이 작품은 OTKA (헝가리어 과학 연구 기금) D45933, T049621, TÉT (헝가리어 과학 재단) A4/04, Arg-17/2006과 죄, Bolyai, Öveges의 장학금과 TÁMOP 지원했습니다 4.2.2-08 / 1 / KMR - 2008-0004 및 4.2.1 / B 09/1/KMR-2010-0001. OTKA 83803. 우리는 오버 음성을 제공하는 윌리엄 Gesztes 감사하고 싶습니다.

1. H9c2 cardiomyoblast 전지를 준비 H9c2 쥐 cardiomyoblasts는 ATCC (Wesel, 독일)에서 얻은 높은 포도당 (4.5 g / L) DMEM 10 % 태아 소 혈청, 4 MM의 L - 글루타민, 100 U / ML 페니실린과 100 μg / ML 스트렙토 마이신을 포함하는으로 확장되었습니다. H9c2 myoblast 전지 라인이 배아 쥐의 심장에서 파생된 것입니다, 그것은 골격과 심장 근육 8,9 모두 체외 모델로 사용됩니다. H9c2 세포의 12 잘 접시를 준비 : <ol><li> H9c2 세포의 배양 접시에서 문화 매체를 제거합니다. </li><li> 2-3 ML 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 5 ML PBS 소화와 두 번 세포를 씻으십시오. </li><li> 37 ° C 배양기에서 5 분 페트리 접시 놓습니다. </li><li> 10 ML DMEM 문화 매체와 트립신을 억제. </li><li> 팔분을 위해 1200 RPM에서 세포를 다운 스핀. </li><li> Trypan 블루 염색법을 사용하여 hemocytometer 1 ML DMEM 배양 매체와 카운트 세포의 표면에 뜨는, resuspend 세포를 제거 후.</li><li> 이 ML DMEM에 따라 잘 12 잘 접시에 씨앗 30,000 H9c2 세포. </li><li> 24 시간 동안 37 ° C CO 2 배양기에 플레이트를 놓습니다. </li></ol> 2. 모의 국소 빈혈 / reperfusion 국소 빈혈 / reperfusion는 H9c2 세포 배양에서 산소 포도당 박탈 (OGD)를 수행하여 체외에서 모의​​했다. 이 절차는 OGD 동안 세포의 관찰을 허용 PeCon 세포 배양 시스템 (Erbach, 독일)와 자이스 혈구 공촛점 현미경의 무대에서 수행되었다. <ol><li> H9c2 세포의 12 잘 접시에서 열망으로 매체를 제거합니다. </li><li> PBS로 두 번 세포를 씻으십시오. </li><li> 물론 당 3 ML 포도당 무료 매체를 추가합니다. </li><li> 세포 배양 시스템에 접시를 놓습니다. </li><li> 배양 시스템의 밖으로 산소를 정화하기 위해 질소 가스를 시작합니다. </li><li> 다음 타이머를 시작 산소 수준이 0.5 %에 도달할 때까지 기다리십시오. 0.5 %의 산소가 약 3 mmHg 부분 긴장을 의미합니다. 1 제목 셀모의 국소 빈혈의 50 분. </li><li> 모의 국소 빈혈의 끝에 세포에서 포도당 무료 매체를 제거합니다. </li><li> 우물에 피펫이 ML 일반 문화 매체. </li><li> 37 ° C CO 2 배양기에서 30 분 12 잘 접시를 놓고, 이것은 &quot;reperfusion 기간&quot;입니다. </li></ol> 3. 준비 구조 mesenchymal 줄기 세포 인간의 근본 골수는 T75 flasks 고려하고 Dulbecco의 변형 이글 배지 (DMEM) 10 % 태아 송아지 혈청 (FCS), 100 U / ML 페니실린과 100 μg / ML 스트렙토 마이신을 포함하는 문화 매체, 4 MM의 L - 글루타민과 함께 희석되었다 1g / L 포도당. flasks 3 일 동안 5 % CO 2의 완전 humidified 분위기에서 37 ° C에서 incubated했다. 잠복기 후 flasks 및 골수의 나머지 구성 요소의 표면에 붙어 BMSCs은 PBS로 세척하여 제거되었습니다. 사용 BMSCs은 실험 1 5 구절 사이에 있었다. iden세포의 tity는 유동세포계측법와 혈통 특정 세포 표면 마커의 존재에 의해 확인되었다. 조혈 행수 특정 표면 마커 (CD34, CD45)와 mesenchymal 표면 마커 (CD73, CD90, CD105과 CD166)을 조사했다. 모의 국소 빈혈 동안 이식을위한 mesenchymal 줄기 세포를 구출 준비합니다. <ol><li> Vybrant는 DMEM 문화 매체 1:200 비율에서 한 형광 염료를 희석. </li><li> 구조 세포에서 매체를 기음과 Vybrant했던 300 μl DMEM를 추가합니다. </li><li> 37 ° C CO 2 배양기에서 30 분 장소 페트리 접시. </li><li> mesenchymal 줄기 세포의 배양 접시에서 문화 매체를 제거합니다. </li><li> 2-3 ML 0.05 % 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 5 ML PBS 소화와 두 번 세포를 씻으십시오. </li><li> 37 ° C 배양기에서 5 분 페트리 접시 놓습니다. </li><li> 10 ML DMEM 문화 매체와 트립신을 억제. </li><li> 팔분을 위해 1200 RPM에서 세포를 다운 스핀. </li><li> supe을 제거한 후한 ML DMEM 배양 매체와 Trypan 블루 염색법을 사용하여 hemocytometer로 카운트 세포에 rnatant, resuspend 세포. </li><li> reperfusion의 끝에 30 분 postischemic H9c2 심장 myoblasts마다 잘 20,000 mesenchymal 줄기 세포를 추가한 후. </li><li> 24 시간 동안 37 ° C CO 2 배양기에 장소 세포의 공동 문화. </li></ol> 4. 공촛점 현미경과 분석 모의 국소 빈혈 / reperfusion 상해 후, 전지가 24 시간 동안 일반 문화 매체 incubated 수 있습니다. <ol><li> 24 시간 PBS로 두 번 세포를 씻어 후. </li><li> 30 분 (5 NM calcein 400 nm의 PBS에서 Ethidium - homodimer - 2) 500 μl 죽은 / 라이브 염료 용액 (5x10 4 셀 / ML)에 세포를 얼룩. </li></ol> 형태학 및 형광 선정 살고 죽은 세포에 대한 공촛점 이미지의 평가는 ImageJ 소프트웨어 (보건 국립 연구소, 미국)과 함께 수행할 수 있습니다. C의 경우O - 문화, MSCS들은 Vybrant나요 셀 라벨로 인해 H9c2 세포에서 구분할 수 있습니다. 죽은 세포의 비율은 맹인 패션 10의 각 문화보기 4 독립적인 필드 (10X 목적)에서 평가할 수 있습니다. 42mm 유리 coverslips에 교양 H9c2 cardiomyoblasts도 공촛점 현미경으로 세포 배양 시스템을 사용하여 국소 빈혈 / reperfusion 상해시 후 시간 저속 비디오 현미경과 함께 조사하실 수 있습니다. 이러한 세포 융합과 세포 나노튜브의 형성과 같은 휴대 전화의 상호 작용은 시간이 지남에 따라 공부하실 수 있습니다. 5. 유동세포계측법와 분석 모의 국소 빈혈 / reperfusion 상해 후, 전지가 24 시간 동안 일반 문화 매체 incubated 수 있습니다. <ol><li> 24 시간 후에, 문화 미디어를 수확. 어떤 죽은 세포는 24 시간 incuba 동안 문화 접시 표면에서 분리 할 수​​ 있기 때문에 그것은 또한 문화 매체를 수집하는 것이 매우 중요합니다기. </li><li> 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)과 PBS 소화와 두 번 세포를 씻으십시오. </li><li> DMEM 문화 매체와 트립신을 억제. </li><li> 팔분위한 1,200 RPM (약 150-200그램)에서 세포와 수확 문화 매체를 돌린다. </li><li> 뜨는 폐기하십시오. </li><li> 30 분 (5 NM calcein 400 nm의 PBS에서 Ethidium - homodimer - 2) 500 μL 죽은 / 라이브 염료 용액 (5x10 4 셀 / ML)에 세포를 일시 중지합니다. </li><li> cytometry 튜브를 흐름 세포를 전송합니다. </li><li> CellQuest Pro 소프트웨어를 시작합니다. </li><li> 측정 흐름 cytometer를 설정하려면, 살고 죽은 세포의 샘플을 제어할 준비합니다. <ol class="lalpha"><li> 12 잘 접시에 3x10 4 밀도에 플레이트 H9c2 세포는 지점 1에 설명되어 있습니다. </li><li> 포인트 1 설명된대로 단일 trypsinisation로 셀 컨트롤을 살 준비합니다. </li><li> 1 시간 10 μm의 H 2 O 2와 치료에 의해 ​​죽은 세포 컨트롤을 준비합니다. </li><li> trypsinisation 후, resuspen500 μl 죽은 / 라이브 염료 용액 (5 NM calcein - AM 400 nm의 PBS에서 Ethidium - homodimer - 2)에서 D 살고 죽은 세포 제어합니다. </li></ol></li><li> 죽은 세포가 homodimer 긍정적인 calcein 부정하고 ethidium하는 동안 살아있는 세포가 calcein 긍정적이고 부정적인 homodimer ethidium되도록 악기 설정을 조정해야합니다. </li><li> 그래프 및 통계 분석 바, 이러한 가치에 수행할 수 있습니다 이러한 인구의 비율은 표현할 수있다. </li><li> 다른 실험 그룹을 측정합니다. </li></ol> 6. 대표 결과 : 우리의 결과는 추가 mesenchymal 줄기 세포가 심장 세포 postischemic의 생존을 향상시킬 수 있다고했다. 공촛점 현미경 이미지 아마도 관찰된 보호 10 중요한 역할을 이러한 부분 멤브레인 연결 또는 세포 나노튜브로 직접 휴대 전화의 상호 작용을, 발표했다. 이러한 나노튜브는 여러 셀 D의 거리 스팬iameters, 그들의 직경은 200, 500 nm의 (흰색 화살표) 사이에 있었다. <img src="/files/ftp_upload/3575/3575fig1.jpg" alt="그림 1" /> 그림 1. 세포 사이의 나노튜브 형성. 나노튜브 형성 (흰색 화살표)가 Vybrant DIO 표시 cardiomyoblasts 및 Vybrant나요 표시 mesenchymal 줄기 세포 사이에 관찰되었다. 유동세포계측법 분석은 생존의 인구 및 괴사성 cardiomyoblasts 및 mesenchymal 줄기 세포를 보여주었다. 흥미롭게도, 우리는 또한 calcein를 포함 세번째 &#39;부상&#39;cardiomyoblast 세포 인구를 관찰뿐만 아니라 죽은 세포, ethidium homodimer (그림 2)의 지표를 포함. <img src="/files/ftp_upload/3575/3575fig2.jpg" alt="그림 2" /> 그림 2. 모의 국소 빈혈 후 다른 세포 집단의 대표 도트 플롯. 몇 가지 실험 슈의 평가postischemic H9c2의 cardiomyoblast에 mesenchymal 줄기 세포뿐만 아니라이 국소 빈혈 후 혼자 교양 심장 myoblasts (그림 3)에 비해 크게 살아있는 세포의 비율이 증가되는 결혼. <img src="/files/ftp_upload/3575/3575fig3.jpg" alt="그림 3" /> 그림 3. ., 또한 이후 증가 : 살아있는 세포의 postischemic cardiomyoblasts에 대한 줄기 세포 치료의 효과 비율이 크게 (90.36 ± 2.60 대 10.52 ± 0.48, ** P&gt; 0.001 제어 대 IR 모델) 컨트롤에 비해 모의 국소 빈혈 이후 감소되었다 mesenchymal 줄기 세포 (IR 모델 대 IR + BMSC : 10.52 ± 0.48 대 25.21 ± 2.13, ** P&gt; 0.001). 데이터 말은 ± SEM을 나타냅니다. 데이터의 통계 분석은 Tukey의 다중 비교 게시물을 임시 시험 분산의 한 방법으로 분석을 사용하여 실시되었다.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+a+revolution+in+therapeutics--recent+advances+in+stem+cell+biology+and+their+therapeutic+applications+in+regenerative+medicine+and+cancer+therapies.">Stem cells: a revolution in therapeutics--recent advances in stem cell biology and their therapeutic applications in regenerative medicine and cancer therapies.</a> Clinical Pharmacology &#38; Therapeutics. 82, 252-252 (2007).</li><li>Angoulvant, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+conditioned+media+attenuates+in+vitro+and+ex+vivo+myocardial+reperfusion+injury.">Mesenchymal stem cell conditioned media attenuates in vitro and ex vivo myocardial reperfusion injury.</a> J Heart Lung Transplant. 30, 95-95 (2010).</li><li>Lim, Y. J., Zheng, S., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+Preconditions+Purkinje+Cells+against+Cell+Death+under+In+Vitro+Simulated+Ischemia/Reperfusion+Conditions.">Morphine Preconditions Purkinje Cells against Cell Death under In Vitro Simulated Ischemia/Reperfusion Conditions.</a> Anesthesiology. 100, 562-562 (2004).</li><li>Guo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen-receptor-mediated+protection+of+cerebral+endothelial+cell+viability+and+mitochondrial+function+after+ischemic+insult+in+vitro.">Estrogen-receptor-mediated protection of cerebral endothelial cell viability and mitochondrial function after ischemic insult in vitro.</a> J Cereb Blood Flow Metab. 30, 545-545 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

시연 프로토콜은 같은 모델의 모든 장점과 단점과 심근 경색에 줄기 세포 치료의 훨씬 더 복잡한 문제 체외 접근 방식에있다. 분명히 그것은 심근 경색 동안 후 일어나는 복잡한 (예 : 면역) 이벤트를 반영하지 수 있지만 postischemic 세포에 대한 추가 세포의 직접적인 효과에 초점을 맞출 수 있습니다. H9c2 cardiomyoblasts에 대한 시뮬레이션 국소 빈혈의 효과는 매우 사용되는 세포의 통과 번호와 O 2<...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> calcein - AM </td><td> 분자 프로브 </td><td> L3224, C3099 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> ethidium homodimer - 2 </td><td> 분자 프로브 </td><td> L3224, E3599 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr><tr><td> Vybrant가나요 </td><td> 분자 프로브 </td><td> V22887 </td><td> <a href="http://www.invitrogen.com">http://www.invitrogen.com</a> </td></tr></tbody></table> 표 1. 시약. <table border="1"><tbody><tr><td> 장비의 명칭 </td><td> 회사 </td><td> 댓글 (옵션) </td></tr><tr><td> 자이스 혈구 현미경에 대한 PeCon 세포 배양 시스템 </td><td> PeCon GmbH의 </td><td> <a href="http://www.pecon.biz/">www.pecon.biz/</a> </td></tr></tbody></table> 표 2. 장비.

stem cell transplantation in an in vitro simulated ischemia/reperfusion model

줄기 세포 이식 프로토콜은 임상 연습 1,2,3에 그들의 방법을 찾는 수 있습니다. 더 나은 결과를 얻기 프로토콜보다 강력한 만들기, implantable 세포에 대한 새로운 소스를 찾는 것이 4,5 최근 연구의 초점입니다. 세포 요법의 효과를 조사하는 것은 쉬운 일이 아니므로 새로운 도구는 치료 과정 6 관련된 메커니즘을 조사하기 위해 필요합니다. 우리는 국소 빈혈 / reperfusion 상해시와 줄기 세포 이식 후 휴대폰 연결 심지어 subcellular 구조의 관찰을 허용하고 세포 요법의 효능을 평가하기 위해 국소 빈혈 / reperfusion의 실험 프로토콜을 설계했습니다. H9c2 cardiomyoblast 세포는 세포 배양 접시 7,8에 배치했다. 국소 빈혈은 0.5 % 이하의 산소 수준 포도당 무료 매체 150 분 모의했다. 그렇다면 정상적인 미디어와 산소 수준은 reperfusion을 시뮬레이트하기 위해 reintroduced되었습니다. 산소 포도당 박탈 후,손상된 세포는 문화에 그들을 추가하여 mesenchymal 줄기 세포를 파생 표시된 인간 골수의 이식과 함께 치료를했다. 그들이 최소한의 침략 수술 쉽게 쉽게 확장하고 autologous되기 때문에 Mesenchymal 줄기 세포는 임상 실험에서 원하는 있습니다. 공동 배양 24 시간 후 세포가 살고 죽은 세포를 구분하는 calcein하고 ethidium - homodimer로 얼룩진했다. 이 설정은 우리가 공촛점 형광 현미경을 사용하여 세포 연결을 조사하고 유동세포계측법에 의해 postischemic 세포의 생존 비율을 정할 수있었습니다. 공촛점 현미경은 세포 융합과 세포 나노튜브의 형성과 같은 두 가지 세포 집단의 상호 작용을 보여주었다. 유동세포계측법 분석 그래프를 꾸몄다 그리고 통계적으로 분석할 수 손상된 세포의 3 클러스터를 공개했다. 이러한 집단은 별도로 조사하고 결론은 시뮬레이션의 효과에 이러한 데이터를 발행한 수치료 접근.

Watch this Scientific Journal Video about Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model at JoVE.com

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

우리는을 설정하는 방법을 보여줍니다<em&gt; 체외에서</em&gt; 국소 빈혈 / reperfusion 모델과 방법을 postischemic 심장 세포에서 줄기 세포 치료의 효과를 평가할 수 있습니다.

에서 간세포 이식 체외에서 시뮬레이션 국소 빈혈 / Reperfusion 모델

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

stem-cell-transplantation-an-vitro-simulated-ischemiareperfusion

Research

JoVE Journal

Medicine

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

14.0K 조회수.  Semmelweis University. 우리는을 설정하는 방법을 보여줍니다 체외에서 국소 빈혈 / reperfusion 모델과 방법을 postischemic 심장 세포에서 줄기 세포 치료의 효과를 평가할 수 있습니다.

비디오: Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

의 마우스 모델 Utero에 이식

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

연구

의학

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

경쟁 손쥐 골수 이식 모델에서 기증자 세포 Engraftment를 확인하려면 수량 실시간 PCR을 사용하여

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

척수 손상의 실험 Contusive 모델에서 신경 줄기 세포 이식

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

창자 허혈 - 재관류 손상의 쥐 모델

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

생성 및 당뇨병 치유 하는 부상을 모델에서 줄기 세포 지방이 많은 파생 (ASC) 시트의 이식

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

대뇌 국 소 빈 혈 reperfusion의 2 배 폐색 마우스 모델

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

재건 미세 수술에서 허혈 재관류 손상의 연구를위한 전 임상 쥐 모델

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...