עבודה זו נתמכת על ידי RO1 DK064938 (עד TH).

<ol>
	<li>Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+as+a+treatment+for+diabetes.">Islet transplantation as a treatment for diabetes.</a> J Am Optom Assoc. 69, 727-732 (1998).</li><li>Gangemi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+for+brittle+type+1+diabetes:+the+UIC+protocol.">Islet transplantation for brittle type 1 diabetes: the UIC protocol.</a> Am J Transplant. 8, 10-1111 (2008).</li><li>Zmuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+ATF3,+an+adaptive-response+gene,+protects+islets+and+ameliorates+inflammation+in+a+syngenic+transplantation+model.">Deficiency of ATF3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngenic transplantation model.</a> Diabetologia. , (2010).</li><li>Bottino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+Islets+to+Isolation+Stress+and+the+Effect+of+Antioxidant+Treatment.">Response of Human Islets to Isolation Stress and the Effect of Antioxidant Treatment.</a> Diabetes. 53, 2559-2568 (2004).</li><li>Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+islet+cell+culture+in+vitro+at+24+degrees+C+on+graft+survival+and+MHC+antigen+expression.">The effect of islet cell culture in vitro at 24 degrees C on graft survival and MHC antigen expression.</a> Transplantation. 49, 272-277 (1990).</li><li>Terasaka, R., Lacy, P. E., Hauptfeld, V., Bucy, R. P., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cyclosporin-A,+low-temperature+culture,+and+anti-Ia+antibodies+on+prevention+of+rejection+of+rat+islet+allografts.">The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.</a> Diabetes. 35, 83-88 (1986).</li><li>Ricordi, C., Lacy, P. E., Sterbenz, K., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+culture+of+human+islets+or+in+vivo+treatment+with+L3T4+antibody+produces+a+marked+prolongation+of+islet+human-to-mouse+xenograft+survival.">Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.</a> Proc Natl Acad Sci U S A. 84, 8080-8084 (1987).</li><li>Schnedl, W. J., Ferber, S., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STZ+transport+and+cytotoxicity.+Specific+enhancement+in+GLUT2-expressing+cells.">STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.</a> Diabetes. 43, 1326-1333 (1994).</li><li>Eckhard, M., Brandhorst, D., Brandhorst, H., Brendel, M. D., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+in+osmolality+and+range+of+density+of+a+continuous+ficoll-sodium-diatrizoate+gradient+for+isopycnic+purification+of+isolated+human+islets.">Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.</a> Transplantation Proceedings. 36, 2849-2854 (2004).</li><li>Gores, P. F., Rabe, F., Sutherland, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+survival+of+intraportal+versus+subrenal+capsular+transplanted+islet+allografts.">Prolonged survival of intraportal versus subrenal capsular transplanted islet allografts.</a> Transplantation. 43, 747-749 (1987).</li><li>Nasr, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+Immune+Privilege+Promotes+Transplantation+Tolerance+by+Altering+the+Balance+between+Memory+and+Regulatory+T+Cells.">Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells.</a> J Immunol. 174, 6161-6168 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95, 1449-1461 (2008).</li><li>Inoue, M., Maeno, T., Hatchell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+allografted+pancreatic+islets+in+the+subretinal+space+in+rats.">Survival of allografted pancreatic islets in the subretinal space in rats.</a> Ophthalmic Res. 35, 48-53 (2003).</li></ol>

אין ניגודי אינטרסים הכריז.

בסעיפים לעיל, תיארנו צעדים מפורטת בידוד השתלת הלבלב איים העכבר. כאן, אנו בקצרה להדגיש את הצעדים הם קריטיים להצלחת ההליכים. 1. איילט בידוד השלבים העיקריים הם מזהים זמן אופטימלי עיכול אנזימטי (1.4), מיצוב מהדק hemostat על פתיחת התריסריון של צינור המרה המשותף (2.2.3), cannulating את צינור המרה המשותף (2.3), רועד נמרץ של הלבלב לאחר 37 ° C לעכל (3.2), והסרת זיהום תא אקסוקרינית (3.3.7 ו - 3.3.16). צעדים אלה יש השפעה דרמטית על התשואה איכות, טוהר איים. אולי הצעד הכי מאתגר מבחינה טכנית הוא cannulation של צינור המרה המשותף. בעכברים רבים, את צינור המרה המשותף יכול להיות קשה לדמיין בשל fascia שמסביב. לכן, מקובל כי ניסיונות cannulate צינור המרה המשותף לגרום לחדירה של הרקמה fascia בלבד. במקרים אלה, Liberase מתחיל למלא את רקמת החיבור סביב ואינו perfuse את הלבלב. אם זאת הוא ציין, לעצור את זרימת Liberase למקם את המחט כך שהוא חודר לתוך לומן של צינור המרה. בעזרת תרגול, אפשר לזהות במהירות את המיקום האופטימלי עבור מיקום המחט כדי להשלים את cannulation מבלי לפגוע בצינור פריך. 2. השתלת איילט השלבים הקריטיים הגיוס STZ של סוכרת (5.1) לבין אספקה ​​יעילה של איים על הכיס subcapsular (5.3.14-5.3.16). STZ הוא אנלוגי גלוקוז באופן טבעי כי הוא רעיל באופן סלקטיבי את הפרשת האינסולין בתאי β-תאים של איי 9. עם זאת, רעילות סלקטיבית מתרחשת בטווח ריכוז צר יחסית. עדות לכך היא העובדה כי מינון גבוה של STZ יכול לגרום הקטלניות (2-3 ימים) מהיר בהעדר היפרגליקמיה. לכן, יש להקפיד לזהות את המינון האופטימלי של STZ עבור זן של עכברים וגיל בשימוש לפני כל ניסויים בקנה מידה גדול הם יזמו. המסירה הפיזית של איים על הכיס subcapsular גם צעד מפתח. כדי להשיג תוצאות עקביות, הכרחי כדי לצמצם את אובדן איים במהלך הלידה שלהם. זה יכול להתרחש אם יש פגמים או דמעות הקפסולה כליה מכסה את כיס או אם נפח מוזרק גדול מדי. נזק הקפסולה יכול להימנע על ידי מניפולציה עדינה וזהירה, ועל ידי שימוש בדיקה זכוכית חלקה להכין את הנרתיק subcapsular. בנוסף, נפח הזרקה ניתן למזער בהסרה זהירה של supernatant בשלב 5.3.6.3. אם supernatant יוסר עד לרמה של איים משקעים, יש בדרך כלל יותר מאשר מספיק מקום בכיס subcapsular למקום 400-500 איים. עם זאת, אם נפח הזרקה גדול מדי, איים נוטים לדלוף החוצה של כיס במהלך תהליך ההשתלה, להתפשר על שחזור של הניסוי. במסגרת פרוטוקול זה, אפשר לשנות כמה צעדים, ועדיין להשיג תוצאות משביעות רצון. אלה כוללים את הבאים: (א) השימוש אנזים שונה לעכל את הלבלב, (ב) שיטה אחרת לאספקת האנזים על הלבלב, (ג) שימוש מתמשך Ficoll-נתרן diatrizoate (FSD) שיפוע במקום צעד אחד צפיפות Histopaque1077, (ד) השתלת איון למקומות אחרים מאשר subcapsule את הכליה. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בפרוטוקול זה, Liberase משמש כדי להחליש את הקשר תאים תאים בתוך הלבלב. Liberase היא למעשה תערובת של מטוהרים מאוד collagenases כי הוכיחו שימושי בשחרור איים מהלבלב. עם זאת, collagenases מטוהרים פחות מסוגלים להשיג תוצאה דומה. לכן, ניתן להשתמש אחר ההכנות אנזים זמינים מסחרית עבור לעכל את הלבלב, כל עוד התנאי לעיכול מותאם להשיג איון איכות גבוהה, תשואה וטוהר. </li><li style=";text-align:right;direction:rtl"> כמו כן ניתן לעכל את הלבלב ללא זלוף ductal של האנזים. מסירת Liberase דרך בצינור המרה המשותף מאפשר חשיפה מרבית של פני השטח כדי הלבלב האנזים. התוצאה היא מערכת העיכול עוד יותר של הלבלב ולשחרר יותר של איים שלמים. עם זאת, צעדים נוספים כדי להגדיל את שטח הפנים של הלבלב כדי האנזים יכול לשמש. לדוגמה, הלבלב מיותרות לפני דוגרים אותו Liberase או ישירות הזרקת Liberase באמצעות הקפסולה הלבלב ניתן להשתמש כדי לבודד איים. עם זאת, אין שיטה אחרת של משלוח Liberase יעיל כמו זלוף דרך בצינור המרה המשותף. לכן, הזרקה ישירה מיותרות או צריכים להישמר כמוצא אחרון, אם את צינור המרה המשותף פגומה ולא perfusable. </li><li style=";text-align:right;direction:rtl"> שיטה חלופית עבור הפרדת איים מתאי אקסוקרינית כרוךהשימוש שיפוע FSD במקום Histopaque1077 10. הפרדה יעילה של איים מתאי אקסוקרינית בפרוטוקול זה בין השאר בשל צפיפות ההפרש בין שני סוגי התאים. Histopaque1077 יש צפיפות של 1.0771 גרם / מ&quot;ל ​​ב 25 ° C. בטמפרטורה זו, Histopaque הוא צפוף יותר מאשר איים אבל פחות צפוף מאשר תאים אקסוקרינית. לכן, תחת כוח של צנטריפוגה, Histopaque1077 כדורי התאים אקסוקרינית תוך הרמת איים על פני השטח. בעיקרון, כל חומר אחר עם צפיפות כי ניתן להפריד איים מתאי אקסוקרינית ניתן להשתמש בשלב זה ועל שיפוע FSD היא אחת חומר כזה. </li><li style=";text-align:right;direction:rtl"> עם כל הכבוד למיקום עבור השתלת איונים, הקפסולה כליות להשתמש בפרוטוקול זה הוא אחד האתרים אפשרי מעטים. בעוד subcapsule כליה הוא האתר הנפוצות ביותר שדווחו של השתלה בעכברים, אתרי השתלת אחרים נמצאו כיעילים והם כוללים הפורטל הכבד וריד, מרחב subretinal, testis, כרית שומן epididymal, הטחול, הלבלב אפילו 11-14. בכל המקומות הללו יש יתרונות משלהם ואתגרים. לכן, הבחירה של אתר עבור איון השתלה צריך להיקבע על ידי שאלות פנה ואת הנסיבות הספציפיות של הניסויים. </li></ol> הוא האמין כי אסטרטגיות שיכולות להתגבר על המגבלות על איון השתלה באופן דרמטי לשפר את הפוטנציאל הטיפולי שלה. לכן, השימוש של מודל Murine כמפורט על ידי פרוטוקול זה מציע גישה אטרקטיבי עבור זיהוי אסטרטגיות כאלה.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI 1640</td>
<td>Reagent</td>
<td>Gibco</td>
<td>31800-022</td>
<td>&nbsp;</td>
<tr>
<td>Liberase TL</td>
<td>Reagent</td>
<td>Roche</td>
<td>05401020001</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Histopaque1077</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>10771</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Citrate</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S4641</td>
<td>&nbsp;</td>
<tr>
<td>Streptozotocin</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>S-0130</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>A144-212</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>Reagent</td>
<td>Vedco</td>
<td>ISOSOL</td>
<td>&nbsp;</td>
<tr>
<td>Glass Capillary Tubes</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22362574</td>
<td>&nbsp;</td>
<tr>
<td>Insulin Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>329461</td>
<td>&nbsp;</td>
<tr>
<td>27 Gauge Syringe Needle</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton Syringe #1702</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14813133</td>
<td>&nbsp;</td>
<tr>
<td>10cm Petri Plates</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>0875712</td>
<td>&nbsp;</td>
<tr>
<td>6cm Petri Plates</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>07770212</td>
<td>&nbsp;</td>
<tr>
<td>6-0 Suture</td>
<td>Equipment</td>
<td>Ethicon</td>
<td>8726H</td>
<td>&nbsp;</td>
<tr>
<td>PE 50 Flexible Tubing</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>NC9083963</td>
<td>&nbsp;</td>
<tr>
<td>5ml Disposable Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>309603</td>
<td>&nbsp;</td>
<tr>
<td>50ml Conical Tube</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15ml Conical Tube</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>352097</td>
<td>&nbsp;</td>
<tr>
<td>0.1mm Nylon Mesh</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22363549</td>
<td>&nbsp;</td>
<tr>
<td>0.419mm Wire Mesh</td>
<td>Equipment</td>
<td>Newark Wire Cloth Company</td>
<td>0300241</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath Incubator</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Equipment</td>
<td>Zeiss</td>
<td>Stemi SV6</td>
<td>&nbsp;</td>
<tr>
<td>Inverted Microscope</td>
<td>Equipment</td>
<td>Nikon</td>
<td>TMS</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Incubator</td>
<td>Equipment</td>
<td>Napco</td>
<td>6300</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Scissors</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS14393</td>
<td>&nbsp;</td>
<tr>
<td>McPherson-Vannas Scissors</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS500260</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS15915</td>
<td>&nbsp;</td>
<tr>
<td>Hemostatic Forceps</td>
<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS 500451</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane Nebulizer</td>
<td>Equipment</td>
<td>Smiths Medical Surgivet</td>
<td>ISOTech 4 OHMEDA</td>
<td>&nbsp;</td>
<tr>
<td>Swing Bucket Centrifuge</td>
<td>Equipment</td>
<td>Dupont &#x2013; Sorvall</td>
<td>RT6000D</td>
<td>&nbsp;</td>
<tr>
<td>Swing Bucket Rotor</td>
<td>Equipment</td>
<td>Dupont &#x2013; Sorvall</td>
<td>H1000B</td>
<td>&nbsp;</td>
<tr>
<td>pH Meter</td>
<td>Equipment</td>
<td>Mettler Toledo/ Fisher Scientific</td>
<td>09313509</td>
<td>&nbsp;</td>
<tr>
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a method for murine islet isolation and subcapsular kidney transplantation

מאז עבודתו החלוצית מוקדם של Ballinger ו Reckard הוכחת כי השתלת איי לנגרהנס לתוך מכרסמים סוכרת יכול לנרמל את רמת הגלוקוז בדם, איון השתלה הוצע להיות טיפול פוטנציאלי עבור 1,2 סוכרת מסוג 1. לאחרונה, ההתקדמות האנושית איון השתלה חיזקו עוד יותר את הדעה הזאת 1,3. עם זאת, שתי המגבלות העיקריות למנוע איון השתלה מלהיות מציאות קליני נרחב: (א) את הדרישה של מספר גדול של איונים לכל מטופל, אשר קשה מפחית את מספר מקבלי פוטנציאליים, (ב) לצורך דיכוי חיסוני כבד, אשר משפיע באופן משמעותי אוכלוסיית הילדים המטופלים בשל פגיעותם החיסון לטווח ארוך. אסטרטגיות שיכולות להתגבר על המגבלות האלה יש פוטנציאל לשפר את התועלת הטיפולית של איון השתלה. איילט השתלת כליה תחת הקפסולה עכבר הוא מודל מקובל לחקור אסטרטגיות שונות כדי לשפר את איון השתלה. ניסוי זה מצריך את בידודו של איים באיכות גבוהה השתלת איים לנמענים סוכרתית. נהלים שניהם דורשים צעדים כירורגית כי ניתן להדגים טוב יותר מאשר באמצעות טקסט על ידי וידאו. הנה, אנחנו המסמך צעדים מפורטים נהלים אלה על ידי וידאו והן בפרוטוקול כתוב. אנחנו גם בקצרה על השתלת מודלים שונים: syngeneic, allogeneic, אוטואימוניות syngeneic ו allogeneic אוטואימוניות.

Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

השתלה של איים מבודדים הוצע להיות טיפול פוטנציאלי עבור סוכרת מסוג 1. כאן אנו מתארים שיטה לבודד איים מהעכבר pancreata ואת השתלת אותם למרחב subcapsular של הכליה.

שיטה בידוד איילט Murine והשתלות כליה Subcapsular

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

a-method-for-murine-islet-isolation-subcapsular-kidney

Research

JoVE Journal

Medicine

62.9K Views.  The Ohio State University. השתלה של איים מבודדים הוצע להיות טיפול פוטנציאלי עבור סוכרת מסוג 1. כאן אנו מתארים שיטה לבודד איים מהעכבר pancreata ואת השתלת אותם למרחב subcapsular של הכליה.

Video: A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

פיתוח Bronchiolitis סותם במודל Murine של השתלת ריאות Orthotopic

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

טכניקת השתלות Murine Heterotopic לב

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

השתלת כליה עכבר: מודלים של דחיית השתל

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

טכניקת השתלת כליה בעכברים

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normothermic<em&gt; Ex Vivo</em&gt; זלוף כליות לשימור הכליה שתלים לפני השתלה

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

הרומן כיתה קלינית שיטת בידוד תאי סטרומה Perivascular כליה אנושית

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

אורתובנושא חולדה השתלת כליה: מגישת רומן וכירורגית פשוטה

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

השתלת כליה אורתוטופית במודל פורצין באמצעות 24 שעות שימור איברים וטלמטריה רציפה

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

השתלת איון מכוסה שומן באמצעות רקמת שומן לבן אפידידימלית

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

השתלת כליה בסיוע רובוט

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...