The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.

Le procedure che utilizzano soggetti animali sono stati approvati dal Comitato di Cura e uso istituzionale degli animali della University of Texas di Austin. 1. Preparazione preoperatoria <ol><li> Registrare il peso corporeo di ogni coniglio. Come per tutti gli studi preclinici, monitorare i pesi degli animali regolarmente per valutare la potenziale tossicità. </li><li> Prima di anestetizzare il coniglio, riempire una provetta da 50 ml con gel per ultrasuoni disponibili in commercio e far girare a 500 xg per 30 s; ci dovrebbero essere visibili bolle nel gel al termine della centrifugazione. </li><li> Somministrare glicopirrolato per via sottocutanea alla dose di 0,1 mg / kg e acepromazine intramuscolare alla dose di 0,75 mg / kg. Attendere 15-20 minuti per il sedativo abbia effetto, mentre il monitoraggio dei segni vitali e il comportamento. NOTA: Glicopirrolato è un agente anti-colinergico che impedisce bradicardia e riduce respiratorie e GI secrezioni. Acepromazine è un sedativo che funge da premedication per l&#39;anestesia. </li><li> Somministrare 35 mg / kg di ketamina e 5 mg / kg di xylazina sottocutanea come anestetico. Tuttavia, come l&#39;operatore diventa più esperto e può compiere la consegna intraduttale più rapidamente, diminuire i dosaggi farmaceutiche a 15 mg / kg di ketamina e 3 mg / kg di xylazina sottocutanea; questo ridurrà il tempo di anestesia e il tempo richiesto per l&#39;animale per recuperare da anestesia. </li><li> Controllare e documentare la frequenza cardiaca, SPO 2, temperatura, frequenza respiratoria, e il colore membrana mucosa ogni 15 min. Applicare il lubrificante occhio in entrambi gli occhi. NOTA: Solo il personale che hanno subito la formazione adeguata e sono stati approvati dalla IACUC della loro istituzione dovrebbe amministrare o monitorare l&#39;anestesia. L&#39;uso di un monitor anestesia veterinario può aiutare ad acquisire segni vitali ed è consigliato. Il monitor anestesia veterinaria, tuttavia, non sostituisce la necessità di controllare manualmente l&#39;animale ogni 15 min. Vedere le istruzioni del produttore Fo l&#39;uso corretto di un monitor veterinaria. </li><li> Verificare l&#39;insorgenza di anestesia da un pinch punta dolce; il coniglio deve essere non reattivi prima di continuare. </li><li> radersi con cura l&#39;addome caudale del coniglio nella zona attorno al terzo e quarto coppie di mammelle inguinali. </li><li> Con la maggior parte dei capelli rimossi, applicare una crema depilatoria over-the-counter all&#39;area rasata. Rimuovere il min crema 10 dopo l&#39;applicazione usando tovaglioli di carta umidi bagnate con acqua tiepida. NOTA: la pelle di coniglio è molto fragile / sensibile. La crema depilatoria deve essere utilizzato per non più di 10 min. In realtà, è più sicuro provare un &quot;punto di prova&quot; a 5 min e lasciare solo il più a lungo se posto test indica un tempo più lungo è necessario periodo. </li><li> Pulire l&#39;area con tamponi di garza imbevuta di alcol per pulire il sito di iniezione. </li><li> Posizionare il coniglio sul suo dorso in un recipiente a forma di v foderato con un ricircolo coperta acqua calda e un tampone assorbente. </li></ol> 2. Preparation del mezzo di contrasto <ol><li> Ricostituire il reagente non mirati contrasto in base alle istruzioni del produttore. Pipetta su e giù delicatamente per miscelare. NOTA: L&#39;agente di contrasto utilizzato in questo protocollo è stabile a temperatura ambiente per 4 - 6 h dopo la ricostituzione. oscillare delicatamente il flaconcino tra ogni recupero. NOTA: Il volume di soluzione necessario dipende dal numero di condotti da iniettare. Il coniglio ha 4 aperture duttale per tettarella, e 0,2 ml di soluzione è sufficiente a riempire un albero duttale di un adulto Nuova Zelanda White Rabbit (Oryctolaguscuniculus). Così, un volume totale di 0,8 ml può essere consegnato ad una ghiandola mammaria. </li></ol> 3. La consegna intraduttale <ol><li> Individuare i capezzoli appropriate da iniettare; i 3 ° e 4 ° coppie di mammelle inguinali sono raccomandati, poiché essi vengono facilmente visualizzate quando l&#39;animale è posizionato sul suo dorso. </li><li> Carico 0,2 mL di sterile salina allo 0,9% inuna siringa da tubercolina da 1 ml Luer-serratura con un ago 22 G. Smaltire l&#39;ago G 22 una volta che la soluzione salina è nella siringa e sostituirlo con un ago calibro 25 sterile. Pulire delicatamente l&#39;area con 85% alcol isopropilico ed un tampone di garza. </li><li> Con la smussatura dell&#39;ago e la siringa parallelo al corpo dell&#39;animale, inserire la smussatura dell&#39;ago nel lato del capezzolo e iniettare lentamente 0,1-0,2 mL di soluzione fisiologica; questo permetterà per una migliore visualizzazione delle aperture duttale. </li><li> Carico 0,2 ml di soluzione per l&#39;iniezione in una siringa da tubercolina 1 ml Luer-lock. </li><li> Mantenere la tettarella delicatamente con il pollice e l&#39;indice e sollevarla leggermente per posizionarlo per l&#39;iniezione intraduttale; una lente di ingrandimento oculare indossabile può essere di aiuto nel visualizzare le aperture duttale. </li><li> Pur mantenendo la posizione sollevata del capezzolo, cannulate accuratamente il condotto di interesse utilizzando un ago smussato punta 25 G. </li><li> Dopo l&#39;incannulamento, torcere delicatamente la siringa luer-lock sul mozzo della smussato-tip ago infusione fino a quando non si blocca in posizione. </li><li> Sollevare la tettarella e iniettare la soluzione lentamente per ridurre al minimo i potenziali danni causati dal rapido movimento fluido all&#39;interno della condotta; in nessun momento ci dovrebbe essere la resistenza quando si inietta la soluzione. </li></ol> 4. Ultrasound Imaging <ol><li> Applicare una quantità di gel ultrasuoni centrifugato alla pelle della zona di interesse. Verificare che non vi siano bolle nel gel, in quanto questi saranno compromettere la qualità dell&#39;immagine. </li><li> Impostare la profondità di imaging a 6 mm. Posizionare il trasduttore 21 MHz in contatto con il gel e la scansione dell&#39;area di interesse in B-mode. Osservare il mezzo di contrasto nella regione scansionata tra cui l&#39;apertura duttale e nel condotto. NOTA: Queste impostazioni sono stati sviluppati per l&#39;utilizzo con una specifica macchina ad ultrasuoni fotoacustica. Fare riferimento alla tabella dei materiali per maggiori dettagli. Può essere necessario regolare la potenza di trasmissione e la profondità di imaging di altri sistemi di imaging per ottimizzare mammariavisualizzazione della ghiandola. </li><li> Rimuovere il gel per ultrasuoni dalla pelle dell&#39;animale con un tampone di garza. </li></ol> 5. Cura postoperatoria <ol><li> Osservare il sito di iniezione: non ci dovrebbero essere i segni di un trauma alla regione del capezzolo o al tessuto circostante, e gonfiore nella zona circostante la tettarella probabilmente indica una mammaria iniezione cuscinetto adiposo, piuttosto che una iniezione di intraduttale successo. </li><li> Posizionare il coniglio in una posizione sternale. Se necessario, dare 0,2 mg / kg per via endovenosa di yohimbina alla vena marginale dell&#39;orecchio; ciò invertire l&#39;effetto della xilazina e permettere all&#39;animale di recuperare più rapidamente da anestesia. </li><li> Monitorare il coniglio ogni 15 minuti per tutto il periodo di recupero fino a quando l&#39;animale è vigile, reattivo, e mantiene una posizione sternale. NOTA: Questa procedura non dovrebbe causare danni ai tessuti o gonfiore. Se rossore o gonfiore si osservano, somministrare una dose di meloxicam 0.1-0.2 mg / kg PO una volta che l&#39;animale è vigile e in gradodi assumere farmaci per via orale. Contattare personale veterinario dell&#39;istituto per ulteriori indicazioni. </li></ol>

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The authors have nothing to disclose.

Questo modo di somministrazione intraduttale alla ghiandola mammaria coniglio può essere utilizzato per i reagenti di contrasto ecografici e molte altre soluzioni acquose, compresi coloranti vitali e terapeutica. Studi precedenti hanno dimostrato la consegna intraduttale di ormoni 17, 18, 19. Nei modelli di roditori, la consegna intraduttale di acidi nucleici 8, chemioterapici 6,<...

<html> La consegna intraduttale di agenti terapeutici è stata studiata in modelli di roditori e in fase iniziale sperimentazione umana 3, 4, 5, 6, 11, 12. Un recente studio di fase I ha dimostrato la sicurezza e la fattibilità di carboplatino intraduttale o intraduttale doxorubicina liposomiale pegilata in donne in attesa di mastectomia per il trattamento del cancro invasivo 2. Precedenti protocolli per la consegna intraduttale sono stati sviluppati per topo e ratto mammarie ghiandole 6, 7, 8, 9. Per scopi di ricerca, iniezioni di cellule tumorali intraduttale e la consegna lentivirale vettore di oncogeni sono stati anche eseguiti in modelli di roditoriref &quot;&gt; 13, 14, 15, 16. Tuttavia, un ideale modello in vivo del processo di consegna intraduttale dovrebbe consentire lo sviluppo di nuove classi di composti terapeutici e facilitare la valutazione preclinica. differenze anatomiche tra i roditori e gli esseri umani hanno complicato la traduzione di questi studi. Diversamente topi, in cui ciascun condotto termina in un capezzolo separata, il seno umano è costituito da 5 a 9 sistemi duttali indipendenti, ognuno con un&#39;apertura separata termina al capezzolo. Coniglio ghiandole mammarie porto quattro sistemi duttale indipendenti, ciascuna separatamente, accessibile attraverso uno dei quattro orifizi in una sola tettarella. Un modello di coniglio più si avvicina al anatomia umana e consente lo studio di drug delivery intraduttale in un contesto più rilevante. Qui, usiamo due tecniche per valutare la consegna intraduttale. La co-somministrazione di uncolorante vitale permette la visualizzazione attraverso la pelle e fornisce una conferma semplice e rapida del metodo. Per alcune applicazioni, la mappatura risoluzione maggiore dei condotti può essere preferito. Presentiamo qui un protocollo per l&#39;imaging ad ultrasuoni dei condotti attraverso la consegna intraduttale di un reagente non mirato contrasto.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MicroMarker non-targeted contrast reagent</td>
			<td>VisualSonics</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock 1mL Syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate 0.2mg/mL</td>
			<td>Wedgewood Compounding Pharmacy</td>
			<td>GLYCOP-INJ013VC</td>
			<td>6 month shelf life, supply may be limited.&#160;</td>
		</tr>
		<tr>
			<td>Atropine Sulfate 0.5 mg/mL</td>
			<td>Animal Health International</td>
			<td>15320764</td>
			<td>If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.</td>
		</tr>
		<tr>
			<td>Ketamine HCL 100mg/mL</td>
			<td>Animal Health International</td>
			<td>21250699</td>
			<td><a href="http://www.animalhealthinternational.com/">http://www.animalhealthinternational.com/</a></td>
		</tr>
		<tr>
			<td>Acepromazine 10mg/mL</td>
			<td>Animal Health International</td>
			<td>17640541</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20mg/mL</td>
			<td>Animal Health International</td>
			<td>20101547</td>
			<td></td>
		</tr>
		<tr>
			<td>Yohimbine 0.2mg/mL</td>
			<td>Animal Health International</td>
			<td>14588965</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removing Cream</td>
			<td>Veet</td>
			<td></td>
			<td>Sensitive skin solution. Available through local retailers.</td>
		</tr>
		<tr>
			<td>Blunt tip infusion needles</td>
			<td>Sai Infusion Technology</td>
			<td>B14-50</td>
			<td><a href="http://www.sai-infusion.com/collections/blunt-needles">http://www.sai-infusion.com/collections/blunt-needles</a></td>
		</tr>
		<tr>
			<td>Veterinary Pulse Oximeter</td>
			<td>EdanUSA</td>
			<td>VE-H100B</td>
			<td><a href="http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html">http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html</a></td>
		</tr>
		<tr>
			<td>Warm Water Pump</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Blanket</td>
			<td>Animal Health International</td>
			<td>21232696</td>
			<td>Maxi-Therm Lite Warming Pads</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
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intraductal delivery to the rabbit mammary gland

Trattamenti intraduttale localizzati per il cancro al seno offrono potenziali vantaggi, compresa la lettura efficiente al tumore e ridotta tossicità sistemica e effetti avversi 1, 2, 3, 4, 5, 6, 7. Tuttavia, rimangono molte sfide prima di questi trattamenti possono essere applicati più ampiamente. Lo sviluppo e la validazione di terapie intraduttale in un modello animale adeguato facilitano lo sviluppo di strategie terapeutiche per i pazienti intraduttale. Mentre la ghiandola mammaria topo è stato ampiamente utilizzato come sistema modello di sviluppo mammario e tumorigenesi, l&#39;anatomia è distinta dalla ghiandola umana. Un modello animale più grande, come il coniglio, può servire come un modello migliore per la struttura della ghiandola mammaria e sviluppo terapeutico intraduttale. in contrast a topi, in cui dieci alberi dotto sono spazialmente distribuiti lungo l&#39;asse del corpo, ciascuna terminante in un capezzolo a parte, la ghiandola mammaria di coniglio assomiglia più da vicino la ghiandola umana, con più sistemi duttale sovrapposti che escono attraverso le aperture separate in una tettarella. Qui, vi presentiamo i metodi minimamente invasive per la fornitura di reagenti direttamente nel condotto mammaria coniglio e per la visualizzazione della stessa consegna con ecografia ad alta risoluzione.

Qui mostriamo che la consegna intraduttale di reagenti contrasto ai dotti mammari di un coniglio può essere raggiunta senza traumi al tessuto (Figura 2). Nei conigli, quattro sistemi duttale separati convergono in una sola tettarella e, quindi, possono essere consultati e ripreso individualmente con questo metodo. Aperture individuali duttale sono facilmente visualizzati; notare la marcatura di una seconda apertura duttale adiacente al condotto cannulata in Figu...

Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

Intraductal Delivery to the Rabbit Mammary Gland

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Consegna intraduttale al Coniglio ghiandola mammaria

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

intraductal-delivery-to-the-rabbit-mammary-gland

Research

JoVE Journal

Bioengineering

9.2K Views.  The University of Texas at Austin. Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents. 

Video: Intraductal Delivery to the Rabbit Mammary Gland

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparametrica mappatura ottica del Langendorff-perfuso Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

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Bioingegneria

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Costruire un idrogel collagene per la fornitura di Stem Cell-loaded Chitosan Microsfere

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Microscala Vortex-assistita Elettroporatore per sequenziale molecolare di consegna

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello<em&gt; In Vitro</em&gt; Modello della parete delle vie aeree

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Una cultura Modello 3D Ormone-sensibile della Human ghiandola mammaria Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

La consegna dei terapeutica siRNA al SNC Utilizzando cationici e anionici liposomi

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabbricazione di Schiume matrice extracellulare-derivati ​​e microcarriers come tessuto-specifica delle cellule, Cultura e piattaforme di distribuzione

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Iniezione delle nanoparticelle Retroductal alla ghiandola sottomandibolare murina

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Crescita e caratterizzazione di organoidi irradiati da ghiandole mammarie

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...