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

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The authors have nothing to disclose.

המחקר של התפשטות β-cell בתרבות נפגע בדרך כלל על ידי מספר קשיים. ראשית, קווי β-cell הנציח מאופיינים לתארים גבוהים של התפשטות ממה נמצא β-תאי אנדוגני איים חי. בנוסף, שורות התאים הנציחו אלה חסרות את הארכיטקטורה הנורמלית חיונית לתפקוד β-תא נורמלי. שתי עובדות אלו מקשות על מנת לקב...

β-תאי לבלב הם התאים היחידים המייצר אינסולין בגוף הם קריטיים לשמור על הומאוסטזיס הגלוקוז בדם. בעוד אנשים בריאים הם בעלי מסת β-cell מספיק ולתפקד להסדיר דם גלוקוז כראוי, אנשים עם סוכרת מתאפיינים המוני β-cell מספיק ו / או פונקציה 1,2. הוצע כי גרימת התפשטות β-cell יכול בסופו של דבר להגדיל β-cell המוני ולשחזר הומאוסטזיס הגלוקוז בחולי סוכרת 3. עם זאת, הערכה ואימות של תרכובות שגשוג פוטנציאל β-תא איי שלמים הכרחי לפני ניתן לפתח טיפולים יעילים. השתלת איי אדם cadaveric לתוך אנשים עם סוכרת משחזר הומאוסטזיס הגלוקוז בדם במשך זמן מה, אבל זמינויות ההצלחה של הליך ניסיוני זה הפריעו מחסור איי אדם זמינים להשתלה ועל ידי מוות בטא תא ומאחור האייםאה השתלת 4. אפילו עם גילוי הגורמים לגרום הכפל של תאים מייצרי אינסולין, אתגר גדול עדיין קיים במתן הגורמים הללו לאתרים רלוונטיים in vivo. אסטרטגיה אחת למסירה מקומית מתמשכת של תרכובות שגשוג β-cell היא פולי (לקטית-שיתוף גליקולית) חומצה (PLGA). יש PLGA היסטוריה של שימוש ב FDA אישר מוצרים משלוח סמים בשל בטיחות גבוהה, פריקות ביולוגית, קינטיקה שחרור המורחבת 5. באופן ספציפי, PLGA הוא קופולימר של lactide ו glycolide הזה מבזה באמצעות הידרוליזה עם מים או in vivo או בתרבות לחומצה לקטית וחומצה גליקולית, אשר מתרחשים מטבוליטים באופן טבעי בגוף. מתחם התרופה הכמוס יכול להשתחרר הסביבה על ידי שתי דיפוזיה ו / או מנגנוני שחרור מבוקר שפלים. Encapsulation של COI מספק הגנה מפני השפלה אנזימטי, שיפור הזמינות הביולוגית של מגיב לעומת unencapsulaטד COI 5. אנו ממליצים microspheres PLGA ניתן להשתמש כדי לנהל תרכובות המועמד איים שלמים בתרבות, ובסופו של דבר in vivo. בדיקת יעילותם של PLGA לנהל mitogens β-cell כדי איי vivo לשעבר היא קריטית לפני פרוטוקולי השתלת נחקרות. נכון לעכשיו, אין טכניקה למדוד התפשטות β-cell בבעלי חיים. ניסויים להעריך את האפקטיביות של תרכובות שגשוג פוטנציאל in vivo ולכן יש לתת לכלב של תרכובות אלה לחיות חיים, עם דיסקציה עוקבות ועיבוד של pancreata עבור immunolabeling. פרוטוקולים כאלה הם יקרים ומייגעים, ודורשים המתחם להיות מערכתיים, ללא כל ערובה לכך שהם יגיעו האי. לעומת זאת, כמה הנציח קווי β-cell זמינים לחקר תאים מייצרי אינסולין בתרבות, אבל שורות תאים אלה חסרים את הארכיטקטורה איון environment נמצא אורגניזמים חיים 6. קווי β-cell הונצח גם מאופיינים כבעלי מידה רבה יותר של שכפול מ β-תאי אנדוגני in vivo, ובכך סיבכה ניתוח של תרכובות המשרים התפשטות. במחקר זה, אנו מתארים פרוטוקול המשתמשת איים שלמים מבודדים עכברים בוגרים. בניגוד קווי β-cell, איים שלמים שומרים על ארכיטקטורת איון נורמלית. כמו כן, בניגוד הניסויים שנערכו in vivo, מתן תרכובות שגשוג ישירות איים שלמים תרבותיים מפחית באופן משמעותי את כמות ריאגנטים כי יש צורך למדוד במדויק התפשטות β-cell. המחקר הנוכחי מנצל PLGA כדי לנהל COI, בדוגמה זו, גורם הגדילה רקמת חיבור אנושי רקומביננטי (rhCTGF). השיטה המתוארת כאן מקנה יתרון משמעותי על פני הממשל של מתחם גלם האי בתרבית שכן היא מאפשרת שחרור מתמשך של מתחם לתוך התקשורת דואר. יש לציין, assay זה יכול להיות שונה כדי לנהל מגוון רחב של חלבונים ונוגדנים מעניינים איים שלמים. השפעות על סוגי תאים אנדוקריניות אחרות, כולל תאי α, ניתן לנתח גם.

<table><tbody><tr><td>Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td><td>ThermoFisher Scientific</td><td>O6149</td><td>For labeling COI with fluorophore</td></tr><tr><td>DMSO Dimethyl Sulfoxide</td><td>Fisher BioReagents</td><td>BP231-1</td><td>For dissolving fluorophore in step 1</td></tr><tr><td>Disposable PD-10 Desalting Columns</td><td>GE Healthcare</td><td>17-0851-01</td><td>Desalting column used in step 1</td></tr><tr><td>Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000 - 69,000</td><td>Sigma-Aldrich</td><td>739960</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>Poly(vinyl alcohol) molecular weight 89,000 - 98,000</td><td>Sigma-Aldrich</td><td>341584</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>RPMI 1640</td><td>Thermo Scientific</td><td>11879-020</td><td>For culturing islets</td></tr><tr><td>Dextrose Anhydrous</td><td>Fisher BioReagents</td><td>200-075-1</td><td>Supplement for islet media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>Antibiotics for islet media</td></tr><tr><td>Normal horse serum</td><td>Jackson ImmunoResearch</td><td>008-000-121</td><td>Supplement for islet media</td></tr><tr><td>96-well tissue culture plate</td><td>Corning</td><td>3603</td><td>For culturing islets</td></tr><tr><td>Ethylene glyco-bis(2-aminoethylether)-&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E4378</td><td>Supplement for pre-assay islet media</td></tr><tr><td>Cytospin 4 Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>For spinning cells onto microscope slides</td></tr><tr><td>EZ Single Cytofunnel</td><td>Thermo Scientific</td><td>A78710020</td><td>For spinning cells onto microscope slides</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher BioReagents</td><td>BP118-500</td><td>Used in dissociating islets</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>For fixing cells</td></tr><tr><td>Triton&amp;nbsp; X-100</td><td>Fisher BioReagents</td><td>BP151</td><td>For permeabilizing cells</td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td>Blocking reagents for immunofluorescence</td></tr><tr><td>Anti-Ki67 antibody</td><td>abcam</td><td>ab15580</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Polyclonal Guinea Pig Anti-Insulin</td><td>Dako</td><td>A0564</td><td>For insulin immunofluorescence</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Guinea Pig</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td>For insulin immunofluorescence</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>ThermoFisher Scientific</td><td>D1306</td><td>For nuclei visualization in immunofluorescence</td></tr><tr><td>Aqua-Mount</td><td>Lerner Laboratories</td><td>13800</td><td>Fast drying mounting media</td></tr><tr><td>FreeZone -105 &amp;deg;C 4.5 Liter Cascade Benchtop Freeze Dry System</td><td>Labconco</td><td>7382020</td><td>For lyophilization of microspheres</td></tr></tbody></table>

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

פיתוח חומרים ביולוגיים גדל באופן משמעותי את פוטנציאל שיגור תרופות למגוון של סוגי תאים ורקמות, כולל β-תאי הלבלב. בנוסף, חלקיקים ביולוגיים, הידרוג, ופיגומים גם מספקים הזדמנות ייחודית לניהול מתמשך, משלוח סמים לשליטה על בטא תאים בתרבית ו במודלי רקמה מושתלות. טכנולוגיות אלו מאפשרות בחקר גורמי התפשטות β-cell המועמד באמצעות איי שלמים ומערכת רלוונטית translationally. יתר על כן, בקביעה האפקטיבית ואת הכדאיות של גורמי מועמד בהמרצת ההתרבות β-cell במערכת תרבות היא קריטית לפני לנוע קדימה מודלי in vivo. בזאת, אנו מתארים שיטת שיתוף תרבות איי עכבר שלמים עם מתחם מתכלה של עניין (COI) פולי -loaded (לקטית-שיתוף גליקולית וחומצה) (PLGA) microspheres לצורך הערכת ההשפעות של שנגרם o שחרור באתרוגורמים mitogenic f על התפשטות β-cell. טכניקה זו מתארת ​​בפירוט כיצד ליצור microspheres PLGA המכיל מטען רצוי באמצעות ריאגנטים זמינים מסחרי. בעוד הטכניקה המתוארת משתמשת גורם הגדילה רקמת החיבור אנושי רקומביננטי (rhCTGF) כדוגמה, מגוון רחב של COI יכול לשמש בקלות. בנוסף, שיטה זו מנצלת 96-גם צלחות כדי למזער את כמות ריאגנטים צורך להעריך התפשטות β-cell. פרוטוקול זה ניתן להתאים בקלות כדי להשתמש בביו-חומרים אלטרנטיביים אופייני תא אנדוקריניות אחרות כגון הישרדות תא ומצב בידול.

איור 1 הוא ייצוג חזותי של microspheres שנוצר באמצעות פרוטוקול לעיל. הפרוטוקול המתואר כאן תשואות microspheres הטעון rhCTGF בגדלים שונים. השבר הגדול של microspheres יהיה בין 1 ל -10 מיקרומטר קוטר, אם כי microspheres מסוים עשוי להיות גדול יותר (איור 2). אם ?...

Watch this Scientific Journal Video about Sustained Administration of Î²-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Polylactic-co-glycolic acid Microspheres at JoVE.com

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

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

מנהל מתמשך של mitogens β-cell כדי איי עכבר Intact<em&gt; Ex Vivo</em&gt; שימוש פוליפוני מתכלה (לקטית-שיתוף גליקולית וחומצה) מיקרוסכמות

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

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

Research

JoVE Journal

Bioengineering

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

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

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

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.

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 ...

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; עכבר דגם של סוכרת מסוג 2: בידוד לבלב וLive- תא 2-פוטון הדמיה של איי שלמים

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, ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

הנדסה משטח של לנגרהנס עם StarPEG Nanocoating Heparinized

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

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

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

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

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

A-תכולה גבוהה<em&gt; במבחנה</em&gt; פלטפורמת Discovery הלבלב איילט β-cell שכפול

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

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 ...

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.

שיטה לבידוד עכבר הלבלב איילט וקביעת תאית cAMP

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

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

במבחנה הלבלב Organogenesis ממפוזר אבות עכבר עובריים

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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 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.

מנהל מתמשך של mitogens β-cell כדי איי עכבר Intact

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