Il lavoro è stato sostenuto dalle concessioni 1P01DA028555-01A1, NS034239 2R01, 2R37 NS36126, P01 NS31492, P20RR 15635, P01MH64570 e P01 NS43985 (a HEG) dal National Institutes of Health. Gli autori ringraziano la signora Robin Taylor per la lettura critica del manoscritto ed eccezionale supporto grafico e letterario. Vorremmo ringraziare Steve per la sua Grzelak bagnato fresatura competenza. Vorremmo anche ringraziare il dottor Han Chen dell&#39;Università del Nebraska-Lincoln impianto nucleo microscopia elettronica per la fornitura la scansione e la trasmissione delle immagini microscopia elettronica. Infine, vorremmo ringraziare Megan Marquart per la sua esperienza dal vivo con microscopia confocale.

<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>

Nessun conflitto di interessi dichiarati.

Nanoarte sono progettati per migliorare l&#39;HIV-1 terapie. Abbiamo proposto un monociti-macrofagi sistema di consegna della droga come primo passo per sviluppare queste tecnologie per uso clinico e come sistema di laboratorio per lo sviluppo di farmaci nanoformulated. I nostri lavori passati hanno dimostrato che il sistema è vitale per una tale applicazione 5,6. Nella presente relazione, abbiamo illustrato i metodi per la sintesi di NP comunemente usati farmaci HIV-1 (indinavir, IDV; ritonovir, RTV, efavirenz, EFV e atazanavir, ATV). Questa è stata ottenuta mediante fresatura da bagnato, omogeneizzazione e ultrasuoni. È importante sottolineare che il nostro approccio permette di modifiche di Nanoarte caratteristiche fisiche come la dimensione, forma e carica 5,6. Da un&#39;attenta selezione di tensioattivi, si può controllare la carica della particella da altamente negativo neutro altamente positivo. Modificando il tempo di elaborazione e l&#39;intensità, si può controllare la dimensione e il rendering delle particelle piccole come ≤ 200 nm o grande come ≥ 3 micron. La forma della particella può essere controllata, ma in misura minore rispetto alle altre caratteristiche fisiche. Per esempio, particelle sferiche possono essere effettuate tramite sonicazione con polimeri, mentre le forme geometriche più lati possono essere prodotti tramite macinazione ad umido o omogeneizzazione. Macinazione ad umido e omogeneizzazione produrre particelle di forma diversa, ma questo processo dipende dal metodo di frazionamento e non possono essere direttamente controllata per. Queste proprietà dei metodi di fabbricazione può consentire ricerche per produrre facilmente NanoArt alle specifiche di progetto preciso. Ciò è di vitale importanza dato che le proprietà fisico-chimiche di Nanoarte hanno un potente effetto su entrambi la loro stabilità e come interagiscono con le cellule. Per esempio, abbiamo dimostrato che nanoART che sono circa 1 micron di grandezza, hanno una forte carica positiva, e hanno una forma geometrica più lati sono più rapidamente ripreso da macrofagi, più stabile, una volta all&#39;interno delle cellule, e rilasciato per un lungo periodo di tempo 6. Un metodo per il test è stato sviluppato NanoArt che permette semplici per lo screening della capacità delle particelle &#39;a essere ripreso, trattenuto e rilasciato dai macrofagi, una delle cellule bersaglio del principio di HIV-1, oltre alla loro capacità di inibire la replicazione virale. Questo permette di differenziare facilmente NanoArt in base alle prestazioni e identificare quelli che hanno le migliori possibilità di successo in un modello in vivo, in tal modo, aumentando sia l&#39;efficienza e la velocità di sviluppo di Nanoarte per uso umano. E &#39;stato dimostrato che i macrofagi del midollo osseo del mouse quando caricato ex vivo con NanoArt e adoptively trasferito per via endovenosa in umanizzato da HIV-1 nei topi infettati possono viaggiare a siti di infezione attiva, compreso il sistema nervoso centrale, e la droga di rilascio per fino a 2 settimane per inibire la replicazione virale 2-4. Inoltre, queste cellule NanoArt caricato stati anche in grado di ridurre efficacemente il numero di cellule infettate dal virus nel plasma, linfonodi, milza, fegato e polmone oltre che proteggere le cellule CD4 + 2. Questi studi, anche se preliminari, dimostrano che una cellula-mediata sistema di consegna NanoArt possibile distribuire quantità clinicamente significative di farmaco sia per sangue e nei tessuti infetti. A causa dei risultati preliminari incoraggianti da HIV-1 studi sui topi infetti, stiamo attualmente sviluppando un modello di immuno deficienza di scimmie infette da virus macaco per testare ulteriormente il potenziale di Nanoarte per uso clinico.

Abbreviazioni utilizzate nella tabella: IDV: indinavir; RTV: ritonavir; ATV: atazanavir; EFV: effavirenz; PVA: polivinilalcool; SDS dodecil solfato di sodio; P188: poloxamer 188 (detta anche Pluronic F68); mPEG 2000-DSPE: metil-poli (glicole etilenico) 1,2-distearoyl-fosfatidil-etanolammina; DOTAP: (1-oleoyl-2-[6 - [(7-nitro-21-benzoxadiazol ,3-4-il) ammino] Hexanoyl] - 3-trimetilammonio propano); PLGA: poli (lattico-co-glicolico acido); CTAB: bromuro cetyltrimethyl di ammonio; DMEM: Dulbecco modificato Aquila media; MCSF: macrofagi fattore stimolante le colonie; HPLC: alta pressione cromatografia liquida; HIV-1 : Human Immunodeficiency Virus-1

methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs

Nanoformulated farmaci in grado di migliorare la farmacodinamica e la biodisponibilità mentre serve anche a ridurre la tossicità di una terapia antiretrovirale (ART) i farmaci. A tal fine, il nostro laboratorio ha applicato i principi della nanomedicina per semplificare i regimi ARTE e come tale ridurre tossicità migliorando al contempo la conformità e la farmacocinetica della droga. Metodi semplici e affidabili per la produzione d&#39;arte nanoformulated (NanoArt) sono mostrati. Le particelle di droga pura sono incapsulati da un sottile strato di rivestimento surfattante lipidi e prodotto da frazionamento cristalli di farmaco più grandi in quelle più piccole da una fresatura bagnata o ad alta pressione di omogeneizzazione. In un metodo alternativo farmaco libero è sospeso in una goccia di un polimero. Qui, farmaco si dissolve in un polimero poi agitato da ultrasuoni fino a quando i singoli si formano goccioline di dimensioni nanometriche. Dispersione della luce dinamica e l&#39;esame microscopico caratterizzare le proprietà fisiche delle particelle (dimensioni delle particelle, di carica e forma). Le loro proprietà biologiche (assorbimento delle cellule e la ritenzione, citotossicità ed efficacia antiretrovirale) sono determinati con umane derivate da monociti macrofagi (MDM). MDM sono derivati ​​da monociti umani isolati da sangue periferico leukopacks utilizzando elutriation centrifughe per la depurazione. Tali ematica macrofagi possono essere utilizzati come trasportatori cellulari per la distribuzione NanoArt del virus dell&#39;immunodeficienza umana (HIV) gli organi infetti. Noi ipotizzare che il riconfezionamento dei farmaci antiretrovirali disponibili clinicamente in nanoparticelle per l&#39;HIV-1 trattamenti può migliorare la compliance e influenzare positivamente gli esiti della malattia.

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

Nanoparticelle di indinavir, ritonavir, atazanavir e efavirenz sono stati realizzati con macinazione ad umido, omogeneizzazione e ultrasuoni. Queste nanoformulations, denominati collettivamente nanoformulated terapia antiretrovirale (NanoArt), valutata macrofagi basato consegna della droga. Monociti-macrofagi derivati ​​NanoArt assorbimento, ritenzione e rilascio prolungato sono stati determinati. Questi studi preliminari suggeriscono il potenziale di Nanoarte per uso clinico.

Sviluppo metodi per il trasporto dei macrofagi del sangue Borne Nanoformulated farmaci antiretrovirali

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

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JoVE Journal

Bioengineering

13.1K Views.  University of Nebraska Medical Center. Nanoparticelle di indinavir, ritonavir, atazanavir e efavirenz sono stati realizzati con macinazione ad umido, omogeneizzazione e ultrasuoni. Queste nanoformulations, denominati collettivamente nanoformulated terapia antiretrovirale (NanoArt), valutata macrofagi basato consegna della droga. Monociti-macrofagi derivati ​​NanoArt assorbimento, ritenzione e rilascio prolungato sono stati determinati. Questi studi preliminari suggeriscono il potenziale di Nanoarte per uso...

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.

Metodi di rilevamento a fluorescenza per piattaforme goccia microfluidica

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

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Bioingegneria

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.

Migliorata metodo per la preparazione di un cell-based, Contact modello umano della barriera sanguigna del cervello

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.

Metodi per caratterizzare il co-sviluppo di biofilm e Habitat Eterogeneità

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.

Modello in vitro di fisiologiche e patologiche flusso sanguigno con applicazione di indagini di rimodellamento vascolare cellulare

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.

Completamente dispositivo automatizzato centrifuga Microfluidic per il rilevamento di proteine ​​Ultrasensitive da sangue intero

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.

Sviluppo di un multicellulare tridimensionale Organotipica Modello della mucosa intestinale umana coltivate in microgravità

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 permeazione di ferritine FITC-caricati attraverso un Rat barriera emato-encefalica: un modello per studiare la consegna di Nanoformulated Molecole

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.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

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.

Non distruttive monitoraggio dello sviluppo del vaso sanguigno tessutale basati su Scaffold degradabile usando la tomografia a coerenza ottica

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.

Catetere leggero intra-cardiaco per il monitoraggio del metabolismo cellulare utilizzando la spettroscopia transmurale di riambente di cuori mammiferi perfusi

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