우리는이 프로젝트의 여러 단계에 도움, 조언과 중요한 입력을 제공하는 모든 동료에게 감사하고 싶습니다. 이 비디오 문서의 생산은 IMIDIA (의 자금이 지원 된 <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ), 교육 및 당뇨병 연구 독일 센터 (DZD로 연구 (BMBF)에 대한 독일의 성 <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd - ev.de</a> ) 기술 드레스덴 (Dresden)의 대학에서 대학 병원 칼 구스타프 이카루스 호. 이 책자에 이르는 작품 부여 계약을 N ° 155005 (IMIDIA), 유럽 연합 (EU)의 7 번째 프레임 워크 프로그램 (FP7/2007-2013)와 EFPIA에서 금전적 인 기부로 구성되어있는 자원을 아래에 혁신적인 의약품 사업 공동 인수의 지원을 받고 있습니다 회사의 종류 기여 인치

1. 인간 췌장 조직의 동결 <ol><li> 지방 조직, 혈관, 신경 및 메스와 핀셋과 비 parenchymal 조직을 제거하고 조각 (~ 0.5-1 cm 조각)로 췌장 조직을 잘라. </li><li> Cryomold의 중심에 하나 췌장 조직 조각을 놓고, 차가운 조직 - 테크 10월 컴파운드로 완전히 커버. 그의 아래 액체 질소에 미리 냉각 2 Methylbutane와 스냅 - 동결로 덮여 있습니다 병에 Cryomold을 놔. -80 ° C.에있는 냉동 샘플을 저장 </li></ol> 2. 냉동 섹션의 작성 <ol><li> RNA의 변성을 피하기 위해 모든 단계에 걸쳐 장갑을 착용한다. </li><li> 멀리 차가운 100 % 에탄올과 RNase와 그라 이오 스탯 나이프, 표본 홀더와 붓를 청소합니다. 그라 이오 스탯 내부의 냉동 조직을 배치합니다. </li><li> 조직, 칼과 붓 -20 ° C.에 도달 할 때까지 기다리 기다리는 동안 그라 이오 스탯 챔버 내부 SuperFrost 플러스 슬라이드와 사전 시원한 그들을 레이블을조직 -20 ° C.에 도달 할 수에 대한 </li><li> 표본 홀더에있는 조직 - 테크 10월 컴파운드를 적용하고 상단에 고정 된 조직을 배치합니다. </li><li> 조직 - 테크 10월 컴파운드이 고정되면 cryoblock을 다듬과 면도날과 조직 - 테크 10월 컴파운드의 초과를 제거합니다. </li><li> 췌장 조직 블록에서 40 연속 10 μm 슬라이스 총 자른다. 또한 우리는 microdissection 과정에서 조각 안에 섬의 식별을 용이하게 할 수있는 췌장 섹션의 개요 도면에 저장되는 제 1 및 중간 조각을 절단하는 것이 좋습니다. </li><li> 칼 블레이드의 섹션 준수하여야 할 만 지역을 따뜻하게하기 위해 손가락 (장갑으로 덮여)으로 슬라이드의 뒷면을 터치하여 SuperFrost 플러스 슬라이드의 중앙에 섹션을 전송합니다. </li><li> -20에서 그라 이오 스탯 내부의 섹션 1-2 분 ° C, 당신이 드라이 아이스에 배치 슬라이드 상자에 넣어 그 이후에 말린다. 섹션을 저장-80에서 ° C. </li><li> 필요한 경우, 종이 수건과 차가운 100 % 에탄올로 칼 블레이드를 청소. </li></ol> 3. 냉동 섹션 탈수 <ol><li> 시약 준비 : 30 ML 신선하게 조리 된 70 % 에탄올, 30 ML DEPC - 처리 물, 2x30 ML 100 % 에탄올과 50 ML Cellstar 튜브 (팔콘)로 30 ML의 크실렌을 흘려, 오한과 얼음에있는 모든 시약을 (크실렌 제외) 유지. </li><li> 100 % 에탄올과 RNaseZAP와 후드 작업 공간을 청소하십시오. </li><li> 다음과 같은 과정이 cryosections가 : 얼음 차가운 DEPC - 처리 물에 5-6 빠른 딥을 씻고, 30 초에 얼음 차가운 70 % 에탄올에 해결, 크실렌의 4 분에 품다, 얼음 차가운 100 % 에탄올에 1 분에 두 번 탈수 실내 온도 (25 ° C), 3 ~ 5 분에 대한 건조 공기에서. cryosections 또 한 쌍의로이 단계를 반복합니다. </li><li> 빛과 습기에서 보호하기 위해 알루미늄 호일로 싼 50 ML Cellstar 관으로 백업 뒤로 두 건조 cryosections를 삽입합니다. </li></ol> &lt;P 클래스 = &quot;jove_title&quot;&gt; 4. PALM MicroBeam, Zeiss와 베타 세포의 레이저 Microdissection <ol><li> RNaseZAP과 현미경을 청소하고 RoboMover의 접착제 모자를 탑재합니다. </li><li> 필터 세트 09 (여기 : 450-490 nm의, 방출&gt; 515 nm 정도), AutoLPC 모드, 50 %의 레이저 에너지 65 %, 레이저 초점, 100 % 레이저 속도, AutoLPC 샷 사이에 5 μm 거리, 6 μm 거리 다음 설정을 설정합니다 -10,100 : 선에 높이를 작동. </li><li> 무대에 하나의 슬라이드를 삽입합니다. </li><li> 미세 시각화 (5 배 또는 10 배 렌즈) 아래 autofluorescent 베타 세포를 찾습니다. </li><li> 40x 렌즈로 전환, &quot;자유형&quot;선택 도구를 사용하여 베타 세포를 선택하고 레이저를 사용하여 사진을 microdissect. </li><li> 한 AdhesiveCap로 4 탈진 cryosections의 조직을 캡처합니다. 1 주석 : 한 건조 cryosection의 베타 세포를 해부 할 수있는 시간이 될 것입니다 조직 섹션의 rehydration을 피하​​기 위해 10-20 분 이상이 아니어야합니다저하 RNA. 2 의견 : 검사 점으로 무대를 이동 한 후 육안 검사에 의해 성공적으로 microdissection 프로세스를 확인합니다. </li></ol> 5. Microdissected 베타 세포 농축 조직의 용해 <ol><li> RoboMover에서 AdhesiveCap를 제거합니다. </li><li> AdhesiveCap의 뚜껑과 42에서 거꾸로을 길러 ° C를 30 분에에 Pipet 10 μl 추출 버퍼 (XB, PicoPure RNA 분리 키트, 악튜러스). </li><li> 10,000 XG에서 스핀 다운 및 드라이 아이스에 lysate를 넣어. RNA 추출이 수행 될 때까지 -80 ° C에 저장합니다. </li><li> 반복 3 단계를 반복합니다.) 5.) 다른 모든 섹션과. </li></ol> 6. RNA 추출 <ol><li> 10 개의 10 μl lysates을 결합 </li><li> 70 % 에탄올 (DNase 처리 포함)에 1:1 비율 추출 버퍼 (XB)를 사용하여 PicoPure RNA 분리 키트, 악튜러스의 프로토콜에 따라 실온에서 협력하여 RNA 격리와 함께 진행합니다. </li></ol> 7. RNA 분석 <ol><li> 애질런트 2100 Bioanalyser (PicoChip)을 사용하여 품질과 수량의 분석에 대해 1 μl RNA를 사용합니다. 정량 평가는 칩 병렬로로드 표준 RNA를 참조로 이루어집니다. </li></ol>

<ol>
	<li>Bonner, R. F., Emmert-Buck, 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. 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=Laser+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. 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=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, 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=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. C., 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=Laser+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

우리는 수술 췌장 표본의 인간 섬의 레이저 microdissection (LMD)에 대한 신뢰할 수있는 접근 방법을 설명합니다. LMD 현미경을 사용할 수 있다고합니다,이 절차는이를 모두 비 당뇨병과 2 형 당뇨병 환자 과목에서 인간의 작은 섬 소재에 대한 액세스를 증가, 부분 pancreatectomies를 수행하는 연구 기관에서 구현 될 수 있습니다. 이 작은 섬 고립에 제공 pancreata의 소수 주어진 특히 해당됩니다. 의 Favourable ?...

<table border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 2 Methylbutane (이소 펜탄) </td><td> 로스 </td><td> 3927.1 </td><td></td></tr><tr><td> AdhesiveCap (불투명, 500 μl) </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td> Cellstar 튜브 (50 ML) </td><td> greiner 바이오 - 원 </td><td> 210 261 </td><td> 지원 스커트로 </td></tr><tr><td> Cellstar 튜브 (50 ML) </td><td> greiner 바이오 - 원 </td><td> 227.261 </td><td></td></tr><tr><td> 디 에틸 pyrocarbonate (DEPC) </td><td> 시그마 </td><td> D 5758 </td><td></td></tr><tr><td> 드라이 아이스 </td><td></td><td></td><td></td></tr><tr><td> 절대 에탄올 </td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td> 액체 질소 </td><td></td><td></td><td></td></tr><tr><td> 브러쉬를 페인트 </td><td></td><td></td><td></td></tr><tr><td> PALM MicroBeam </td><td> ZEISS </td><td></td><td></td></tr><tr><td> 필 - 편도 삽입 금형 (립니다), 12 X 12mm </td><td> ProSciTech </td><td> RR12 </td><td> 탑 내부 22x22 mm, 깊이 21mm </td></tr><tr><td> 악튜러스 PicoPure 냉동 RNA 분리 키트 </td><td> 응용 Biosystems </td><td> KIT 0,204 </td><td></td></tr><tr><td> 플라스틱 클램프 </td><td></td><td></td><td></td></tr><tr><td> 면도칼 </td><td></td><td></td><td></td></tr><tr><td> RNase 무료 DNase 세트 </td><td> Qiagen </td><td> 79,254 </td><td></td></tr><tr><td> RNaseZAP </td><td> 시그마 </td><td> R 2020 </td><td></td></tr><tr><td> 메스 /외과 블레이드 </td><td> 테크노는 잘라 내기 </td><td> 2800111 </td><td></td></tr><tr><td> SuperFrost 플러스 부착 현미경 슬라이드 </td><td> 열 과학 </td><td> J1800AMN​​Z </td><td> 25x75x1.0 mm </td></tr><tr><td> 조직 - 테크 10월 컴파운드 </td><td> 사쿠라 </td><td> 4583 또는 0807000022 </td><td></td></tr><tr><td> 족집게 </td><td> 브라운 </td><td> BD168R </td><td></td></tr><tr><td> 크실렌 </td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

레이저 microdissection (LMD)는 실질 1,2의 미세한에서 선택한 셀과 조직의 복구를 할 수있는 기술입니다. 해부 세포는 이러한 transcriptomic 또는 proteomic 연구, DNA 평가 나 염색체 분석 2,3 등 조사의 다양한 사용할 수 있습니다. LMD의 특히 어려운 응용 프로그램은 RNA (4)의 lability로 인해 셀이 같은 췌장 등의 RNases의 풍부 조직 분석하던 때 특히 눈에 띄는 될 수 transcriptome 분석이다. 대상 세포의 빠른 식별 및 수집을 가능하게 microdissection 프로토콜은 조직 처리 시간을 단축 할 수 있으며, 결과적으로, RNA의 보존을 보장하기 위해이 설정에 필수적입니다. 여기 transcriptomic 연구 5에 사용되는 수술 표본에서 인간의 췌장 베타 세포를 획득을위한 프로토콜을 설명합니다. 0.5-1에 대한 C의 췌장의 작은 조각m 3 sectioning 때까지 즉시 2 Methylbutane 차가운에 냉동 조직 - 테크 10월 컴파운드에 포함 resected의 췌장의 표본의 건강 나타나는 여백,에서 절단하고, -80에 저장 ° C 있었다. 10 μm 두께의 40 시리얼 섹션은 1-2 분의 그라 이오 스탯 내부 건조하고, -80에 저장 유리 슬라이드, ° C.에 개별적으로 전송, -20 ° C 설정에 따라 그라 이오 스탯을 삭감했다 즉시 레이저 microdissection 절차 전에 섹션은 얼음 차가운 백퍼센트에서 두 1 분 incubations하여 얼음처럼 차가워에서 수정 갓 30 초에 70 %의 에탄올을 준비, 얼음 차가운 DEPC - 처리 물에 5-6 딥으로 세척하고, 건조 된 에탄올은 4 분 동안 크실렌 (조직 탈수에 사용되는)에 의해 다음, 조직 섹션 3-5 분 동안 나중에 한 후 공기 건조했다. 중요한 것은, 모든 단계는 크실렌의 부화를 제외하고 얼음처럼 차가운 시약을 사용하여 수행되었습니다 - 이전에 설명 된 프로토콜을 6 이상 수정. UT얼음 차가운 시약의 ilization는 베타 세포의 고유 autofluorescence의 발음 증가로 이어진, 그들의 인정을 촉진. microdissection를 들어, 4 개의 섹션은 각 시간 건조했다 : 두 수분과 표백에서 조직을 보호하기 위해 호일로 싼 50 ML 튜브에 배치 된, 나머지 두 사람은 즉시 microdissected되었습니다. 이 절차는 (AutoLPC) 모드를 Catapulting 자동 레이저 압력을 채용 PALM MicroBeam 악기 (Zeiss)를 사용하여 수행되었습니다. 40-60 분보다 더 이상 필요 넷 cryosections에서 베타 세포 / 작은 섬의 해부의 완성. 셀은 한 AdhesiveCap으로 수집, 10 μl 용해 버퍼로 lysed되었습니다. transcriptomic 분석을 위해 각 단일 RNA 시료는 10 셀 microdissected 샘플, 피코 순수 RNA 절연 키트 (악튜러스)를 사용하여 RNA 추출 다음을 조합하여 얻은 것입니다. 이 프로토콜은 따라서 자신의 신속하고 정확한 인식과 C을 촉진, 인간의 베타 세포의 고유 autofluorescence을 향상ollection. 이 절차의 추가 개선 2 형 당뇨병과 관련된 변경 사항을보다 잘 이해 가능한 의미와 함께, phenotypically 다른 베타 세포의 절개를 사용하도록 설정할 수도 있습니다.

그림 1에 도시 된 바와 같이, 수정 건조 프로토콜은 이전의 게시 된 프로토콜 6에 비해 베타 세포의 autofluorescence의 개선을 이끌었다. 설명 프로토콜 적용, 39 수술 췌장 표본의 각 31&#39;544&#39;704 μm 세 조직 / 췌장 표본의 평균 (: - 81&#39;522&#39;153 μm 3 8&#39;742&#39;390 범위) 40 시리얼 cryosections를 생성하는 데 사용 된 표 1에 표시된. 이 150 μm의 직경, 또는 3...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

레이저 microdissection은 실질의 미세한에서 선택한 셀의 복구를 할 수있는 기술입니다. 여기 transcriptomic 연구에 사용되는 수술 표본에서 인간의 췌장 섬을 획득을위한 프로토콜을 설명합니다. 우리 프로토콜은 따라서 자신의 컬렉션을 촉진 인간의 베타 세포의 고유 autofluorescence을 향상시킵니다.

수술 표본에서 인간 췌장 섬의 레이저 Microdissection을위한 개선 된 프로토콜

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

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14.3K 조회수.  Paul Langerhans Institute Dresden. 레이저 microdissection은 실질의 미세한에서 선택한 셀의 복구를 할 수있는 기술입니다. 여기 transcriptomic 연구에 사용되는 수술 표본에서 인간의 췌장 섬을 획득을위한 프로토콜을 설명합니다. 우리 프로토콜은 따라서 자신의 컬렉션을 촉진 인간의 베타 세포의 고유 autofluorescence을 향상시킵니다.

비디오: Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

외과 종양 표본의 Explants를 사용하여 소설 안티 - 암 의약품 개발을위한 방법


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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 ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

인간의 췌장을위한 컬렉션 프로토콜

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

인간의 췌장 Islets위한 염색법 프로토콜

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

간 간선 재건과 부분 Orthotopic 간 이식의 쥐 모델에 대한 외과 절차

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

저널을 사용하여 인간의 망막 수술 표본의 Transcriptomic 분석

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

임상적으로 관련동물 모델 및 3D 면역화학에 사용하기 위한 외과 표본에서 고학년 신경교종 세포 배양의 최적화

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

자궁 적출술 검체에서 차 인간의 자궁 내막 기질 세포를 구축하는 두 가지 방법

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

RNA 분석을위한 인간의 전립선 상피 세포의 레이저 캡처 이외에 Microdissection

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

레이저 도플러: 췌 장 섬 Microvascular Vasomotion에 Vivo에서 측정 하기 위한 도구

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...