The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

El procedimiento detallado para operar con la sonda optoacústica volumétrica manual se describe a continuación. Este procedimiento se realiza de acuerdo a las regulaciones institucionales aprobados en relación con los experimentos en animales y humanos. 1. Sistema de Preparación <ol><li> Encender el láser durante un período de calentamiento de ~ 15 min antes de la operación para la estabilización del haz de luz de salida. </li><li> Coloque la parte de agua que encierra con la membrana de aislamiento que está en contacto con la piel (Figura 1). NOTA: La distancia entre la membrana de aislamiento (en contacto con la piel) y la región con la máxima sensibilidad del transductor (centro de la sonda esférica) establece la profundidad de formación de imágenes eficaz. </li><li> Llenar todo el volumen de aproximadamente 100 ml de aislamiento entre la membrana y la superficie del transductor con agua desionizada por medio de una bomba. </li><li> Asegúrese de que no hay fugas de agua ni burbujas de aire son present. Por otra parte, evitar las burbujas de aire, proporcionando la recirculación de agua. </li><li> Realizar los experimentos a temperatura ambiente y asegurar el medio de acoplamiento (agua) se mantiene a esta temperatura. </li></ol> 2. Preparación Imaging <ol><li> Preparación de imágenes humano. </li><li> Eliminar el vello de la parte al-fotografiada-estar con una loción de depilación con el fin de evitar un fondo no deseado en las imágenes (este paso es opcional). </li><li> Aplicar gel de ultrasonido sobre la piel alrededor de la región para formar una imagen con el fin de proporcionar un acoplamiento acústico eficiente. Coloque la sonda optoacústica en la región de interés. Asegúrese de que no haya burbujas de aire en el gel de acoplamiento de ultrasonido. </li><li> Preparación de imágenes de animales. </li><li> Asegúrese de que los procedimientos de atención y experimentales con animales están de acuerdo con las normas y reglamentos institucionales y gubernamentales. </li><li> Retire la piel del animal en la región para obtener imágenes con una loción de afeitar. Proteger los ojos del animal conungüento veterinario, lo que evita la sequedad y los daños causados ​​por la exposición a la radiación láser pulsada intensa. </li><li> Anestesiar al animal mediante el uso de la inyección intraperitoneal (IP) de ketamina / xilazina (100 mg / kg de ketamina kg + 5 mg / kg kg de xilazina) antes del experimento o utilizar anestesia con isoflurano (2-3% (en volumen) con 0,9 l / flujo de gas min) durante el experimento. Confirmar anestesia comprobando el reflejo de la extremidad posterior del animal. </li><li> Aplicar gel de ultrasonido sobre la piel alrededor de la región a ser fotografiado con el fin de proporcionar un acoplamiento acústico eficiente y colocar la sonda optoacústica en la región de interés. Asegúrese de que no haya burbujas de aire en el gel de acoplamiento de ultrasonido. </li></ol> 3. Pre-vista del modo de funcionamiento <ol><li> Establecer la longitud de onda de formación de imágenes (s) entre 690 nm y 900 nm y la tasa de repetición de impulsos entre 10 y 50 Hz. Seleccione los parámetros para el sistema de adquisición de datos acústicos - 1 MWimpedancia de entrada. Adquirir 2.030 muestras para cada pulso de láser a una frecuencia de muestreo de 40 megamuestras por segundo y 12 bits de resolución vertical. Desencadenar la adquisición con la salida Q-switch del láser. </li><li> Asegúrese de que tanto el operador y el paciente utilizar gafas de protección adaptadas a la longitud de onda (s) de excitación óptica. Ajustar la potencia del láser de manera que se mantiene la fluencia de luz en la superficie del tejido por debajo de 20 mJ / cm 2 durante el experimento para longitudes de onda del infrarrojo cercano con el fin de satisfacer los límites de exposición de seguridad para los experimentos humanos 19 y para evitar tensión térmica y daños en la piel en los animales . </li><li> Iniciar el software pre-view con una implementación de algoritmos de procesamiento de GPU para permitir la visualización de imágenes en tres dimensiones a una velocidad correspondiente a la tasa de repetición de impulsos del láser. </li><li> Mueva la sonda y / o el objeto a ser reflejado con el fin de optimizar el rendimiento de visualización y localización de las estructuras de interés. </li></ol> 4. Adquisición de Datos <ol><li> La adquisición de datos para el modo de exploración (de mano). </li><li> Si es necesario, inyectar un agente de contraste antes de la adquisición de enriquecer el contraste en la región de interés. NOTA: En nuestros experimentos no hemos realizado la imagen humana con contraste. Sin embargo, diversos agentes de contraste pueden ser potencialmente utilizados para este propósito. Verde de indocianina (ICG) es un ejemplo de agente de contraste óptico aprobado clínicamente que se puede utilizar para la mejora del contraste en dosis máxima recomendada de 2 mg / kg de peso corporal en los adultos. </li><li> Iniciar el hardware para la adquisición de datos con los parámetros descritos en 3,1 manteniendo la ejecución del software de vista previa. Mueva suavemente la sonda alrededor de la región fotografiada para realizar un seguimiento de las estructuras de interés. NOTA: Cuando las imágenes en múltiples longitudes de onda láser se adquieren simultáneamente, la velocidad del movimiento de la sonda en el modo de mano tiene que ser disminuido significativamente (preferiblemente por debajo de 2 mm / segpara una tasa de repetición de impulsos de láser de 50 Hz) con el fin de evitar artefactos relacionados con el movimiento en las imágenes espectralmente sin mezclar. </li><li> La adquisición de datos para el modo estacionario. </li><li> Monte el objeto fotografiado (ej., Los animales) y la sonda de mano en el soporte e iniciar la adquisición con los parámetros descritos en el punto 3.1, manteniendo la ejecución del software pre-vista. </li><li> Mantener la sonda optoacústica y la parte de formación de imágenes en la misma posición durante el experimento para visualizar los eventos biológicos dinámicos en la región de interés. </li><li> Inyectar un agente de contraste para rastrear su distribución dinámica en la región de interés. NOTA: En nuestros experimentos con ratones, verde de indocianina (ICG) se utilizó para la mejora del contraste. Como pauta general, una cantidad de 10 nmol o 0,4 mg / kg de ICG tiene que ser introducido en la circulación del ratón con el fin de crear un contraste detectable con optoacoustics multi-espectrales en vivo. NOTA: El agente de contraste debe seraprobado para uso humano y / o animal por la autoridad respectiva. </li></ol> 5. Finalización del Experimento <ol><li> Detenga el láser. </li><li> Retire la sonda optoacústica de la región fotografiada. Para el estudio de los animales, detener el suministro de anestesia. </li><li> Colocar el animal en un calentador de infrarrojos para mantener el calor y evitar el contacto con otros animales hasta que se recuperó de la anestesia. No deje que el animal sin vigilancia durante la recuperación de la anestesia. </li></ol> 6. Fuera de línea de procesamiento de datos <ol><li> Cargue el archivo (s) que contiene las señales optoacústica adquiridas en la aplicación de software utilizadas para el procesamiento de datos. </li><li> Utilice un algoritmo de reconstrucción para obtener una matriz de matriz tridimensional correspondiente a una imagen volumétrica de la absorción óptica de cada fotograma y cada longitud de onda. NOTA: Para la reconstrucción, es preferible utilizar un algoritmo que representa factores de distorsión, como heterogéneodades y la atenuación en el objeto fotografiado, efectos de ancho de banda final y forma geométrica de los elementos de detección y variaciones de la luz de fluencia, a fin de obtener una representación más cuantitativa de la distribución de la energía absorbida. </li><li> Usa un algoritmo de desmezcla para obtener, a partir de cada trama de múltiples longitudes de onda, un nuevo conjunto de matrices de la matriz tridimensional que representa la absorción óptica para cada sustancia absorbente presente en la muestra. </li><li> Si es necesario, procesar aún más los conjuntos de matriz que representa la distribución de absorción óptica para facilitar la visualización y la lectura de los parámetros biológicamente relevantes. </li></ol>

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The authors have nothing to disclose.

Las ventajas únicas que ofrece técnicas de imagen optoacústica en la investigación con animales pequeños han creado una fuerte motivación para la traducción de la tecnología en la práctica clínica, con una serie de diagnósticos y aplicaciones de monitoreo de tratamiento previstas por ejemplo., En el cáncer de piel de la mama y, inflamación o enfermedades vasculatura periférica. Sin embargo, a diferencia de los ratones o animales más pequeños, que pueden estar rodeado de un número suficiente de ...

Imágenes (fotoacústica) optoacústica atrae cada vez mayor interés por parte de las comunidades de investigación biológica y médica, tal como se manifiesta por el cada vez mayor número de publicaciones que abarcan gran variedad de nuevas aplicaciones que aprovechan las ventajas únicas que ofrece la tecnología de 1-5. En particular, la capacidad de imagen espectral distintivos agentes de absorción de fotos con resolución espacio-temporal de alto a profundidades que van más allá del límite de difusión de la luz se abre capacidades sin precedentes para la proyección de imagen funcional y molecular 6-10. De hecho, la traducción de la tecnología en la práctica clínica optoacústica viene con perspectivas prometedoras en el diagnóstico y la monitorización del tratamiento de muchas enfermedades. Sin embargo, la limitada propagación de fotones en la dispersión ópticamente y tejidos absorbentes y las respuestas débiles generalmente asociados con el fenómeno optoacústica limitar la profundidad de aplicación del método. Como resultado, optoa de manosondas Coustic se han intentado partes de imagen accesible desde el exterior del cuerpo 11,12 mientras que los sistemas endoscópicos se utilizan para proporcionar imágenes del interior del cuerpo mediante la inserción de ellos a través de orificios naturales 13. Algunas partes de baja absorción del cuerpo humano, tales como la mama de la mujer, también se puede acceder por los escáneres tomográficos optoacústica 14,15. De particular interés es el enfoque de mano, ya que permite gran versatilidad, de manera similar a la ecografía. Aquí, la adaptación de los ultrasonidos común gama de sondas lineales para imágenes optoacústica sigue siendo un reto, debido principalmente a diferencias fundamentales en los requisitos de formación de imágenes tomográficas entre ultrasonido y optoacoustics. Mientras altas velocidades de cuadro en la ecografía estándar están habilitadas por secuencial de transmisión-recepción esquemas que emplean frecuencias de repetición de pulso de alta en el rango de kHz, optoacústica de imágenes en tiempo real en tres dimensiones se consigue por la recolección simultánea de datos de tomografía volumétrica de una sola interrogating pulso de láser. Por lo tanto, la imagen optoacústica de alta calidad implica la adquisición de datos tridimensionales a partir de la más grande de ángulo sólido posible alrededor del objeto fotografiado. Recientemente, se introdujo la primera sonda optoacústica de mano para tres dimensiones (volumétrica) de imágenes en tiempo real 16. El sistema se basa en una matriz bidimensional de elementos piezoeléctricos 256 dispuestos sobre una superficie esférica (puntos azules en la figura 1A) que cubren un ángulo de 90 °. El tamaño de los elementos individuales de aproximadamente 3 x 3 mm 2, así como su orientación y ancho de banda de frecuencia (aproximadamente 2-6 MHz) garantizan colección señal efectiva de un volumen centímetro escala que rodea el centro de la esfera (cubo negro en la figura 1A). Excitación óptica de la región de formación de imágenes está provisto de un haz de fibras insertado a través de una cavidad cilíndrica central de la matriz, de modo que cualquier longitud de onda SUSCeptible de ser transmitida a través del haz de fibras se puede utilizar para la imagen. Una imagen real de la matriz de transductores junto con el haz de fibras ópticas se muestra en la Figura 1B. La excitación eficiente y la detección simultánea de las señales permite formación de imágenes de tejido profundo con excitación de disparo único (un pulso láser), de modo que imágenes en tiempo real a una velocidad determinada por la frecuencia de repetición de impulsos del láser se activa adicionalmente con un Graphics- procesamiento de unidades (GPU) aplicación del procedimiento de reconstrucción 17. Una carcasa cilíndrica con una membrana de polietileno transparente (Figura 1C) se une a la matriz de transductores para encerrar un medio líquido acústicamente transmitir (agua). La membrana está acoplado además al tejido por medio de gel acústico. Una imagen de la sonda optoacústica como se utiliza en el modo de operación de mano se muestra en la Figura 1D. La THR demostradoee de imágenes de mano dimensional optoacústica combinada con la capacidad de formación de imágenes en tiempo real funcional venir con ventajas importantes para el diagnóstico clínico y una serie de aplicaciones potenciales se prevé para diversas indicaciones, tales como la enfermedad vascular periférica, trastornos del sistema linfático, cáncer de mama, lesiones de la piel, inflamación o artritis 18. Además, la capacidad de formación de imágenes permite la visualización rápida de acontecimientos biológicos dinámicos con la sonda dispuesta en una posición estacionaria. Combinado con la longitud de onda de ajuste rápido oscilador paramétrico óptico (OPO) la tecnología láser, este enfoque permite la formación de imágenes en tiempo real de la biodistribución de agentes de absorción de fotos. De esta manera, las nuevas posibilidades pueden surgir igualmente en pequeñas aplicaciones de imágenes de animales, por ejemplo., En el estudio de la hemodinámica del tejido, el seguimiento in vivo de células, la visualización de la farmacocinética, la perfusión de órganos, dirigido imagen molecular de los tumores y el sistema cardiovascular, o la neuroimagen. <p class = &quot;jove_content&quot;&gt; En este trabajo se realiza una descripción detallada del protocolo de imagen experimental para operar con el rendimiento de la matriz esférica optoacústica de mano sonda y escaparate en varios escenarios clínicos por imágenes de animales y pequeñas típicas.

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

Los resultados representativos, lo que demuestra la capacidad de la sonda optoacústica de mano volumétrico descrito, se exhiben en esta sección. En todos los casos, la fluencia de la luz en la superficie de la piel se mantuvo por debajo del límite de exposición seguridad de 20 mJ / cm 2 19. El rendimiento de la sonda en tiempo real el seguimiento de la vasculatura periférica humana se mostró en la Figura 2. Durante el curso de este experimento, la sonda se ...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Universal de mano-tridimensional optoacústica Imaging Sonda de Tejido Profundo Angiografía Humano y Estudios preclínicos funcionales en Tiempo Real

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K vistas.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

Video: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensional óptico resolución microscopía fotoacústica

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Investigación

Bioingeniería

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impresión plana y tridimensional de tintas conductoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Un dispositivo de cámara de flujo tridimensional Novel para el Estudio de quimioquinas-dirigida extravasación de células circulantes en condiciones de flujo fisiológicas

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

El cartílago humano Tissue Fabrication Usando tridimensional Tecnología de impresión inyección de tinta

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Desarrollo de un Modelo Organotípicos multicelular tridimensional de la mucosa intestinal humanas cultivadas en condiciones de microgravedad

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Ingeniería tisular tridimensional alineado astrositos redes para recapitular los mecanismos del desarrollo y facilitar la regeneración del sistema nervioso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Escritura de Electrospinning de andamios Poly(ε-caprolactone) tridimensional con morfologías controlables para aplicaciones de ingeniería de tejidos del derretimiento

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

Fotoacústica clínica mano sistema de imagen por imagen no invasiva en tiempo real de Animal pequeño

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamífero en andamios tridimensionales péptido

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...