El trabajo fue apoyado por becas 1P01DA028555-01A1, 2R01 NS034239, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570 y P01 NS43985 (a HEG) de los Institutos Nacionales de Salud. Los autores agradecen a la Sra. Robin Taylor para la lectura crítica del manuscrito y el excelente gráfica y literaria. Nos gustaría agradecer a Steve por su Grzelak molienda húmeda experiencia. También nos gustaría agradecer al Dr. Han Chen de la Universidad de Nebraska-Lincoln instalaciones básicas de microscopía electrónica para el suministro de la exploración y las imágenes de microscopía electrónica de transmisión. Por último, queremos agradecer a Megan Marquart por su experiencia mediante microscopía confocal en vivo.

<ol>
	<li>Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+and+propagation+of+human+immunodeficiency+virus+on+recombinant+colony-stimulating+factor+1-treated+monocytes.">Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.</a> J Exp Med. 167 (4), 1428-1441 (1988).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+macrophage-based+nanoparticle+platform+for+antiretroviral+drug+delivery.">Development of a macrophage-based nanoparticle platform for antiretroviral drug delivery.</a> Blood. 108 (8), 2827-2835 (2006).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+investigations+for+the+morphologic,+pharmacokinetic,+and+anti-retroviral+properties+of+indinavir+nanoparticles+in+human+monocyte-derived+macrophages.">Laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages.</a> Virology. 358, 148-158 (2007).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+delivery+of+nanoformulated+antiretroviral+drug+to+the+brain+in+a+murine+model+of+neuroAIDS.">Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS.</a> J Immunol. 183 (1), 661-669 (2009).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoformulated+Antiretroviral+Drug+Combinations+Extend+Drug+Release+and+Antiretroviral+Responses+in+HIV-1-Infected+Macrophages:+Implications+for+NeuroAIDS+Therapeutics.">Nanoformulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV-1-Infected Macrophages: Implications for NeuroAIDS Therapeutics.</a> J Neuroimmune Pharmacol. , (2010).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoART+synthesis,+characterization,+uptake,+release+and+toxicology+for+human+monocyte-macrophage+drug+delivery.">NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.</a> Nanomed. 4 (8), 903-917 (2009).</li><li>Kalter, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+HIV+replication+in+macrophage+colony-stimulating+factor-treated+monocytes.">Enhanced HIV replication in macrophage colony-stimulating factor-treated monocytes.</a> J Immunol. 146 (1), 298-306 (1991).</li></ol>

Nanoarte están diseñados para mejorar la terapéutica del VIH-1. Hemos propuesto un monocito-macrófago sistema basado en la administración de fármacos, como primer paso para desarrollar esas tecnologías para uso clínico y como un sistema de pruebas de laboratorio para el desarrollo de fármacos nanoformulated. Nuestros trabajos anteriores han demostrado que el sistema es viable para este tipo de aplicación 5,6. En el presente informe, se han puesto de manifiesto los métodos para la síntesis de NP de uso común del VIH-1 medicamentos (indinavir, IDV, ritonovir, RTV, efavirenz, EFV y atazanavir, ATV). Esto se logró mediante la molienda húmeda, la homogeneización y ultrasonidos. Es importante destacar que nuestro enfoque permite que las modificaciones de Nanoarte características físicas tales como tamaño, forma y carga 5,6. Mediante una cuidadosa selección de los tensioactivos, se puede controlar la carga de la partícula de muy negativo a neutro de muy positiva. Al alterar el tiempo de procesamiento y la intensidad, se puede controlar el tamaño y hacer que partículas tan pequeñas como ≤ 200 nm o tan grandes como ≥ 3 micras. La forma de las partículas también se puede controlar, pero en menor medida que las características físicas. Por ejemplo, las partículas esféricas se pueden hacer a través de ultrasonidos con polímeros, mientras que varias caras formas geométricas se pueden producir a través de la molienda húmeda o la homogeneización. Molienda húmeda y la homogeneización producir partículas de diferente forma, pero este proceso depende del método de fraccionamiento y no pueden ser directamente controlados. Estas propiedades de los métodos de fabricación pueden permitir las investigaciones para producir fácilmente Nanoarte a las especificaciones exactas. Esto es de vital importancia ya que las propiedades físico-químicas de Nanoarte tener un efecto poderoso en tanto su estabilidad y la forma en que interactúan con las células. Por ejemplo, hemos demostrado que Nanoarte que son aproximadamente 1 micra de tamaño, tienen una fuerte carga positiva, y tienen una forma geométrica varios lados son más rápidamente por los macrófagos, más estable, una vez dentro de las células, y puesto en libertad durante un largo periodo de tiempo 6. Un método para probar Nanoarte fue desarrollado que permite la detección simple de la capacidad de las partículas que deben abordarse, retenidos y liberados por los macrófagos, una de las celdas principio objetivo de VIH-1, además de su capacidad para inhibir la replicación viral. Esto permite diferenciar fácilmente Nanoarte basado en el rendimiento e identificar aquellas que tienen la mejor oportunidad de tener éxito en un modelo in vivo, por lo tanto, el aumento de la eficiencia y la velocidad de desarrollo Nanoarte para uso humano. Se ha demostrado que los macrófagos de médula ósea del ratón cuando se cargan ex vivo con Nanoarte y adoptively transferido por inyección intravenosa en humanizada del VIH-1 ratones infectados pueden viajar a los sitios de infección activa, incluyendo el sistema nervioso central, y las drogas de liberación de hasta 2 semanas para inhibir la replicación viral 2-4. Además, estas células Nanoarte carga también fueron capaces de reducir de manera efectiva el número de células infectadas por el virus en el plasma, los ganglios linfáticos, bazo, hígado y pulmón, así como proteger a las células CD4 + 2. Estos estudios, aunque preliminares, demuestran que un sistema Nanoarte mediada por células de entrega puede distribuir cantidades clínicamente significativas de la droga a la sangre y los tejidos infectados. Debido a los alentadores resultados preliminares de VIH-1 en estudios con ratones infectados, actualmente estamos desarrollando un simio de inmunodeficiencia infectados con el virus modelo de macacos para poner a prueba el potencial de la Nanoarte para uso clínico.

Abreviaturas utilizadas en la tabla: IDV: indinavir, RTV: ritonavir, ATV: atazanavir, EFV: effavirenz; PVA: alcohol polivinílico, de sodio SDS dodecil sulfato; P188: poloxámero 188 (también llamado Pluronic F68), MPEG-2000 DSPE: metil-poli (etileno-glicol) 1,2-distearoil-fosfatidil-etanolamina; DOTAP: (1-oleoil-2-[6 - [(7-nitro-21 ,3-benzoxadiazol-4-il) amino] hexanoilo] - 3-trimetilamonio propano); PLGA: poli (láctico-co-glicólico); CTAB: bromuro de amonio cetiltrimetil; DMEM: Dulbecco modificado de Eagle Medium, MCSF: macrófagos factor estimulante de colonias, HPLC: alta presión, cromatografía líquida, el VIH-1 : virus de inmunodeficiencia humana-1

methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs

Drogas Nanoformulated puede mejorar la farmacodinamia y biodisponibilidad, mientras que sirven también para reducir la toxicidad de los medicamentos para el tratamiento antirretroviral (ART) los medicamentos. Con este fin, nuestro laboratorio ha aplicado los principios de la nanomedicina a simplificar los regímenes de terapia antirretroviral y, como tal, reducir la toxicidad al tiempo que mejora el cumplimiento y la farmacocinética de la droga. Métodos simples y fiables para la fabricación nanoformulated ART (Nanoarte) se muestran. Las partículas de la droga pura se encapsulan por una fina capa de surfactante lípidos y producida por el fraccionamiento de los cristales más grandes de drogas en los más pequeños ya sea por vía húmeda o la homogeneización de alta presión. En un método alternativo libre de drogas se ha suspendido en una gota de un polímero. En esto, la droga se disuelve dentro de un polímero se agita por ultrasonidos hasta que las gotas individuales de tamaño nanométrico se forman. Dispersión de luz dinámica y el examen microscópico caracterizar las propiedades físicas de las partículas (tamaño de partícula, de carga y forma). Sus propiedades biológicas (la captación celular y la retención, la citotoxicidad y la eficacia antirretroviral) se determinan con humanos macrófagos derivados de monocitos (MDM). MDM se derivan de los monocitos de sangre periférica humana aislada de leukopacks con elutriación centrífuga para la purificación. Tal transmitidas por la sangre los macrófagos se pueden utilizar como transportadores celulares para la distribución de Nanoarte al virus de inmunodeficiencia humana (VIH) órganos infectados. Postulamos que el reenvasado de los medicamentos antirretrovirales disponibles clínicamente en nanopartículas para el VIH-1 tratamientos pueden mejorar el cumplimiento e influir positivamente en el desarrollo de enfermedades.

Watch this Scientific Journal Video about Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs at JoVE.com

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Las nanopartículas de indinavir, ritonavir, efavirenz y atazanavir se fabrican con molienda húmeda, la homogeneización y ultrasonidos. Estos nanoformulations, denominados colectivamente nanoformulated la terapia antirretroviral (Nanoarte), evaluaron los macrófagos basados ​​en la administración de fármacos. Derivados de los monocitos macrófagos Nanoarte la captación, retención y liberación sostenida se han determinado. Estos estudios preliminares sugieren que el potencial de Nanoarte para uso clínico.

Métodos de desarrollo para el transporte de macrófagos transmitidas por la sangre de los medicamentos antirretrovirales Nanoformulated

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

methods-development-for-blood-borne-macrophage-carriage

Research

JoVE Journal

Bioengineering

13.1K vistas.  University of Nebraska Medical Center. Las nanopartículas de indinavir, ritonavir, efavirenz y atazanavir se fabrican con molienda húmeda, la homogeneización y ultrasonidos. Estos nanoformulations, denominados colectivamente nanoformulated la terapia antirretroviral (Nanoarte), evaluaron los macrófagos basados ​​en la administración de fármacos. Derivados de los monocitos macrófagos Nanoarte la captación, retención y liberación sostenida se han determinado. Estos estudios preliminares sugieren qu...

Video: Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detección de fluorescencia para las plataformas de las gotas de microfluidos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Investigación

Bioingeniería

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Método mejorado para la preparación de una base de células, Contacto Modelo Humano de la barrera hematoencefálica

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Los métodos para la caracterización de la Co-desarrollo de la biopelícula y Hábitat Heterogeneidad

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

En Vitro Modelo de fisiológicos y patológicos de flujo de sangre con aplicación a las investigaciones de remodelado vascular celular

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Totalmente automatizada de dispositivos de microfluidos para centrífuga Ultrasensible detección de proteínas a partir de sangre entera

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

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., ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

In vitro de permeación de ferritinas cargados con FITC través de una barrera sangre-cerebro de rata: un modelo para estudiar la entrega de moléculas Nanoformulated

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dos métodos de descelularización de tejidos vegetales para aplicaciones de ingeniería de tejidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Monitoreo no destructivos del desarrollo Degradable andamio base ingeniería de tejido vascular mediante tomografía de coherencia óptica

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catéter de luz intracardiaca para monitorear el metabolismo celular utilizando espectroscopia de absorbancia transmural de corazones de mamíferos perfundidos

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...