A novel mea bioreactor measuring neural network activity continuously over long periods to study synaptic plasticity and pharmacological outcomes

The ability of culturing neurons for a long time on MicroElectrode Array (MEA) devices plays a critical role in understanding some long-term behaviors of a neuronal network, such as the long-term synaptic plasticity. Moreover, pharmacological outcomes usually requires long recordings to evaluate the complete effects in the culture activity. Applications involving MEAs in long-term analysis suffer from some limits imposed by the current experimental setup. The neurons, cultured on the MEA slices, are housed in an incubator; then they are extracted to record network electrical activity. This procedure can damage the cells (i.e. pH variation, sterility problems, medium evaporation) causing a gradual decline in the health of these cultures and, eventually, the cells apoptosis. Therefore experiments must be limited in time. Lots of solutions are in literature: some of them improve the cells survival but they need of an external incubator; others try to realize independent systems, able to autonomously control temperature, humidity and sterility but not gasses. In this work we present the technological development of an experimental system to measure neural network activity with MEAs continuously over long periods in a controlled atmosphere. The bioreactor described aimed to overcome the above-mentioned limits, in order to provide a single tool that record and process on-line neuronal action potentials without the need of an external incubator. The incubating chamber prototype was designed with Pro-Engineer Wildfire. It was produced, from a cylinder block in polymethylmethacrylate (PMMA - Plexiglas), using rapid prototyping method (Roland Modela MDX-40). The MEA housing and a symmetrical small chamber, the temperature reference, were realised in the bigger one. The heating was obtained bathing a termoresistance in a water bath surrounding the incubating chamber. The gasses and the humidity control (95% Air, 5% CO2; 95% steam) were developed using a commercial CO2 and bubbling module. The electronic for the activity recording was placed on the external top of the device. The whole system was sterilized with Ethylene Oxide (ETO) in order to assure sterility requirement. Software simulations were used to optimize the on-line spike detection and clustering for future implementation on hardware.Preliminary prototype validation on biological environment shows a good capability of cells growth and preservation for long time; moreover, MEA’s electrodes seem not to be spoiled by the incubating environment

A novel mea bioreactor measuring neural network activity continuously over long periods to study synaptic plasticity and pharmacological outcomes

The ability of culturing neurons for a long time on MicroElectrode Array (MEA) devices plays a critical role in understanding some long-term behaviors of a neuronal network, such as the long-term synaptic plasticity. Moreover, pharmacological outcomes usually requires long recordings to evaluate the complete effects in the culture activity. Applications involving MEAs in long-term analysis suffer from some limits imposed by the current experimental setup. The neurons, cultured on the MEA slices, are housed in an incubator; then they are extracted to record network electrical activity. This procedure can damage the cells (i.e. pH variation, sterility problems, medium evaporation) causing a gradual decline in the health of these cultures and, eventually, the cells apoptosis. Therefore experiments must be limited in time. Lots of solutions are in literature: some of them improve the cells survival but they need of an external incubator; others try to realize independent systems, able to autonomously control temperature, humidity and sterility but not gasses. In this work we present the technological development of an experimental system to measure neural network activity with MEAs continuously over long periods in a controlled atmosphere. The bioreactor described aimed to overcome the above-mentioned limits, in order to provide a single tool that record and process on-line neuronal action potentials without the need of an external incubator. The incubating chamber prototype was designed with Pro-Engineer Wildfire. It was produced, from a cylinder block in polymethylmethacrylate (PMMA - Plexiglas), using rapid prototyping method (Roland Modela MDX-40). The MEA housing and a symmetrical small chamber, the temperature reference, were realised in the bigger one. The heating was obtained bathing a termoresistance in a water bath surrounding the incubating chamber. The gasses and the humidity control (95% Air, 5% CO2; 95% steam) were developed using a commercial CO2 and bubbling module. The electronic for the activity recording was placed on the external top of the device. The whole system was sterilized with Ethylene Oxide (ETO) in order to assure sterility requirement. Software simulations were used to optimize the on-line spike detection and clustering for future implementation on hardware.Preliminary prototype validation on biological environment shows a good capability of cells growth and preservation for long time; moreover, MEA’s electrodes seem not to be spoiled by the incubating environment

Lab-on-Chip for testing myelotoxic effect of drugs and chemicals

In the last 20 years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods has approved the colony-forming unit granulocytes–macrophages test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime

A compact and automated ex vivo vessel culture system for the pulsatile pressure conditioning of human saphenous veins

Saphenous vein (SV) graft disease represents an unresolved problem in coronary artery bypass grafting (CABG). After CABG, a progressive remodelling of the SV wall occurs, possibly leading to occlusion of the lumen, a process termed 'intima hyperplasia' (IH). The investigation of cellular and molecular aspects of IH progression is a primary end-point toward the generation of occlusion-free vessels that may be used as 'life-long' grafts. While animal transplantation models have clarified some of the remodelling factors, the pathology of human SV is far from being understood. This is also due to the lack of devices able to reproduce the altered mechanical load encountered by the SV after CABG. This article describes the design of a novel ex vivo vein culture system (EVCS) capable of replicating the altered pressure pattern experienced by SV after CABG, and reports the results of a preliminary biomechanical conditioning experimental campaign on SV segments. The EVCS applied a CAGB-like pressure (80-120\u2009mmHg) or a venous-like perfusion (3\u2009ml/min, 5\u2009mmHg) conditioning to the SVs, keeping the segments viable in a sterile environment during 7\u2009day culture experiments. After CABG-like pressure conditioning, SVs exhibited a decay of the wall thickness, an enlargement of the luminal perimeter, a rearrangement of the muscle fibres and partial denudation of the endothelium. Considering these preliminary results, the EVCS is a suitable system to study the mechanical attributes of SV graft disease, and its use, combined with a well-designed biological protocol, may be of help in elucidating the cellular and molecular mechanisms involved in SV graft disease

Advanced culture systems for ex vivo human vascular tissue conditioning

Our experience shows that using bioengineering approaches facilitates the understanding of vascular physio-pathological mechanisms and, in perspective, will speed up the development of new life-saving treatments. The use of human samples, particularly operating room-derived samples, which would have been otherwise discarded, is a very valuable approach. In line with the 3Rs principles, this methodology is worth the cost of being set up and managed, wherever and whenever possibl

Monophasic and Biphasic Electrical Stimulation Induces a Precardiac Differentiation in Progenitor Cells Isolated from Human Heart

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α(1G)) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype

Does the type of suture technique affect the fluid-dynamic performance of bioprostheses implanted in small aortic roots? Results from an in vitro study.

Background: The in vivo hemodynamic performance of a bioprosthesis implanted in an aortic position is affected by the characteristics of the prosthesis and the sizing strategy adopted. Recently, it has been hypothesized that the type of suture used to implant the prosthesis might influence hemodynamics. Methods: Bioprostheses with labeled sizes of 19 mm and 21 mm were implanted in 2 groups of 5 porcine aortic roots, with native annuli of 19 mm and 21 mm, by means of 2 different suture techniques: simple interrupted and noneverting mattress with pledgets. The aortic roots were tested in an in vitro mock loop. The stroke volume imposed by the mock loop was set at 40 mL, and was increased by steps of 15 mL until a stroke volume of 100 mL was attained. Main fluid-dynamic parameters were analyzed. Results: At each level of stroke volume, ie, 40 mL, 55 mL, 70 mL, 85 mL, and 100 mL, the mean and peak pressure drops were significantly greater with the noneverting mattress suture with pledgets than with the simple interrupted suture. The effective orifice area behaved accordingly, being significantly smaller in the former case. Conclusions: Our data show that the type of suture technique can influence bioprosthesis performance and that it is reasonable to assume that this is especially true in small annuli (<= 21 mm). Thus, to optimize prosthesis performance and reduce the incidence of patient-prosthesis mismatch, the role of the suture technique should not be disregarded

Adventitial vessel growth and progenitor cells activation in an ex vivo culture system mimicking human saphenous vein wall strain after coronary artery bypass grafting

Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-β1 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22α markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation