Name: a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy
Uploaded: 2015-04-30T15:00:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

The authors have no acknowledgements.

주 : 동물의 사용과 관련된 모든 절차는 로컬 입법에 따라 실시된다. 우리의 작업은 시드니 지역 보건 지구 동물 복지위원회에 의해 승인됩니다. 허가 절차는 프로젝트 라이센스 문서에 설명 된 모든 시간에 동물의 복지를 보장 지침을 준수하고 있습니다. 작업이 수행되는 나라의 동물 실험에 법령 준수를 확인합니다. 1. 프로토콜 EM 정착액을 준비하기위한 <ol><li> 정착액 100 ㎖를 들어, 65 ºC로 삼각 플라스크에 증류수 25 ml의 열에 파라 포름 알데히드 분말 2g을 추가 자석 교반기로 일정하게 교반 파라 포름 알데히드를 준비합니다. 참고 : 글루 타르 알데히드, 파라 포름 알데히드 나트륨 cacodylate 버퍼가 모든 유해 물질이며 항상 fumehood 처리해야합니다. </li><li> 다음 냉각 할 수 있도록 65 ºC 분 2에서 열. 필요한 경우 수산화 나트륨 용액을 추가 dropwISE는 완전히 솔루션을 취소 교반하면서. 참고 : 수산화 나트륨이 위험합니다, 취급에주의. </li><li> EM 등급 스톡 글루 타르 알데하이드 10 ㎖, 50 0.2 M 나트륨 cacodylate 버퍼 ml의 자당의 2g을 추가하고 자석 교반기로 저어. 1.0 M CaCl2를 2 ㎖를 추가합니다. </li><li> 증류수 100 ml의 용액을 만들기 이전에 pH를 7.4로 조정한다. 최종 정착은 2.5 % 글루 타르 알데히드, 2 % 파라 포름 알데히드, 2 밀리몰 염화칼슘 2, 0.1 M cacodylate 버퍼에 2 % 자당이 포함되어 있습니다. 총 삼투압이 440 mOsmol / L 있는지 확인하십시오. 준비 24 시간 내에 사용하십시오. </li></ol> 간 2. 관류 고정 <ol><li> 37 ºC - 35 따뜻한 관류 버퍼와 정착액. 방혈 버퍼와 정착의 정확한 온도는 조직의 무결성을 보장하는 데 도움이됩니다. </li><li> 50 밀리그램 / kg 케타민, 5 밀리그램 / kg 자일 라진 단일 복강 내 주사하여 동물을 마취. </li><li> 동물하에되면뒷다리의 철수와 꼬리 핀치에 의해 평가 깊은 마취는, Y 절개 간 포털 정맥을 노출 동물의 복부에 무딘 종료 가위로 이루어집니다. 참고 :이 장비는 쉽게 현미경에서 볼 수됩니다 미생물 및 일반 쓰레기와 시료의 오염을 방지하기 위해 깨끗해야합니다. </li><li> 다른 더 원심 간으로, 간으로 포털 정맥 주위에 한 근위 두 개의 매우 느슨한 봉합을 묶어. </li><li> 캐 뉼러를 고정 .Tighten 봉합사 (쥐 래트 18 G 22 G) 적절한 크기의 IV 캐 뉼러와 문맥을 Cannulate. </li><li> 인산염 완충 식염수로 관류를 시작 37 ºC로 가열하고, H 2 0 10 cm의 압력. PBS 5 내지 20㎖의 보통 간에게서 피를 뽑다 것이 요구된다. 참고 : 기포들이 색전술과 압력 차 유물을 유도으로 정맥에 연결된 튜브를 통해 간을 입력 할 수 있도록하지 마십시오 간세포 손상, SI관류 압이 너무 높으면 nusoids 및 LSEC의 fenestrations 발생한다. </li><li> 복부 및 흉부 대정맥은 버퍼가 간에서 자유롭게 종료 할 수 있도록 절단. 이 LSECs 높은 배압 손상을 방지 할 수 있습니다. </li><li> 간은 혈액의 무료되면, EM 정착액으로 PBS를 교체하고 간 경화 매우 창백한 될 때까지 약 5 분 동안 관류. </li><li> 관류를 중단하고 1로 고정 간을 잘라 - 날카로운 메스를 사용하여 2 mm의 3 블록. </li><li> 4 ºC에서 72 시간 - 24 EM 정착액의 포스트 수정 조직. 참고 : 일반적으로, underfixation는 overfixation보다 더 많은 유물이 발생합니다. </li><li> 4 ºC에서 저장을위한 0.1 M 나트륨 cacodylate 버퍼 후 수정 기간 다음 정착액 변경합니다. 참고 :이 방법으로 저장 시편 (최대 12 개월) 시간의 상당한 기간, 그러나 적절한 보조 고정 보존되며, 시험은 최상의 결과를 권장합니다. </li></ol> 3. 니들 Perfusion 참고 : 니들 고정 직접 생검 간 조직에 정착의 주입을 포함 관류 고정의 변종이다. 정착액 그들을에게서 피를 뽑다 및 정착액하는 조직의 모든 블록을 노출하기 위해 혈관을 플러시하는 목적이다. 케어는 압력 부상 (18, 19)을 피하기 위해 낮은 주입 압력을 유지하기 위해주의해야합니다. <ol><li> 동물이나 피사체 간의 작은 조각을 제거하고 혈액과 이물질을 제거하기 위해 식염수에 헹군다. </li><li> 미세 게이지 바늘 (일반적으로 인슐린 주사기)를 장착 주사기로 생리 식염수를 그립니다. </li><li> 혈액이 조직에서 플러시 될 때까지 간으로 매우 천천히 식염수를 주입한다. 여러 주사가 필요할 수 있습니다. </li><li> 미세 게이지 바늘 (일반적으로 인슐린 주사기)를 장착 다른 주사기로 EM 정착액을 그립니다. </li><li> 이 단단하고 색 황갈색이 될 때까지 간으로 매우 천천히 정착액을 주입한다. 다중 분사S가 필요할 수 있습니다. </li><li> 날카로운 메스를 사용하여 2 mm의 3 블록 - (1)에 고정 간을 잘라. 72 시간 4 ° C의 - 24 EM 정착액에 품어. 차 고정을 시작하기 전에 0.1 M 나트륨 cacodylate 버퍼에 3 회 반복한다. </li></ol> 주사 전자 현미경 4. 준비 <ol><li> 워시 샘플 0.1 M 나트륨 cacodylate 버퍼에 3 회. 지질를 해결하려면 포스트는 2 시간 동안 0.1 M 나트륨 cacodylate 버퍼에 2 %의 산화 오스뮴의 샘플을 수정합니다. 참고 : 글루 타르 알데히드와 오스뮴 테트 록 사이드 크로스가 반응 SEM에 인공물을 생성 완전한 세척은 매우 중요합니다. </li><li> 탈수 개시 에탄올 농도 증가에 샘플을 헹군다. 5 분 동안 50 % 에탄올에서 우선 린스; 70 % 에탄올로 한 후 5 분 동안 3 회; 90 % 에탄올로 5 분간 3 회 씻어; 100 %의 에탄올을 5 분 동안 2 회 씻어; 최종적으로 100 % 에탄올 (분 자체)에서 10 분 동안 2 회 헹군다. </li><li> 샘플에서 모든 에탄올을 제거<html> 및 fumehood에 헥사 메틸 디 실라 잔 (HMDS)로 교체하고 10 분 동안 둡니다. 조직 샘플에서 HMDS를 제거하고 증발 할 수 있습니다. 주 : 따라서 최종 탈수 공정에 사용하기 전에 RT에 도달하도록 냉간시 HMDS 높은 흡습성이다. </li><li> 선택적으로, 즉시 샘플을 장착하거나 SEM 마운트 할 준비가 될 때까지 데시 케이 터에 배치합니다. </li></ol> 5. 장착 참고 : 올바른 표본 장착이 현미경에서 분석 할 수 명확하게 묘사 사인 곡선의 수를 최대화합니다. <ol><li> 금속 현미경 스텁의 기본 레이블과 스텁의 상단에 양면 전도성 카본 테이프를 적용합니다. </li><li> 후속 SEM위한 최선 정현파와 표면을 식별하기 위해 해부 현미경을 사용하여 샘​​플을 시각화. 그루터기의 탄소 테이프 표면에 위쪽으로 그 표면을 놓습니다. </li><li> 스틱 표본 단단히 그루터기에, 표본 지금 스퍼터 코팅에 대한 준비가되어 있습니다. </li></ol>ove_title &quot;&gt; 6. 코팅 참고 : 스퍼터 코터 경내에서 표본을 전도성 금속 (금 또는 백금)의 벌금 필름 시편 코팅 및 전자 빔의 손상으로부터 보호한다. 코팅이 경우 관심 너무 두꺼운 구조 모호 할 수있다. <ol><li> 자동 미세 스퍼터 코터의 진공 챔버에 배치 할 스텁은 45 초 동안 회전 모드와 자동 스퍼터 코팅을 설정합니다. 그러나 금도 적합, 미세한 디테일에 대한 백금 코팅을 사용합니다. </li><li> 마운트 된 간 표본의 외부 표면에 탄소 페인트의 얇은 층을 적용 코트가 아닌 사인 표면을 돌보는. </li><li> 시험편을 백금으로 코팅 탄소 페인트가 건조되면 그들은 현미경에서 관찰을위한 준비가되어 있습니다. 샘플은 데시 케이 터에 저장 될 수있다. </li></ol> 7. 스캐닝 전자 현미경을 사용하여 <ol><li> 장소는 주 사형 전자 현미경으로 표본을 현미경으로 진공을 반환. &lt;/ 리&gt; <li> 낮은 배율에서 시작하여 시험편의 시각화를위한 현미경 표준 작동 절차를 사용하여, 서서히 배율을 증가시킨다. </li><li> 현미경 거리, 장소의 크기와 난시를 작업에 맞게 조정되어 있는지 확인합니다. </li><li> 고정 무결성을 확인합니다. LSEC이 틈에서 대부분 무료이며, 30 측정 fenestrations 포함되어있는 경우 - 250 nm의 직경이 일반적으로 그 시편 준비는 성공적이었다 나타냅니다. </li><li> 정현파는 간극의 전체 또는 fenestrations없는 경우 표본 준비 성공되지 않았거나 중요한 병리학있다. 이 경우, 매우 미세한 면도날 샘플을 슬라이스하고, 절단면을 재 도장 성공적 고정 정현파 분석. </li><li> 약 15,000-20,000 × 배율 좋은 고정 및 fenestrations 명확하게 (볼 수 있습니다 경우 일반적으로 적어도 10 fenestrations를 포함와 파편의 무료 평면 LSEC 표면을 선택). </li><li> 간 당 적어도 두 개의 블록을 사용하여, 블록의 다른 영역에서, 간 당 적어도 10 정현파 이미지를 저장한다. 15,000에서 10 현미경을 샘플링 - fenestrations의 정량은 전체 간을 대표하는 동물 20,000 × 배율은 일반적으로 충분합니다. </li></ol> 8. 분석 참고 : NIH로부터 다운로드 할 수없는 ImageJ에 소프트웨어는 창호 직경 및 주파수 (정량화하기 위해 이용된다 <a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij을</a> ). <ol><li> 이미지 열기 J 및 사진 (화면 1 샷)에 포함 된 스케일 막대를 사용하여 스케일을 설정합니다. </li><li> 유창하고 defenestrated 지역을 포함 LSEC의 전체 평면 주변 다각형 ImageJ에있는 도구, 추적을 사용하여. 이 거짓 기공을 팽창하므로 ​​바로 체 플레이트를 추적하지 마십시오. obscuri있을 세포 표면에있는 임의의 부스러기가 큰 조각을 제외NG의 fenestrations. </li><li> , 창호 직경을 측정하는 줄 도구를 선택하여 각 창호의 가장 긴 직경을 통해 라인을 추적하고, 라인을 측정하기 위해 &quot;M&quot;을 누르고, &quot;D&quot;를 영구적으로 사진에 선을 그릴 (그릴). 이 라인은 창호 직경으로서 정의된다. 줄의 길이는 지금 ImageJ에 결과 상자 (화면 2 샷)에 사용할 수 있습니다. </li><li> 큰 250 nm를 초과 LSEC 세포질 구멍뿐만 아니라 fenestrations 모든 갭을 측정한다. 간격은 종종 격차는 질병과 독성의 결과로 발생할 수 있지만 가난한 관류 또는 고정을 반영하는 유물이다. 간격에 대한 데이터는 나중에 분석 창호 파라미터 계산에서 제외된다. </li><li> 원형하지 않은 갭 영역은 간극 주위 추적 및 ImageJ에 그들의 면적을 계산에 의해 산출한다. </li><li> furthe에 대한 Excel 스프레드 시트로 ImageJ에의 결과 상자에서 데이터를 붙여 넣기R 분석. </li></ol> 9. 계산 <ol><li> (간격이 ≥ 250 nm의 아르 격차, 포함하지 않음) 모든 창호 직경의 평균 창호 직경 = 평균. </li><li> R, 반경, 개별 fenestrations 직경 (R = D / 2)을 포함하지 않는 갭으로부터 계산된다 창호 면적 = πr 2. </li><li> 다공성 (%) = (Σ (πr 2) / 분석 전체 면적 - 갭의 Σ (영역 =)) (μm의 2)) × 100. </li><li> 창호 주파수 = fenestrations의 총 수 / (전체 면적 (갭 영역) (μm의 2) - Σ 분석했다. </li></ol> 창호 데이터의 프리젠 테이션 (10) 주 : 창호 데이터 정량화를 포함한 가능한 한 간행물은 다음과 같은 정보를 포함해야 <ol><li> 사용 경계 직경은 일반적으로 (fenestrations을 정의하는 것을 확인하는 문 창호 직경,창호 주파수 (수 / μm의 2) 및 다공성 (%)) 나노 50-250 사이. </li><li> 갭이 분석 창호 직경 간, 블록, 이미지 및 fenestrations 수의 도수 분포 그래프 분석에 포함되어야에 포함되었는지 여부를 확인하는 문. </li></ol>

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The authors have nothing to disclose.

정확하고 재현성 간 정현파 내피의 상태를 측정하는 기능이 고도로 전문화 된 세포의 생물학을 이해하는데 중요한 단계이다. 구조화 조명 현미경 (32), 원자력 현미경 (33) 및 D-STORM (직접 확률론 광학 Reconstrucion 현미경) (34) 같은 새로운 기술은 시험 관내에서 이러한 세포의 형태에 관한 중요한 정보를 부여하지만 SEM 시각화하고 자신의 구조를 측정하는 주 방?...

간 사인 내피 세포 (LSECs)는 매우 간 사인 곡선의 벽을 일렬로 차별화 된 내피 세포 수 있습니다. LSECs가 아닌 diaphragmed 있습니다 fenestrations으로 천공되어, 세포 횡단는 직경이 50 ~ 250 nm의 모공. LSECs의 표면의 20 %까지 체 플레이트 1-3 (도 1)라고 수십 내지 수백 그룹 보통 fenestrations 의해 덮여있다. Fenestrations는 고효율 한외 여과 시스템을 구축, 및 혈장 혈액 간세포 nanosubstrates 사이의 전송을 허용한다. Fenestrations 동적 구조입니다 - 크기 및 / 또는 번호가 모두 다양한 생리 학적 상태, 약물, 질병에 대한 응답으로 변경 될 수 있습니다. 예를 들어, fenestrations는 공급 상태 4보다 금식에 큰; 세포 노화와 여러 질병 상태 7-13에서 발생 당 5, 6 및 크기의 감소 및 fenestrations 수 2 디 iodoamphetamine는 창호 수를 증가 </sup&gt;. fenestrations의 크기 및 수의 정확한 측정 LSEC 모폴로지는 다양한 질환, 독성, 및 생리적 상태에 의해 영향을받는 방법을 이해하는 것이 중요하다; 간 기능에 미치는 영향; 개발에 대한 치료 적 개입 1 창호를 변조. fenestrations의 연구는 어렵다. 그대로 간 조직 또는 배양 LSECs 모두 이전에는 관찰하여 전자 현미경이 가능했습니다 있도록 fenestrations의 직경은, 기존의 광학 현미경의 해상도 아래에 자리 잡고 있습니다. 주사 전자 현미경 (SEM)을 자주, SEM은 수천 내피 표면 및 측정의 큰 영역의 관찰이 가능하기 때문에 창호의 크기, 주파수 및 공극률 (fenestrations 의해 천공 LSEC 막의 백분율)를 연구하기 위해 사용되어왔다 하지 수만 fenestrations 수천의 경우. 그 유틸리티에도 불구하고,에서보고 된 결과에 대한 연구를 SEM을 기반이러한 창호 크기, 수, 빈도와 같은 다공성 LSEC 파라미터는 문헌 (표 1)에서 광범위하게 다양하다. Fenestrations 및 체 플레이트 계약, 휴식 팽창 또는 표본 준비 중 합체 깨지기 쉬운 구조입니다, 따라서 신중한 처리를 자신의 무결성을 유지하기 위해 필요합니다. 상승 관류 압 (14); 정착 및 버퍼 (15)의 잘못된 삼투압; 부적절한 고정 또는 고정 시간; 및 사후 고정 탈수 및 건조 속도는 미세 구조의 보존을 방해 유물 (그림 2)을 생성 할 수 현미경에 대한 처리의 모든 영역입니다. fenestrations 손실 ( &#39;defenestration&#39;) 및 창호 수축 저감 창호 직경 셀 다공성 결과 불량한 고정의 결과로서 발생할 수있다. SEM 분석을위한 시료의 보존을 개선하기위한 방법은 앞에서 설명 15-17되었고 논의 될여기에 표본 보존을 개선하는 방법에 대한 팁. 시험편 보존 주요 목표는 LSEC의 표면이 가시화 될 수 있도록 정현파에서 혈액을 제거하고, 고압 또는 지연 고정 하나에서 LSEC 손상을 방지한다. 포털 정맥을 통해 정착의 항목 간 관류 간 고정을 위해 선호하는 방법입니다. 상세하게 설명한 바와 같이 다른 곳에서 16, 18 관류가 낮은 압력에서 수행되어야한다 (예를 들면 H 2 0 10cm) LSEC에 압력 관련 관류 유물과 손상을 방지하려면, 일반적으로 세포막에 내 큰 격차를 나타내. 다른 19 상세히 설명하지만, 적절한 정착 종종, 사람과 동물에서 간 생검 바늘 관류를 사용하여 얻을 수있다. 혈액 샘플에서 플러시 될 때까지이 방법은 조직으로 직접 주사를 포함 고정액과 조직은 견고하고 고정된다. 전자 현미경 샘플의 고정이 반환 한 할 필요가간은 매우 신속한자가 분해 공정의 결과로서 발생하는 미세 구조적 변화를 방지하는 혈류의 중단 다음 최대한 빨리 ORMED. 또한 인공물의 포함을 최소화하고 fenestrations의 측정을 표준화 이미지 분석의 방법을 제시한다. 다공성 및 창호 주파수 현미경에 대한 사인 곡선의 선택의 변화, 인공물의 이미지 분석, 셀 면적의 측정은 발표 결과에 큰 차이를 주도했다. 평가 및 fenestrations 및 데이터 프리젠 테이션에 대한 최소 요구 사항을 측정하는 표준화 된 접근 방식은 분명히 이전 4,10,20-31 문헌에 언급되지 않았다.

<table><tbody><tr><td>25% EM grade Glutaraldehyde</td><td>ProSciTech</td><td>C001</td><td>Store stock at -20 C until needed, avoid refreeze</td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma Aldrich</td><td>158127</td><td>Always prepare Paraformaldehyde fresh</td></tr><tr><td>Sodium Cacodylate powder</td><td>Sigma Aldrich</td><td>C0250</td><td>Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O, used mostly at 0.1 M by preparing 1:2 dilution</td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>C1016</td><td>Prepare 1 M CaCl2by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Osmium tretroxide</td><td>ProSciTech</td><td>C011</td><td>Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td></tr><tr><td>Ethanol- Absolute</td><td>Sigma Aldrich</td><td>459836&amp;nbsp;</td><td>100 % Ethanol must be high grade and stored with Molecular Sieve</td></tr><tr><td>Other grades of Ethanol</td><td>Labtech</td><td>EL5</td><td>Prepare graded Ethanols with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma Aldrich</td><td>52619&amp;nbsp;</td><td>Allow to reach room temperature before use</td></tr><tr><td>Cannulas</td><td>Terumo</td><td>TSROX1832C, TSROX2225C, TSROX2419C</td><td>18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td></tr><tr><td>Conductive Carbon tape</td><td>ProSciTech</td><td>IA0201</td><td></td></tr><tr><td>Carbon Paint</td><td>ProSciTech</td><td>I003</td><td></td></tr><tr><td>Ketamine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Xylazine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Molecular Sieve</td><td>Sigma Aldrich</td><td>208647</td><td>Removes water from the 100 % Ethanol</td></tr></tbody></table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

주 사형 전자 현미경 저배율에서의 초기 시각화는 많은 간 큰 용기 및 정현파 (도 1a)을 관찰하기에 충분히 큰 노출 영역 간 시편의 평평한 표면을 보여준다. 장착 스텁에 올바른 간 블록 배치를 보장하는 것은 이러한 이유 때문에 (그림 1B)를 위해 피해야한다 사인 곡선과 간 Glisson의 캡슐의 선명한 이미지를 얻기 위해 필수적이다. 배율을 높이면 간 조밀 맥관계의 가까이 ?...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

주사 전자 현미경으로 간 사인 내피 세포의 분석 및 그들의 Fenestrations위한 표준화 된 방법

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

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JoVE Journal

Biology

14.4K 조회수.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

비디오: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

Hepatocytes에서 마우스 간 사인 내 피 세포의 정화 관류

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

연구

생화학

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

제브라피쉬에서 비알코올성 지방간 질환 관련 간세포암의 초기 진행 중 간 미세환경 분석

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

암 연구학

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

전단 응력의 인간 간 사인 내피에서 조건에 백혈구 모집을 연구하는 흐름 접착 분석

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

면역학 및 감염병학

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

간암 조직에서 유래한 작은 세포외 소포체의 농축 방법

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

간 재관류에 의한 간 세포의 정화를 얹는 생체내 간 Endocytosis에서

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

생물학

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

본래 동맥의 내피 층에서 산화 질소와 활성 산소의 욕실 얼굴 인식

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

혈관 내피 세포를 직접 관찰하기 위해 En Face 면역 형광 염색사용

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

생물학적 조직 샘플의 직렬 블록-얼굴 스캐닝 전자 현미경 검사(SBF-SEM)

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

마우스 일차 간 정현파 내피 세포의 분리 및 특성화

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...