우리는 유동세포계측법와 도움 마르셀 토로 인정 싶어요. 이 연구는 스웨덴어 심장과 폐 재단, 유럽위원회는 FP7 &quot;CardioCell&quot;, 스웨덴어 연구 협의회, AFA 보험 및 ALF에 의해 지원되었다. OB는 도이치 Forschungsgemeinschaft에 의해 지원되었다.

1. 심장 핵​​의 분리 <ol><li> 1퍼센트 BSA / PBS 코팅 용액의 10 ML과 코트 ultracentrifuge 튜브 (Beckman의 원심 분리기 튜브 # 363664). 튜브를 모자하고 그들 튜브 회전에서 30 분 동안 회전하자. 코팅 용액을 제거하고 원심 분리기 튜브 공기 건조 (마우스 심장 당 하나의 튜브가 다른 종류의 단일 마우스 하트, 번갈아 최대 5 마우스의 마음이나 심장 조직의 1g의 분석에 필요한하게 (예 : 인간) 처리할 수 있습니다 연결한 튜브에서). </li><li> 모두 다음 단계는 얼음에서 수행되어야합니다. 메스 신선한 또는 스냅인 냉동 마우스 심장의 좌심실 해부. 참고,이 프로토콜은 마우스 마음에 최적화되어 있습니다뿐만 아니라 쥐 또는 사람의 마음에 맞게 할 수 있습니다. 또는 다른 종의에서 심장 조직의 1g까지 사용합니다. </li><li> 때론 작은 큐비클로 표본을 트림. </li><li> 용해 완충액의 15 ML 가득한 50 ML 팔콘 튜브에 조직 조각을 전송합니다. </li><li> 를 Homogenize10 초위한 24,000 rpm으로 T-25 울트라 Turrax 프로브 균 질기 (이카)과 심장 조직. </li><li> 30 ML에 용해 버퍼의 동일한 볼륨으로 homogenate를 희석. </li><li> 더욱 조직과 무료 핵 homogenize하는 유리 douncer (40 ML)를 사용합니다. 대형 통관 유봉들과 8 스트로크를 수행합니다. </li><li> 원유 핵는 100 μm의 70 μm의 나일론 메쉬 세포 스트레이너 (BD Biosciences), 연속적으로 분리할 전달합니다. </li><li> 원유 핵은 10 분 700 XG에서 냉장 원심 분리기 (4 ℃)에서 분리 내려 봐. </li><li> 반전 튜브에 의해 신중하게 뜨는을 제거하고 종이 타월로 튜브의 안쪽을 닦으십시오. 핵 펠렛을 방해하지 않도록주의하십시오. </li><li> 원유 핵이 솔루션은 여러 번 최대 pipetting 및 아래쪽으로 자당 버퍼의 5ml에 격리 디졸브. 녹아 펠렛으로 자당 버퍼의 추가 25 ML을 추가합니다. </li><li> 코팅 ultracentrifuge 튜브 (단계 1.1을 참조) 신선한 자당 버퍼의 10 ML을 추가합니다. &lt;/ 리&gt; <li> 조심스럽게 단계 1.9에서 자당 버퍼에 녹아있는 resuspended 핵 펠렛과 자당 버퍼의 부가 10 ML 입혀라. </li><li> JS13.1 무료 스윙 로터에 그들을 배치하기 전에 원심 분리기 튜브의 균형과 고속 원심 분리기 (Beckman 아벤티 S-25)에 날개를 놓습니다. </li><li> 60 분 동안 4 ° C에서 13,000 XG에 핵 샘플을 봐. </li><li> 스핀이 완료되면 로터에서 신중하게 튜브를 제거하고 반전 튜브에 의해 표면에 뜨는를 버리고 종이 타월로 튜브의 안쪽에서 남아있는 파편을 닦아. </li><li> 핵 저장 버퍼 (NSB 플러스 버퍼)의 1ml에 핵 펠렛를 해산. 참고 : NSB 플러스 DNA를 안정제로 1.5 MM spermine이 포함되어 있습니다. </li><li> cardiomyocyte 핵의 immunostaining 단계 2.1 진행합니다. </li></ol> 2. 유동세포계측법 위해 Immunostaining <ol><li> immunostaining에 대한 부정적인 제어를 준비합니다. 핵 샘플 20 μl를 벗어 나누어지는와보세요NSB 플러스 버퍼의 DD 980의 μl. </li><li> immunolabel cardiomyocyte 핵에 1:500의 희석에 핵 샘플로 백신 pericentriolar 소재 한 항체를 (토끼 안티 PCM-1, 아틀라스 항체)를 추가합니다. 단계 2.1 준비 부정적인 컨트롤에 대한 안티 PCM-1 항체와 같은 희석에 isotype 항체를 추가합니다. </li><li> 4 ° C에서 하룻밤 사이에 부정적인 제어 및 샘플 튜브를 품어. </li><li> NSB 플러스 버퍼 (10 분 700 XG에서 냉장 원심 분리기 (4 ℃)에서 튜브를 돌려서. 뜨는 취소하고 NSB 플러스 버퍼의 1 ML에 핵 펠렛를 해산)에 적어도 한 번 이상 부정적인 제어 및 샘플을 씻으십시오. </li><li> 1:1000의 희석에 부정적인 제어 및 샘플 튜브에 안티 - 토끼 형광 이차 항체 (FITC 또는 APC)를 추가합니다. </li><li> 한 H에 대해 4 ° C에서 제외어 제어 및 샘플 튜브를 품어. </li><li> NSB 플러스 버퍼 (10 분 700 XG에 냉장 원심 분리기 (4 ℃)에서 튜브를 다운 스핀과 함께 적어도 한 번 이상 부정적인 제어 및 샘플을 씻으. 뜨는을 무시하고) NSB 플러스 버퍼의 1 ML에 핵 펠렛를 해산. </li><li> 흐름 cytometric 분석과 정렬을 진행합니다. </li></ol> 3. 유동세포계측법 <ol><li> 당신이 단계 1.1에 설명된대로 정렬 유동세포계측법를 시작하기 전에 1퍼센트 BSA / PBS 용액 코트 핵 컬렉션 튜브 (팔콘 15 ML). </li><li> 샘플 및 30 μm의 셀 스트레이너를 통해 부정적인 제어를 필터링하고 (BD의 유입) 먼저 흐름 cytometer에 부정적인 컨트롤을로드합니다. 전방 산란 (FSC), 전방 산란 펄스 폭 (FS 펄스 폭)와 사이드 분산형 (SSC) (그림 2A와 B)을 바탕으로 핵 및 singlets (단일 핵)을 정의하는 첫 번째와 두 번째 게이트를 정의합니다. 샘플로 얼룩의 DNA (DRAQ5 (1:500)를) 추가하면 처음에 핵 인구를 식별하는 데 도움이 될 수 있습니다. </li><li> immunolabeled 샘플을로드 및 비 cardiomyocyte 핵 (PCM-1-음수)에서 cardiomyocyte 핵 (PCM-1-positve)을 분리하기 위해 세 번째 게이트를 정의합니다. 정렬을 시작합니다(그림 2C와 D). </li></ol> 옵션 : 핵의 DNA 콘텐츠 (ploidy)를 분석하고 핵 (예 : Hoechst 33342 또는 DRAQ5) (그림의 2e)에 얼룩 적절한 DNA를 추가 세포주기 분석을 수행하기 위해서는. <ol start="4"><li> 유동세포계측법 정렬 후, 얼음 핵을 배치하고 정렬 순도 (그림 3A와 B)를 확인하기 위해 다시 분석합니다. </li><li> 15 분 대한 냉장 원심 분리기 1500 XG에서 수집 튜브의 정렬된 핵을 봐. </li><li> 다운 스트림 응용 프로그램과 호환 버퍼에 핵 펠렛를 해산. </li></ol> 4. 대표 결과 핵의 형태 및 무결성의 DNA 얼룩에 의해 평가 및 현미경 (그림 1)에 의해 시각하실 수 있습니다. 성공적인 PCM-1이 라벨은 epifluorescence 현미경 의해 유동세포계측법 (그림 1 및 그림. 2C와 d)에 의해 평가됩니다. PCM-1-positi같군과 부정적인 집단이 아니라 (그림 2C와 D) 서로 구분해야합니다. murine 좌심실에서 모든 핵의 약 30 %는 (그림 2D) cardiomyocyte 핵 있어야합니다. 순결을 정렬하는 것은 정렬 핵 (그림 3A와 B에게) 다시 분석하여 평가 할 수 있습니다. 두 핵의 인구는 95 %를 초과 정렬 순결이 있어야합니다. <img alt="그림 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> 1 그림. PCM-1 cardiomyocyte 핵을 식별합니다. 심장 핵 (가) PCM-1 (B) 및 성인 쥐 심장에서 Nkx2.5 (C)에 대한 항체 물들일 수 있습니다. (D) PCM-1-라벨이 핵은 cardiomyocyte 세포질 (마이 오신 중쇄 (MHC))으로 둘러싸여 있으며, PCM-1 염색법 (스케일 바, 20 μm의 및 10에 의해 cardiomyocyte 핵의 정확한 신원을 문서화, 전사 인자 Nkx2.5를 표현하고 있습니다 μm의 (D, 삽입된 페이지)). (E) DNA와 시각 심장 핵의 분리는 DRAQ5에 얼룩. (F와 G) Cardiomyocyte 핵은 PCM-1 (스케일 바 10 μm의)에 대한 항체로 분류하고 있습니다. 참고 조직 섹션에 myocyte 핵 및 절연 핵 (화살표)에서 PCM-1 epinuclear 염색법 패턴. <img alt="그림 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> 그림 2. cardiomyocyte 핵의 흐름 cytometric 정렬. () 심장 핵은 전방 산란 (FSC)와 사이드 분산형 (SSC)에 의해 식별됩니다. (b)는 두 번째 게이트는 FSC와 FS 펄스 폭 5에 하나의 핵을 식별합니다. (C, D) 형​​광 게이팅은 심장 조직에서 cardiomyocyte 핵의 분리 (PCM-1-양성) 및 비 cardiomyocyte (PCM-1-음수)이 핵 수 있습니다. (마) 마우스 cardiomyocyte이 대부분 (&gt; 80 %) diploid (2N)이며, 오직 작은 부분 집합은 (4N) 6 tetraploid입니다. 참고, 인간 cardiomyocytes는 polyploidy 핵의 높은 주파수 (&gt; 2N) 7,8가 포함되어 있습니다. <img alt="그림 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <stroNG&gt; 그림 3. 정렬 cardiomyocyte 및 비 cardiomyocyte 핵의 순도 분석. 정렬 비 cardiomyocyte ()와 cardiomyocyte 핵 (B)의 재 분석. 두 인구 99 %를 초과 정렬 순결을 보여줍니다.

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Vis. Exp. (20), e914 (2008).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

cardiomyocyte 핵의 정확한 신원은 myocardium 2,3의 재생 과정의 분석을 위해 매우 중요합니다. 신선한 조직에서 cardiomyocytes를 분리하기위한 종래의 기술은 주로 세포외 기질 단백질의 소화 효소 및 저속 원심 분리에 의한 간질 세포에서 이후의 정화를 기준으로합니다. 배아 줄기 세포 (ESC)에서 생활 cardiomyocytes의 자세한 정화는 같은 SIRPA 9 또는 mitochondrial 염료 10 표면 마커와 immunolabeling하여 수행할 수, 고정 cardiomyocytes 같은 마이 오신 중쇄 (MHC) 또는 같은 핵 단백질로서 세포질 마커에 의해 확인할 수 있습니다 GATA4 또는 Nkx2.5. 그러나, Nkx2.5과 GATA4의 표현은 성숙 cardiomyocytes에 국한되지 않고, 또한 개발 도중 심장 경색 11,12 후에 전구 세포에서 감지가 가능하다. cardiomyocytes을 식별하는 유전자 전략은 가장 높은 특이성하지만, C를 표시annot은 대형 동물 모델이나 사람 1에 적용됩니다. 기존 전략의 한계는 증식, apoptosis와 chimerism 1,13 등 희귀 사건의 부량에 의존 논란이 결론을 이끌어있다. 따라서 성숙한 차별 cardiomyocyte의 핵을 파악하고 정화 정확한 전략을 수립하는 것이 필요가있다. 여기서는 pericentriolar 자료 1 (PCM-1)을 기반으로 정화 기법을 설명합니다. 이전에, 우리는 세 가지 독립적인 핵 마커, PCM-1, 심장 트로포닌 T와 나는 같은 cardiomyocyte 인구를 식별하는 것을 보여줌으로써 우리의 핵 절연의 유효성을 강화. 2,3 정화 인구가 cardiomyocyte 특정 유전자 제품의 높은 농축을 보여줬 등 심장 troponins, 마이 오신 중쇄 단백질과 전사 인자 Nkx2.5과 GATA4 2,3 등. PCM-1을 기반으로 현재의 프로토콜은, 성공적으로 F를 적용되었습니다혹은 인간 및 마우스 cardiomyocyte 매출 2,4뿐만 아니라 cardiomyocyte transcriptional 프로파일 2의 분석의 분석. 우리는 제시된 전략은 또한 다른 종의에서 cardiomyocytes의 고립 적응 수 있다고 기대하고 있습니다. PCM-1, centrosome 단백질은 그것이 누적 핵 막에 다시 localizes만이 차별화된 myocytes 2,14 (그림 1)에서 불용성 매트릭스를 형성합니다. 유동세포계측법는 apoptosis와 세포주기 재입국 15 cardiomyocytes에서는 드문 이벤트를 계량하는 데 필요한 세포의 높은 숫자의 급속한 편견 분석이 가능합니다. 또한, 염기 analogues는 (예 : BrdU) 기반의 확산 assays는 쉽게 우리의 제시 방법 4를 사용하여 적용할 수 있습니다. 우리의 기술은 단일 cardiomyocyte의 핵 (원자력 ploidy)의 DNA 내용이 중요한 정보를 제공 multinuclear cardiomyocytes 16,17, 분석할 수있다는 장점이 있습니다cardiomyocyte 중복 및 polyploidisation 6-8 구별합니다. 그러나 우리의 전략의 성격으로 인해, cardiomyocyte multinucleation의 특성화는 불가능합니다. 제시된 방법의 추가 응용 프로그램은 epigenetic 염색질의 변화, nucleoproteins, protein-DNA/RNA 상호 작용와 핵 transcriptome 18,19에 초점을 맞춘 연구입니다. 때문에 동결 - 해동 조직의 핵의 안정성, 제시 핵 격리 전략도 biobanks에서 보관된 자료를 활용하는 것이 가능하게, 냉동 조직에 적용할 수 있습니다.

<table border="1"><tbody><tr><td> 1. 용해 완충액 </td></tr><tr><td> 시약의 이름 </td></tr><tr><td> 0.32 M의 자당 </td></tr><tr><td> 10 MM 트리스-HCL (산도 = 8) </td></tr><tr><td> 5 MM CaCl 2 </td></tr><tr><td> 5 밀리미터 마그네슘 아세테이트 </td></tr><tr><td> 2.0 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산) </td></tr><tr><td> 0.5 MM EGTA </td></tr><tr><td> 1 ㎜ DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. 자당 버퍼 </td></tr><tr><td> 시약의 이름 </td></tr><tr><td> 2.1 M의 자당 </td></tr><tr><td> 10 MM 트리스-HCL (산도 = 8) </td></tr><tr><td> 5 밀리미터 마그네슘 아세테이트 </td></tr><tr><td> 1 ㎜ DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. 핵 저장 버퍼 (NSB 플러스) </td></tr><tR&gt; <td> 시약의 이름 </td></tr><tr><td> 0.44 M의 자당 </td></tr><tr><td> 10 MM 트리스-HCL (산도가 = 7.2) </td></tr><tr><td> 70 MM KCl </td></tr><tr><td> 10 밀리미터 MgCl 2 </td></tr><tr><td> 1.5 MM spermine </td></tr></tbody></table><table border="1"><tbody><tr><td> 시약 및 장비 </td><td> 회사 </td></tr><tr><td> Isotype 토끼 IgG 칩 학년, # ab37415 </td><td> Abcam </td></tr><tr><td> 토끼 반 PCM-1 항체, # HPA023374 </td><td> 아틀라스 항체 </td></tr><tr><td> 돈키 초. 항체, 안티 토끼 알렉사 488 형석, #-21206 또는 이와 동등한 초. 형광 항체 </td><td> 생명 기술 </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> 세포 strainers 30 μm의, 70 μm의 및 100 μm의 </td><td> BD Biosciences </td></tr><tr><td> 유리 douncer (40 ML)과&quot;L&quot;을 유봉 </td><td> VWR (Wheaton의 공업 주식 회사) </td></tr><tr><td> T-25 울트라 Turrax 균 질기 </td><td> 이카 독일 </td></tr><tr><td> 공구 S25 N-18 G를 분산 </td><td> 이카 독일 </td></tr><tr><td> Beckman 아벤티의 원심 분리기 </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td></tr><tr><td> 팔콘 튜브 15 ML 50 ML </td><td> VWR </td></tr><tr><td> Beckman의 원심 분리기 튜브 # 363664 </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td></tr><tr><td> JS13.1 무료 스윙 로터 </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td></tr><tr><td> 유입의 cytometer </td><td> Beckman 보습 바로 앞에 달린 풀베는 날 </td></tr><tr><td> 튜브 회전 </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

대부분의 전략에만 세포질 마커 단백질 1에 의존로 cardiomyocyte 핵의 식별은 조직 섹션에 도전했습니다. 이러한 증식과 apoptosis와 같은 심장 myocytes에서는 드문 행사 심장 myocyte 핵의 정확한 신원은 항상성 및 병리 학적 조건 2의 세포 갱신을 분석해야합니다. 여기서는 pericentriolar 자료 1 (PCM-1)와 후속 유동세포계측법 정렬에 대한 항체가있는 밀도의 침강 및 immunolabeling으로 해부 조직에서 cardiomyocyte 핵을 분리하는 방법을 제공합니다. 이 전략은 신선한 조직 및 냉동 보관 물질에 동일하게 작업의 장점과 높은 처리량 분석 및 절연을 허용합니다. 이렇게하면 이미 biobanks에서 수집한 자료를 공부할 수 있습니다. 이 기술 적용과 종의 광범위한 테스트와 같은 탄소-14 데이트 3 셀 - 사 등 여러 다운 스트림 애플 리케이션에 적합하다CLE 분석 4, thymidine의 analogues의 시각화 (예 : BrdU와 이두) 4, transcriptome 및 epigenetic 분석.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

심장 핵​​은 밀도 침강 통해 격리 및 유동세포계측법 의해 cardiomyocyte 핵 파악하고 정렬하는 pericentriolar 소재 1 (PCM-1)에 대한 항체로 immunolabeled있다.

사후 조직에서 Cardiomyocyte 핵의 분리

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Nanyang Technological University

Research

JoVE Journal

Medicine

16.9K 조회수.  University of Lund. 심장 핵​​은 밀도 침강 통해 격리 및 유동세포계측법 의해 cardiomyocyte 핵 파악하고 정렬하는 pericentriolar 소재 1 (PCM-1)에 대한 항체로 immunolabeled있다.

비디오: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

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 ...

연구

의학

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

전체 조직과 조직 검사에서 인간 상피 Enteroids과 Colonoids 설립

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

격리 및 프로파일 링 마이크로 RNA-포함하는 인간의 담즙에서 엑소 좀을

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

샘플링 전략 및 돼지 생물 의학 모델에서 Biobank 조직 샘플의 처리

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

기간 Placentae의 태아 세포 막에서 기본 인간 Decidual 세포의 고립

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

성인 마우스에서 심방 근세포의 격리

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

광유전학적으로 변조된 심근세포 활동의 전기기계적 평가

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

허혈성 심장 질환 및 심장 근막 수축의 비디오 기반 비교 모델

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

RNA 분석 및 대식세포 표현형에 적합한 인간 내장 지방 조직에서 생존 가능한 지방 세포 및 기질 혈관 분획 분리

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...