저자는 기술 지원을위한 조시 Basford 박사 샤오 분 리튬 감사드립니다. 이 작품은 NIH 보조금 (DK70992와 ML에 DK92779)에 의해 부분적으로 지원되었다.

1. 준비 모든 버퍼는 갓 무균 기술을 사용하여 준비하고 코닝 0.22 μm의 필터를 사용하여 소독을 필터링합니다. <ol><li> 행크의 균형 소금 솔루션에 다음을 추가하여 전을 버퍼링 재관류 준비 (HBSS, CA이없이 + 및 MG 2 +, 표 1 참조) : 25에 0.5 밀리미터, 그리고 HEPES에 0.9 밀리미터, EDTA (에틸렌 다이아 민 테트라 초산)으로 MG 2 + (MgCl 2) 음. </li><li> (HBSS에 다음을 추가하여 관류 버퍼 II를 준비 칼슘 2 + 및 MG 2 +, 25 MM에 HEPES : 표 1)을 참조하십시오. </li><li> 재관류 버퍼 II 플러스 collagenaseII 준비 : collagenase II (1000 U) 300 ML 재관류 버퍼 II와를 해산하고 재관류 전에 물을 욕조에 솔루션 보온. collagenase II의 활동이 시간과 함께 감소하기 때문에이 솔루션은 30 분 이내에 사용해야합니다. </li><li> 윌리엄의 전체 매체를 준비하려면 다음을 추가; 인슐린 ~ 100 nm의, dexamethasone ~ 100 nm의 페니실린을 100 IU / ML 및 스트렙토 마이신 100 밀리그램 / ML로 L-글루타민이 MM, 태아 소 혈청 (FBS) 5 %까지 : 윌리엄스 &#39;보통 E됩니다. </li><li> 이러한 버퍼는 42에서 물을 욕조에 30 분간 예열해야 ° C, 37 정맥의 콘센트 온도에 대응하는 최적의 온도 ° C.가 </li></ol> 2. 간 절연을위한 쥐 재관류 <ol><li> 관류 시스템은 펌프, autoclavable silastic 관 그리고 물 목욕 (그림 1 참조)으로 구성되어 있습니다. 10 ML / 분 연동 관류 펌프의 미리 설정된 유량. </li><li> intraperitoneal (IP) 케타민 (87 밀리그램 / kg 체중) 플러스 xylazine (13 밀리그램 / ㎏)과 성인 쥐 (300g의 체중)을 마취. 마취 심도는 발가락 핀치에 의해 감시되어야한다. 쥐가 더 이상 유해 자극에 응답하지되면 복부 머리 사립 betadine와 에탄올과 복부 면도. 중간선 절개를 통해 입력합니다. </li><li> 폭로신중하게 복강의 오른쪽 바깥으로 내장을 이동하고, 간장 포털 정맥 (그림 1 참조)에 18 게이지 angiocath을 삽입하여 간장 포털 정맥. </li><li> 바늘로 perfusate 튜빙을 연결하고 사전 예열 (37 ° C) 재관류 버퍼 내로 낮은 유속 (10 ML / 분)에서 원래의 장소에 주입을 시작합니다. </li><li> 제대로 수행한 경우, 간은 즉시 희게하기 시작한다. 성공 cannulation이 확정되면, 하부 대정 맥 (IVC)에서 잘라내기가 유출 (그림 1)을 허용합니다. 성공 cannulation을위한 추가 시험은 IVC에 멸균 면봉으로 가벼운 압력을 적용하여 수행할 수 있으며, 간장의 모든 돌출부가 빠르게 팽창하기 시작한다. </li><li> 25 ML / 분 흐름의 속도를 높이십시오. 간장은 색이 창백 수있게됩니다. </li><li> 추가 육분에 대한 흐름의 중단없이 함께 관류 버퍼 II 플러스 collagenase II에 대한 재관류 솔루션을 전환합니다. </li> &lt;리&gt; 주기적으로 (소화 동안 5-10 번) 5 초 간격 위해 IVC에 면봉으로 압력을 적용합니다. 간은 차례 총 소화 시간을 절감하고, 최종 수확량 증가, 향상된 간장 세포 분열로 이어지는, 팽창할 것입니다. </li><li> collagenase 재관류 후, 간 부드러운보고 시작합니다. 20 ML 윌리엄의 전체 매체와 미리 냉장 무균 비커에 간 무료로 장소를 해부하고 조직 세포 배양 후드로 가져가라. </li></ol> 3. Hepatocyte 세포 격리 <ol><li> 세포 배양 후드 내에서 부드럽게 멸균 배양 접시 내에서 윌리엄의 전체 매체에 세포를 분산하기 위해 셀 스크레이퍼를 사용합니다. </li><li> 결합 조직 및 소화되지 않은 조직 파편을 제거하기 위해 50 ML 원뿔 튜브에 100 μm의 기공 크기의 셀 스트레이너를 통해 세포 분산을 필터링합니다. </li><li> 4 ° C.에서 3 분 50 XG 40 ML 윌리엄의 전체 매체와 원심 분리기로 세포를 일시 중지 </li><li> 뜨는을 대기음, 그리고부드럽게 세포를 씻어 40 ML 감기에 윌리엄의 전체 매체에 세포를 다시 일시 중지합니다. 원심 분리를 반복합니다. </li><li> 뜨는을 대기음, 가볍게 25 ML 윌리엄의 완전한 매체로 다시 중지 세포. 튜브에 PBS에서 25 ML 90 % Percoll 용액을 넣고 가볍게 섞는다. </li><li> 4 ° C.에 10 분 200 XG에 원심 분리기 실행 가능한 세포가 Percoll 기울기의 하단에 남아 있기 때문에 그라데이션의 상단에서 죽은 세포를 기음. </li><li> 30 ML 따뜻한 윌리엄의 전체 매체에 세포 펠렛을 일시 중단하고 원심 분리를 반복 20 ML 따뜻한 윌리엄의 전체 매체에 세포 펠렛을 다시 일시 중지합니다. </li><li> hemocytometer를 사용하여 세포 현탁액 내에서 세포를 카운트하고 trypan 블루 염색법에 의해 세포 생존을 결정합니다. </li></ol> 4. Hepatocyte 문화 <ol><li> 원하는 농도에 대한 따뜻한 윌리엄의 완전한 매체로 세포를 희석, 예를 들어 2.5 × 10 5 세포 / ML. 세포 배양 접시에 원하는 볼륨에서 플레이트 세포, 예를 들어. 5 × 10 5 세포 / 2 ML / 음, 6 웰스 / 판, 또는 2.5 자 × 10 5 세포 / 1.5 ML / 12 웰스 / 접시. 취소 코팅 플레이트는 간장 세포 배양에 대한 벌금입니다. </li><li> hepatocytes가 전체 셀 크기로 성장 및 최종 합류를 반환하기위한 충분한 공간을 유지하면서&gt; 85% 가능한 세포를 가진 전형적인 준비에서 세포 밀도는 세포 - 세포 접촉을 허용 약 60~70%의 합류를 도달한다 90~95%. </li><li> 잘의 중심 영역에서 총체적으로 세포에 대한 경향을 최소화하기 위해, 즉, hepatocytes의도 단일층를 형성하기 위해 (세포 배양기의 공기 흐름 안쪽에 의한 가능성이 높습니다), 접시 안에 남아있게 인큐베이터에 그들을 배치하기 전에 30 분 세포 배양 후드. </li><li> 문화는 37 전지 95 %의 공기 5 % CO 2의 humidified 분위기 속에서 ° C에서. 4-H 문화 후에는 세포도 동일한 혈청 함유 배지에 남아있을이나 혈청-f 옵션과 함께 매체를 교체ree 매체, 예를 들어 HepatoZYME-SFM (표 1 참조). 혈청이없는 매체는 혈청이없는 배지가 사용되는 호르몬로부터 악영향으로 세포 형태를 유지하는 데 도움이됩니다. </li><li> 세포 실험에 대한 사전을 복구하고 적어도 하룻밤 성장하도록 허용합니다. 이것이 중요한 효소 (예, P450s)의 기능을 보존하는 데 도움이 될 수 있기 때문에 우리는 24 시간 이내에 검사를위한 세포를 사용하는 것이 좋습니다. </li><li> 필요한 경우, 2 일 간격으로 성장 매체를 교체합니다. </li></ol> 5. 대표 결과 조건은 정기적으로 쥐를 간부터 준비 당 1.0 X 10 8 세포의 세포 수확을 생성 설명했다. trypan 파랑 제외로 측정 hepatocytes의 생존 능력 88 ~ 96 % 범위 내에서 일관했습니다. 그림 2, 4 시간의 시딩 후 hepatocytes의 집계 및 양식 클러스터에 묘사된. 대부분 절연 세포는 전형적인 단일층 성장에 평평하고 확산. 24 시간으로, 세포의 가장자리가 정의되고, 세포의 표면은 매우 부드럽고이며, 지질 방울은 볼 수 있습니다. 세포 한시 세 세포의 중앙에 위치한 원형이다 nucleoli,,, 그리고 세포 간의 핵 디스플레이 일관성있는 크기 (그림 2)가. <img alt="그림 1" src="/files/ftp_upload/3917/3917fig1.jpg" /> 그림 1. 간 재관류의 도표. 18 게이지 angiocath가 간 포털 정맥에 삽입되는 다음 perfusate 튜브는 바늘에 연결됩니다. 성공 cannulation이 확정되면, 하부 대정 맥 (IVC)에서 잘라내기가 유출 허용합니다. <img alt="그림 2" src="/files/ftp_upload/3917/3917fig2.jpg" /> 그림 2. 시간이 지남에 따라 교양 hepatocytes (4 시간 ~ 24 시간), 배율 X 200 형태론. 24 시간 동안 배양해 후 세포는 전형적인 단일층 성장에 확산하고, 세포 사이의 분기점은 선형입니다. <table bordeR = &quot;1&quot;&gt; <tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> HBSS (CA이없는 이상, MG 2 +) </td><td> Invitrogen </td><td> 14,174 </td></tr><tr><td> HBSS (CA 2 +, 그리고 MG 2 +) </td><td> Invitrogen </td><td> 14,025 </td></tr><tr><td> 윌리엄스의 중간 E </td><td> Invitrogen </td><td> 12551-032 </td></tr><tr><td> Collegenase II </td><td> 워딩턴 </td><td> LS004176 </td></tr><tr><td> 셀 스트레이너 (100 μm의) </td><td> BD </td><td> 352,360 </td></tr><tr><td> HepatoZYME-SFM </td><td> Invitrogen </td><td> 17,705 </td></tr><tr><td> Percoll </td><td> 시그마 </td><td> P4937 </td></tr><tr><td> Angiocath (18 게이지) </td><td> BD </td><td> 381,705 </td></tr></tbody></table> 표 1.

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+a+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study.</a> J. Cell Biol. 43, 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Saito, K., Kobayashi, K., Mizuno, Y., Fukuchi, Y., Furihata, T., Chiba, K., Schmidt, M., Schmitz, H. J., Baumgart, A., Guedon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+alpha+(PPARalpha)+agonists+induce+constitutive+androstane+receptor+(CAR)+and+cytochrome+P450+2B+in+rat+primary+hepatocytes.">Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists induce constitutive androstane receptor (CAR) and cytochrome P450 2B in rat primary hepatocytes.</a> Drug Metab. Pharmacokinet. 25, 108-111 (2010).</li><li>Gomez-Lechon, M. J., Donato, M. T., Castell, J. V., Jover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatocytes+in+primary+culture:+the+choice+to+investigate+drug+metabolism+in+man.">Human hepatocytes in primary culture: the choice to investigate drug metabolism in man.</a> Curr. Drug Metab. 5, 443-462 (2004).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malar. J. 6, 169 (2007).</li><li>Bu, S. Y., Mashek, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+long-chain+acyl-CoA+synthetase+5+mediates+fatty+acid+channeling+between+anabolic+and+catabolic+pathways.">Hepatic long-chain acyl-CoA synthetase 5 mediates fatty acid channeling between anabolic and catabolic pathways.</a> J. Lipid Res. 51, 3270-3280 (2010).</li><li>Aiken, J., Cima, L., Schloo, B., Mooney, D., Johnson, L., Langer, R., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+in+rat+liver+perfusion+for+optimal+harvest+of+hepatocytes.">Studies in rat liver perfusion for optimal harvest of hepatocytes.</a> J. Pediatr. Surg. 25, 140-144 (1990).</li><li>Jauregui, H. O., McMillan, P. N., Hevey, K., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+lectin+binding+to+adult+rat+hepatocyte+cell+surfaces.">A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces.</a> In Vitro Cell. 24, 401-412 (1988).</li><li>Klaunig, J. E., Goldblatt, P. J., Hinton, D. E., Lipsky, M. M., Trump, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+liver+cell+culture.+II.+Primary+culture.">Mouse liver cell culture. II. Primary culture.</a> In Vitro. 17, 926-934 (1981).</li><li>Hay, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operator-induced+contamination+in+cell+culture+systems.">Operator-induced contamination in cell culture systems.</a> Dev. Biol. Stand. 75, 193-204 (1991).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

hepatocytes의 차 문화가 널리 간 생리와 병리의 다양한 측면을 연구하는 데 사용되는 체외 모델입니다. 예를 들어, 주 문화는 P450 cytochromes, 약물 대사, 약물 - 약물 상호 작용 및 세포 독성 및 genotoxicity 3-7의 메카니즘을 포함한 마약 metabolizing 효소의 표현과 기능을 평가하는 데 사용됩니다. 쥐의 간장 세포의 격리와 문화를 설명하는 프로토콜은 8. Aiken 외의 이전 보고서?...

isolation and primary culture of rat hepatic cells

기본 hepatocyte 문화는 광범위 간기능, 질병, pathophysiology, 약리학 및 기타 관련 과목의 기초 연구에 사용되고 유용한 도구입니다. 그대로 hepatocytes의 절연을위한 2 단계 collagenase 재관류를 기반 방법은 먼저 1,969 1 베리와 친구에 의해 소개되었으며, 그 이후 많은 수정 작업을 거쳐왔다. 가장 일반적으로 사용되는 기술은 Seglenin 1,976 2로 설명되었다. 기본적으로 hepatocytes는 포털 정맥을 통해 비 순환 collagenase 재관류에 의한 anesthetized 성인 쥐에서 dissociated 있습니다. 분리된 세포 후 100 μm의 기공 크기의 메쉬 나일론 필터를 통해 여과하고, 접시에 배양해 있습니다. 4 시간 배양 후, 매체는 문화에 대한 추가적인 시간 동안 혈청 함유 또는 혈청 프리 매체, 예를 들어 HepatoZYME-SFM,로 대체됩니다. 이러한 절차는 더 나은 텍스트로보다 영상에 의해 입증하실 수 있습니다 외과 및 무균 배양 단계를 필요로합니다. H오히려, 우리는 다수의 유력한 hepatocytes의 생성에 항상 있도록 비디오 및 서면 프로토콜 모두에서 이러한 절차에 대한 자세한 단계를 문서화.

Watch this Scientific Journal Video about Isolation and Primary Culture of Rat Hepatic Cells at JoVE.com

Isolation and Primary Culture of Rat Hepatic Cells

기본 hepatocytes는 생리학 관련 실험 시스템의 생화학, 분자, 그리고 신진 대사 기능을 평가하는 유용한 도구를 제공합니다. 우리는 지속적으로 to1.0 × 10 백업 실행 가능한 hepatocytes를 생성 원위치 간 재관류에 쥐에 대한 신뢰할 수있는 프로토콜을 설명<sup&gt; 8</sup88 ~ 96% 사이의 세포 생존 능력과 준비를 당&gt; 세포.

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-and-primary-culture-of-rat-hepatic-cells

Research

JoVE Journal

Biology

55.4K 조회수.  University of Cincinnati College of Medicine. 기본 hepatocytes는 생리학 관련 실험 시스템의 생화학, 분자, 그리고 신진 대사 기능을 평가하는 유용한 도구를 제공합니다. 우리는 지속적으로 to1.0 × 10 백업 실행 가능한 hepatocytes를 생성 원위치 간 재관류에 쥐에 대한 신뢰할 수있는 프로토콜을 설명 8 세포.

비디오: Isolation and Primary Culture of Rat Hepatic Cells

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

성인 쥐 심장 Myocytes의 차 문화

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

연구

생물학

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

일차 종양에서 면역 세포의 분리

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

마우스 기본 췌장 포상 세포의 분리 및 문화

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

쥐 Adrenocortical 셀 및 Steroidogenic 기능의 분석 실험의 기본 문화

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

소형 돼지에서 1 차 대동맥 내피 세포의 격리 및 배양

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

성인 마우스및 쥐 심근세포의 격리, 트랜스페션 및 장기 문화

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

비 바이러스 유전자 치료를 위한 포유류 1차 안료 상피 세포의 격리, 배양 및 유전 공학

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

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 ...