Este trabajo fue apoyado por AAUW internacional de becas para Adeola Adewola, NIH / CNRR (U42RR023245) a José Oberholzer, y el Proyecto de Diabetes de Chicago.

A. microfabricación de dispositivos de 3 capas perifusion microfluidos Fondo del pozo de protocolo maestro (150 micras pozos profundos) <ol><li> Limpie la oblea con una cuchilla de afeitar, si es necesario. Limpie con acetona, metanol, y el IPA. Realizar el tratamiento de plasma de 50 vatios durante 30 s. </li><li> Giro SU8-100 @ 2000 rpm. [Paso 1: 500 rpm, 10 s, 100 rpm / s, Paso 2: 2000 rpm, 30 s, 300 rpm / s]. [NOTA: no tienen la oblea con pinzas después de girar SU8] </li><li> Soft hornear la oblea a 65 ° C durante 20 min y 95 ° C durante 50 min. </li><li> La oblea se expone a la luz UV a través de una máscara que desee. Dosis de 150 m de altura es de 650 mJ / cm 2. </li><li> Después de la exposición hornear la oblea a 65 ° C durante 1 minuto y 95 ° C durante 12 min. </li><li> Desarrollar la oblea en SU8 desarrollador durante 15 minutos. </li></ol> Microcanal Maestro Protocolo (500 m de pozos profundos) <ol><li> Limpie la oblea con una cuchilla de afeitar, si es necesario. Limpie con acetona, metanol, y el IPA. Realizar el tratamiento de plasma de 50 vatios durante 30 s. </li><li> Giro SU8-2150 @ 1000 rpm. [Paso 1: 500 rpm, 10 s, 100 rpm / s, Paso 2: 1000 rpm, 30 s, 300 rpm / s]. </li><li> Soft hornear la oblea a 65 ° C durante 15 min y 95 ° C durante 2 horas y 30 minutos. </li><li> La oblea se expone a la luz UV a través de una máscara que desee. Dosis de exposición para la altura es de 685 micras 650 mJ / cm 2. </li><li> Después de la exposición hornear la oblea a 65 ° C durante 5 minutos ya 95 ° C durante 35 min. </li><li> Desarrollar la oblea en SU8 desarrollador durante 20-30 min. </li></ol> PDMS preparación de la solución <ol><li> Polidimetilsiloxano (PDMS) solución se prepara mediante la mezcla de 10 partes de elastómero de silicona con una parte del agente de curado de un kit estándar Sylgard 184. </li><li> Las burbujas generadas en la solución de PDMS durante el proceso de mezcla se eliminan usando un secador de vacío. </li><li> La burbuja de una solución libre de PDMS es lenta dispensado sobre la SU8 maestros y un vacío placa de Petri para la tercera capa. </li><li> La temperatura de la placa caliente se establece en 75 ° C y el PDMS se cura a esta temperatura durante 2 horas </li><li> Entradas, salidas y los pozos de cambio se troquelan con el golpeador tamaño del orificio adecuado. </li><li> Las capas están unidas entre sí en un portaobjetos de vidrio (tamaño 0,1 mm) utilizando un dispositivo de plasma de mano. </li></ol> B. Arreglo experimental <ol><li> Etanol al 70% el flujo a través del dispositivo de micro-para esterilizar. DI flujo de agua para lavar el etanol. Perfusión de 50 ml de 0,5% de BSA a través del dispositivo para prevenir la no específica de adsorción de la insulina a las paredes de microcanales. </li><li> El gradiente de rampa de la glucosa y otros pendientes relacionadas generados por el software de LabVIEW que se comunica con la jeringa bombas se ponen a prueba para asegurarse de que los gradientes son estables. </li><li> 25-30 islotes ratones fueron incubadas con Fura-2/AM M 5 (un indicador de calcio, sondas moleculares, CA) y 2,5 M rodamina 123 (Rh123, un indicador de potencial mitocondrial, Sigma, MO) durante 30 minutos a 37 ° C en buffer Krebs-Ringer (KRB) que contiene 2 mM de glucosa </li><li> Los islotes de los ratones son cuidadosamente pipeta en el dispositivo de microfluidos perifusion a través del puerto de entrada. </li><li> La red de microfluidos es la configuración de la conexión por la entrada de las bombas de jeringa con tubo Tygon y un conector en la salida al colector de fracciones. El dispositivo se encuentra en una fase de calentamiento (37 ° C) en el microscopio y el tubo de admisión se calienta en un hornillo para mantener la temperatura de la cámara y la solución a 37 ° C. </li><li> Inmediatamente después de la instalación, los islotes de los ratones son perifused con KRB conteniendo 2 mM de glucosa durante 10 minutos y luego una rampa de glucosa (2 mM 25 mM) durante 25 min. </li><li> Lapso de tiempo las imágenes se recogen y analizan cada 15 s por el software SimplePCI. El perifusate también se recoge cada minuto con un colector de fracciones para analizar la secreción de insulina por ELISA kit. </li></ol> Resultados representante Islotes de ratón se perifused con un gradiente lineal de 2-25 mM de glucosa. Como se muestra en la Figura 1, la entrada de calcio y la secreción de insulina se activan después de unos 13 minutos de perifusion, lo que corresponde a la glucosa de 6 mm. Cambios en los potenciales mitocondriales son visto antes como era de esperar, a unos 11 minutos. Estos datos demuestran la ventaja de utilizar esta red de microfluidos para caracterizar la fisiología del islote. <img src="/files/ftp_upload/1649/1649fig1.jpg" alt="Figura 1" /> Figura 1. Las respuestas fisiológicas a la estimulación de células islote.

<ol>
	<li>Mohammed, J. S., Wang, Y., Harvat, T. A., Oberholzer, J., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+multimodal+characterization+of+pancreatic+islets.">Microfluidic device for multimodal characterization of pancreatic islets.</a> Lab Chip. 9, 97-106 (2009).</li></ol>

Los sistemas tradicionales de perifusion islote (macroescala y microescala) tienen algunas limitaciones, incluyendo complejos de configuración y diseño, altos requerimientos técnicos, y la dificultad para crear prescrito por el usuario gradientes químicos en el sistema. El sistema de microfluidos perifusion descrito aquí supera estas limitaciones con la simple geometría de diseño y fabricación. Más importante aún, este sistema puede ser integrado con el enfoque de imágenes de fluorescencia que proporcionan una herramienta única para estudiar la fisiología del islote. El sistema ha demostrado con una alta relación señal-ruido y espacio-temporal de la resolución de estas señales de fluorescencia. El prototipo actual puede tener múltiples configuraciones perifusion en un chip y ofrecen distintos tipos de microambientes para fines de aplicación generalizada.

<table><tbody><tr><td>60ml Syringes</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fura-2 fluorescence dye</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>Rhodamine123 Fluorescence dye</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>30&quot; Silicone tubings</td><td>Cole-Parmer</td><td></td><td>1/16 x 1/8in</td></tr><tr><td>1.5ml Eppendorf tubes</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Y-connectors</td><td>Cole-Parmer</td><td></td><td>1/16&amp;#8221; &amp;amp; 4mm</td></tr><tr><td>Syringe connectors</td><td>Cole-Parmer</td><td></td><td>female luer plug 1/16&amp;#8221;</td></tr><tr><td>Straight connectors</td><td>Cole-Parmer</td><td></td><td>1/16&amp;#8221;</td></tr><tr><td>Elbow connector</td><td>Cole-Parmer</td><td></td><td>1/16&amp;#8221;</td></tr><tr><td>Havard syringe pump</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Perifusion device</td><td></td><td></td><td></td></tr><tr><td>Hot plate</td><td>PMC</td><td></td><td></td></tr><tr><td>Thermometer</td><td>Omega Engineering, Inc.</td><td></td><td></td></tr><tr><td>Fraction collector</td><td>Gibson</td><td></td><td></td></tr><tr><td>Pippettor</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Inverted epiflorescence microscope</td><td>Olympus Corporation</td><td>IX71</td><td></td></tr><tr><td>Charge-coupled device</td><td>QImaging</td><td></td><td>Retiga-SRV, Fast 1394</td></tr></tbody></table>

a multi-parametric islet perifusion system within a microfluidic perifusion device

Un dispositivo de microfluidos perifusion islote fue desarrollado para la evaluación de la dinámica de la secreción de insulina de los islotes y varias imágenes simultáneas de fluorescencia de la entrada de calcio mitocondrial y los posibles cambios. El dispositivo consta de tres capas: la primera capa contiene una matriz de micro pozos (500 m de diámetro y 150 m de profundidad) que ayudan a inmovilizar a los islotes durante la exposición al flujo y maximizar la superficie expuesta de los islotes, la segunda capa contiene una circular perifusion cámara (3 mm de profundidad, 7 mm de diámetro), y la tercera capa contiene un canal de entrada de mezcla que los aficionados a cabo antes de la inyección en la cámara de perifusion (2 mm de ancho, 19 mm de longitud y 500 m de altura) para la optimización de la eficiencia en la mezcla antes de entrar en la cámara de perifusion. La creación de los gradientes de la glucosa, asimismo, de una forma lineal, campana, y las formas cuadradas también se pueden crear en la red perifusion de microfluidos y se ha demostrado.

Watch this Scientific Journal Video about A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device at JoVE.com

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Un dispositivo de microfluidos perifusion islote fue desarrollado para la evaluación de la dinámica de la secreción de insulina de los islotes y varias imágenes simultáneas de fluorescencia de la entrada de calcio mitocondrial y los posibles cambios.

A multi-paramétricos Islet Sistema Perifusion dentro de un dispositivo de microfluidos Perifusion

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

a-multi-parametric-islet-perifusion-system-within-microfluidic

Nanyang Technological University

Research

JoVE Journal

Biology

11.8K vistas.  University of Illinois, Chicago. Un dispositivo de microfluidos perifusion islote fue desarrollado para la evaluación de la dinámica de la secreción de insulina de los islotes y varias imágenes simultáneas de fluorescencia de la entrada de calcio mitocondrial y los posibles cambios.

Video: A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Los chips de microfluidos controlado con matrices elastoméricas microválvula

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Investigación

Biología

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

Estimulación Cerebral sector mediante una red de microfluidos y de la Cámara de perfusión estándar

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Microfluídica endotelizado para estudiar las interacciones microvasculares en Enfermedades Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Bioingeniería

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Control cuantitativo y temporal del Microambiente oxígeno en el Islote de un solo nivel

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable Red Vascular con un modelo de tejido en un dispositivo de microfluidos

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una plataforma microfluídica multicapa para la conducción de la expresión génica sin células prolongada

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generación de condiciones ambientales dinámicas utilizando un dispositivo microfluídico de alto rendimiento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Biorreactor de perfusión multiflujo integrado con fraccionamiento de salida para cultivo celular dinámico

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

Cultivo de flujo multicanal sin bomba para mantener y evaluar tejidos y células

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Autor destacado: Avance de la investigación en fisiología ocular a través de un cultivo de flujo multicanal para un mantenimiento óptimo del tejido y una evaluación en tiempo real 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.