The authors would like to thank Bethany Carboneau (Vanderbilt University) for critical reading of this manuscript. We also thank Anastasia Coldren (Vanderbilt University Medical Center Islet Procurement and Analysis Core) for islet isolations, and Dr. Alvin C. Powers (Vanderbilt University Medical Center) and Dr. David Jacobson (Vanderbilt University) for use of their centrifuge and tissue culture facility. This research involved use of the Islet Procurement and Analysis Core of the Vanderbilt Diabetes Research and Training Center supported by NIH grant DK20593. This work was supported by an American Heart Association Postdoctoral Fellowship (14POST20380262) to R.C.P., and grants from the Juvenile Diabetes Research Foundation (1-2011-592), and Department of Veterans Affairs (1BX00090-01A1) to M.A.G.

Tutte le procedure sono state approvate e eseguite in conformità con il Comitato cura e l&#39;uso Vanderbilt Istituzionale Animal. 1. Etichettatura COI con Fluoroforo (opzionale) <ol><li> Scegliere un colorante fluorescente che reagisce con un&#39;ammina primaria libera (ad esempio, su una proteina), come esteri o derivati succinimidyl fluoresceina, per visualizzare microsfere carico. Sciogliere 8x eccesso molare (rispetto alla moli di COI) di fluoroforo in 200 ml di dimetilsolfossido (DMSO). </li><li> Risospendere 50 mg di COI fino ad un volume finale di 800 microlitri in una soluzione veicolo (concentrazione finale di 62,5 ng / ml). La soluzione di veicolo varierà a seconda della fonte del COI. NOTA: veicolo tipico soluzioni includono tampone fosfato salino (PBS) o DMSO. </li><li> Aggiungere tutta la soluzione fluoroforo / DMSO dal punto 1.1 e risospendere il COI. Vortex notte a 4 ° C. In alternativa, agitare la miscela di reazione a temperatura ambiente(RT) per 4 ore. </li><li> Dopo la reazione di marcatura, rimuovere l&#39;eccesso fluoroforo utilizzando una colonna dissalazione. <ol><li> Scolare buffer di storage da colonna dissalazione e risciacquare con 16 ml di acqua deionizzata. Gettare tutto il flusso attraverso. </li><li> Aggiungere il COI fluorescente alla colonna di dissalazione. Eluire con 1,2 ml di acqua deionizzata e raccogliere il flusso attraverso. </li><li> step eluizione Ripetere altre quattro volte, ogni volta aggiungendo 1,2 ml di acqua deionizzata e di raccolta del flusso attraverso come campione separato. </li><li> Congelare tutto il flusso raccolte attraverso i campioni a -80 ° C e liofilizzare secondo le istruzioni del produttore per 24 ore. </li></ol></li></ol> 2. COI-caricato microsfere Preparazione tramite l&#39;acqua acqua-in-olio-in-emulsione di solventi evaporazione Metodo <ol><li> Aggiungere 1 mg di fluorescente COI a 100 ml di acqua deionizzata per formare la prima fase acquosa (W1). </li><li> Sciogliere 65 mg di poli (aci lattico-co-glicolicod) (50:50 Lactide: Glycolide, peso molecolare 54.000 - 69.000) in 750 ml di diclorometano in una provetta. Ultrasonicate per 10 - 30 sec (a 160 W) per sciogliere completamente il PLGA. Ciò costituisce la fase olio (O). </li><li> Aggiungere tutti fase W1 generato nel passaggio 2.1 alla fase O in maniera goccia a goccia. Emulsionare usando un omogeneizzatore portatile a 20.000 rpm per 30 secondi per formare la fase W1 / O. </li><li> Aggiungere tutti fase / O W1 in maniera goccia a goccia a 15 ml di una soluzione all&#39;1% (PVA) (peso / volume) poli acquosa (vinil alcool) ed emulsionare usando un omogeneizzatore portatile a 20.000 rpm per 30 sec . </li><li> Trasferire tutto dell&#39;emulsione generato nel passaggio da 2.4 a 200 ml pallone a fondo tondo e soggetto ad un 635 millimetri Hg di vuoto usando un evaporatore rotante per 1 ora per rimuovere il solvente e generare la fase acquosa. </li><li> Aliquota 1 ml di fase acquosa generati in fase di 2,5 a quattordici provette da microcentrifuga. La centrifugazione della soluzione acquosa in tubi da microcentrifuga a 7.500xg per 8 min. NOTA: A questo punto, le microsfere sono presenti nella soluzione acquosa rimanente e si concentrano sul fondo della provetta. </li><li> Rimuovere con cautela 900 ml di soluzione acquosa dai tubi microcentrifuga con una micropipetta, pipettaggio in modo da non disturbare le microsfere sul fondo della provetta. <ol><li> Lavare le microsfere di un eccesso di PVA con l&#39;aggiunta di 1 ml di acqua deionizzata ad ogni provetta. Centrifugare a 7500 xg per 8 minuti, e ancora rimuovere con cautela 900 ml di soluzione acquosa dai tubi microcentrifuga con una micropipetta, pipettaggio in modo da non disturbare le microsfere sul fondo della provetta. NOTA: La soluzione acquosa 100 microlitri rimanente contiene microsfere generate. </li></ol></li><li> Congelare la soluzione acquosa microsfera generato nel passaggio 2,7 a -80 ° C. </li><li> microsfere lyophilize utilizzando un liofilizzatore secondo il fornitore dile istruzioni e conservare a -20 ° C. NOTA: Misura efficienza carico del COI sciogliendo completamente microsfere di PLGA e determinare la concentrazione di proteine rispetto ad una curva standard della proteina free 7. </li><li> Come controllo negativo, genera una serie di microsfere &quot;in bianco&quot; (di seguito denominati microsfere di controllo). NOTA: La generazione di microsfere di controllo è identica alla generazione di idrofili microsfere COI caricati, senza aggiunta COI a 800 microlitri della soluzione di veicolo in fase 1.2. particelle di controllo Partita a concentrazione di massa di PLGA rispetto a microsfere PLGA COI-caricati per il saggio mitogeno β-cellule. </li></ol> 3. Preparazione di Islet cultura dei media e media pre-test <ol><li> Preparare 200 ml media per la cultura di isole di topo intatte: RPMI 1640 multimediale integrato con 11 mm di glucosio, il 10% siero di cavallo, 100 U / ml di penicillina G e 100 mg / ml di streptomicina (di seguito indicato come èlasciare terreni di coltura). NOTA 1: A differenza di siero fetale bovino (FBS), siero di cavallo manca lattogeno placentare, un induttore della proliferazione β-cellule, che confonde l&#39;analisi del saggio di proliferazione. NOTA 2: Poiché le microsfere di PLGA non vengono sterilizzati prima dell&#39;uso, l&#39;aggiunta di antibiotici ai terreni di coltura è fondamentale per evitare contaminazione microbiologica. </li><li> Rimuovere 25 ml di terreni di coltura isolotto con una pipetta elettronica e posto in un tubo conico da 50 ml. Supplemento 25 ml di mezzo di coltura isolotto nel tubo conico 50 ml con una concentrazione finale di 0,2 mM EGTA per generare il supporto pre-test. NOTA: L&#39;aggiunta di EGTA allenta leggermente contatti cellula-cellula nelle isole senza alterare l&#39;architettura isolotto. Questo aiuta a garantire la COI può raggiungere le cellule interne del isolotto e aiuta a prevenire la necrosi del isolotto centrale. </li></ol> 4. coltura di Intact isole di topo <ol><li> Isolare isolotti intatti da un ceppo preselezionato, il sesso, age, e il genotipo di topi dopo una digestione collagenasi di routine del 8,9 pancreas. Pool insieme isolotti da come campioni. </li><li> Aliquota 40 isolotti in un pozzetto di una piastra di coltura tissutale a 96 pozzetti in 200 ml di terreni di coltura isolotto. Visivamente isolotti size-partita per bene (una precisa valutazione di equivalenza isolotto (IEQ) tra i campioni non è necessario). Riempire i pozzi vuoti immediatamente adiacenti ai pozzi con isolotti con 200 ml di acqua sterile per fornire un buffer di evaporazione. Culture isolotti in mezzi notte a 37 ° C in atmosfera di 95% CO aria e 5% 2. </li></ol> 5. risospensione del COI-caricati e Controllo PLGA microsfere <ol><li> Dopo il recupero isolotto overnight, microsfere risospendere aggiungendo supporti pre-test per un&#39;aliquota di liofilizzati microsfere di PLGA COI caricati tale che la concentrazione finale del COI nella provetta è di 10 ng / ml (la quantità di COI presente in una data quantità di le micro generatiatmosfere sono stati determinati in fase 2.9). Sonicare le microsfere COI-caricati risospeso in un bagno di acqua ghiacciata per 10 minuti a 160 W con impulsi lunghi 10 sec a 4 ° C. </li><li> controllo Risospendere PLGA microsfere aggiungendo lo stesso volume di supporti pre-test per un&#39;uguale massa di controllo liofilizzato microsfere di PLGA. Sonicare i risospesa microsfere di controllo PLGA in un bagno di acqua ghiacciata per 10 minuti a 160 W con impulsi lunghi 10 sec a 4 ° C. </li><li> Visivamente confermano microsfere sono dispersi pipettando 2 ml di microsfere risospese tra due vetrini. Visualizza microsfere utilizzando un obiettivo 40X su un microscopio a epifluorescenza o campo chiaro. </li><li> Se significativa aggregazione delle microsfere è ancora visibile, ultrasuoni in un bagno di acqua ghiacciata per altri 10 minuti a 160 W con impulsi lunghi 10 sec a 4 ° C e la visualizzazione di ripetizione di microsfere risospesa. </li></ol> 6. Il trattamento di isolotti con COI-caricati o controllo PLGA microsfere &lt;/ P&gt; <ol><li> Per ciascun pozzetto da trattare con microsfere COI-caricati, preparare supporti dosaggio diluendo il 10 ng / ml di microsfere COI risospesa con mezzi di pre-test per un volume finale di 100 microlitri e una concentrazione finale predeterminata. NOTA: La concentrazione finale ottimale della proteina può variare per ogni COI impiegato per questo dosaggio. Idealmente, un intervallo di concentrazioni finali sono testati. </li><li> Per ogni bene per essere usato come controllo, preparare il controllo dei media dosaggio diluendo il controllo microsfere risospesa PLGA generati al passo 5.2 con lo stesso volume di diluizione utilizzato per diluire le microsfere COI-caricati al punto 6.1. NOTA: Isolotti trattati con mezzi saggio di controllo serviranno come controllo negativo per consentire l&#39;individuazione di eventuali effetti delle microsfere vuote possono avere sui risultati sperimentali. </li><li> Rimuovere con cautela 100 ml di terreni di coltura isolotto da ogni pozzetto con una micropipetta tale che non isolotti sono sloggiati dai pozzi. Delicatamente aggiungere 100 μl di mezzi di analisi o 100 ml di mezzi saggio di controllo in ciascun pozzetto. </li><li> Incubare isolotti a 37 ° C in atmosfera di 95% CO aria e 5% 2 per tre giorni. </li></ol> 7. Dispersione Isolotti intatte Onto vetrini da microscopio <ol><li> Dopo tre giorni, utilizzare una micropipetta per rimuovere con attenzione e gettare i media saggio da tutti i pozzetti in modo da non rimuovere isolotti. Delicatamente aggiungere 200 ml di PBS a isolotti di lavarle dei mezzi di test. Rimuovere con cautela e scartare PBS con una micropipetta e lavare delicatamente isolotti di nuovo con altri 200 ml di PBS. </li><li> Rimuovere e gettare PBS con una micropipetta e aggiungere 100 ml di 0,025% tripsina, 2 mM EDTA soluzione. Incubare a temperatura ambiente per 3 min. Pipettare isolotti nella soluzione tripsina-EDTA su e giù ogni 2 minuti fino a quando isolotti sono confermati visivamente di essere disperso nelle singole cellule (si noti che alcuni piccoli grumi di cellule possono essere ancora presenti) utilizzando un microscopio ottico. Trasferire l&#39;intero volume di ciascun pozzetto singolamente in microprovette labeled-. </li><li> Aggiungere 400 ml di terreni di coltura isolotto a ciascuna provetta per fermare la dissociazione delle cellule tripsina-mediata. </li><li> campioni di centrifugazione per 5 min a 100 xg a 4 ° C a pellet cellule insulari al fondo dei tubi microcentrifuga. </li><li> Rimuovere delicatamente il surnatante con una micropipetta, e risospendere pellet in 200 terreni di coltura isolotto ml fresca. </li><li> Utilizzando un cito-centrifuga, centrifuga dissociato isolotti a 140 xg per 3 minuti su vetrini da microscopio cariche. </li><li> Air diapositive a secco a temperatura ambiente per 10 minuti, quindi disegnare una casella intorno agli isolotti dissociate con un pennarello idrofoba. </li><li> Fissare le cellule con 75 microlitri 4% paraformaldeide (PFA) a temperatura ambiente per 10 min. Rimuovere 4% PFA e lavare delicatamente le cellule in 75 ml di PBS due volte. Dopo il lavaggio, permeabilize cellule con 75 microlitri 0,2% Triton X-100 in PBS per 10 min. Quindi, lavare le cellule con 75 ml di PBS. Attenzione: PFA è noto per essere allergenico, cancerogeno e tossico. </li></ol&gt; 8. immunofluorescenza Etichettatura di dissociato isolotti per l&#39;insulina e la proliferazione cellulare Marker, Ki67 <ol><li> Preparare una camera umida posizionando due asciugamani di carta bagnata sul fondo di una scatola vetrino da microscopio capacità 100 diapositiva. </li><li> Collocare i vetrini piatto verso il basso (cellule verso l&#39;alto) in camera umida. Preparare i campioni per l&#39;etichettatura aspirando il PBS dalla fase di lavaggio precedente utilizzando una micropipetta e l&#39;aggiunta di 75 ml di soluzione bloccante (5% Normal Donkey Serum (NDS) in PBS) per ogni diapositiva per bloccare legame non specifico di anticorpi contro i campioni. Incubare i vetrini in soluzione bloccante per 1 ora a RT. </li><li> Delicatamente aspirato soluzione bloccante utilizzando una micropipetta e aggiungere 75 ml di soluzione di anticorpo primario contenente cavia anti-insulina e coniglio anticorpi anti-Ki67, diluito 1 mg / ml a 5% NDS in PBS. Incubare a temperatura ambiente per 1 ora. NOTA: gli indicatori alternativi di proliferazione delle cellule proliferanti includono l&#39;antigene nucleare delle cellule (PCNA) e phosphohistone H3 (PH3), </li><li> Aspirare delicatamente soluzione di anticorpo primario con una micropipetta e lavare le cellule con 75 ml di PBS. Aspirare PBS usando una micropipetta e lavare le cellule due volte, ogni volta con 75 ml di PBS. Incubare ciascun campione con l&#39;anticorpo secondario 75 microlitri anti-guinea pig Cy5 e anti-rabbit Cy3 anticorpo secondario diluito a 2 ug / ml in soluzione bloccante per 1 ora a RT. NOTA: non utilizzare anticorpi secondari marcati con lo stesso o spettralmente sovrapposizione fluoroforo che è stato già utilizzato per etichettare le microsfere. </li><li> soluzione di anticorpo secondario Aspirare utilizzando una micropipetta e incubare in 75 ml di 300 nM DAPI in PBS per 3 minuti a RT. Aspirare DAPI utilizzando una micropipetta e sciacquare in acqua deionizzata per 5 min. </li><li> Aspirare delicatamente l&#39;acqua da campioni utilizzando una micropipetta. Spot una goccia (circa 100 ml) di soluzione di montaggio rapida essiccazione contenente antifade reagente su un vetrino di vetro. Invertire il lato del microscopio del campione scivolo verso il basso, onto la soluzione di montaggio. Premere delicatamente il vetrino contro il vetrino con una punta di pipetta per rimuovere le bolle e spazzare via il liquido in eccesso con un panno di pulizia morbido. Lasciare coprioggetto asciugare per una notte a temperatura ambiente. </li></ol> 9. Acquisizione di immagini e analisi <ol><li> Utilizzare un microscopio a epifluorescenza con una fotocamera collegata per l&#39;acquisizione delle immagini. Per ogni fluoroforo, determinare il momento ottimale di esposizione (in genere 20 msec per DAPI, 40 - 80 msec per l&#39;insulina, e 250 msec per Ki67). Assicurarsi parametri di acquisizione delle immagini sono uguali per tutti i campioni. </li><li> Per ogni campione, contare manualmente 3.000 cellule insulino-positive e di quelle contare quante sono anche Ki67-positivi. contare soltanto cellule insulino-positive che hanno un nucleo ben definito e ben visibile. Calcolare la percentuale di proliferazione cellule beta per ogni campione dividendo il numero di cellule Ki67-positive / insulino-positive per il numero totale di cellule insulino-positive e moltiplicando per 100. </li><li> dopo determining la percentuale di proliferazione cellule beta per ciascun campione, determinare se una differenza statisticamente significativa nella proliferazione delle cellule β-esiste tra controllo e campioni sperimentali, analizzando i risultati con un one-way ANOVA utilizzando il software statistico disponibile in commercio. </li></ol>

<ol>
	<li>Butler, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+deficit+and+increased+beta-cell+apoptosis+in+humans+with+type+2+diabetes.">Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes.</a> Diabetes. 52 (1), 102-110 (2003).</li><li>Levy, J., Atkinson, A. B., Bell, P. M., McCance, D. R., Hadden, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+deterioration+determines+the+onset+and+rate+of+progression+of+secondary+dietary+failure+in+type+2+diabetes+mellitus:+the+10-year+follow-up+of+the+Belfast+Diet+Study.">Beta-cell deterioration determines the onset and rate of progression of secondary dietary failure in type 2 diabetes mellitus: the 10-year follow-up of the Belfast Diet Study.</a> Diabet Med. 15 (4), 290-296 (1998).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85 (4), 1255-1270 (2005).</li><li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harb Perspect Med. 2 (7), 007823 (2012).</li><li>Danhier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA-based+nanoparticles:+an+overview+of+biomedical+applications.">PLGA-based nanoparticles: an overview of biomedical applications.</a> J Control Release. 161 (2), 505-522 (2012).</li><li>Skelin, M., Rupnik, M., Cencic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta+cell+lines+and+their+applications+in+diabetes+mellitus+research.">Pancreatic beta cell lines and their applications in diabetes mellitus research.</a> ALTEX. 27 (2), 105-113 (2010).</li><li>Rui, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+of+vascular+endothelial+growth+factor+using+poly-lactic-co-glycolic+acid+microspheres:+in+vitro+characterization+and+application+in+polycaprolactone+fumarate+nerve+conduits.">Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: in vitro characterization and application in polycaprolactone fumarate nerve conduits.</a> Acta Biomater. 8 (2), 511-518 (2012).</li><li>Lacy, P. E., Kostianovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+isolation+of+intact+islets+of+Langerhans+from+the+rat+pancreas.">Method for the isolation of intact islets of Langerhans from the rat pancreas.</a> Diabetes. 16 (1), 35-39 (1967).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> J Vis Exp. (7), e255 (2007).</li><li>Mukherjee, B., Santra, K., Pattnaik, G., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization+and+in-vitro+evaluation+of+sustained+release+protein-loaded+nanoparticles+based+on+biodegradable+polymers.">Preparation, characterization and in-vitro evaluation of sustained release protein-loaded nanoparticles based on biodegradable polymers.</a> Int J Nanomedicine. 3 (4), 487-496 (2008).</li><li>Riley, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+growth+factor+modulates+adult+beta-cell+maturity+and+proliferation+to+promote+beta-cell+regeneration+in+mice.">Connective tissue growth factor modulates adult beta-cell maturity and proliferation to promote beta-cell regeneration in mice.</a> Diabetes. 64 (4), 1284-1298 (2015).</li><li>Mosser, R. E., Gannon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+small+scale+screening+of+candidate+beta+cell+proliferative+factors+using+intact+islets.">An assay for small scale screening of candidate beta cell proliferative factors using intact islets.</a> Biotechniques. 55 (6), 310-312 (2013).</li><li>Carvell, M. J., Marsh, P. J., Persaud, S. J., Jones, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+interactions+regulate+beta-cell+proliferation+in+islet-like+structures.">E-cadherin interactions regulate beta-cell proliferation in islet-like structures.</a> Cell Physiol Biochem. 20 (5), 617-626 (2007).</li><li>Wakae-Takada, N., Xuan, S., Watanabe, K., Meda, P., Leibel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+the+regulation+of+islet+beta+cell+mass+in+mice:+the+role+of+E-cadherin.">Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin.</a> Diabetologia. 56 (4), 856-866 (2013).</li><li>Gavrieli, Y., Sherman, Y., Ben-Sasson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+programmed+cell+death+in+situ+via+specific+labeling+of+nuclear+DNA+fragmentation.">Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.</a> J Cell Biol. 119 (3), 493-501 (1992).</li><li>Kuo, L. J., Yang, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+-+a+novel+biomarker+for+DNA+double-strand+breaks.">Gamma-H2AX - a novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-309 (2008).</li><li>Daoud, J., Rosenberg, L., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+culture+and+preservation+strategies:+advances,+challenges,+and+future+outlook.">Pancreatic islet culture and preservation strategies: advances, challenges, and future outlook.</a> Cell Transplant. 19 (12), 1523-1535 (2010).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biol Proced Online. 11, 3-31 (2009).</li><li>Xu, Q., He, C., Xiao, C., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Oxygen+Species+(ROS)+Responsive+Polymers+for+Biomedical+Applications.">Reactive Oxygen Species (ROS) Responsive Polymers for Biomedical Applications.</a> Macromol Biosci. 16 (5), 635-646 (2016).</li><li>Makadia, H. K., Siegel, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+Lactic-co-Glycolic+Acid+(PLGA)+as+Biodegradable+Controlled+Drug+Delivery+Carrier.">Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier.</a> Polymers (Basel). 3 (3), 1377-1397 (2011).</li><li>Cui, F., Shi, K., Zhang, L., Tao, A., Kawashima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanoparticles+loaded+with+insulin-phospholipid+complex+for+oral+delivery:+preparation,+in+vitro+characterization+and+in+vivo+evaluation.">Biodegradable nanoparticles loaded with insulin-phospholipid complex for oral delivery: preparation, in vitro characterization and in vivo evaluation.</a> J Control Release. 114 (2), 242-250 (2006).</li><li>Kavanaugh, T. E., Werfel, T. A., Cho, H., Hasty, K. A., Duvall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-based+technologies+for+osteoarthritis+detection+and+therapy.">Particle-based technologies for osteoarthritis detection and therapy.</a> Drug Deliv Transl Res. , (2015).</li><li>Joshi, R. V., Nelson, C. E., Poole, K. M., Skala, M. C., Duvall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+pH-+and+temperature-responsive+microparticles+for+protein+delivery+to+ischemic+tissues.">Dual pH- and temperature-responsive microparticles for protein delivery to ischemic tissues.</a> Acta Biomater. 9 (5), 6526-6534 (2013).</li><li>Poole, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS-responsive+microspheres+for+on+demand+antioxidant+therapy+in+a+model+of+diabetic+peripheral+arterial+disease.">ROS-responsive microspheres for on demand antioxidant therapy in a model of diabetic peripheral arterial disease.</a> Biomaterials. 41, 166-175 (2015).</li></ol>

The authors have nothing to disclose.

Lo studio della proliferazione β-cellule in coltura è tipicamente ostacolato da diverse difficoltà. In primo luogo, le linee β-cellule immortalati sono caratterizzati da elevati livelli di proliferazione di quello che si trova in endogene cellule beta nelle isole dal vivo. Inoltre, queste linee cellulari immortalizzate mancano della normale architettura critica per la normale funzione β-cellule. Questi due fatti rendono difficile determinare se i risultati ottenuti utilizzando immortalati linee β-cellule terrà ve...

Pancreatiche beta-cellule sono le uniche cellule che producono insulina nel corpo e sono fondamentali per mantenere l&#39;omeostasi del glucosio nel sangue. Mentre gli individui sani hanno massa β-cellule sufficienti e la funzione di regolare correttamente il glucosio nel sangue, le persone con diabete sono caratterizzati da massa β-cellule insufficiente e / o la funzione 1,2. E &#39;stato proposto che induce la proliferazione delle cellule β-in ultima analisi può aumentare la massa β-cellulare e ripristinare l&#39;omeostasi del glucosio nei soggetti con diabete 3. Tuttavia, la valutazione e la validazione di potenziali composti proliferative β-cellule in isolotti intatto è necessario, prima di terapie efficaci possono essere sviluppate. Il trapianto di isole umane cadavere in individui con diabete di ripristinare l&#39;omeostasi del glucosio nel sangue per un certo tempo, ma la disponibilità e il successo di questa procedura sperimentale è ostacolato da una carenza di isole umane disponibili per il trapianto e la morte delle cellule beta nelle isole di poppaER trapianto 4. Anche con la scoperta di fattori che inducono la moltiplicazione delle cellule che producono insulina, una sfida importante esiste ancora nella realizzazione di questi fattori per siti rilevanti in vivo. Una strategia per la consegna locale sostenuto di composti proliferative β-cellule è il poli (lattico-co-glicolico) Acido (PLGA). PLGA ha una storia di uso in prodotti approvati dalla FDA di consegna della droga a causa della sua elevata sicurezza, biodegradabilità, e cinetica di rilascio prolungato 5. In particolare, PLGA è un copolimero di lattide e glicolide che degrada per idrolisi con acqua in vivo o in coltura in acido lattico e acido glicolico, che sono naturalmente presenti metaboliti nel corpo. Il composto farmaco incapsulato può essere rilasciato nell&#39;ambiente circostante sia diffusione e / o meccanismi di rilascio di degradazione controllata. Incapsulamento di COI fornisce protezione contro la degradazione enzimatica, migliorando la biodisponibilità del reagente rispetto a unencapsulaTed COI 5. Si consiglia di microsfere di PLGA può essere utilizzato per amministrare composti candidati a isolotti intatti nella cultura, e in ultima analisi in vivo. Testare l&#39;efficacia di PLGA per amministrare mitogeni β-cellule di isolotti ex vivo è fondamentale prima di protocolli di trapianto sono esplorate. Attualmente, non esiste una tecnica per misurare la proliferazione β-cellule di animali vivi. Gli esperimenti per valutare l&#39;efficacia di potenziali composti proliferative in vivo quindi richiedere la somministrazione di questi composti per animali vivi, con successiva dissezione e lavorazione di pancreas per immunomarcatura. Tali protocolli sono costosi e laboriosi e richiedono il composto deve essere somministrato per via sistemica, senza alcuna garanzia che raggiungeranno gli isolotti. Viceversa, diverse linee β-cellulari immortalizzate sono disponibili per lo studio delle cellule che producono insulina in coltura, ma queste linee cellulari mancano l&#39;architettura isolotto e environment trovato negli organismi viventi 6. Linee β-cellulari immortalizzate sono anche caratterizzati come aventi un più alto grado di replicazione di endogene beta cellule in vivo, complicando l&#39;analisi di composti che inducono la proliferazione così. In questo studio, si descrive un protocollo che utilizza isolotti intatti isolati da topi adulti. A differenza di linee di β-cellule, isolotti intatti conservano normale architettura isolotto. Allo stesso modo, a differenza di esperimenti condotti in vivo, il funzionamento composti proliferative direttamente isolotti intatti coltura riduce significativamente la quantità di reagenti che è necessario misurare con precisione la proliferazione β-cellule. L&#39;attuale studio utilizza PLGA per amministrare un COI, in questo esempio, fattore ricombinante umano del tessuto connettivo della crescita (rhCTGF). Il metodo qui descritto conferisce un vantaggio significativo rispetto la somministrazione del composto grezzo di isolotti coltivate in quanto consente un rilascio continuo di composto in the media. In particolare, questo dosaggio può essere modificato per gestire un&#39;ampia varietà di proteine ​​e anticorpi di interesse per isolette intatte. Effetti su altri tipi di cellule endocrine, tra cui alfa-cellule, possono anche essere analizzati.

<table border="0" cellpadding="0" cellspacing="0" width="819">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">O6149</td>
			<td style="width:307px;">For labeling COI with fluorophore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO Dimethyl Sulfoxide</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP231-1</td>
			<td>For dissolving fluorophore in step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;">17-0851-01</td>
			<td>Desalting column used in step 1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000-69,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">739960</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(vinyl alcohol) molecular weight 89,000-98,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">341584</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">11879-020</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose Anhydrous</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">200-075-1</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td>Antibiotics for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal horse serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">008-000-121</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well tissue culture plate</td>
			<td style="width:179px;">Corning</td>
			<td align="right" style="width:119px;">3603</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethylene glyco-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td>Supplement for pre-assay islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytospin 4 Cytocentrifuge</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78300003</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EZ Single Cytofunnel</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78710020</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td>BP118-500</td>
			<td>Used in dissociating islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>For fixing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton&#160; X-100</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP151</td>
			<td>For permeabilizing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal donkey serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">017-000-121</td>
			<td>Blocking reagents for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Ki67 antibody</td>
			<td style="width:179px;">abcam</td>
			<td style="width:119px;">ab15580</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyclonal Guinea Pig Anti-Insulin</td>
			<td style="width:179px;">Dako</td>
			<td style="width:119px;">A0564</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy3 AffiniPure Donkey Anti-Rabbit</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">711-165-152</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy5 AffiniPure Donkey Anti-Guinea Pig</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">706-175-148</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>For nuclei visualization in immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aqua-Mount</td>
			<td style="width:179px;">Lerner Laboratories</td>
			<td align="right" style="width:119px;">13800</td>
			<td>Fast drying mounting media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">FreeZone -105&#176;C 4.5 Liter Cascade Benchtop Freeze Dry System</td>
			<td style="width:179px;">Labconco</td>
			<td align="right" style="width:119px;">7382020</td>
			<td>For lyophilization of microspheres</td>
		</tr>
	</tbody>
</table>

sustained administration of &#946;-cell mitogens to intact mouse islets ex vivo using biodegradable poly(lactic-co-glycolic acid) microspheres

Lo sviluppo di biomateriali ha aumentato significativamente il potenziale per la consegna mirato di farmaci ad una varietà di tipi di cellule e tessuti, incluse le cellule beta del pancreas. Inoltre, biomateriale particelle, idrogel, e ponteggi offrono anche l&#39;opportunità unica di amministrare sostenuta, controllabile somministrazione di farmaci ai beta-cellule in coltura e nei modelli di tessuto trapiantato. Queste tecnologie permettono lo studio dei fattori di proliferazione candidato β-cellule utilizzando isolotti intatti e un sistema di traslazione rilevante. Inoltre, determinare l&#39;efficacia e la fattibilità di fattori candidati per stimolare la proliferazione delle cellule β-in un sistema di coltura è fondamentale prima di andare avanti a modelli in vivo. Qui, si descrive un metodo di co-coltura isole di topo intatte con composti biodegradabili di interesse (COI) poli MOLLA (acido lattico-co-glicolico) (PLGA) microsfere al fine di valutare gli effetti della sostenuta in situ rilascio ofattori mitogeni F sulla proliferazione β-cellule. Questa tecnica viene descritto in dettaglio come generare microsfere di PLGA contenenti un carico desiderato utilizzando reagenti commercialmente disponibili. Mentre la tecnica descritta utilizza fattore ricombinante umano connettivo crescita tissutale (rhCTGF), come esempio, potrebbe essere facilmente utilizzata un&#39;ampia varietà di COI. Inoltre, questo metodo utilizza piastre a 96 pozzetti per minimizzare la quantità di reagenti necessarie per valutare la proliferazione β-cellule. Questo protocollo può essere facilmente adattato per usare biomateriali differenti e altre caratteristiche cellulari endocrine come la sopravvivenza delle cellule e lo stato di differenziazione.

La figura 1 è una rappresentazione visiva delle microsfere generate utilizzando il protocollo di cui sopra. Il protocollo qui descritto produce microsfere rhCTGF-caricati di varie dimensioni. La frazione più grande di microsfere sarà compreso tra 1 e 10 micron di diametro, anche se alcune microsfere possono essere più grandi (Figura 2). Se lo si desidera, dimensioni microsfere può essere sintonizzato ed ottimizzato sulla base di par...

Watch this Scientific Journal Video about Sustained Administration of ÃŽÂ²-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Polylactic-co-glycolic acid Microspheres at JoVE.com

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

Amministrazione sostenuta di Mitogeni β-cellule per Intact isole di topo<em&gt; Ex Vivo</em&gt; Uso biodegradabile poli (acido lattico-co-glicolico) Microsfere

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

sustained-administration-cell-mitogens-to-intact-mouse-islets-ex-vivo

Research

JoVE Journal

Bioengineering

7.2K Views.  Vanderbilt University Medical Center. Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.

Video: Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Ad alta risoluzione di immagini 3D Ex-Vivo I campioni biologici da Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Ricerca

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 ...

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Ex vivo Mimicry di emopoiesi normale e anormale umana

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Optical Frequency Domain Imaging di<em&gt; Ex vivo</em&gt; I campioni polmonari Resezione: Ottenere One to One immagine per Istopatologia Correlazione

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinazione del tasso di trasporto di xenobiotici e nanomateriali attraverso la placenta con il<em&gt; Ex vivo</em&gt; Umano Modello perfusione placentare

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Cellule staminali per la programmazione terapeutica angiogenesi Uso biodegradabili polimerico Nanoparticelle

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Iniezione intra-linfonodo di particelle polimeriche biodegradabili

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

L&#39;uso della<em&gt; Ex Vivo</em&gt; Chandler Loop Apparecchio per valutare la biocompatibilità di Modificati Condotti sangue Polimerici

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Chiarificazione primaria del fluido di coltura cellulare raccolto da CHO utilizzando un separatore acustico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

Preparazione, somministrazione e valutazione dell'assorbimento cellulare tessuto-specifico in vivo di liposomi marcati con coloranti fluorescenti

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...