Name: scaffold-supported transplantation of islets in the epididymal fat pad of diabetic mice
Uploaded: 2017-07-23T13:00:00.000+00:00
Duration: 11 min 57 s
Description: Watch this Scientific Journal Video about Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice at JoVE.com

המחברים מבקשים להודות ויי ג &#39;אנג מ Guanhao ביוטק למתן את הפיגומים decellularized. אנו מודים ל- Xiao-hong Peng על השיחות המועילות. מחקר זה נתמך מבחינה כספית על ידי הקרן הלאומית למדעי הטבע של סין (פרויקט מס &#39;3212021).

<ol>
	<li>Shapiro, A. 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=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>Shapiro, A. M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Trial+of+the+Edmonton+Protocol+for+Islet+Transplantation.">International Trial of the Edmonton Protocol for Islet Transplantation.</a> N Engl J Med. 355, 1318-1330 (2006).</li><li>Ryan, E. 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=Five-year+follow-up+after+clinical+islet+transplantation.">Five-year follow-up after clinical islet transplantation.</a> Diabetes. 54, 2060-2069 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., 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=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> Br J Surg. 95, 1449-1461 (2008).</li><li>Schmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islets+find+a+new+transplant+home+in+the+omentum.">Pancreatic islets find a new transplant home in the omentum.</a> Nat Biotechnol. 35 (1), (2017).</li><li>Dufour, 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=Development+of+an+ectopic+site+for+islet+transplantation,+using+biodegradable+scaffolds.">Development of an ectopic site for islet transplantation, using biodegradable scaffolds.</a> Tissue Eng. 11, 1323-1331 (2005).</li><li>Weaver, J. 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=Controlled+Release+of+Dexamethasone+from+Organosilicone+Constructs+for+Local+Modulation+of+Inflammation+in+Islet+Transplantation.">Controlled Release of Dexamethasone from Organosilicone Constructs for Local Modulation of Inflammation in Islet Transplantation.</a> Tissue Eng Part A. 21, 2250-2261 (2015).</li><li>Wang, K., 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=From+Micro+to+Macro:+The+Hierarchical+Design+in+a+Micropatterned+Scaffold+for+Cell+Assembling+and+Transplantation.">From Micro to Macro: The Hierarchical Design in a Micropatterned Scaffold for Cell Assembling and Transplantation.</a> Adv Mater. 29, (2017).</li><li>Blomeier, H., 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=Polymer+Scaffolds+as+Synthetic+Microenvironments+for+Extrahepatic+Islet+Transplantation.">Polymer Scaffolds as Synthetic Microenvironments for Extrahepatic Islet Transplantation.</a> Transplantation. 82, 452-459 (2006).</li><li>Gibly, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrahepatic+islet+transplantation+with+microporous+polymer+scaffolds+in+syngeneic+mouse+and+allogeneic+porcine+models.">Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models.</a> Biomaterials. 32, 9677-9684 (2011).</li><li>Moskalewski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+the+Islets+of+Langerhans+of+the+Guinea+Pig.">Isolation and Culture of the Islets of Langerhans of the Guinea Pig.</a> Gen Comp Endocrinol. 5, 342-353 (1965).</li><li>Lacy, P. E., Kostianovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+isolation+of+intact+islets+of+Langerhans+from+the+rat+pancreas.">Method for the isolation of intact islets of Langerhans from the rat pancreas.</a> Diabetes. 16, 35-39 (1967).</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>Li, D. S., Yuan, Y. H., Tu, H. J., Liang, Q. L., Dai, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+islet+isolation+from+mouse+pancreas.">A protocol for islet isolation from mouse pancreas.</a> Nat Protoc. 4, 1649-1652 (2009).</li><li>Stull, N. D., Breite, A., McCarthy, R., Tersey, S. A., Mirmira, 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=Mouse+Islet+of+Langerhans+Isolation+using+a+Combination+of+Purified+Collagenase+and+Neutral+Protease.">Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease.</a> J Vis Exp. (67), (2012).</li><li>Sakata, N., Yoshimatsu, G., Tsuchiya, H., Egawa, S., Unno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+diabetes+mellitus+for+islet+transplantation.">Animal models of diabetes mellitus for islet transplantation.</a> Exp Diabetes Res. , 256707 (2012).</li><li>Schmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islets+find+a+new+transplant+home+in+the+omentum.">Pancreatic islets find a new transplant home in the omentum.</a> Nat Biotech. 35, 8-8 (2017).</li><li>Londono, R., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+scaffolds+for+regenerative+medicine:+mechanisms+of+in+vivo+remodeling.">Biologic scaffolds for regenerative medicine: mechanisms of in vivo remodeling.</a> Ann Biomed Eng. 43, 577-592 (2015).</li></ol>

למחברים אין מה לחשוף.

לבלב הלבלב זמן העיכול הם שני פרמטרים מרכזיים המשפיעים על התשואה איון ואיכות. Moskalewski הראשון דיווח על שימוש בתערובת collagenase גולמי לעכל טחון הלבלב גינס 11 . Lacy et al. דיווחו על הזרקה של אנזימים למערכת צינורית כדי לבלבל את הלבלב, אשר הגדילו מאוד את התשואה איון...

סוג 1 סוכרת (T1D) היא הפרעה אנדוקרינית אוטואימונית שבה תאי איון הם ablated על ידי המערכת החיסונית, וטיפול בחולים תלוי הזרקת אינסולין אקסוגני לכל חייהם. פרוטוקול אדמונטון מהווה ציון דרך במחקרים קליניים על השתלת איון; איים היו infused דרך הווריד הפורטל והושתלו באתר intrahepatic 1 . עם זאת, שני מכשולים עיקריים - מקורות לא מספיקים של איים התורם ואת engletment איון המסכן, למנוע את ההצלחה הגדולה של השתלת איון 2 . בדרך כלל, איים צריכים להיות שנאספו משלושה תורמים cadaveric כדי להפוך את מצב hyperglycemic של חולה אחד; זאת בשל התשואה הנמוכה של הליכי בידוד איון ואת איון הפסד לאחר ההשתלה. בפרט, למרות שאיים שלאחר ההשתלה היו שטופים דם עשיר בחמצן, המגע הישיר עם הדם עורר לעיתים קרובות את הדלקת המייצרת דם מיידיתתגובה של טורי (IBMIR), אשר עלולה לגרום לאובדן חריף של האיים. בטווח הארוך, נראה כי ההפסד ההדרגתי של איים בחולים היווה ירידה של שיעורי היפוך הסוכרת בקבוצות הקליניות, אשר יכול להגיע ל 90% בשנה הראשונה ו ירד ל 30% ו 10% על ידי 2 ו 5 שנים לאחר ההשתלה, בהתאמה 3 . השתלת Islet באתרים extrahepatic כבר אסטרטגיה אטרקטיבית כדי להפחית את הקשר הישיר של איים עם דם תוך הגבלת השתלות למיקומים מוגדרים יותר לעומת עירוי intrahepatic. מחקרים בוצעו בקפסולת הכליה, העין, השרירים, רפידות השומן וחללים תת-עוריים במהלך השנים האחרונות, מה שמראה כי איים באתרים אלה מסוגלים לשרוד ולתפקד כדי לשחזר את הנורמוגליזציה 4 . בנוסף, איים באתרים אלה הם לשחזור, מה שמאפשר ביופסיה או אפילו להליכי החלפת נוספים. אקסטרהפטיתזה ולכן להפגין פוטנציאל גדול להשתלה קלינית 5 . Biomaterials מבוססי פיגומים נבדקו באופן אינטנסיבי עבור השתלת תאים והנדסת רקמות. תלת מימדי (3D) פיגומים בדרך כלל מכילים מבנים נקבובי יכול לשמש תבניות הסלולר כדי ליצור מבנה מרחבי / ארגון של תאים או מאגרים לספק שחרור מבוקר של רמזים ביו. פיגומים יש גם מפוברק מחומרים פולימריים, כגון פולי (glycolide-L- לקטט) 6 , פולי (dimethylsiloxane) 7 , ו תרמופלסטיים פולי (urethane) 8 , כדי להשתלות איים EFP. בהשוואה להשתלה ישירה של איים, השימוש פיגומים נמצא להפחית את איים הפסד על ידי מניעת דליפה של איים לתוך חלל intraperitoneal 9 , 10 , מתן הגנה מכנית moduמניעת תגובה דלקתית מקומית. פיגומים ובכך ניתן יהיה לפתח לקדם engraftment איון באתרים להשתלה 7. במחקר זה, אנו מתכוונים להפגין פרדיגמה של השתלת איון ב EFP, מתבצעת מודלים עכברים באמצעות DCS. פיגומים נגזר מטריצות תאיים משכו עניין רב בשנים האחרונות בשל biocompatibility מעולה מבנים נקבוביים טבעיים יותר לעומת מוצרים סינתטיים. כאן, אנו מתארים פרוטוקול בידוד חזק כדי להשיג איים הלבלב בתשואות גבוהות של עכברים C57BL / 6J. DCSs מעובד מן קרום הלב קרום זרעו אז עם איים, ואת השתלות הושתלו EFP במודלים סוכרתית סינרגנית. נורמוגליקמיה בעכברים הושגה בתוך 10 ימים ונשמרה עד 100 ימים, עד להסרת השתלות.

<table><tbody><tr><td>Dissecting scissor</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Forceps</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>0.5 mm diameter wire mesh</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Artery hemostatic clamp</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Microscopic hemostatic clamp</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Hemostatic forceps</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Absorbable 6-0 PGLA sutures&amp;nbsp;</td><td>JINHUAN</td><td></td><td>With needle</td></tr><tr><td>Wound clip</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Gauze</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>Sterile drapes</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>10mL syringe</td><td>JINGHUAN</td><td></td><td></td></tr><tr><td>1 mL syringe</td><td>JINGHUAN</td><td></td><td></td></tr><tr><td>27G intravenous needle</td><td>JINGHUAN</td><td>0.45x15 RWSB</td><td></td></tr><tr><td>1.5 mL Eppendorf tube</td><td>Axygen</td><td></td><td></td></tr><tr><td>15mL conical tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50mL conical tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>35mm Non-treated&amp;nbsp; Peri-dishes</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3422</td><td></td></tr><tr><td>0.22 &amp;mu;m filter</td><td>Pall</td><td>PN4612</td><td></td></tr><tr><td>10 mL serological pipet</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Pipet filler S1</td><td>Thermo Scientific</td><td>9501</td><td></td></tr><tr><td>Pipette (2-20&amp;mu;L)</td><td>Axygen</td><td>AP-20</td><td>AXYPET&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Gibco</td><td>C14175500CP</td><td></td></tr><tr><td>Collagenase P</td><td>Roche</td><td>COLLP-RO</td><td></td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11879-20</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>D-glucose</td><td>Gibco</td><td>A24940-01</td><td></td></tr><tr><td>Glucose meter</td><td>Roche</td><td></td><td>ACCU-CHEK</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Streptozotocin</td><td>Sigma</td><td>V900890</td><td>Vetec&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>Chloral hydrate</td><td>J&amp;amp;K</td><td>C0073</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>71497</td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C2404</td><td></td></tr><tr><td>Iodophors</td><td>Ningbo Medical</td><td></td><td></td></tr><tr><td>C57BL/6J, 10-12 weeks old</td><td>VitalRiver</td><td></td><td>Beijing, China</td></tr><tr><td>Decellularized scaffold</td><td>Guanhao Biotec</td><td>131102</td><td>Guangzhou, China</td></tr></tbody></table>

scaffold-supported transplantation of islets in the epididymal fat pad of diabetic mice

השתלת Islet הוכח קלינית להיות יעיל בטיפול סוכרת סוג 1. עם זאת, האסטרטגיה הנוכחית השתלת intrahepatic עלול לגרום לתגובות דם שלמים חריפים ולגרום engraftment איון עני. כאן, אנו מדווחים על פרוטוקול איתן להשתלת איים באתר ההשתלות האקסטרפטית - משטח השומן האפידידימלי (EFP) - במודל עכבר סוכרתי. פרוטוקול לבודד ולטהר איים בתפוקה גבוהה של עכברים C57BL / 6J מתואר, כמו גם שיטת השתלת מבוצע על ידי זריעת איים על גבי פיגום decellularized (DCS) ו השתלת אותם באתר EFP בעכברים C57BL / 6J סינתטי שניתנו סוכרת על ידי streptozotocin. השתל DCS המכיל 500 איים הפך את מצב היפרגליקמיה בתוך 10 ימים, בעוד איים חופשי ללא DCS נדרש לפחות 30 ימים. הנורמוגליקמיה נשמרה עד 3 חודשים עד שהשתלפה. לסיכום, DCS שיפרה את engraftment של איים לתוך tהוא אתר extrahepatic של EFP, אשר יכול בקלות להיות מאוחזר עשוי לספק פלטפורמה לשחזור ושימושי לחקר חומרים פיגומים, כמו גם פרמטרים השתלה אחרים הנדרשים engraftment איון מוצלח.

השיטה שלנו clamping, המבוצעת באמצעות מהדק הרדמה מיקרוסקופיים, הוא פשוט וחוסך זמן לעומת טכניקה קשירת תפר. זה לקח בערך 4 שעות כדי לבודד ולטהר כ 1,200 איים מ 6 עכברים. איים מבודדים טרי בדרך כלל היה פריפריה מחוספס תחת מיקרוסקופ אופטי ( איור 3 א ). ל?...

Watch this Scientific Journal Video about Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice at JoVE.com

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

פרוטוקול זה מדגים בידוד איים Murine וזרע על פיגום decellularized. איים נתמך פיגומים הושתלו לתוך כרית שומן האפידידימלי של streptozotocin (STZ), המושרה עכברים סוכרתית. איים שרדו באתר ההשתלות והפכו את מצב ההיפרגליקמיה.

פיגום נתמך השתלת איים בשומן השומן האפידידימלי של עכברים סוכרתית

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

scaffold-supported-transplantation-islets-epididymal-fat-pad-diabetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

10.0K Views.  Peking University.  פרוטוקול זה מדגים בידוד איים Murine וזרע על פיגום decellularized. איים נתמך פיגומים הושתלו לתוך כרית שומן האפידידימלי של streptozotocin (STZ), המושרה עכברים סוכרתית. איים שרדו באתר ההשתלות והפכו את מצב ההיפרגליקמיה. 

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

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.

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 ...

Medicine

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

השתלת איי הלבלב לקופסית הכליה בעכברים סוכרתיים של

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Biology

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

כימות של פונקציה הגלובלית הדיאסטולי על ידי ניתוח המבוסס על דוגמנות Kinematic של Transmitral זרימה באמצעות הפורמליזם מילוי Parametrized הדיאסטולי

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG (hOMING)

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.

Intra-Omental Islet Transplantation Using h-Omental Matrix Islet filliNG hOMING

Here, we present a protocol for in vivo validation of hydrogel-based cell therapy, illustrated by the example of islet transplantation. h-Omental Matrix Islet filliNG (hOMING) implantation allows implantation of a cell-hydrogel mixture between the omental layers, near to blood vessels, to maximize engraftment in a proper metabolic environment.

השתלת איון עם פגיעה אינטרה באמצעות h-עם פגיעה מטריקס איון מילוי (יונת)

Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the ...

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.

השתלת intraportal של לנגרהנס במודל עכבר

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

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

השתלת מודל שומן לבן תת-עורית (ISWAT) מדגם ההשתלה של איויין מורנה

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site 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 ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

מזעור דימום וריד פורטל לאחר עירוי במהלך השתלת איים תוך-הפטיים בעכברים

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

שיטת השתלת איון כדי Omentum העכבר

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Murine Model of Islet Transplantation: A Surgical Method to Infuse Pancreatic Islets in Portal Vein and Prevention of Postoperative Bleeding

Source: Gou, W. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62530">Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.</a> J. Vis. Exp. (2021)
In this video, we describe a procedure to infuse pancreatic islet cells into the&#160;hepatic portal vein, followed by application of gel foam to successfully prevent bleeding. Postoperative bleeding is a common cause of high mortality during islet transplantation.

מודל מורין להשתלת איילט: שיטה כירורגית להחדיר איי לבלב בווריד הפורטל ומניעת דימום לאחר הניתוח

Source: Gou, W. et al. Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice. J. Vis. Exp. (2021)
In this video, ...