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;utilisation de l&#39;animal suit les procédures spécifiées dans le protocole M / 11/181 approuvé par le Vanderbilt Institutional Animal Care et l&#39;utilisation Comité pour Gu. souris CD1 ou CBA / BL6 ont été achetés auprès de fournisseurs commerciaux et croisées dans l&#39;animalerie Vanderbilt pour obtenir des souris néonatales. 1. Préparation de la souris, Stock Solutions et équipements <ol><li> Pour la croix de la souris: mettre en place une croix de la souris et enregistrer les dates de colmatage à l&#39;aide de planification expérimentale. souris CD1 donnent généralement naissance autour du jour 19 après l&#39;accouplement. Utilisez CD1 ou CBA / BL6 croix pour obtenir CD1 ou CBA / BL6 neonates, respectivement. Intercroisements entre les deux lignes utilisent les femelles et les mâles CD1 CBA / BL6. </li><li> Pour la collagénase stock: peser 200 mg Type de collagénase IV avec une balance de précision élevé. Transférer dans un tube de 50 ml de centrifugeuse. Dissoudre dans 40 ml de HBSS avec Ca 2+ / Mg 2+ (1,26 et 0,5 mM, respectivement) pour faire un / ml de solution mère de 5 mg. <ol><li> Autoriser la collagénase àdissoudre pendant ~ 30 min en agitant doucement. Aliquoter et maintenir la solution congelée dans -20 ° C en tant que stock, qui reste actif pendant au moins un an. </li></ol></li><li> Autoclave pour stériliser plusieurs paires de pinces, des ciseaux et des conseils de P20 pour la dissection et l&#39;îlot cueillette. </li><li> Pour le 1 M de glucose stock: peser 9 g de glucose dans un tube de 50 ml de centrifugation, ajouter 50 ml du milieu RPMI 1640 pour dissoudre le glucose. Maintenir à 4 ° C jusqu&#39;à son utilisation, mais pas plus de 6 mois. </li></ol> 2. Solutions de travail NOTE: Le jour de l&#39;isolement des îlots, préparer les réactifs suivants. <ol><li> Pour la solution IV de travail de type collagénase: dégel et diluer collagénase stocks sur la glace. Distribuer 0,15 ml disponible dans chaque ml tube de 1,7 microfuge. Ajouter 1,35 ml HBSS avec Ca 2+ / Mg 2+. Inversez le tube 5 fois. Laisser sur la glace pendant 15 min. <ol><li> Spin à grande vitesse pendant 3 min dans une microcentrifugeuse. Transférer le surnageant dans un nouveau tube. Laisser sur la glace jusqu&#39;à utilisation. Jeter le tube avec des débris insolubles. </li></ol></li><li> Pour RPMI complet 1640: ajouter 2,5 mL 1 M de glucose, 50 ml inactivé par la chaleur FBS, et 5 100x ml Stock Pen-streptocoque dans 500 ml du milieu RPMI 1640. Laisser sur la glace jusqu&#39;à utilisation. </li></ol> 3. Isolement et Pancréas Digestion <ol><li> Euthanasier neonates avec isoflurane, suivis par décapitation selon des protocoles approuvés. </li><li> Poser la souris avec le ventre vers le haut. Asperger avec de l&#39;éthanol à 70% pour la stérilisation. </li><li> Soulevez la peau du ventre avec des pinces et couper les couches de la peau et des muscles longitudinalement le long de la ligne médiane avec une paire de ciseaux, de la zone génitale à la cage thoracique. Cela ouvre la cavité abdominale pour exposer tous les organes internes. </li><li> Transférez tous les organes internes à un plat de 100 cm avec une paire de pinces. Localisez le pancréas, qui est un organe dispersé avec une partie dorsale qui s&#39;accroche à l&#39;estomac et de la rate et une partie ventrale qui adhère au duodénum 11. </li&gt; <li> Ajouter HBSS dans le plat pour submerger les organes. En solution, les tissus pancréatiques accrochent plus au duodénum, ​​de l&#39;estomac, ou de la rate. Il est facilement reconnaissable sous un champ de dissection en raison de sa couleur blanche typique. Utilisez une paire de pinces pour éplucher les tissus pancréatiques loin de tissus et de transfert entourant à une nouvelle boîte de 60 mm avec HBSS. </li><li> Couper chaque pancréas avec une paire de ciseaux en morceaux plus petits que 5 mm à travers toutes les dimensions. Transférer dans un tube de microcentrifugation de 1,5 ml. Jusqu&#39;à cinq pancreata (moins de P7) peuvent être digérés dans un seul tube. Pour les souris P8-P16, utiliser un tube par le pancréas. </li><li> Ajouter 0,5 - solution de travail 1 mL de collagénase à chaque tube (0,2 ml pour chaque pancréas P1-P7 Utiliser 0,5 ml pour chaque pancréas P8-P16.). Laisser le tube dans un incubateur à 37 ° C pendant jusqu&#39;à 15 min. Inversez le tube deux fois par min. </li><li> Après environ 5 minutes, appuyez sur le tube pour surveiller l&#39;état de la digestion du tissu pancréatique. Poursuivre le processus de digestion jusqu&#39;à ce que la majeure partie du pancréastissu apparaît amas de cellules fragmentées, avec un diamètre &lt;2 mm. Le lysat apparaît trouble, qui est due à l&#39;autolyse des cellules acineuses normal. </li></ol> 4. Lysate laver <ol><li> Spin lysat à 500 xg pendant 10 s dans une microcentrifugeuse. Retirer la couche de surnageant avec un Pipetman P1,000. Le surnageant doit apparaître turbide mais dépourvue d&#39;amas cellulaires. Ne pas utiliser de l&#39;aspiration, ce qui peut rapidement aspirer à une distance de fragments de tissus en bas en raison de la présence d&#39;un ADN collant. </li><li> Ajouter 1 RPMI mL 1640 complète. Appuyez doucement le tube pour remettre en suspension le lysat fragmenté. Inversez haut et en bas 10 fois. Répétez l&#39;étape 4.1. </li><li> Répétez l&#39;étape 4.2 plus de deux fois ou jusqu&#39;à ce que le surnageant apparaît clairement. Re-suspendre des fragments pancréatiques lavés dans 1 ml complets RPMI 1640 médias. </li></ol> 5. Islet Isolation NOTE: Pour un petit nombre de pancreata, comme ci-dessous (5.1) directe ramasser la main peut être utilisé pour l&#39;îlot isolation. Un grand nombre de pancreata (&gt; 6), la méthode décrite au point 5.2 est préféré. <ol><li> Direct cueillette à la main: <ol><li> Transfert lysat de l&#39;étape 4.3 dans un plat de 60 mm. Ajouter 5 ml complets RPMI 1640. Mettre sous un microscope à dissection. </li><li> Mettre en place l&#39;éclairage lumineux sur le terrain sur le microscope. Réglez l&#39;intensité lumineuse à ~ 50% de la puissance maximale. Sous cette condition, les îlots apparaissent comme légèrement grappes roses, tandis que les cellules acineuses grappes de forme irrégulière sombres (figure 1A). Un grossissement de ~ 50X (combinant la puissance de l&#39;objectif et l&#39;oculaire) est recommandé d&#39;offrir la meilleure façon d&#39;îlots de visualisation et l&#39;ouverture de la pointe de la pipette en même temps pour la cueillette. </li><li> Pick-up îlots avec une pointe de pipette P20 tout en évitant les clusters acineuses. Fractions Distribuer îlots-enrichi à un nouveau plat de 60 mm avec RPMI milieu complet (figure 1B). </li><li> Répétez le processus cueillette à la main deux fois de plus. Laissez îlots purs dans un nouveau 60plat mm avec 4 ml du milieu RPMI complet (Figure 1C). </li></ol></li><li> centrifugation Gradient: <ol><li> Préparer un tube de 15 ml de centrifugeuse qui est préchargé avec 2 ml polysucrose et de sodium diatrizoate à une densité de 1,077 g / ml. Laisser le tube sur la glace jusqu&#39;à utilisation. </li><li> Transférer la suspension de l&#39;étape 4.3 sur la partie supérieure de la solution de diatrizoate de sodium, le polysucrose et avec une pipette Pasteur. Ne pas déranger la couche inférieure. Tissues de jusqu&#39;à 10 nouveau-né ou 2 P17 pancreata peuvent être chargés dans chaque tube de 15 ml. </li><li> Spin le tube à ~ 600 xg dans un rotor oscillant pendant 15 min à 4 ° C. Utiliser les paramètres de frein-off. </li><li> Après centrifugation, transférer les médias et les couches de interphase (noter que dans des conditions de fonctionnement idéales, les îlots seront situés dans l&#39;interphase entre le polysucrose et la culture média après centrifugation) dans un nouveau tube de 15 ml. Ajouter 10 ml de milieu, mélanger doucement mais complètement. </li><li> Isoler la fraction d&#39;îlots enrichià 300 xg pendant 5 min. Retirez le support de lavage. Répétez le lavage une fois de plus avec les médias RPMI complet. Pellets Resuspendre dans 4 ml de milieu, qui contient la plupart des îlots et des conduits (figure 1D). </li><li> Hand-cueillir les îlots comme indiqué à la section 5.1. </li></ol></li></ol> 6. GSIS Assays en îlots isolés <ol><li> Incuber les îlots provenant de l&#39;étape 5.1.4 dans un milieu RPMI complet pendant 2 h, dans une culture tissulaire en C incubateur à 37 °. </li><li> Utiliser une pipette pour enlever RPMI sous un microscope de dissection. Ajouter 3 solution KRB mL avec 2,8 mM de glucose à la plaque 3. Agiter 10 fois pour laver les îlots. Retirer solutions KRB. <ol><li> Répétez le processus de lavage une fois. Incuber les îlots avec 3 solution KRB mL à 37 ° C pendant 1 h dans un incubateur. </li></ol></li><li> Aliquoter 1 KRB ml dans chaque puits d&#39;une plaque de 12 puits. Préchauffer le stock KRB et la plaque à 37 ° C dans un incubateur. </li><li> Laver les îlots deux fois avec préchauffée KRB comme dans 6.2. Transfer 10 îlots à chaque puits des plaques préchauffées. Incuber dans un incubateur à 37 °. </li><li> Après 45 minutes, retirer 50 ul de surnageant. Celui-ci contient la solution d&#39;insuline sécrétée sous le glucose basale. </li><li> Ajouter 32 pl 500 mM de glucose. Agiter la plaque 20 fois pour mélanger la solution. Incuber dans un incubateur à 37 °. Agiter la plaque 5 fois, une fois toutes les 5 min. </li><li> Après 45 min, retirer 50 ul surnageant. Ceci est l&#39;insuline sécrétée sous haute glucose. </li><li> Transférez tous les îlots à un tube de 1,5 ml. Congeler à -20 ° C pendant 30 min. Décongeler à la température ambiante pendant 5 min. Répétez le processus de congélation et de décongélation. </li><li> Ajouter 0,5 ml d&#39;éthanol à 70% avec du HCl 20 mM. Laisser reposer jusqu&#39;au lendemain. Ceci est la insuline dans des îlots. </li><li> Utiliser ELISA classique pour tester les niveaux d&#39;insuline, décrit précédemment 3. </li></ol>

<ol>
	<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.

Ici, nous fournissons un protocole étape par étape sur l&#39;isolement des îlots de pancréas qui sont trop petites pour perfusion conventionnelle. Il est prévu pour produire des îlots préparés pour toutes les études en fonction des îlots tels que la purification des cellules bêta, l&#39; analyse de l&#39; expression génique, des îlots de maturation des cellules bêta, la prolifération, la réponse au stress cellulaire, la survie cellulaire, le métabolisme et le maintien fonctionnel GSIS, etc. Ce ...

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

protocoles îlot-isolement à base Perfusion-de grande pancreata mammifères sont bien établis. De tels protocoles sont facilement réalisées dans de nombreux laboratoires en raison de la grande taille du canal pancréatique qui permet une injection de collagénase prête et la perfusion tissulaire subséquente. En revanche, l&#39;isolement des îlots de la petite pancreata, comme celui des souris néonatales, est difficile parce que la perfusion ne sont pas facilement réalisables dans la petite pancreata. Nous décrivons ici une simple procédure détaillée qui récupère un nombre important d&#39;îlots de souris nouveau-nés avec une aide visuelle. Des pancréas entier fraîchement disséqués ont été digérés avec 0,5 mg / mL de collagénase IV dissous dans une solution saline équilibrée de Hanks (HBSS) à 37 ° C dans des tubes de microcentrifugation. Les tubes ont été exploitées régulièrement pour faciliter la dispersion des tissus. Quand la majeure partie du tissu a été dispersé de petits amas d&#39;environ 1 mm, les lysats ont été lavées trois à quatre fois avec un milieu de culture avec 10% de sérum de fœtus bovin (FBS). grappes Islet,dépourvue de tissus acineuses reconnaissables, peut ensuite être récupéré sous la dissection stéréoscope. Cette méthode récupère 20 - 80 petites et grandes îlots par le pancréas de souris nouveau-né. Ces îlots sont appropriés pour des dosages plus en aval concevables, y compris la sécrétion d&#39;insuline, l&#39;expression génique et la culture. Un exemple de dosage de sécrétion d&#39;insuline est présenté pour valider le processus d&#39;isolement. Le fond génétique et le degré de digestion sont les plus grands facteurs qui déterminent le rendement. Fraîchement solution de collagénase avec une activité élevée est préférable, car elle contribue à l&#39;isolement du système endocrinien exocrine. La présence de cations [calcium (Ca 2+) et le magnésium (Mg 2+)] dans toutes les solutions et de sérum bovin fœtal dans le lavage / media cueillette sont nécessaires pour un bon rendement d&#39;îlots avec intégrité adéquate. Un champ de dissection avec un bon contraste et le grossissement aidera également.

Dans des conditions optimales, la méthode présentée peut produire 20 - 80 îlots de pancréas chaque petite souris. Ce nombre dépend de l&#39;arrière-plan génétique, l&#39;âge des souris et la taille des îlots à récupérer. Parmi les couramment utilisés, CD1 out-élevé et / 6J de race pure C57BL produisent moins d&#39;îlots dont la taille est plus petite que les hybrides entre CD1 et C57BL / 6 ou B6CBAF1 commercial / souris J font. Main directe cueillette généralement don...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Nous décrivons ici un protocole pour isoler les îlots intacts de souris néonatales. Pancreata ont été partiellement digéré avec de la collagénase, suivi d&#39;un lavage et cueillette à la main. 20 - 80 amas d&#39;îlots peuvent être obtenus par le pancréas de souris nouveau-nés, qui sont appropriés pour de nombreuses études d&#39;îlots.

Isolation efficace des îlots fonctionnels de néonatales Pancréas souris

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

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Research

JoVE Journal

Developmental Biology

10.0K vues.  Vanderbilt University Medical School.  Nous décrivons ici un protocole pour isoler les îlots intacts de souris néonatales. Pancreata ont été partiellement digéré avec de la collagénase, suivi d'un lavage et cueillette à la main. 20 - 80 amas d'îlots peuvent être obtenus par le pancréas de souris nouveau-nés, qui sont appropriés pour de nombreuses études d'îlots. 

Vidéo: 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.

Une méthode pour isoler l&#39;îlot murin et transplantation rénale sous-capsulaires

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

Recherche

Médecine

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.

L&#39;isolement des îlots humains à partir des patients partiellement pancréatectomisés

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.

Transplantation soutenue d&#39;échafaudage d&#39;îlots dans la pate épididimale de souris diabétiques

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

Bio-ingénierie

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; Modèle de souris de diabète de type 2: pancréatique isolement des îlots et des cellules vivantes 2-Photon Imaging d&#39;îlots intacts

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.

Un îlots pancréatiques Murine Cell-based Screening for diabétogène 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 ...

Immunologie et infection

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.

Un haut contenu<em&gt; In vitro</em&gt; Pancreatic Islet β-cellulaire Plateforme de découverte de la réplication

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.

Souris îlot de Langerhans isolement à l&#39;aide d&#39;une combinaison de la collagénase purifiée et protéase neutre

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 ...

Biologie

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.

Intraporte Transplantation d’îlots pancréatiques dans le modèle murin

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.

Une méthode pour la souris Isolement îlots pancréatiques et AMPc intracellulaire Détermination

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.

Isolation efficace des îlots fonctionnels de néonatales Pancréas souris

Résumé

Explorer plus de vidéos

Chapitres dans cette vidéo

Une méthode pour isoler l'îlot murin et transplantation rénale sous-capsulaires

L'isolement des îlots humains à partir des patients partiellement pancréatectomisés

Transplantation soutenue d'échafaudage d'îlots dans la pate épididimale de souris diabétiques

Lepr

Un îlots pancréatiques Murine Cell-based Screening for diabétogène Environmental Chemicals

Un haut contenu

Souris îlot de Langerhans isolement à l'aide d'une combinaison de la collagénase purifiée et protéase neutre

Intraporte Transplantation d’îlots pancréatiques dans le modèle murin

Une méthode pour la souris Isolement îlots pancréatiques et AMPc intracellulaire Détermination

Un protocole simple à haut rendement pour l'isolement des îlettes pancréatiques des souris