Este trabalho foi parcialmente apoiado pelos governos nacionais e da União Europeia, através do projeto de ENIAC JU DeNeCoR sob o número 324257 do contrato de concessão. Os autores gostaria de agradecer a Prof Giovanni Magenes, Prof Piero Tortoli e Dr. Giosuè Caliano pelo precioso apoio, supervisão e observações perspicazes que tornou este trabalho possível. Agradecemos também a Prof Egidio D'Angelo e seu grupo (Lab. BCC), juntamente com a Fondazione Istituto Neurologico C. Mondino, por fornecer o rastreamento de movimento e instrumentação do senhor e Giancarlo Germani senhor aquisições. Finalmente, gostaríamos de agradecer ao Dr. Nicoletta Caramia, Dr. Alessandro Dallai e Sra. Barbara Mauti seu valioso apoio técnico e Sr. Walter Volpi para fornecer o cérebro bovino.

<ol>
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S., Caliano, G., Magenes, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth-of-field+enhancement+in+Filtered-Delay+Multiply+and+Sum+beamformed+images+using+Synthetic+Aperture+Focusing.">Depth-of-field enhancement in Filtered-Delay Multiply and Sum beamformed images using Synthetic Aperture Focusing.</a> Ultrasonics. 75, 216-225 (2017).</li><li>Boni, E., Cellai, A., Ramalli, A., Tortoli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+performance+board+for+acquisition+of+64-channel+ultrasound+RF+data.">A high performance board for acquisition of 64-channel ultrasound RF data.</a> Proc. IEEE Int. Ultrasonics Symp. , 2067-2070 (2012).</li><li>Matrone, G., Savoia, A. 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Os autores declaram que têm sem interesses financeiros concorrentes.

Vários trabalhos foram apresentados na literatura descrevendo técnicas que são semelhantes ou relacionados ao protocolo apresentado. Estas técnicas baseiam-se também sobre o uso de metas realistas, incluindo animal fixo ou cérebros de cadáveres, mas eles são principalmente concebidos para testes dos métodos de registo digital de vários tipos.
O protocolo descrito aqui, no entanto, tem a finalidade específica de testes dos EUA sondas em configurações diferentes nos estágios inicia...

<table border="0" cellpadding="0" cellspacing="0" width="925">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">ULA-OP</td>
			<td style="width:167px;">University of Florence</td>
			<td style="width:104px;">N/A</td>
			<td style="width:415px;">Ultrasound imaging research system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">3D imaging piezeoelectric probe</td>
			<td style="width:167px;">Esaote s.p.a.</td>
			<td style="width:104px;">9600195000</td>
			<td>Mechanically-swept 3D ultrasound probe, model BL-433</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Linear-array piezoelectric probe</td>
			<td style="width:167px;">Esaote s.p.a.</td>
			<td style="width:104px;">122001100</td>
			<td>Ultrasound linear array probe, model LA-533</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">CMUT probe</td>
			<td style="width:167px;">University Roma Tre</td>
			<td style="width:104px;">N/A</td>
			<td>Ultrasound linear array probe based on CMUT technology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">MAGNETOM Skyra 3T MR scanner</td>
			<td style="width:167px;">Siemens Healthcare</td>
			<td style="width:104px;">N/A</td>
			<td>MR scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Head coil</td>
			<td style="width:167px;">Siemens Healthcare</td>
			<td style="width:104px;">N/A</td>
			<td>32-channel head coil for MR imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">NDI Polaris Vicra</td>
			<td style="width:167px;">NDI Medical</td>
			<td style="width:104px;">8700335001</td>
			<td>Optical motion tracking system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Pointer tool</td>
			<td style="width:167px;">NDI Medical</td>
			<td style="width:104px;">8700340</td>
			<td>Passive pointer tool with 4 reflecting markers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Clamp-equipped tool</td>
			<td style="width:167px;">NDI Medical</td>
			<td style="width:104px;">8700399</td>
			<td>Rigid body with 4 reflecting markers and a clamp to be connected to the US probe handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Bovine brain</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Brain of an adult bovine, from food suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Formalin solution</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>10% buffered formalin solution for bovine brain fixation - CAUTION, formalin is a toxic chemical substance and must be handled with care; specific regulations may also apply (see for instance US OSHA Standard 1910.1048 App A)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Plastic container for anatomical parts</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Cilindrical plastic container with lid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Glass spheres</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>3mm diameter spheres of Flint glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Agar</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>30 g, for phantom preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Glycerine</td>
			<td style="width:167px;">AEFFE Farmaceutici</td>
			<td style="width:104px;">A908005248</td>
			<td>100 g, for phantom preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Distilled water</td>
			<td style="width:167px;">Solbat Gaysol</td>
			<td style="width:104px;">8027391000015</td>
			<td>870 g, for phantom preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Beaker</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Beaker used for the diluition of glycerine and agar in distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Lysoform</td>
			<td style="width:167px;">Lever</td>
			<td style="width:104px;">8000680500014</td>
			<td>A benzalkonium chloride and water solution was used for the agar phantom preservation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Polystyrene mannequin head</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Polyestirene model which was cutted and used to design the configuration of spheres&#39;patterns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Green tissue marking dye for histology</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Colour used to mark the glass spheres&#39; positions on the bovine brain surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Yellow enamel</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Enamel used to colour the glass spheres implanted in the agar phantom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Water tank</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>50x50x30 cm plastic tank filled degassed water up to a 15cm height&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Mechanical arm</td>
			<td style="width:167px;">Esaote s.p.a.</td>
			<td style="width:104px;">N/A</td>
			<td>Mechanical arm clamped to the water tank border and used to held the probe in fixed positions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Plate of synthetic resin</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Plate used as a support for the bovine brain positioning in the water tank</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Sewing threads</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Sewing thread segments used to immobilize the brain on the resin plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Adhesive tape</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Adhesive tape used to fix the sewing thread extremities onto the resin plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Plastic food container</td>
			<td style="width:167px;">N/A</td>
			<td style="width:104px;">N/A</td>
			<td>Sealed food container used for the agar phantom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Notebook</td>
			<td style="width:167px;">Lenovo</td>
			<td style="width:104px;">Z50-70</td>
			<td>Lenovo&#160; Z50-70, Intel(R) Core i7-4510U @ 2.0 GHz, 8 GB RAM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Workstation</td>
			<td style="width:167px;">Dell Inc.</td>
			<td style="width:104px;">T5810</td>
			<td>Intel(R) Xeon(R) CPU E3-1240v3 @ 3.40 GHz, 16 GB RAM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Matlab</td>
			<td style="width:167px;">The MathWorks</td>
			<td style="width:104px;">R2013a</td>
			<td>Software tool, used for space transformation computation and 3D reconstruction from image planes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Paraview</td>
			<td style="width:167px;">Kitware Inc.</td>
			<td style="width:104px;">v. 4.4.1</td>
			<td>Open-source software for 3D image processing and visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">NDI Toolbox - ToolTracker Utility</td>
			<td style="width:167px;">NDI Medical</td>
			<td style="width:104px;">v. 4.007.007</td>
			<td>Software for marker position visualization and tracking in the NDI Polaris Vicra measurement volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">C++ data-logging software</td>
			<td style="width:167px;">NDI Medical</td>
			<td style="width:104px;">v. 4.007.007</td>
			<td>Software for marker position recording on a text log file</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">ULA-OP software&#160;</td>
			<td style="width:167px;">University of Florence</td>
			<td style="width:104px;">N/A</td>
			<td>Software for real-time display and control of the ULA-OP system</td>
		</tr>
	</tbody>
</table>

an experimental protocol for assessing the performance of new ultrasound probes based on cmut technology in application to brain imaging

A possibilidade de realizar uma avaliação precoce e repetível do desempenho de imagem é fundamental no design e processo de desenvolvimento de novo ultra-som (US) sondas. Particularmente, uma análise mais realista com alvos de imagens específicas do aplicativo pode ser extremamente valiosa para avaliar o desempenho esperado de sondas dos EUA em seu campo clínico potencial de aplicação.
O protocolo experimental apresentado neste trabalho foi propositadamente concebido para fornecer um processo de avaliação de específicas do aplicativo para recém-desenvolvido nos sonda protótipos baseados na tecnologia capacitiva microusinado transdutor ultra-sônico (CMUT) em relação a imagens do cérebro.
O protocolo combina o uso de um cérebro bovino fixado em formalina como o destino da imagem latente, o que garante tanto realismo e repetibilidade dos procedimentos descritos e das técnicas de neuronavigation emprestadas de neurocirurgia. A sonda dos EUA na verdade está ligada a um sistema que adquire os dados de posição e permite a superposição de imagens dos Estados Unidos para fazer referência a imagens de ressonância magnética (RM) do cérebro de rastreamento de movimento. Isto fornece um meio para peritos humanos para realizar uma avaliação qualitativa visual da sonda E.U. desempenho de imagem e comparar as aquisições feitas com diferentes sondas. Além disso, o protocolo baseia-se na utilização de um sistema completo e aberto de pesquisa e desenvolvimento para aquisição de imagens dos Estados Unidos, ou seja, o scanner de ultrassom avançado abre plataforma (ULA-OP).
O manuscrito descreve detalhadamente os instrumentos e procedimentos envolvidos no protocolo, em particular para a calibração, aquisição de imagens e registro de imagens dos EUA e senhor. Os resultados obtidos provam a eficácia do protocolo geral apresentado, que é inteiramente aberto (dentro dos limites da instrumentação envolvido), repetíveis e abrange todo o conjunto de atividades de aquisição e processamento para imagens dos EUA.

O principal resultado alcançado através do protocolo descrito é a validação experimental de um procedimento de avaliação eficazes e repetíveis para o 2D e 3D capacidades de imagem de nós sonda protótipos baseados na tecnologia CMUT, na aplicação em potencial ao cérebro imagem latente. Depois de implementar todas as etapas do protocolo descrito, um perito pode então aplicar as funções de software de visualização (por exemplo, cortar a orientação livre extraçã...

Watch this Scientific Journal Video about An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging at JoVE.com

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

O desenvolvimento de novas sondas de ultra-som (US), baseado na tecnologia capacitiva microusinado transdutor ultra-sônico (CMUT) requer uma avaliação realista antecipada dos recursos de imagem. Descreveremos um protocolo experimental repetível para aquisição de imagens de US e comparação com imagens de ressonância magnética, usando um cérebro bovino ex vivo como um alvo de imagem.

Um protocolo Experimental para avaliar o desempenho de novas sondas de ultrassom baseado na tecnologia CMUT na aplicação de imagens do cérebro

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

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10.3K visualizações.  University of Pavia. O desenvolvimento de novas sondas de ultra-som (US), baseado na tecnologia capacitiva microusinado transdutor ultra-sônico (CMUT) requer uma avaliação realista antecipada dos recursos de imagem. Descreveremos um protocolo experimental repetível para aquisição de imagens de US e comparação com imagens de ressonância magnética, usando um cérebro bovino ex vivo como um alvo de imagem.

Vídeo: An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

Alto rendimento, extração automatizada de DNA e RNA de amostras clínicas utilizando a Tecnologia TruTip em Robots Liquid Handling comuns

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Bioengenharia

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Um Experimental e Finite Element protocolo para investigar o transporte de solutos neutras e carregadas em toda a cartilagem articular

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

Um sistema de tomografia fotoacústica compacta de alto desempenho para<em&gt; In Vivo</em&gt; Imagem de cérebro de animais pequenos

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabricação e validação de um sistema de órgãos-em-microplaqueta com eléctrodos integrados para quantificar diretamente a resistência elétrica de Transendothelial

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

Baseado em agarose tecido imitando Phantoms ópticos por espectroscopia de reflectância difusa

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Uma nova técnica cirúrgica como fundamento na Vivo parcial engenharia fígado de rato

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Um novo modelo Ex Vivo para a avaliação de desempenho de Material de injeção submucosa endoscópica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Uma plataforma de tecnologia de código aberto para fabricar modelos de cultura 3D à base de hidrogel de forma automatizada e padronizada

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...