Name: effective isolation of functional islets from neonatal mouse pancreas
Uploaded: 2017-01-06T11:30:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

l&#39;utilizzo di animali segue le procedure di cui al protocollo M / 11/181 approvato dalla Vanderbilt Institutional Animal Care e del Comitato Usa per Gu. topi CD1 o CBA / BL6 sono stati acquistati da fornitori commerciali e attraversato nella struttura animale Vanderbilt per ottenere topi neonatali. 1. Preparazione di topi, soluzioni di riserva, e attrezzature <ol><li> Per la croce del mouse: impostare una croce del mouse e registrare le date di tamponamento per aiutare la pianificazione sperimentale. topi CD1 di solito partoriscono intorno al giorno 19 dopo l&#39;accoppiamento. Utilizzare CD1 o CBA / BL6 croci per ottenere CD1 o CBA / BL6 neonati, rispettivamente. Incrociati tra le due linee utilizzano femmine e maschi CD1 CBA / BL6. </li><li> Per la collagenasi magazzino: pesare 200 mg di collagenasi di tipo IV con un saldo di alta precisione. Trasferire in un tubo da 50 ml centrifuga. Sciogliere in 40 mL HBSS con Ca 2+ / Mg 2+ (1,26 e 0,5 mm rispettivamente) per fare un / magazzino soluzione 5 mg ml. <ol><li> Consentire a collagenasisciogliere per ~ 30 minuti agitando delicatamente. Aliquotare e conservare la soluzione congelata nel -20 ° C come magazzino, che rimane attivo per almeno un anno. </li></ol></li><li> Autoclave per la sterilizzazione diverse paia di pinzette, forbici, e suggerimenti P20 per la dissezione e isolotto raccolta. </li><li> Per il glucosio magazzino 1 M: pesare 9 g di glucosio in un tubo da 50 ml centrifuga, aggiungere 50 ml di RPMI 1640 media per sciogliere il glucosio. Tenere a 4 ° C fino al momento dell&#39;uso, ma non più di 6 mesi. </li></ol> 2. Soluzioni di lavoro NOTA: Il giorno di isolamento delle isole, preparare i seguenti reagenti. <ol><li> Per la soluzione IV di lavoro tipo collagenasi: disgelo e diluire collagenasi magazzino dal ghiaccio. Dispensare 0,15 mL magazzino in ciascuna 1,7 ml provetta per microcentrifuga. Aggiungere 1,35 ml HBSS con Ca 2+ / Mg 2+. Invertire la provetta per 5 volte. Lasciare in ghiaccio per 15 min. <ol><li> Girano ad alta velocità per 3 minuti in una microcentrifuga. Trasferire il surnatante in una nuova provetta. Lasciare in ghiaccio fino al momento dell&#39;uso. Eliminare il tubo con residui insolubili. </li></ol></li><li> Per un completo RPMI 1640 supporti: aggiungere 2,5 ml 1 M di glucosio, 50 mL di calore inattivato FBS, e 5 ml 100x Pen-strep magazzino in 500 ml RPMI 1640 mezzi. Lasciare in ghiaccio fino al momento dell&#39;uso. </li></ol> 3. Isolamento e Pancreas Digestione <ol><li> Euthanize neonati con isoflurano, seguita da decapitazione secondo protocolli approvati. </li><li> Posare il mouse con la pancia rivolta verso l&#39;alto. Spruzzare con il 70% di etanolo per la sterilizzazione. </li><li> Sollevare la pelle pancia in su con una pinzetta e tagliare gli strati di pelle e muscoli longitudinalmente lungo la linea mediana, con un paio di forbici, dalla zona genitale alla gabbia toracica. Questo apre la cavità addominale per esporre tutti gli organi interni. </li><li> Trasferire tutti gli organi interni di un piatto 100 centimetri con un paio di pinzette. Individuare il pancreas, che è un organo sparso con una porzione dorsale che si aggrappa allo stomaco e la milza e una parte ventrale, che si aggrappa al duodeno 11. </li&gt; <li> Aggiungere HBSS nel piatto di sommergere gli organi. In soluzione, tessuti pancreatici aggrappano più al duodeno, stomaco, milza. E &#39;facilmente riconoscibile in un ambito dissezione causa del suo colore bianco tipico. Utilizzare un paio di pinzette per sbucciare i tessuti pancreatici di distanza dal tessuto circostante e trasferimento in un nuovo piatto 60 millimetri con HBSS. </li><li> Tagliare ogni pancreas con una forbice in pezzi più piccoli di 5 mm di diametro qualsiasi dimensione. Trasferimento in una provetta da microcentrifuga da 1,5. Fino a cinque pancreas (più giovane di P7) possono essere digeriti in un tubo. Per i topi P8-P16, utilizzare un tubo per il pancreas. </li><li> Aggiungere 0.5 - soluzione di lavoro 1 ml di collagenasi ad ogni provetta (0,2 ml per ogni pancreas P1-P7 Utilizzare 0,5 ml per ogni pancreas P8-P16.). Lasciare la provetta in un incubatore a 37 ° fino a 15 min. Invertire il tubo due volte ogni min. </li><li> Dopo circa 5 minuti, toccare il tubo per monitorare lo stato di digestione del tessuto pancreatico. Continuare il processo di digestione finché la maggior parte del pancreastessuto appare come gruppi di cellule frammentati, con diametro &lt;2 mm. Il lisato apparirà nuvoloso, che è dovuto alla normale autolisi delle cellule acinose. </li></ol> 4. lisato di lavaggio <ol><li> Spin il lisato a 500 g per 10 s in una microcentrifuga. Rimuovere lo strato di surnatante con una Pipetman P 1.000. Il surnatante deve apparire torbida, ma privo di gruppi di cellule. Non usare l&#39;aspirazione, che può rapidamente aspirare via i frammenti di tessuto in basso a causa della presenza di alcuni DNA appiccicoso. </li><li> Aggiungere 1 ml di RPMI 1640 completo dei media. Toccare il tubo delicatamente per risospendere il lisato frammentato. Invertire su e giù per 10 volte. Ripetere il punto 4.1. </li><li> Ripetere il punto 4.2 altre due volte o fino a quando il surnatante appare evidente. Risospendere frammenti pancreatiche lavati in 1 ml completo RPMI 1640 multimediali. </li></ol> 5. L&#39;isolamento Islet NOTA: per un piccolo numero di pancreas, diretta a mano raccogliendo, come di seguito (5.1) può essere utilizzato per isolotto ISOLzione. Per grandi numeri di pancreas (&gt; 6), il metodo descritto in 5.2 è preferito. <ol><li> Diretto raccolta manuale: <ol><li> Trasferire lisato dal punto 4.3 a un piatto di 60 millimetri. Aggiungere 5 ml completi RPMI 1640 mezzi. Portare sotto un microscopio da dissezione. </li><li> Impostare l&#39;illuminazione in campo chiaro sul microscopio. Regolare l&#39;intensità della luce a ~ 50% della potenza massima. In questa condizione, isolotti appaiono come un po &#39;grappoli di colore rosa, mentre le cellule acinari scuri cluster di forma irregolare (Figura 1A). Si raccomanda un ingrandimento di 50X ~ (combinando la potenza dell&#39;obiettivo e il pezzo occhio) per offrire il miglior modo di isolotti visualizzazione e l&#39;apertura punta della pipetta simultaneamente per il prelievo. </li><li> Pick up isolotti con un puntale P20 evitando cluster acinar. Frazioni dispensare isolotti arricchita di un nuovo piatto 60 millimetri con RPMI completo supporto (Figura 1B). </li><li> Ripetere il processo di raccolta manuale altre due volte. Lasciare isolotti pure in una nuova 60mm piatto con 4 ml di mezzi RPMI completi (Figura 1C). </li></ol></li><li> centrifugazione in gradiente: <ol><li> Preparare una provetta da centrifuga 15 che viene precaricato con 2 mL polysucrose e sodio diatrizoato ad una densità di 1.077 g / mL. Lasciare la provetta in ghiaccio fino al momento dell&#39;uso. </li><li> Trasferire la sospensione dal punto 4.3 sulla parte superiore della soluzione diatrizoato polysucrose e sodio con una pipetta Pasteur. Non disturbare il livello inferiore. I tessuti di fino a 10 appena nato o 2 P17 pancreas possono essere caricati in ciascuna provetta 15 ml. </li><li> Spin la provetta a ~ 600 xg in un rotore oscillante per 15 min a 4 ° C. Utilizzare le impostazioni dei freni-off. </li><li> Dopo la centrifugazione, trasferire i mezzi di comunicazione e strati interfase (si noti che in condizioni di esercizio ottimali, le isole saranno situati nella interfase tra i media e la cultura polysucrose dopo la centrifugazione) in un nuovo tubo 15 ml. Aggiungere 10 ml di mezzi, Mescolare con cura. </li><li> Spin giù la frazione isolotto arricchitaa 300 xg per 5 min. Rimuovere il supporto di lavaggio. Ripetere il lavaggio ancora una volta con i media RPMI completo. Pellet Risospendere in a 4 ml media, che contiene per lo più isolotti e condotti (Figura 1D). </li><li> Selezionare manualmente le isole come descritto nel paragrafo 5.1. </li></ol></li></ol> 6. GSIS saggi in isole isolate <ol><li> Incubare le isolette dal punto 5.1.4 nei media RPMI completo per 2 ore in un C coltura tissutale incubatore a 37 °. </li><li> Usare una pipetta per rimuovere RPMI sotto un microscopio da dissezione. Aggiungere 3 mL di soluzione KRB con 2,8 mm di glucosio alla piastra 3. Agitare 10 volte per lavare gli isolotti. Rimuovere soluzioni KRB. <ol><li> Ripetere il processo di lavaggio una volta. Incubare le isolette con 3 mL di soluzione KRB a 37 ° C per 1 ora in un incubatore. </li></ol></li><li> Aliquotare 1 mL KRB in ciascun pozzetto di una piastra 12 pozzetti. Pre-riscaldare il brodo KRB e la piastra a 37 ° C in un incubatore. </li><li> Lavare gli isolotti due volte con KRB preriscaldata come in 6.2. Transfer 10 isolette a ciascun pozzetto delle piastre preriscaldate. Incubare in un incubatore a 37 °. </li><li> Dopo 45 minuti, ritirare 50 ml di surnatante. Questo contiene la soluzione di insulina secreta sotto glucosio basale. </li><li> Aggiungere 32 ml 500 mm di glucosio. Agitare la piastra 20 volte per miscelare la soluzione. Incubare in un incubatore a 37 °. Agitare la piastra 5 volte una volta ogni 5 min. </li><li> Dopo 45 minuti, ritirare 50 microlitri surnatante. Questo è l&#39;insulina secreta sotto alta glucosio. </li><li> Trasferire tutte le isolette in una provetta da 1,5 ml. Congelare in -20 ° C per 30 min. Scongelare a temperatura ambiente per 5 min. Ripetere il processo di congelamento e scongelamento. </li><li> Aggiungere 0,5 ml di etanolo al 70% con 20 mM HCl. Lasciar riposare. Questo è l&#39;insulina nella isolotti. </li><li> Utilizzare ELISA convenzionale per saggiare i livelli di insulina, descritto in precedenza 3. </li></ol>

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	<li>Matsumoto, S., 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=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. (50), (2011).</li><li>Zhao, A., 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=Galphao+represses+insulin+secretion+by+reducing+vesicular+docking+in+pancreatic+beta-cells.">Galphao represses insulin secretion by reducing vesicular docking in pancreatic beta-cells.</a> Diabetes. 59 (10), 2522-2529 (2010).</li><li>Planas, R., 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=Gene+expression+profiles+for+the+human+pancreas+and+purified+islets+in+type+1+diabetes:+new+findings+at+clinical+onset+and+in+long-standing+diabetes.">Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.</a> Clin Exp Immunol. 159 (1), 23-44 (2010).</li><li>Tonne, J. 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=Global+gene+expression+profiling+of+pancreatic+islets+in+mice+during+streptozotocin-induced+beta-cell+damage+and+pancreatic+Glp-1+gene+therapy.">Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced beta-cell damage and pancreatic Glp-1 gene therapy.</a> Dis Model Mech. 6 (5), 1236-1245 (2013).</li><li>London, N. J., Swift, S. M., Clayton, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+functional+evaluation+of+islets+of+Langerhans.">Isolation, culture and functional evaluation of islets of Langerhans.</a> Diabetes Metab. 24 (3), 200-207 (1998).</li><li>Rorsman, P., 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=Failure+of+glucose+to+elicit+a+normal+secretory+response+in+fetal+pancreatic+beta+cells+results+from+glucose+insensitivity+of+the+ATP-regulated+K++channels.">Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.</a> Proc Natl Acad Sci U S A. 86 (12), 4505-4509 (1989).</li><li>Hellerstrom, C., Swenne, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+and+proliferation+of+fetal+pancreatic+beta-cells.">Functional maturation and proliferation of fetal pancreatic beta-cells.</a> Diabetes. 40 (Suppl 2), 89-93 (1991).</li><li>Blum, B., 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=Functional+beta-cell+maturation+is+marked+by+an+increased+glucose+threshold+and+by+expression+of+urocortin+3.">Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.</a> Nat Biotechnol. 30 (3), 261-264 (2012).</li><li>Hegre, O. D., 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=Nonenzymic+in+vitro+isolation+of+perinatal+islets+of+Langerhans.">Nonenzymic in vitro isolation of perinatal islets of Langerhans.</a> In Vitro. 19 (8), 611-620 (1983).</li><li>Dolen&#353;ek, J., Rupnik, M. A., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), e102445 (2015).</li><li>White, J., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Source Book of Enzymes, Source Book of Enzymes. , (1997).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54 (1), 133-137 (2005).</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></ol>

The authors declare no competing financial interests for the described work.

Qui, forniamo un protocollo step-by-step su isolamento delle isole dal pancreas che sono troppo piccoli per la perfusione convenzionale. Si prevede di produrre isolotti pronti per tutti gli studi isolotto-based come la purificazione delle cellule beta, analisi di espressione genica, isolotto la maturazione delle cellule beta, la proliferazione, risposte allo stress cellulare, la sopravvivenza delle cellule, il metabolismo, e funzionale manutenzione GSIS, ecc. Questo sarà, al meglio delle nostre conoscenze, il ...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table><tbody><tr><td>Collagenase Type IV</td><td>Sigma Aldrich, St Louis, MO</td><td>C5138</td><td></td></tr><tr><td>1x HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech/Cellgro, Manassas, VA</td><td>MT21020CV</td><td>If HBSS wihout Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; is obtained, CaCl&lt;sub&gt;2&lt;/sub&gt; and MgSO&lt;sub&gt;4&lt;/sub&gt; can be added to 1.26 and 0.5 mM, respectively.&amp;nbsp;</td></tr><tr><td>RPMI 1640 w/o glucose</td><td>Thermo Fisher Scientific/Life Technologies, Waltham, MA</td><td>11879-020</td><td>Glucose needs to be added to specific levels to not interfere with subsequent islet usage.&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Sigma Aldrich, St Louis, MO</td><td>G-7021&amp;nbsp;</td><td></td></tr><tr><td>polysucrose and sodium diatrizoate solution</td><td>Sigma Aldrich, St Louis, MO</td><td>Histopaque-10771</td><td></td></tr><tr><td>Stereoscope</td><td>Carl-Zeiss, Oberkochen, Germany</td><td>Stemi2000</td><td></td></tr><tr><td>Stereoscope</td><td>Leica, Wetzlar, Germany</td><td>Leica M165</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5417C</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>Centrifuge 5810R</td><td></td></tr><tr><td>15-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-666</td><td></td></tr><tr><td>50-mL centrfuge tubes</td><td>VWR, Radnor, PA</td><td>89039-658</td><td></td></tr><tr><td>Precision balance</td><td>VWR, Radnor, PA</td><td>VWR-225AC</td><td></td></tr><tr><td>Microfuge tubes</td><td>VWR, Radnor, PA</td><td>87003-294</td><td></td></tr><tr><td>Pipetman P1000</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123602</td><td></td></tr><tr><td>Pipetman P20</td><td>Fisher Scientific,&amp;nbsp; Waltham, MA</td><td>F123600</td><td></td></tr><tr><td>100 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-088</td><td></td></tr><tr><td>60 x 15 mm dish</td><td>VWR, Radnor, PA</td><td>25384-168</td><td></td></tr><tr><td>12-well plates</td><td>VWR, Radnor, PA</td><td>665-180</td><td></td></tr><tr><td>Scissor</td><td>Fine Scientific Tools, Foster City, CA</td><td>14080-11</td><td></td></tr><tr><td>Tweezers</td><td>Fine Scientific Tools, Foster City, CA</td><td>5708-5</td><td></td></tr><tr><td>CD1 mice</td><td>Charles River Laboratories, Wilminton, MA&amp;nbsp;</td><td>CD-1</td><td></td></tr><tr><td>C57BL/6J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>C57BL/6J</td><td></td></tr><tr><td>B6CBAF1/J</td><td>The Jackson laboratory, Farmington, CT&amp;nbsp;</td><td>B6CBAF1/J</td><td></td></tr></tbody></table>

effective isolation of functional islets from neonatal mouse pancreas

protocolli isolotto-isolamento di perfusione a base di grandi dimensioni pancreas dei mammiferi sono ben stabiliti. Tali protocolli sono facilmente effettuate in molti laboratori causa delle grandi dimensioni del dotto pancreatico che permette iniettabile collagenasi pronta e successiva perfusione tissutale. Al contrario, isolamento delle isole dal pancreas piccolo, come quello dei topi neonatali, è impegnativo, perché la perfusione non è facilmente realizzabile nel piccolo pancreas. Qui si descrive una semplice procedura dettagliata che recupera un numero considerevole di isolotti da topi appena nati con assistenza visiva. Appena sezionato intero pancreas sono stati digeriti con 0,5 mg / ml di collagenasi IV disciolto in soluzione salina bilanciata Hanks &#39;(HBSS) a 37 ° C, in tubi da microcentrifuga. I tubi sono stati sfruttato regolarmente per aiutare la dispersione dei tessuti. Quando la maggior parte del tessuto è stata dispersa per piccoli gruppi circa 1 mm lisati sono stati lavati tre a quattro volte con mezzo di coltura con siero fetale bovino 10% (FBS). cluster Islet,privo di tessuti acinar riconoscibili, possono poi essere recuperati sotto dissezione stereoscope. Questo metodo recupera 20 - 80 piccole e grandi dimensioni isolotti per pancreas di topo appena nato. Questi isolotti sono adatti per saggi a valle più immaginabili, tra cui la secrezione di insulina, l&#39;espressione genica, e la cultura. Un esempio di saggio secrezione di insulina è presentato per convalidare il processo di isolamento. Il background genetico e il grado di digestione sono i maggiori fattori che determinano il rendimento. soluzione di collagenasi Appena fatto con alta attività è preferito, in quanto aiuta in isolamento endocrino-esocrina. La presenza di cationi [calcio (Ca 2+) e magnesio (Mg 2+)] in tutte le soluzioni e siero bovino fetale al lavaggio / sono necessari per una buona resa di isolotti con integrità supporto corretto prelievo. Un ambito dissezione con un buon contrasto e l&#39;ingrandimento aiuterà anche.

In condizioni ottimali, il metodo presentato può produrre 20 - 80 isolotti da ogni piccolo pancreas mouse. Questo numero dipende dal background genetico, età dei topi, e la dimensione di isolotti da recuperare. Tra il comunemente usato, CD1 out-allevato e C57BL / 6J pura producono meno isolotti con dimensioni inferiori a ibridi tra CD1 e C57BL / 6 o B6CBAF1 commerciale / topi J fanno. Mano diretto raccogliendo in generale ha dato un numero minore di isolotti, probabilmente a causa dell...

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Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Descriviamo qui un protocollo per isolare isolotti intatti da topi neonati. Pancreas sono stati parzialmente digerito con collagenasi, seguita da lavaggio e raccolta manuale. 20 - 80 clusters insule possono essere ottenuti per pancreas da topi appena nati, che sono adatti per diversi studi insulari.

Efficace isolamento di isole funzionali da neonatali mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

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10.0K Views.  Vanderbilt University Medical School.  Descriviamo qui un protocollo per isolare isolotti intatti da topi neonati. Pancreas sono stati parzialmente digerito con collagenasi, seguita da lavaggio e raccolta manuale. 20 - 80 clusters insule possono essere ottenuti per pancreas da topi appena nati, che sono adatti per diversi studi insulari. 

Video: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Ricerca

Medicina

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolamento di isole umane da pazienti parzialmente Pancreatectomized

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Il trapianto di isole nel supporto del grasso epididimico dei topi diabetici

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioingegneria

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; Db</sup&gt; Mouse Modello di diabete di tipo 2: pancreas isolamento Islet e live-cella 2-Photon Imaging di isolotti Intact

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Uno Screening basati su cellule dell'isolotto pancreatico murino per prodotti chimici ambientali diabetogeni

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Immunologia e infezioni

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A High-contenuti<em&gt; In Vitro</em&gt; La replica della piattaforma Discovery isole pancreatiche β-cellule

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Mouse Isolotto di Langerhans isolamento utilizzando una combinazione di Collagenasi depurato e proteasi neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Biologia

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal trapianto di isole pancreatiche nel modello del topo

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Un metodo per l&#39;isolamento del mouse isole pancreatiche e intracellulare cAMP Determinazione

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Efficace isolamento di isole funzionali da neonatali mouse Pancreas

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

Isolamento di isole umane da pazienti parzialmente Pancreatectomized

Il trapianto di isole nel supporto del grasso epididimico dei topi diabetici

LEPR

Uno Screening basati su cellule dell'isolotto pancreatico murino per prodotti chimici ambientali diabetogeni

A High-contenuti

Mouse Isolotto di Langerhans isolamento utilizzando una combinazione di Collagenasi depurato e proteasi neutra

Intraportal trapianto di isole pancreatiche nel modello del topo

Un metodo per l'isolamento del mouse isole pancreatiche e intracellulare cAMP Determinazione

Un semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi