Questo studio è stato supportato dal US Department of Veterans Affairs (Washington DC), le sovvenzioni 0852048 e 0731201, 0854430 e dalla National Science Foundation (Arlington, VA), e Grant RO1 DK080897 dal National Institutes of Health (Bethesda, MD) . Mr. Khanna ricevuto il sostegno di una generosa donazione da Mr. Edward Ross e il dottor Monica Moya dalla Bill &amp; Melinda Gates Foundation (Seattle, WA).

Il protocollo qui descrive una procedura in tre fasi per la generazione di microperle alginato multistrato (Figura 1). In primo luogo, microperle alginato sono formati (Figura 2A). Questa procedura è descritta nella sezione 1 di seguito. Cellule o proteine ​​può essere aggiunta alle microperline in questa fase in modo da agire come un sistema di consegna. Il passo successivo comporta la formazione di uno strato di permeazione selettiva delle microperline e viene descritto nella sezione 2. La fase finale consiste nella formazione di uno strato supplementare e alginato è descritto nella sezione 3. Questa forma uno strato all&#39;esterno della superficie dei granuli (Figura 2B) può essere utilizzata per incapsulare e fornire molecole terapeutiche (Figura 2C) per dirigere risposta cellulare al trapianto seguente sistema. 1. Alginato microsfere Preparazione <ol><li> Preparare una 1,5% (w / v) soluzione di alginato LVM sciogliendo 15 g di alginato LVM in 1 mL dialginato soluzione strato interno (25 mM tampone HEPES, 118 mM NaCl, 5,6 mM KCl, e 2,5 mM MgCl 2 in acqua deionizzata, regolato a pH 7,4). Miscelare con un vortex finché la potenza alginato è completamente disciolta per formare una soluzione limpida, viscosa. Nota: Questo protocollo descrive condizioni ottimali per l&#39;incapsulamento isoletta entro microcapsule alginato 1 La concentrazione e la composizione del alginato microsfere può essere alterato per regolare le proprietà per altre applicazioni (per esempio farmaci consegna, ingegneria dei tessuti, ecc) Nota:. Per incapsulamento isolotto gli isolotti possono essere aggiunti alla soluzione di alginato in questa fase prima di caricare nel microencapsulator. </li><li> Preparare la soluzione di reticolazione (22 mM CaCl 2 in acqua DI) disciogliendo 100 mM CaCl 2 e 10 mM di tampone HEPES in acqua deionizzata e regolando a pH 7,4. Nota: altri cationi bivalenti come Br 2 +, Sr 2 + etc può essere usato al posto di Ca 2 +, a seconda della natura di gelificazione d alginatoesired. </li><li> Impostare due canali microencapsulator giacca aria alginato aggiungendo un ago calibro 25 ad una siringa, e regolare le valvole su ogni lato per assicurarsi che l&#39;ago si trova al centro della giacca aria. Aghi di calibro differenti possono essere utilizzati per questa fase, a seconda delle dimensioni delle microsfere mirate alginato. Questa fase può anche essere effettuata con una siringa, non dovrebbe essere uno microencapsulator disponibile. Aggiungere la soluzione di alginato alla siringa, e selezionare un ago calibro sulla base delle dimensioni di microperle desiderati. </li><li> Inserire un pallone contenente 10 ml di soluzione di reticolazione direttamente sotto l&#39;ago. Mettere una barra di agitazione nella soluzione. </li><li> Iniettare goccioline direttamente nella soluzione di CaCl 2 per reticolare il alginato per formare microsfere. Incubare le perle nella soluzione di reticolazione per la cura per almeno quindici minuti, mescolando continuamente. </li><li> Trasferire le microsfere una provetta da centrifuga da 15 mL. Rimuovere la soluzione residua,ed eseguire tre lavaggi con CaCl 2 0.2% in soluzione fisiologica per due minuti ciascuno. </li></ol> 2. Rivestimento delle microsfere con poli-L-ornitina <ol><li> Preparare 3 mL di una soluzione 0,1% (w / v) di poli-L-ornitina (OLP) in soluzione fisiologica. Posizionare la soluzione su un vortice finché non è completamente sciolto, formando una soluzione limpida. Poly-L-lisina (PLL) può essere utilizzato al posto di OLP per questa fase, che risulta in livelli simili di permselectivity. </li><li> Trasferire le microsfere di alginato nella soluzione OLP. Inserire un Vortex per 30 minuti per permettere il tempo OLP sufficiente per interagire con l&#39;alginato formando un policatione-polianione complesso. Alla fine di 30 minuti, dovrebbe esserci un rivestimento bianco chiaramente visibile attorno alle microperline alginato. </li><li> Rimuovere la soluzione OLP, ed eseguire tre lavaggi con lo 0,2% CaCl 2 in soluzione salina normale per due minuti ciascuno. </li></ol> 3. Creazione del alginato esterno Layer <ol><li> Preparare una soluzione di alginato di concentrazione desiderata che deve essere utilizzato per creare lo strato esterno. La soluzione viene preparata come descritto in (1,1) sopra. La dimensione dello strato esterno è influenzato dalla composizione e la concentrazione di alginato usato (Figura 3). </li><li> Trasferire le microsfere di alginato a una cella-filtro. Utilizzare un Kimwipe per assorbire soluzione in eccesso in modo da asciugare le microperline per quanto possibile. </li><li> Trasferire le microsfere ad una superficie Parafilm. Altre superfici lisce, come vetro o addirittura una capsula Petri di plastica può essere sostituito Parafilm, se desiderato. </li><li> Trasferire la soluzione preparata in alginato (3.1) sulle microsfere di alginato. Il volume di soluzione di alginato essere deve coprire completamente le microperline alginato. Sia le microsfere rimangono nella soluzione di alginato per 45 minuti. </li><li> Usare una pipetta per rimuovere l&#39;eccesso di soluzione di alginato non è vincolata alle microsfere. </li><li> Trasferire le microsfere inad una soluzione di CaCl 2 22mm. Questa reticola soluzione di alginato intorno alle microperline, causando la formazione dello strato distinto alginato esterno. </li><li> Eseguire tre lavaggi con 22 mM CaCl 2 in soluzione salina normale per due minuti ciascuno. Visualizzazione le microperline sotto un microscopio dovrebbe consentire uno strato distinto alginato esterno da vedere formato intorno al nucleo microsfere. </li></ol> 4. Risultati rappresentativi <img src="/files/ftp_upload/3388/3388fig1.jpg" alt="Figura 1" /> Figura 1. Uno schema della procedura di creazione microperle alginato multistrato. Riprodotto con il permesso di Khanna et al. J Biomed Mater Res A. Novembre, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig2.jpg" alt="Figura 2" /> Figura 2. (A) e (B) sono immagini contrasto di fase di microperle alginato. (A) mostra una microsferedopo la sintesi fase (1,7), mentre (B) mostra uno strato distinto alginato esterno presenti dopo il completamento della fase (3,7). (C) è una immagine di fluorescenza FITC-marcato proteina BSA incapsulate nello strato esterno. Riprodotto con il permesso di Khanna et al. J Biomed Mater Res A. Novembre, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig3.jpg" alt="Figura 3" /> Figura 3. La dimensione dello strato esterno alginato può essere variata in base alla composizione e concentrazione di alginato usato. I nostri risultati mostrano che, sia per LVM e alginato LVG, le dimensioni esterne di livello aumenta con la concentrazione alginato crescente, e che alginato LVG produce più spessi strati esterni di alginato di LVM a concentrazioni uguali. Riprodotto con il permesso di Khanna et al. J Biomed Mater Res A. Novembre, 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig4.jpg" alt="Figura 4"/&gt; Figura 4. Rilascio di FGF-1, una proteina fattore di crescita angiogenetico, dallo strato esterno alginato variata in base alla concentrazione di LVM e alginato LVG utilizzato. (A) e (B) mostra rilascio cento, e (C) e (D) indica il rilascio corrispondente massa di FGF-1 rispetto al tempo per diverse formulazioni dello strato esterno. C&#39;è una versione scoppio esposti per tutte le condizioni entro i primi 5 h (A e C) e basse dosi di rilascio continuo per un massimo di 30 giorni (B e D). Riprodotto con il permesso di Khanna et al. J Biomed Mater Res A. novembre;. 95 (2), 632-40 (2010) <a href="http://www.jove.com/files/ftp_upload/3388/3388fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

<ol>
	<li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-+Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L- Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Yamagiwa, K., Kozawa, T., Ohkawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alginate+composition+and+gelling+conditions+on+diffusional+and+mechanical+properties+of+calcium-alginate+gel+beads.">Effects of alginate composition and gelling conditions on diffusional and mechanical properties of calcium-alginate gel beads.</a> J. Chem. Eng. Jpn. 28, 462-467 (1995).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65, 605-610 (1999).</li><li>Uriel, S., Brey, E. M., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+low+levels+of+fibroblast+growth+factor-1+promote+persistent+microvascular+network+formation.">Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation.</a> Am. J. Surg. 192, 604-609 (2006).</li><li>Moya, M. L., Lucas, S., Francis-Sedlak, M., Liu, X., Garfinkel, M. R., Huang, J. J., Cheng, M. H., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+FGF-1+increases+vascular+density+in+comparison+to+bolus+administration.">Sustained delivery of FGF-1 increases vascular density in comparison to bolus administration.</a> Microvasc. Res. 78, 142-147 (2009).</li><li>Moya, M. L., Cheng, M. H., Huang, J. J., Francis-Sedlak, M. E., Kao, S. W., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+FGF-1+loaded+alginate+microbeads+on+neovascularization+and+adipogenesis+in+a+vascular+pedicle+model+of+adipose+tissue+engineering.">The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering.</a> Biomaterials. 31, 2816-2826 (2010).</li><li>Khanna, O., Moya, M. L., Greisler, H. P., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayered+Microcapsules+for+the+Sustained-Release+of+Angiogenic+Proteins+From+Encapsulated+Cells.">Multilayered Microcapsules for the Sustained-Release of Angiogenic Proteins From Encapsulated Cells.</a> Am. J. Surg. 200, 655-658 (2010).</li><li>Khanna, O., Moya, M. L., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multilayered+alginate+microcapsules+for+the+sustained-release+of+fibroblast+growth+factor-1.">Synthesis of multilayered alginate microcapsules for the sustained-release of fibroblast growth factor-1.</a> J. Biomed. Mater. Res. A. 95, 632-640 (2010).</li></ol>

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Alginato è un naturale, polisaccaride acido estratto dalle alghe ed è composto di unità di 1,4 &#39;-β-D-mannuronico (M) e α-L-guluronico acido (G) 2,3. Gelazione semplice verifica quando cationi divalenti, quali Ca 2 +, Sr 2 +, Ba 2 + o interagire con G-monomeri che formano ponti ionici tra le catene adiacenti di alginato. Microperle di alginato sono state utilizzate per fornire una varietà di proteine, inclusi fattore di crescita dei fibroblasti-1 (FGF-1), fattore di c...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Pronova ultrapura LVG alginato </td><td> Nova-Matrix </td><td> 4200006 </td><td> Una varietà di formulazioni alginato sono disponibili. La scelta di alginato influenza le proprietà finali dei microperle, tra cui dimensioni, proprietà meccaniche e di trasporto. La composizione utilizzato deve essere ottimizzato per una data applicazione. </td></tr><tr><td> Pronova ultrapura LVM alginato </td><td> Nova-Matrix </td><td> 4200206 </td><td> Una varietà di formulazioni alginato sono disponibili. La scelta di alginato influenza le proprietà finali dei microperle, tra cui dimensioni, proprietà meccaniche e di trasporto. La composizione utilizzato deve essere ottimizzato per una data applicazione. </td></tr><tr><td> Poly-L-ornitina cloridrato </td><td> Sigma-Aldrich </td><td> P2533 </td><td></td></tr></tbody></table>

generation of alginate microspheres for biomedical applications

Materiali a base di alginato hanno ricevuto una notevole attenzione per le applicazioni biomediche a causa della loro natura idrofila, biocompatibilità e architettura fisica. Le applicazioni includono l&#39;incapsulamento delle cellule, la consegna della droga, delle cellule staminali della cultura, e impalcature di ingegneria dei tessuti. In realtà, studi clinici sono attualmente eseguite in cui isolotti sono incapsulati in alginato microperline rivestite PLO come trattamento di diabete tipo. Tuttavia, un gran numero di isolette sono necessari per efficacia a causa trapianto scarsa sopravvivenza seguente. La capacità di stimolare a livello locale la formazione di microvascolare rete intorno alle cellule incapsulate possono aumentare la loro redditività attraverso il miglioramento dei trasporti di ossigeno, glucosio e altri nutrienti vitali. Fibroblast Growth Factor-1 (FGF-1) è un fattore di crescita naturale che è in grado di stimolare la formazione di vasi sanguigni e migliorare i livelli di ossigeno nei tessuti ischemici. L&#39;efficacia di FGF-1 è aumentata quando si è consegnato in una sustained moda piuttosto che un unico grande-bolo amministrazione. Il locale a lungo termine rilascio di fattori di crescita da sistemi di incapsulamento delle isole possano stimolare la crescita dei vasi sanguigni direttamente verso le cellule trapiantate, potenzialmente migliorare i risultati innesto funzionali. In questo articolo, si delineano le procedure per la preparazione di microsfere di alginato per l&#39;uso in applicazioni biomediche. Inoltre, si descrive un metodo che abbiamo sviluppato per la generazione di microsfere di alginato multistrato. Le celle possono essere incapsulati in alginato nucleo interno, e le proteine ​​angiogeniche nello strato esterno alginato. Il rilascio di proteine ​​da questo strato esterno potrebbe stimolare la formazione di reti locali microvascolari direttamente verso le isole trapiantate.

Watch this Scientific Journal Video about Generation of Alginate Microspheres for Biomedical Applications at JoVE.com

Generation of Alginate Microspheres for Biomedical Applications

Nelle sezioni seguenti, si delineano le procedure per la preparazione di microsfere di alginato per l&#39;uso in applicazioni biomediche. Specificatamente illustrano una tecnica per creare multistrato alginato microsfere per il duplice scopo di incapsulamento delle cellule e proteine ​​come potenziale trattamento di diabete di tipo 1.

Generazione di microsfere di alginato per applicazioni biomediche

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

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20.6K Views.  Illinois Institute of Technology. Nelle sezioni seguenti, si delineano le procedure per la preparazione di microsfere di alginato per l'uso in applicazioni biomediche. Specificatamente illustrano una tecnica per creare multistrato alginato microsfere per il duplice scopo di incapsulamento delle cellule e proteine ​​come potenziale trattamento di diabete di tipo 1.

Video: Generation of Alginate Microspheres for Biomedical Applications

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

Generazione di Comprehensive Database Oncologia Toracica - Strumento per la ricerca traslazionale

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

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Medicina

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adattamento del semiautomatico circolanti (CTC) Saggi Tumor Cell per applicazioni cliniche e di ricerca preclinica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Raccolta ed estrazione di Saliva DNA per il sequenziamento di nuova generazione

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Experimental Glaucoma indotta da iniezione oculare di microsfere magnetiche

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generazione di Microtumors Uso 3D Biogel Umano Sistema Cultura e cellule di glioblastoma paziente-derivato per Kinomic Profiling and Drug Testing risposta

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Generazione di Media e Applicazioni organo condizionata per lo studio Influenze organo-specifiche sul cancro al seno metastatico Comportamento

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

Drug Repurposing Ipotesi Generation Con il &quot;RE: Drugs sottili&quot; Sistema

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Strategie di campionamento e trattamento dei campioni di tessuto Biobank Porcine modelli biomedicale

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...