이 작품은 NIH R01 DK05823 (RGW까지), F32 DK083213 (JWW까지), F30 DK081265 (ALO까지)에 의해 그리고 프레 드와 수잔 Biesecker 소아과 간 센터 (RGW까지)에서 교부금에 의해 재정 지원되었다.

달리 명시되지 않는 한 모든 절차는 상온에서 수행됩니다. 1. 효소 솔루션의 작성 <ol><li> 0.22 μm의 필터를 사용하여 표 1과 살균 필터에 따라 효소 솔루션을 준비합니다. 사용 시간까지 용액 1과 4에 실내 온도 및 솔루션 3 용액 2 ° C를 유지. 분리된 세포와 접촉으로 오는 모든 솔루션과 장비는 살균해야합니다. </li></ol> 2. 재관류 장비 준비 <ol><li> HBSS 마이너스 칼슘 2 + / MG 2 프라임 관류 시스템은 +, 37로 예열되어있는 ° C. 참고 : perfusate 그것이 간장에 도착했을 때, HBSS가 37 ° C.로 설정 물을 욕조에 따뜻하게되어 37 ° C 있는지 확인하려면 그것이 관류 펌프 통과 후, 40 ° C (그림 1) 물 순환 장치 세트에 연결되어 외피 유리 콘덴서를 진행합니다. </li><li> 대한 외과 소독70 % 에탄올로 비커에 ols. </li></ol> 3. 간의 재관류 <ol><li> Nembutal의 intraperitoneal 주사 (적절한 마취를 얻을 필요에 50-100 밀리그램 / kg 체중)에 의해 성인 남자 Sprague-Dawley 쥐를 마취. </li><li> 플라스틱 트레이에 위로 향한 자세로 동물을 놓고 테이프 트레이에 팔다리를 채운다. </li><li> 70 % EtOH로 복부를 청소합니다. </li><li> 중간선에서 복부의 피부에 작은 절개를합니다. 복부에 큰 U 형 절단함으로써 멀리 근막으로부터 피부를 해부. 복막 구멍을 노출하기 위해 같은 모양을 따라 가위로 복부 근육을 잘라요. </li><li> 부드럽게 오른쪽 복부 내장을 이동이 면봉을 사용하여 포털 정맥을 쉽게받을 수 있습니다. </li><li> 포털 정맥 밑에 둘 멸균 2.0 실크 봉합을 통과하고, 느슨하게 묶어. </li><li> 16-18 게이지 IV 카테터에 포털 정맥을 Cannulate하고 느슨하게 TI를 강화하여 장소에 카테터를 확보이전 단계 (그림 2)에서 에드 봉합. </li><li> IV 카테터 (HBSS 마이너스 칼슘 2 + / MG 2 + 4 ML에 희석 헤파린 5,000 USP 단위 / ML 중 1 ML)를 통해 희석된 헤파린을 주사. 간은 희게한다. </li><li> 관류 시스템에 정맥 주사 카테터를 연결합니다. + 10 분 20 ML / 분 속도로 HBSS 마이너스 칼슘 2 + / MG 2 Perfuse. 혈액 및 perfusate의 배수를 허용하는 간장으로 IVC는 열등 가로로 쪼개다. 복강에서 풀링된 혈액을 기음. </li><li> 0.3 % collagenase 용액 (500 ML HBSS 플러스 칼슘 2 + / MG 2 +의 제 2 형 collagenase 150 MG) 25 분 동안 perfuse로 관류 액을 변경합니다. 이전에 해부하는 복부 플랩로 덮는으로 간 촉촉한 유지. 약 37에서 내부 복부 온도를 유지하기 위해 영역을 통해 복부 플랩 및 위치 가열 램프를 통해 배양 접시 덮개를 올려 ° C. </li></ol> 4. 회의 작성전자 담즙의 나무 <ol><li> 포셉과 복강으로부터 밖 리프팅하여 간을 제거하고 차가운 Leibovitz의 L-15 미디어로 가득 무균 조직 배양 접시에 넣습니다. </li><li> 간 캡슐 보지, 조직 문화 후드에 근무하고 가볍게 포셉으로 떨어져 간 실질 조직을 괴롭혀. 마지막으로 격리된 담즙의 나무로 남아 때까지 실질 조직을 떠나 애타게 계속하면서 Liebovitz의 L-15 미디어로 가득한 여러 요리를 통해 간을 이동합니다. 남은 담즙의 나무가 흰색이지만 간 실질 조직은 색 황갈색이다. </li><li> 페니실린 / 스트렙토 마이신의 10 ML (10,000 IU / ML 페니실린과 10,000 μg / ML의 스트렙토 마이신)로 가득한 50 ML 팔콘 튜브로 절연 담즙의 나무를 놓고, 15 분 동안 얼음에 둡니다. </li></ol> 5. 포털 Fibroblast 분획의 분리 <ol><li> 멸균 50 ML 팔콘 튜브에 담즙의 나무를 놓고 멸균 가위로 말하다. </li><li> 효소 솔의 작은 금액을 추가튜브에 ution # 1 (약 5 ml) 쇼핑하고 슬러리의 일관성에 도달할 때까지 담즙의 나무를 말하다 것을 계속한다. </li><li> 멸균 유리병에 슬러리 하거라. 남은 해결책 # 1 매는 튜브를 씻으 다음 유리병에 넣어서는. 30 분간 100 rpm으로 흔들어으로 37 ° C에서 품어. </li><li> 나일론 메쉬의 단일 레이어 (30 마이크론 기공 크기)으로 덮여 비커를 통해 슬러리를 필터링합니다. </li><li> 효소 용액 # 2와 메쉬를 세척하여 멸균 유리병에 메쉬 위에 남아있는 모든 조직을 전송합니다. 멸균 15cm 배양 접시에 솔루션의 절반 (25 ML)를 붓는하여 시작하십시오. 조심스럽게 메쉬 반전 후, 중심 지역을 훼손하지 않고 메쉬의 가장자리를 개최하여 비커에서 메쉬를 제거하고 메쉬에있는 모든 남아있는 조직을 제거하기위한 배양 접시에있는 솔루션으로 그것을 찍기. 메쉬를 폐기하십시오. 멸균 유리병에 배양 접시에있는 솔루션을 전송합니다. 레마와 페트리 접시를 린스25 ML 솔루션 # 2와 같은 유리병에 전송을 ining. 30 분간 100 rpm으로 흔들어으로 37 ° C에서 품어. </li><li> 그러는 동안, 비커에 여과물을 타고 50 ML 팔콘 튜브에 붓는다. 상온에서 5 분간 1,600 RPM (460 X G)에서 원심 분리기. </li><li> 미디어를 대기음 및 솔루션 # 3의 25 ML에 펠렛을 resuspend. </li><li> 단계 5.5에서 솔루션을 타고 30 마이크론 메쉬로 덮여 두 번째 무균 비커에 그 파일을 필터링합니다. </li><li> 메쉬를 폐기하십시오. 상온에서 5 분간 1,600 rpm으로 여과물을 원심 분리기. 기음과 스텝 5.7로 이루어졌다대로 해결책 # 3의 25 ML과 펠렛을 resuspend. </li><li> 단계 5.7 및 5.9에서이 분수를 결합하고 실온에서 5 분간 1,600 rpm으로 다시 원심 분리기. </li><li> 미디어를 대기음 및 포털 fibroblast 문화 매체의 10 ML (DMEM/F12 10 % FBS, 1 % 페니실린 / 스트렙토 마이신, 0.3 % gentamycin, 및 0.2 % fungizone과 보완)에 펠렛을 resuspend. </li><li> 생존과 조직 문화 요리 접시 세포를 세어보세요. 원하는 응용 프로그램에 따라 10 cm 조직 배양 플레이트 당 0.5-5 X 10 6 세포가 적합합니다. 성인 Sprague-Dawley 쥐에서, 우리는 일반적으로 2-10000000 가능한 세포를 구하십시오. </li><li> 비 점착 성의 잔해를 제거하려면 다음 날 문화 미디어를 교체합니다. 이후, 모든 이일 문화 미디어를 교체하십시오. </li></ol> 6. 대표 결과 성인 쥐의 간 격리 주요 포털 섬유아 세포는 격리 후 1, 3과 7 일 (그림 3)에 표시됩니다. 세포는 섬유아 세포의 형태학 전형으로 늘어나지되는 것을 확인할 수 있습니다. 세포 순도를 확인하기 위해 안티 - 엘라스틴 항체 (CL55041AP, Cedarlane 연구소, 벌링턴, NC) 물들일 수있다. 포털 섬유아 세포는 문화에 myofibroblastic 차별을 받고있다. 이것은 α-평활근의 actin (; A2547, 시그마, 세인트 루이스, 미주리 α-SMA)에 대해 immunostaining에 의해 입증하실 수 있습니다. <table국경 = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> 0.3 % collagenase 용액 </td><td> 2 형 collagenase </td><td> 150 MG </td></tr><tr><td> 칼슘 2 + / MG 2 HBSS + </td><td> 500 ML </td></tr><tr><td rowspan="7"> 해결 방법 # 1 (Pronase) </td><td> 소의 혈청 알부민 </td><td> 50 MG </td></tr><tr><td> Collagenase의 제 2 형 </td><td> 25 MG </td></tr><tr><td> Pronase </td><td> 18 MG </td></tr><tr><td> DNase </td><td> 3 MG </td></tr><tr><td> DMEM/F12 </td><td> 47.5 ML </td></tr><tr><td> 펜 / Strep </td><td> 한 ML </td></tr><tr><td> 태아 소 혈청 </td><td> 1.5 ML </td></tr><tr><td> 해결책 # 2 </td><td> 소의 혈청 알부민 </td><td> 50 MG </td></tr><tr><td rowspan="6"> (Hyaluronidase) </td><td> Collagenase의 제 2 형 </td><td> 25 MG </td></tr><tr><td> Hyaluronidase </td><td> 22 MG </td></tr><tr><td> DNase </td><td> 3 MG </td></tr><tr><td> DMEM/F12 </td><td> 47.5 ML </td></tr><tr><td> 펜 / Strep </td><td> 한 ML </td></tr><tr><td> 태아 소 혈청 </td><td> 1.5 ML </td></tr><tr><td rowspan="2"> 해결책 # 3 </td><td> DNase </td><td> 3 MG </td></tr><tr><td> RPMI 배지 1,640 </td><td> 50 ML </td></tr></tbody></table> 표 1. 효소 솔루션. <img alt="그림 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> 1 그림. 재관류가 설정할 수 있습니다. A. 온난 칼럼을위한 물 순환 장치. B. 칼럼은 재관류 미디어를 포함하는. C. 꼭지는 펌프 유출을 통제. 디 펌프 유입됩니다. E.은 가변 속도 연동 펌프. 바이오 클린 세제 F. 진공 플라스크. G. 히트 램프. 온난 화 재관류 미디어에 대한 H. 워터는 목욕. <img alt="그림 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> Figu재 2. 현장 재관류합니다. A. IV 카테터는 봉합으로 확보하고 꼭지를 통해 관류 튜빙에 연결, 포털 정맥에 삽입. B. 유리 피펫은 가로로 IVC 밖으로 배수 관류 액을 제거하기 위해 진공 플라스크 및 벽면 흡입에 연결. I.의 창자. 기사를 간. <img alt="그림 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> 그림 3. 위상 콘트라스트와 immunofluorescence는 문화에 쥐 포털 섬유아 세포의 얼룩. 격리 후 1, 3, 7 일간 배양해 포탈 섬유아 세포가 엘라스틴, α-SMA 및 DAPI에 대한 고정 및 물들어 있었다.

<ol>
	<li>Parola, M., Marra, F., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblast+-+like+cells+and+liver+fibrogenesis:+Emerging+concepts+in+a+rapidly+moving+scenario.">Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario.</a> Mol. Aspects Med. 29, 58-66 (2008).</li><li>Dranoff, J. A., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+fibroblasts:+Underappreciated+mediators+of+biliary+fibrosis.">Portal fibroblasts: Underappreciated mediators of biliary fibrosis.</a> Hepatology. 51, 1438-1444 (2010).</li><li>Tuchweber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+phenotypic+modulation+of+portal+fibroblasts+in+the+early+stages+of+cholestatic+fibrosis+in+the+rat.">Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.</a> Lab Invest. 74, 265-278 (1996).</li><li>Beaussier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prominent+contribution+of+portal+mesenchymal+cells+to+liver+fibrosis+in+ischemic+and+obstructive+cholestatic+injuries.">Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.</a> Lab Invest. 87, 292-303 (2007).</li><li>Kinnman, N., Housset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peribiliary+myofibroblasts+in+biliary+type+liver+fibrosis.">Peribiliary myofibroblasts in biliary type liver fibrosis.</a> Front Biosci. 7, d496-d503 (2002).</li><li>Kinnman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblastic+conversion+of+peribiliary+fibrogenic+cells+distinct+from+hepatic+stellate+cells+is+stimulated+by+platelet-derived+growth+factor+during+liver+fibrogenesis.">The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.</a> Lab Invest. 83, 163-173 (2003).</li><li>Uchio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+retinol-binding+protein-1+expression+and+modulation+during+in+vivo+and+in+vitro+myofibroblastic+differentiation+of+rat+hepatic+stellate+cells+and+portal+fibroblasts.">Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts.</a> Lab Invest. 82, 619-628 (2002).</li><li>Kruglov, E. A., Jain, D., Dranoff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+rat+liver+fibroblasts.">Isolation of primary rat liver fibroblasts.</a> J. Investig. Med. 50, 179-184 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+and+substrate+stiffness+regulate+portal+fibroblast+activation+in+culture.">Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.</a> Hepatology. 46, 1246-1256 (2007).</li><li>Bosselut, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+proteomic+features+of+two+fibrogenic+liver+cell+populations:+hepatic+stellate+cells+and+portal+myofibroblasts.">Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts.</a> Proteomics. 10, 1017-1028 (2010).</li><li>Dranoff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecto-nucleoside+triphosphate+diphosphohydrolase+NTPDase2/CD39L1+is+expressed+in+a+novel+functional+compartment+within+the+liver.">The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver.</a> Hepatology. 36, 1135-1144 (2002).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

포털 섬유아 세포는 담즙의 섬유증의 중요한 역할을한다. 자, 쥐의 간 포털에서 섬유아 세포를 분리해 체외에서이 세포 인구를 공부하는 간단한 방법을 제공하는 Kruglov 및 Dranoff 8 시까지 발행된 원본 프로토콜의 수정에 대해 설명합니다. 이러한 접근 방식은 테아제 소화와 크기를 기반 여과를 사용합니다. 방법의 주요 장점은 세포의 비교적 순수한 인구가 유전자 발현이나 며칠이 고립 후 기능성 세포 행동을 연구함으로써, 문화의 통로없이 얻을 수있다는 것입니다. 이 기술은 또한 건강하고 병에 걸린 간은 모두에서 세포를 분리의 가능성을 제공합니다. 이 프로토콜의 가장 중요한 단계 중 하나는 간장 실질 조직의 효소 소화입니다. 성공적인 소화를 위해, 그것은 초기 perfu 때에 혈액이 완전히 간 밖으로 빠져나간 것을 확인하는 것이 중요하다심지어는 간 상태가 창백되어 있는지 확인함으로써 시온. 이것을 촉진하기 위해서는 perfusing하면서 부드럽게 간이 마사지하기 위해 면봉을 사용할 수 있습니다. 실행 가능한 세포의 낮은 수확량의 간 실질 조직 결과의 이상 - 소화, 이하 - 소화가 어려운 담즙의 나무에서 간 실질 조직을 분리하는 것입니다 동안. pronase의 준비가 다른 많은, 사용 금액의 최적화 간의 효소 활동의 다양성을 가지고 있기 때문에 가능한 세포의 낮은 수율이있을 때 필요한 경우가 있습니다. 이 프로토콜은, 포털 정맥을 cannulate하는 작은 게이지 IV 카테터를 사용하여 마우스 간 또는 젊은 쥐의 간장에서 포털 섬유아 세포를 분리 동물의 무게에 비례하여 재관류 솔루션의 볼륨을 줄이는 약 10 간 재관류 율을 감소를 위해 수정할 수 있습니다 ML / 분. 문화 포털 섬유아 세포는 α-SMA의 전남편에 의해 입증, 10-14 일 myofibroblastic 차별을 받다pression 9. 각종 마커는 fibulin-2를 포함한 간장 별 모양의 세포, IL-6, 엘라스틴에서 포털 섬유아 세포를 구별하는 데 사용되었습니다 뉴클레오 사이드 triphosphate diphosphohydrolase-2 (NTPDase2), cofilin-1과 같은 synaptophysin 2, 6, 10, 11 등의 연결 단백질 . 우리는 포털 섬유아 세포 며칠 9시 문화받은 후 NTPDase2이 손실되는 동안 엘라스틴은, 심지어 myofibroblastic 분화 이후, 포털 섬유아 세포에 대한 좋은 마커 것으로 나타났습니다. 따라서 일반적으로 엘라스틴을위한 immunofluorescence 염색법에 의해 포털 섬유아 세포의 절연을 확인합니다. 이 기법의 한계는 바로 포털 fibroblast 절연 후, Kupffer 세포 및 담즙의 세포를 포함한 세포를 오염의 작은 분수가라는 점이다. 포털 섬유아 세포는 비교적 순수한 인구 (&gt; 98%) 9하였으며, 그러나, 2-3 일 이내에 이러한 오염 세포를 자라다 것입니다. 시문화 포털 섬유아 세포는 조직 배양 플라스틱에 myofibroblastic 차별을 받다 제정신, 우리는 일반적으로 고립 7 일 이내에 이들 세포를 공부합니다. 방법 섹션에 설명된대로 전지는 10 % 태아 소 혈청을 포함하는 포털 fibroblast 문화 매체에 보관됩니다. 그러나 이러한 세포는 며칠 동안 2% 태아 소 혈청을 포함하는 성장 미디어에서 살아남을 것입니다. 우리는 일반적으로 분리 후 최소 24 시간 전까지는 낮은 혈청 미디어를 사용하지 마십시오. 포털 섬유아 세포가 myofibroblastic 차별을 받다되면, 그들은 여러 번 passaged 및 myofibroblasts의 냉동 주식으로 보관하실 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 2 형 Collagenase </td><td> 생물 워딩턴 (레이크 우드, 뉴저지) </td><td> LS004177 </td></tr><tr><td> Pronase </td><td> ROCHE (인디애나 폴리스, IN) </td><td> 70290120 </td></tr><tr><td> Hyaluronidase </td><td> 시그마 (세인트 루이스, 미주리) </td><td> H3506 </td></tr><tr><td> Deoxyribonuclease </td><td> 시그마 </td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen (Carlsbad, CA) </td><td> 11,320 </td></tr><tr><td> 칼슘 2 + / MG이없는 HBSS + </td><td> Mediatech (매나 사스, VA) </td><td> 21-021-CM </td></tr><tr><td> 칼슘 2 + / MG 2 HBSS + </td><td> Mediatech </td><td> 21-020-CM </td></tr><tr><td> Leibovitz의 L-15 </td><td> Invitrogen </td><td> 11,415 </td></tr><tr><td> RPMI 중형 1,640 </td><td> Invitrogen </td><td> 11,875 </td></tr><tr><td> 소의 알부민 세럼 </td><td> 시그마 </td><td> A2153 </td></tr><tr><td> 태아 소 혈청 </td><td> 제미 나이 바이오 (서부 새크라멘토, 캘리포니아) </td><td> 900-108 </td></tr><tr><td> Nitex 나일론 메쉬 30 μm의 </td><td> Genesee 과학 (샌디에고, 캘리포니아) </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

간 섬유증이 활성화 myofibroblasts에 의한 세포외 기질의 과도한 증착에 의해 정의됩니다. 간장 별 모양의 세포, 포털 섬유아 세포 및 골수 유래 섬유아 세포 하나를 포함하여 간장 myofibroblasts의 여러 엽 성의 전구 물질이 있습니다. 간장 별 모양의 전지는 최고의 공부 줬어하지만 포털 섬유아 세포가 점점 특히 담즙의 섬유증 2, myofibroblast 수영장에 중요한 기여자로 인정됩니다. 포털 섬유아 세포는 잠재적으로 담즙이 3-5 흉터의 초기 단계에서 핵심적인 역할을 놀고, 담즙 상피 부상에 대한 응답으로 확산을 받고있다. 포털 섬유아 세포를 분리하는 방식이 세포 인구의 체외 연구에서 허용하고 포털 섬유아 세포가 담즙의 섬유증에서 재생 역할의 큰 이해로 이어질 것입니다. 포털 섬유아 세포가 돋아 나옴 6, 7, 리튬 등 다양한 기술을 사용하여 격리되어사이즈 선택 8 시까지 다음 효소 소화와 버전 재관류. 가지 기법에 비해 소화와 크기를 선택 기법의 장점은 세포 문화에 통과의 필요성없이 공부를 할 수있다는 것입니다. 여기서는 주된 세포의 비교적 순수한 인구시킨 결과가되는 쥐 간암에서 포털 섬유아 세포의 격리를 위해 Kruglov 및 Dranoff 8 시까지 설명한 원래 기술의 수정된 버전을 설명합니다.

Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

쥐의 간장에서 포털 섬유아 세포를 분리를위한 기법을 설명합니다. 간은는 perfused과 소화하고<em&gt; 현장에서</em&gt; collagenase으로 이어<em&gt; 예 생체내</em간 슬러리와 세포 크기의 선택&gt; 소화. 이 방법은 문화의 통과를 위해 필요없이 포털 섬유아 세포의 순수 인구를 제공합니다.

쥐 포탈 섬유아 세포의 분리에 의한<em&gt; 현장에서</em&gt; 간 재관류

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

isolation-of-rat-portal-fibroblasts-by-in-situ-liver-perfusion

Research

JoVE Journal

Biology

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K 조회수.  The Children's Hospital of Philadelphia. 쥐의 간장에서 포털 섬유아 세포를 분리를위한 기법을 설명합니다. 간은는 perfused과 소화하고 현장에서 collagenase으로 이어 예 생체내 소화. 이 방법은 문화의 통과를 위해 필요없이 포털 섬유아 세포의 순수 인구를 제공합니다.

비디오: Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

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 ...

연구

생물학

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

쥐 절차<em&gt; 현장에서</em&gt; 골격근 수축 등록

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

2 단계 콜라게나 제 관류 절차에 의한 인간의 간세포의 분리

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

성인 쥐 심장에서 고품질 Cardiomyocytes, 내피 세포 및 섬유 아세포의 동시 격리

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

성인 심장 섬유 아세포 및 근섬유 아세포의 격리 및 특성화

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

골격근 섬유아세포(Skeletal Muscle Fibroblast)에서 엑소좀의 분리 및 특성 분석(Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts)

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

다중 파라미터 관류 조절을 통한 원발성 쥐 간세포의 분리

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...