped with a dedicated large-sensor high-speed camera. Combining these two optical methods the micro-scale approach (stimulation) meets the large-scale approach (recording). This methodology may turn out to be extremely useful for testing the ability of drugs to affect neuronal properties as well as alterations in inter- and intra-neuronal communication

PhotoMEA: A New Step Towards Total Optical Analysis of In Vitro Neuronal Networks

Light stimulation of neurons is a promising approach for investigating the molecular mechanisms at the basis of neuronal physiology and plasticity. In particular, flash photolysis of caged compounds offers the unique advantage of allowing to quickly change the concentration of either intracellular or extracellular bioactive molecules, such as neurotransmitters or second messengers, for the stimulation or modulation of neuronal activity. In this field of research, we describe a simple laser-based set-up for the local activation of caged compounds. The coupling of a UV laser diode to a small-core optical fibre allows to reduce the uncaging area and to quickly change the stimulation point. The actual localisation of the light stimulation is determined using a caged fluorescent compound (dextran, DMNB-caged fluorescein). The efficiency of our set up for neuronal stimulation is tested with a caged neurotransmitter (MNI-caged-L-glutamate). Activation of caged glutamate evokes neuronal responses that are recorded using a MicroElectrode Array system and/or following the variations in the concentrations of the Cai 2+ . This work shows that our laser-based set-up is a powerful tool for local activation of caged compound allowing a unique opportunity to follow the effects of local neuronal pathways on neuronal network activity, for instance during pharmacological and toxicological treatments

A new optical tool to combine optical and electrical analysis in neuronal drug screening

Since their introduction 30 years ago, Micro-Electrode Arrays (MEAs) have been exploited as devices providing distributed information about learning, memory and information processing in a cultured neuronal network, thus changing the field of view from the scale of the single cell (glass pipettes) to the scale of the complex network properties. MEAs represent a growing technology for the study of the functional activity of neuronal networks in a large-scale view providing the possibility (a) to gain information about the spatio-temporal dynamics of the neuronal network, (b) to allow recordings of electrical activity over periods of time not compatible with conventional electrodes and (c) to monitor network activity at several sites in parallel. More recently, according to the trend aimed at the reduction of animal tests, MEAs have been exploited as in vitro biosensors to monitor both acute and chronic effects of drugs and toxins on neuronal networks in physiological or pathophysiological conditions. Now, optical methods for neuronal stimulation, e.g. using caged compounds, represent an useful tools to overcome the limits affecting the MEA technology. Here, local light stimulations were obtained switching caged glutamate in the active form by UV light pulses using optical fibres exactly aligned at the MEA electrodes. This approach allows us to activate the network or to delivery other active compounds in specific regions of the network and to monitor their effects on the overall network functioning. Combining these two optical (stimulation) and electrical (detection) methods a micro-scale approach (stimulation) meets a large-scale approach (detection). This methodology may turn out to be extremely useful for testing the ability of drugs and toxins to affect neuronal properties as well as alterations in inter- and intra-neuronal communication. In this frame, a patent was registered for this novel optoelectronic technological solution for the study of the neuronal activity in culture, in order to understand the physiological and pathological functioning of neuronal networks

PhotoMEA: a new optical biosensor for the study of the functional properties of neuronal networks

Technological innovations in the fields of biomedical optics and electronics have lead to an extremely high level of miniaturization. These steps opened important perspectives to interface optoelectronic instruments with cellular systems. In this frame, a patent was registered for a novel optoelectronic technological solution, named PhotoMEA, for the study of the neuronal activity in culture, in order to understand the physiological and pathological functioning of neuronal networks. At present, the neuronal functional properties are investigated either by a large-scale approach (i.e. MicroElectrode Array devices, MEAs) that enables the study of the general activity of a complex neuronal network, or alternatively by a micro-scale approach (i.e. intracellular or patch electrodes) suitable for the detailed analysis of the molecular mechanisms that actively contribute to the generation and modulation of the single neuron activity. Systems based on electrodes have yielded important results in neurophysiology, but now they start to show some severe limits, such as the possibility of inducing cellular damage in the case of intracellular electrodes and the poor spatial resolution in the case of MEAs. The PhotoMEA device combines two optical tools for studying the functional properties of in-vitro neuronal networks. Light stimulation and optical recording of neuronal activity are promising approaches for investigating the molecular mechanisms at the basis of neuronal physiology. In particular, flash photolysis of caged compounds offers the unique advantage of allowing to quickly change the concentration of either intracellular or extracellular bioactive molecules, such as neurotransmitters or second messengers, for the stimulation or modulation of neuronal activity. Moreover, optical recordings of neuronal activity by Voltage-Sensitive Dyes (VSDs) allow to follow changes of neuronal membrane potential with high-spatial resolution. This enables the study of the sub-cellular responses and that of the entire network at the same time. Combining these two optical methods the micro-scale approach (stimulation) meets the large-scale approach (recording). This methodology may be extremely useful for testing the ability of drugs to affect neuronal properties as well as alterations in inter- and intra-neuronal communication

PhotoMEA: An opto-electronic biosensor for monitoring in vitro neuronal networks activity

PhotoMEA is a biosensor useful for the analysis of an in vitro neuronal network, fully based on optical methods. Its function is based on the stimulation of neurons with caged-glutamate and the recording of neuronal activity by fluorescence Voltage-Sensitive Dyes. The main advantage is that it will be possible to stimulate even at sub-single neuron level and to record with high resolution the activity of the entire network in the culture. A large-scale view of neuronal intercommunications offers a unique opportunity for testing the ability of drugs to affect neuronal properties as well as alterations in the behaviour of the entire network. The concept and a prototype for validation is described here in details