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

동물 사용이 프로토콜 M에 규정 된 절차를 다음 / 구에 대한 밴더빌트 기관 동물 관리 및 사용위원회에 의해 승인 / (181) 11. CD1 또는 CBA / BL6 마우스는 상용 공급 업체에서 구입 및 신생아 쥐를 얻기 위해 밴더빌트 동물 시설에서 교차했다. 마우스 1. 준비, 재고 솔루션 및 장비 <ol><li> 마우스 크로스의 경우 : 마우스 십자가를 설정하고 실험 계획을 돕기 위해 연결 날짜를 기록합니다. CD1 마우스는 보통 짝짓기 후 하루 (19)의 주위에 출산을 제공합니다. 사용 CD1 또는 CBA / BL6은 각각 CD1 또는 CBA / BL6 신생아를 얻기 위해 십자가. 두 선 사이의 Intercrosses는 CD1의 여성과 CBA / BL6 남성을 사용한다. </li><li> 콜라게나 주식에 대한 : 고정밀 밸런스 200 mg의 콜라겐 유형 IV 무게. 50 ML의 원심 분리기 튜브에 전송합니다. 칼슘 40 ㎖의 HBSS에 용해 2+ / 마그네슘 2+ (각각 1.26와 0.5 밀리미터는) 5 ㎎ / ㎖의 원액을 확인합니다. <ol><li> 에 콜라게나 허용부드러운 흔들림과 함께 ~ 30 분 동안 용해. 나누어지는 및 1 년 이상 활성 상태를 유지 재고 한 -20 ° C에 냉동 솔루션을 유지. </li></ol></li><li> 해부 및 섬 따기위한 핀셋, 가위, 및 P20 팁 몇 쌍을 소독하기 위해 압력솥. </li><li> 1 M 포도당 주식에 대한 : 50 mL의 원심 분리기 튜브에 9g 포도당 무게 포도당을 용해 50 mL의 RPMI에게 1640 미디어를 추가 할 수 있습니다. 사용할 때까지 4 ° C에 보관하지,하지만 6 개월 이상. </li></ol> 2. 작업 솔루션 참고 : 섬 분리의 날, 다음과 같은 시약을 준비합니다. <ol><li> 콜라게나 제 타입 IV 가공 솔루션 : 해동 얼음에 콜라게나 주식을 희석. 각각 1.7 mL의 미세 원심 분리 관에 0.15 mL의 주식을 분배. 칼슘 / 마그네슘 2+와 1.35 ㎖의 HBSS를 추가합니다. 튜브 5 번 반전. 15 분 동안 얼음에 둡니다. <ol><li> 미세 원심에서 3 분 동안 고속으로 회전. 새로운 튜브에 뜨는을 전송합니다. 사용할 때까지 얼음에 남겨주세요. 불용성 이물질와 튜브를 폐기하십시오. </li></ol></li><li> 전체 RPMI 1640 미디어 : 500 ㎖의 RPMI 1640 미디어에 2.5 mL의 1 M 포도당, FBS 불 활성화 50ml의 열, 5 ㎖의 100 배 펜 연쇄상 구균 주식을 추가합니다. 사용할 때까지 얼음에 둡니다. </li></ol> 3. 췌장의 분리 및 소화 <ol><li> 승인 된 프로토콜에 따라 잘린 다음 이소 플루 란과 신생아를 안락사. </li><li> 배꼽이 위를 향와 마우스를 놓습니다. 살균 70 % 에탄올로 스프레이. </li><li> 핀셋으로 배꼽 피부를 들어 올려 흉곽의 생식기 영역에서 가위와 정중선을 따라 길이 방향으로 피부와 근육 층을 잘라. 이것은 모든 내부 장기를 노출 복강을 엽니 다. </li><li> 핀셋으로 100cm 접시에 모든 내부 장기를 전송합니다. 위장과 비장과 십이지장 (11)에 달라 붙는 복부 부분에 달라 붙는 지느러미 부분과 분산 - 장기 인 췌장을 찾습니다. </리&gt; <li> 장기 잠수함하기 위해 접시에 HBSS를 추가합니다. 용액에서, 췌장 조직이 더 이상 십이지장, 위, 비장에 집착하지 않는다. 이 때문에 전형적인 화이트 색상의 해부 범위에서 쉽게 인식 할 수있다. HBSS로 새로운 60mm 요리에 떨어져 주변 조직 및 전송에서 췌장 조직을 박리 핀셋을 사용합니다. </li><li> 어떤 차원에서 5mm보다 작은 조각으로 가위 각각의 췌장을 잘라. 1.5 ML의 microcentrifuge 관에 전송합니다. 최대 5 pancreata는 (P7 미만) 하나의 튜브에 소화 할 수있다. P8-P16 마우스의 경우, 췌장 당 하나의 튜브를 사용합니다. </li><li> 각각의 튜브에 1 ML의 콜라게나 작업 용액 (각 P1-P7 췌장 0.2 mL 씩 P8-P16 췌장 0.5 mL로 사용합니다.) - 0.5를 추가합니다. 최대 15 분 동안 37 ° C를 인큐베이터에서 튜브를 남겨주세요. 튜브 두 번 모든 분을 반전. </li><li> ~ 5 분 후, 췌장 조직의 분해 상태를 모니터링하기 위해 상기 튜브를 탭. 췌장의 대부분까지 소화 과정을 계속조직은 직경 &lt;2mm와 같은 파편 세포 클러스터를 나타납니다. 해물 정상 선방 세포자가 분해에 의한 흐림을 나타납니다. </li></ol> 4. 해물 세척 <ol><li> 미세 원심에서 10 초 동안 500 XG에 해물 스핀. P1,000의 pipetman에 뜨는 층을 제거합니다. 상층 액은 혼탁하지만 세포 클러스터없는 나타납니다. 빠르게 인해 일부 끈적 DNA의 존재에 맨 아래에있는 조직 조각을 멀리 대기음 수있는 구토물이기도를 사용하지 마십시오. </li><li> 1 mL의 RPMI 1640 전체 미디어를 추가합니다. 조각난 해물을 재현 탁 부드럽게 튜브를 누릅니다. 최대 반전 10 배 아래로. 단계를 반복 4.1. </li><li> 단계를 반복 4.2 두 번 이상 또는 상등액을 분명히 나타날 때까지. 1 mL의 완전한 RPMI 1640 배지에서 세척 췌장 조각을 다시 일시 중지합니다. </li></ol> 5. 작은 섬 격리 참고 : pancreata의 작은 번호는 직접 손으로 따기 아래 (5.1) 섬의 ISOL 사용할 수 있습니다ATION. pancreata 많은 경우 (&gt; 6), 5.2에서 설명하는 방법이 바람직하다. <ol><li> 직접 손으로 따기 : <ol><li> 60 밀리미터 접시 단계 4.3에서 해물을 전송합니다. 5 ㎖ 전체 RPMI 1640 미디어를 추가합니다. 해부 현미경으로 가져옵니다. </li><li> 현미경의 시야 조명을 설정합니다. 빛의 세기 최대 전력의 ~ 50 %를 조정합니다. 이 상태에서, 독도는 선포 세포와 같은 어두운 불규칙한 모양의 클러스터 반면 (그림 1A)로 약간 핑크 클러스터를 나타납니다. ~ 50X (목적의 힘과 눈 조각을 결합)의 확대가 가시화 섬의 가장 좋은 방법 및 따기위한 동시에 피펫 팁 오프닝을 제공하는 것이 좋습니다. </li><li> 선포 클러스터를 피하면서 P20 피펫 팁과 섬을 선택합니다. 분배 RPMI 전체 미디어 (그림 1B)와 새로운 60mm 요리에 분수를 섬이 풍부한. </li><li> 두 번 더 핸드 피킹 과정을 반복합니다. 새로운 60 순수한 독도를 남겨4 mL의 전체 RPMI 미디어 (그림 1C)와 mm 접시. </li></ol></li><li> 그라데이션 원심 분리 : <ol><li> 1.077 g / ㎖의 밀도로 2 ML의 polysucrose 나트륨 diatrizoate 미리로드 된 15 mL의 원심 분리기 튜브를 준비합니다. 사용할 때까지 얼음 튜브를 남겨주세요. </li><li> 파스퇴르 피펫으로 polysucrose 나트륨 diatrizoate 솔루션의 상단에 단계 4.3에서 정지를 전송합니다. 바닥 층을 방해하지 마십시오. 최대 새로 태어난 10 2 pancreata P17에서 조직은 각각 15 mL의 튜브에로드 할 수 있습니다. </li><li> 4 ° C에서 15 분 동안 스윙 로터에 ~ 600 XG에 튜브를 스핀. 브레이크 오프 설정을 사용합니다. </li><li> 원심 분리 후, 새로운 15ml를 튜브에 미디어 및 계면 층 (이상적인 동작 조건에서 아일렛 원심 분리 후 polysucrose 문화 매체 사이의 계면에 위치 될 것이라는 점에 유의) 옮긴다. 10 mL의 미디어를 추가 부드럽게 잘 섞는다. </li><li> 작은 섬이 풍부한 부분을 스핀 다운5 분 300 XG에. 세척 용지를 제거합니다. 전체 RPMI 매체와 세척을 한 번 더 반복합니다. 대부분 섬 및 덕트 (그림 1D)를 포함 4 mL의 미디어에의를 Resuspend 펠릿. </li><li> 5.1 절에 설명 된대로 독도를 손으로 선택합니다. </li></ol></li></ol> 격리 된 독도 6. 국제 대학원 분석 실험 <ol><li> 37 ° C 조직 문화 인큐베이터에서 2 시간 동안 전체 RPMI 미디어의 단계 5.1.4에서 독도를 품어. </li><li> 해부 현미경 RPMI을 제거하는 피펫을 사용합니다. 플레이트 (3)에 2.8 mM의 포도당과 3 mL의 KRB 솔루션을 추가합니다. 독도를 씻어 10 회 소용돌이 친다. KRB 솔루션을 제거합니다. <ol><li> 한 번 세척 과정을 반복합니다. 인큐베이터에서 1 시간 동안 37 ° C에서 3 ㎖ KRB 용액 독도 부화. </li></ol></li><li> 12 웰 플레이트의 각 웰에 1 mL의 분취 KRB. 인큐베이터에서 37 ° C로 KRB 재고 및 플레이트를 사전 따뜻하게. </li><li> 6.2에서와 같이 미리 예열 KRB 두 번 독도를 씻으십시오. 트랜스FER 미리 예열 플레이트의 각 웰에 10 아일렛. 37 ° C 배양기에서 품어. </li><li> 45 분 후, 상층 액 50 μL를 철회. 이것은 기초 포도당에서 분비되는 인슐린 솔루션이 포함되어 있습니다. </li><li> 32 μL 500 mM의 포도당을 추가합니다. 솔루션을 섞어 판 20 회 소용돌이 친다. 37 ° C 배양기에서 품어. 판을 한 번에 5 시간마다 5 분 소용돌이 친다. </li><li> 45 분 후, 50 μL 상층 액을 철회. 이 높은 포도당에서 분비되는 인슐린이다. </li><li> 1.5 ㎖의 튜브에 모든 섬을 전송합니다. 30 분 동안 -20 ℃에서 동결. 실온에서 5 분 동안 해동. 냉동과 해동 과정을 반복합니다. </li><li> 20 mM의 염산 0.5 mL의 70 % 에탄올을 추가합니다. 하룻밤 둡니다. 이 섬에 남아 인슐린이다. </li><li> 인슐린 수치를 분석하기 위해 기존의 ELISA를 사용하여 이전에 3 설명했다. </li></ol>

<ol>
	<li>Matsumoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. (50), (2011).</li><li>Zhao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galphao+represses+insulin+secretion+by+reducing+vesicular+docking+in+pancreatic+beta-cells.">Galphao represses insulin secretion by reducing vesicular docking in pancreatic beta-cells.</a> Diabetes. 59 (10), 2522-2529 (2010).</li><li>Planas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+for+the+human+pancreas+and+purified+islets+in+type+1+diabetes:+new+findings+at+clinical+onset+and+in+long-standing+diabetes.">Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.</a> Clin Exp Immunol. 159 (1), 23-44 (2010).</li><li>Tonne, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiling+of+pancreatic+islets+in+mice+during+streptozotocin-induced+beta-cell+damage+and+pancreatic+Glp-1+gene+therapy.">Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced beta-cell damage and pancreatic Glp-1 gene therapy.</a> Dis Model Mech. 6 (5), 1236-1245 (2013).</li><li>London, N. J., Swift, S. M., Clayton, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+functional+evaluation+of+islets+of+Langerhans.">Isolation, culture and functional evaluation of islets of Langerhans.</a> Diabetes Metab. 24 (3), 200-207 (1998).</li><li>Rorsman, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+glucose+to+elicit+a+normal+secretory+response+in+fetal+pancreatic+beta+cells+results+from+glucose+insensitivity+of+the+ATP-regulated+K++channels.">Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.</a> Proc Natl Acad Sci U S A. 86 (12), 4505-4509 (1989).</li><li>Hellerstrom, C., Swenne, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+and+proliferation+of+fetal+pancreatic+beta-cells.">Functional maturation and proliferation of fetal pancreatic beta-cells.</a> Diabetes. 40 (Suppl 2), 89-93 (1991).</li><li>Blum, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+beta-cell+maturation+is+marked+by+an+increased+glucose+threshold+and+by+expression+of+urocortin+3.">Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.</a> Nat Biotechnol. 30 (3), 261-264 (2012).</li><li>Hegre, O. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonenzymic+in+vitro+isolation+of+perinatal+islets+of+Langerhans.">Nonenzymic in vitro isolation of perinatal islets of Langerhans.</a> In Vitro. 19 (8), 611-620 (1983).</li><li>Dolen&#353;ek, J., Rupnik, M. A., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), e102445 (2015).</li><li>White, J., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Source Book of Enzymes, Source Book of Enzymes. , (1997).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54 (1), 133-137 (2005).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85 (4), 1255-1270 (2005).</li></ol>

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

여기, 우리는 기존의 관류 너무 작은 pancreata에서 섬 분리에 대한 단계별 프로토콜을 제공합니다. 등과 베타 세포의 정제, 유전자 발현 분석, 췌장 베타 세포의 성숙, 증식, 세포 스트레스 반응, 세포 생존, 대사 및 작용 GSIS 유지, 모든 섬 기반 연구 준비 아일렛을 수득 할 것으로 예상된다. 이것은 우리의 지식, 신생아 쥐에서 섬 격리을 수행하는 새로운 연구를 안내하는 최초의 상세한 영상 ?...

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

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

effective isolation of functional islets from neonatal mouse pancreas

큰 포유 동물 pancreata에서 관류 기반의 섬 - 격리 프로토콜은 잘 확립되어있다. 이러한 프로토콜은 쉽게 인해 준비 콜라겐 주입 및 이후의 조직 관류를 허용 췌관의 큰 크기로 많은 실험실에서 실시하고 있습니다. 관류 쉽게 작은 pancreata에서 얻을 수 없기 때문에 반대로, 작은 pancreata에서 섬 격리는, 신생아 쥐처럼, 도전이다. 여기에서 우리는 시각적 인 도움으로 새로 태어난 쥐에서 독도의 상당한 숫자를 복구 상세한 간단한 절차를 설명합니다. 갓 해부 전체 pancreata는 마이크로 원심 튜브에서 37 ° C에서 행크스 균형 염 용액 (HBSS)에 용해하고, 0.5 ㎎ / ㎖의 콜라게나 제 IV로 분해 하였다. 튜브는 조직의 분산을 돕기 위해 정기적으로 도청했다. 조직의 대부분이 1mm 주위에 작은 클러스터에 분산시킨 경우, 용 해물을 10 % 소 태아 혈청 (FBS)로 배양 배지와 서너 번 세척 하였다. 섬 클러스터,인식 선포 조직의 결여, 다음 입체경을 해부에서 복구 할 수 있습니다. 새로 태어난 마우스의 췌장 당 대형 섬에 80 소형 -이 방법은 (20)를 복구합니다. 이 섬은 인슐린 분비, 유전자 발현, 문화를 포함한 대부분의 생각할 수있는 다운 스트림 분석에 적합하다. 인슐린 분비 분석의 예는 분리 프로세스를 검증하기 위해 제공된다. 유전 적 배경과 소화의 정도는 수익률을 결정하는 가장 큰 요인이다. 이 내분비 - 외분비 격리 에이즈로 높은 활성 갓 만든 콜라겐 용액은 바람직하다. 세척의 모든 솔루션 및 소 태아 혈청에있는 양이온의 존재 [칼슘 (Ca 2+)과 마그네슘 (Mg를 2+)] / 따기 매체는 적절한 무결성 섬의 좋은 수율이 필요하다. 좋은 대조 및 확대와 해부 범위도 도움이 될 것입니다.

각각의 작은 마우스의 췌장에서 아일렛 80 - 최적 조건에서 제시된 방법 (20)을 수득 할 수있다. 이 번호는 마우스의 유전 적 배경, 나이에 의존하고, 섬의 크기를 회복한다. 일반적으로 사용되는 가운데, CD1가에서 자란와 C57BL / 6J 순종 마우스 CD1 및 C57BL / 6 또는 상업적 B6CBAF1 사이의 잡종보다 작은 크기의 작은 섬을 생산 / J 마우스는 않습니다. 일반적으로 따기 직접 손으로 ...

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

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

우리는 신생아 쥐에서 그대로 섬을 분리 본원 프로토콜을 설명합니다. Pancreata 부분적으로 세척하고 손을 따기 다음, 콜라게나로 소화했다. 20-80 아일렛 클러스터 여러 섬 연구에 적합한 새로 태어난 쥐의 췌장에 따라 얻을 수있다.

신생아 마우스 췌장의 기능 독도의 효과적인 분리

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

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.0K 조회수.  Vanderbilt University Medical School.  우리는 신생아 쥐에서 그대로 섬을 분리 본원 프로토콜을 설명합니다. Pancreata 부분적으로 세척하고 손을 따기 다음, 콜라게나로 소화했다. 20-80 아일렛 클러스터 여러 섬 연구에 적합한 새로 태어난 쥐의 췌장에 따라 얻을 수있다. 

비디오: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

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

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

Murine 아일릿 절연 및 Subcapsular 신장 이식을위한 방법

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

연구

의학

Isolation of Human Islets from Partially Pancreatectomized Patients

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

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

부분 Pancreatectomized 환자에서 인간 아일 레츠의 분리

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

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

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

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

당뇨병 쥐의 부고환 지방 세포에 이식 된 지지체 지지대 지지대

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

생체공학

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; 2 형 당뇨병의 마우스 모델 : 본래 독도 췌장 섬 세포의 분리 및 라이브 셀 2 광자 이미징

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

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

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

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

Diabetogenic 환경 화학 물질에 대 한 Murine 췌 장 섬 세포 기반 검사

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

면역학 및 감염병학

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.

높은 콘텐츠<em&gt; 체외</em&gt; 췌장 작은 섬의 β 세포 복제 디스커버리 플랫폼

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

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

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

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

Langerhans 절연의 마우스 작은 섬은 정화의 Collagenase와 중성 단백 분해 효소의 조합을 사용하여

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

생물학

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

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

Intraportal 이식 마우스 모델에서 췌 장 독도의

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.

마우스 췌장 섬의 분리 및 세포 내 cAMP를 결정하는 방법

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

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

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

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

신생아 마우스 췌장의 기능 독도의 효과적인 분리

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