The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

동물 실험 의학 (FEM, 샤리 테, 베를린, 독일)의 시설에 보관하고, 모든 실험 프로토콜을 검토하고, 국가 보건 및 지역 담당 사무소 (LAGeSo, 베를린, 독일의 승인을 하였다. 등록 번호 O 0365 / 11). 1. 간 수확 <ol><li> 수술 전 준비 <ol><li> 고정을 위해 코르크 판을 사용합니다. 접시에 의료 드레이프를 넣어. 네 바늘을 사용하여, 수술 흡입 마취의 코르크 판에 흡입 마스크를 고정한다. </li></ol></li><li> 포털 정맥 정맥의 사전 약속 <ol><li> 세 방향 꼭지에 말초 정맥 카테터 (G 20)을 연결합니다. 정맥 카테터는 약의 길이, 비스듬히 절단한다. 1.5 cm. </li><li> 드 에어 링거 용액으로 가득 10 ML의 주사기를 사용하여 시스템. 그것은 신중하게 해제 공기 가스 색전증 부정적인 관류 탈세 포화에 영향을 미칠 수 있기 때문에 매우 중요하다. </li></ol></li><li> 사전 약속동맥 정맥의 <ol><li> 나비 캐 뉼러를 사용하여 5 mm의 길이로 절단 바늘. 5mm 바늘과 superglue로 수정, 5cm 튜브 (외경 0.96 mm ID 0.58 mm)를 장착한다. 더 큰 튜브에와 superglue로 수정, 2cm 길이의 튜브 (외경 0.61 mm ID 0.28 mm)를 삽입합니다. </li><li> 교대로이 공사를 통해 다른 튜브를 미끄러 superglue로 고정합니다. 드 에어 링거 용액으로 채워진 5 ML의 주사기와 동맥 정맥. </li></ol></li><li> 마취와 진통 <ol><li> 1.5 L / min의 유량으로 산소를 3.5 %의 이소 플루 란으로 마취 유도 챔버에서 래트 마취를 유도한다. </li><li> 운전시 안전 진통을 보장하기 위해, 복강 metamizol (100 ㎎ / ㎏ 신체 질량)과 저용량 케타민 / 메데 토미 (kg의 신체 질량 당 10 밀리그램 / 0.1 mg)을 주입. </li><li> 수술 마취 이후의 유지 보수를 위해 흡입 마스크를 통해 1 L / 분 산소 1.5 % 이소 플루 란를 제공합니다. </li></ol></li><li> 수술 전 약정 &lt;OL&gt; <li> 전기 전단 기계와 자유주의로 복부를 면도. 소독 용 70 % 에탄올로 복부에 면도​​ 절개 사이트를 적셔. </li><li> , 준비된 접시에 앙와위에서 동물을 배치 흡입 마스크에 머리를 삽입하고 의료 테이프와 핀 사지를 고정합니다. 거즈 압축과 초과 에탄올과 모피를 제거합니다. </li></ol></li><li> 마취 평가 <ol><li> 호흡을 보면서 마취 깊이를 평가합니다. 마취가 (40 ~ 60 호흡 / 분)만큼 깊은 것 같다 때, 진통 두 뒷다리의 발가락 사이의 피부를 꼬집어하는 수술 집게를 사용하여 발가락 핀치 반사를 트리거하기 위해 시도하여 충분한 지 여부를 확인합니다. </li></ol></li><li> 복부 접근 <ol><li> 방광 위 꼬리 복부 벽에 중간 절개를합니다. V 자형 방식으로 양측 늑골 아치에이 시점에서 잘라. 다이어프램으로 잘라 않도록주의하십시오. 가슴 위에 잘라 복부 벽을 당깁니다. </li&gt; <li> , overholt 클램프와 칼 모양의 수정 두개쪽으로 당겨과 고무 밴드로 고정합니다. 장을 대피 젖은 면봉을 사용합니다. 젖은 거즈 붕대에 장을 감싸. 또한, 젖은 붕대 모든 복부 마진을 커버합니다. </li></ol></li><li> 간 준비 <ol><li> 낫 모양의 인대를 해부하다. 두개쪽으로 다이어프램의 돔 아래 중간과 왼쪽 간 로브를 개최 젖은 거즈 붕대를 사용합니다. hepatoduodenal 인대와 상부 대망 간 로브 노출됩니다. </li><li> 로브가 이동 될 때까지 조심스럽게 상단 대망 로브에 부착 된 인대를 해부하는 두 microforceps 봄 가위를 사용합니다. 대신에 중간과 왼쪽 엽을 잡고, 거즈 압축에서 젖은 면봉을 사용하여 로브를 이동합니다. </li><li> 위 대망 간 엽의 동원 후, 왼쪽으로 배를 이동합니다. 클램프와 위장을 수정하고 microforceps 및 스프링 가위로 작은 대망을 해부하다. </li><li> 낮은 omenta를 동원L 간 엽이 이동하고 이후에 그 구멍에 다시 로브를 이동시킬 때까지. 십이지장을 유지하기 위해 박하우스 클램프를 사용합니다. 스트레칭과 hepatoduodenal 인대를 노출 왼쪽으로 십이지장을 이동합니다. 뻗어 hepatoduodenal 인대의 담관을 외접. </li><li> 지방 및 췌장 조직에서 담관을 해부하다. 담관에게 담관 분기에서 1.5 cm를 잘라. 주위의 지방 조직에서 문맥을 해부. 십이지장 정맥을 노출. </li><li> 실크 6-0로 두 번 십이지장 정맥을 결찰하고 2 합자 사이의 정맥을 나눕니다. 모든 동맥 라미가 공개 될 때까지 주변 조직에서 복강 트렁크 및 공통 간 동맥을 해부하다. </li><li> 주변 조직으로부터 십이지장 동맥 해방. 서로 멀리 떨어져있는 관계로, 실크 6-0로 두 번 십이지장 동맥을 묶어. 2 합자 사이의 십이지장 동맥을 해부하다. </li><li> 비장 동맥을 해방주변 조직으로부터. 서로 멀리 떨어져있는 관계로, 실크 6-0로 두 번 비장 동맥을 묶어. 2 합자 사이의 비장 동맥을 해부하다. </li><li> 주변 조직으로부터 왼쪽 위 동맥 해방. 서로 멀리 떨어져있는 관계로, 실크 6-0로 두 번 왼쪽 위 동맥을 묶어. 2 합자 사이의 동맥을 해부하다. </li><li> 오른쪽 신장과 오른쪽 측면 간 엽 사이의 열등한 대정맥 정맥을 외접. 복막 공간에서 정맥을 해부하다. 복강 트렁크에 따라 대동맥을 노출. </li><li> 복막 지방 조직을 해부하다. 하대 정맥에 1 ml의 생리 식염수 1,000 IE의 헤파린을 주입한다. 캐 뉼러를 후퇴와 화장 솜에 절개를 닫습니다. 헤파린의 전신 효과를 1-2 분을 기다립니다. </li><li> 집게로 대동맥을 외접. 대동맥을 해부하고 쥐에게서 피를 뽑다. 또한, 신장에서 원심 하대 정맥을 해부하다. </li></ol></li><li> 포털 정맥로 (사용 섹션 1.2에서 제조 된 정맥) <ol><li> 말단 포털 정맥에 물고기 입 절개를합니다. 집게로 절개를 열고 포털 정맥을 cannulate. 간 탈색 될 때까지 20 ㎖ 링거 용액으로 간을 세척 및 합자 (실크 6-0)와 캐 뉼러를 해결. </li></ol></li><li> 동맥 캐 뉼러 (사용 섹션 1.3에서 제조 된 정맥) <ol><li> 복강 트렁크 위의 대동맥을 해부하다. 복막 공간에서 대동맥 세그먼트를 해방. 대동맥 패치를 생성하기 위해 길이 방향으로 대동맥 세그먼트를 잘라. 정적 홀더에 자체 내장 캐뉼라를 삽입합니다. </li><li> 캐 뉼러를 통해 대동맥 패치를 미끄러 2 집게를 사용합니다. 정맥을 통해 실크 6-0로 간 동맥을 결찰하고 지대치에 패치를 수정합니다. </li></ol></li><li> 간 외식 <ol><li> 다이어프램을 열고 원형 절개를 주변 구조에서 간을 해부하다. </li></ol></li><li> 저장 <ol><li> 스토사용할 때까지 350 ml의 링거 용액으로 가득 멸균 500ml의 탱크에 간 재. </li></ol></li></ol> 2. 세제 솔루션 <ol><li> 10 % 트리톤 X-100 원액 <ol><li> 900 ml의 아쿠아 DEST와 1,000 ml의 병을 채 웁니다. 100 ml의 트리톤 X-100 (99,9 %)를 추가합니다. 트리톤 X-100을 용해 교반기를 사용합니다. 아쿠아 DEST와 병을 채 웁니다. 1000 ml의 총 부피에 도달 할 때까지. </li></ol></li><li> 1 % 트리톤 X-100 <ol><li> 1000 ㎖의 병에 스톡 용액 100 ㎖ (10 %)를 첨가. 900 ml의 아쿠아 DEST를 추가합니다. 두 번 병을 흔들어. 멸균 필터를 통해 1 % 트리톤 X-100 용액 필터. </li></ol></li><li> 10 % SDS 원액 <ol><li> 900 ml의 아쿠아 DEST와 1,000 ml의 병을 채 웁니다. 과 66.99 g을 SDS (99.9 %, 밀도 0.67)를 추가합니다. SDS를 용해 교반기를 사용합니다. 아쿠아 DEST와 병을 채 웁니다. 1000 ml의 총 부피에 도달 할 때까지. </li></ol></li><li> 1 % SDS <ol><li> 1000 ml의 봇에 스톡 용액 100 ㎖ (10 %)을 추가TLE. 900 ml의 아쿠아 DEST를 추가합니다. 두 번 병을 흔들어. 멸균 필터를 통해 1 % SDS 용액을 필터. </li></ol></li></ol> 3. 탈세 포화 <ol><li> 관류 장치의 설정 <img alt="그림 1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> 그림 1 :. 진동 압력 조건에서 선택적 동맥과 네 쥐 간을 포털 정맥 관류에 대한 관류 장치의 제도 (. Struecker 등에서 수정 14) <ol><li> 삼방 콕 및 충전 레벨을 조절하고 500 mL의 0.9 % NaCl로 반응기를 채우기 위해 Heidelberger 확장 드레인에 링크. <img alt="그림 2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> 그림 2. (4) 포털 정맥 액세스 통해 버블 트랩 (5) (2) 또는 동맥 AC 통해 선단 관류 셋업의 반응식 관류 챔버를 연결 (1,400cm 3) (1)운 (3). 기포 트랩 (5)의 말단부에서 폐색 (4) 및 배출구 (6)를 구비한다. Heidelberger 확장 (7)에 버블 트랩 (5)를 연결합니다. 펌프 세그먼트 (8)에 Heidelberger 확장을 연결합니다. 또한, 다른 Heidelberger 확장 (9)에 펌프 세그먼트 (8)를 연결합니다. 세제 (10)의 병에 마지막 Heidelberger 확장 (9)를 삽입합니다. (관류 또는 다수의 경우에 압력 유통로) 관류 챔버에 마스크 (11)를 연결한다. 반응기 유체를 배출, 폐기물 용기 (12)에 대한 유출을 연결한다. <ol><li> 펌프로 펌프 세그먼트를 삽입합니다. PBS로주기를 채우기 위해 펌프를 시작, 버블 트랩의 선단을 닫고 환기를 엽니 다. 거품의 바닥 안전하게 PBS (2-3 CM)으로 피복되는 경우, 벤트를 닫고 버블 트랩의 말단부를 연다. </li><li> 간 설치 <ol><li> 관련 반응기 액세스 문맥 캐 뉼러의 삼방 콕 링크. C동맥 액세스 동맥 정맥 connect를. 공기가 시스템에 입력하지 않도록주의하십시오. </li></ol></li><li> 진동 압력 조건의 적용 <ol><li> 15 BPM의 주파수 및 26 밀리바 (분. 4 밀리바, 최대. 30 밀리바)의 진폭 호흡기를 사용합니다. 압력 분포 챔버로 호흡기를 연결하고 반응기 챔버를 연결합니다. 압력 응용 프로그램을 시작합니다. </li></ol></li><li> 탈세 포화 프로토콜 <ol><li> / 분 5 ㎖의 유속으로 관류 단계를 수행한다. 90 분 동안 1 % 트리톤 X-100 간 관류와 탈세 포화를 시작합니다. 관류 챔버 내부 상수 및 관류 된 간 상기 오일 레벨을 유지하기 위해 관류 장치의 유출을 통해 폐기 유출 물을 허용. 90 분 후, 1 % SDS에 세제를 변경합니다. <img alt="그림 3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="표 1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;P&gt; 그림 3 : 모든 실험 그룹의 관류 프로토콜.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+decellularization+for+multi-organ+scaffolds+in+rats.">Systemic decellularization for multi-organ scaffolds in rats.</a> Transplantation proceedings. 44, 1151-1154 (2012).</li><li>Shirakigawa, N., Ijima, H., Takei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+liver+as+a+practical+scaffold+with+a+vascular+network+template+for+liver+tissue+engineering.">Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering.</a> J Biosci Bioeng. , (2012).</li><li>Ren, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+two+decellularization+methods+in+the+development+of+a+whole-organ+decellularized+rat+liver+scaffold.">Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.</a> Liver Int. 33, 448-458 (2013).</li><li>Struecker, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+rat+liver+decellularization+by+arterial+perfusion+under+oscillating+pressure+conditions.">Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions.</a> J Tissue Eng Regen Med. , (2014).</li><li>Struecker, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+Liver+Decellularization+Under+Oscillating+Pressure+Conditions:+A+Technical+Refinement+to+Improve+the+Homogeneity+of+the+Decellularization+Process.">Porcine Liver Decellularization Under Oscillating Pressure Conditions: A Technical Refinement to Improve the Homogeneity of the Decellularization Process.</a> Tissue engineering. Part C, Methods. , (2014).</li></ol>

The authors have nothing to disclose.

쥐의 간 수확 및 탈세 포화에 대한 발표 기술은 쉽게 재현 할 수 있지만, 고려해야 할 중요한 특정 단계가 있습니다 : 이 혈액 응고를 활성화하고 간 내 혈전 형성으로 이어질 수 있기 때문에 간 수확을 준비하는 동안, 그것은 심한 출혈을 방지하는 것이 중요하다. 우리의 의견으로, 그 문맥을 통해 관류 동안 간동맥을 통해 혈액의 유입을 방지하기 위해 포털 정맥 삽관 직전 ?...

탈세 포화하고 recellularization 시험관에 1 작용 성, 이식 가능한 장기의 생성을 가능하게 할 수있다. 제거 세포와 항원 물질에 의해 (예., DNA는 알파 - 갈 에피토프)에서 기관은 이하 비 - 면역 세포 외 기질 (ECM)을 얻을 수있다. 이 행렬은 기관의 입체 microanatomy을 보존하고 다른 가능성이 이종 기원의 세포 다시 채우기위한 이상적인 biomatrix로서 역할을 할 수있다. 따라서, 탈세 포화 된 쥐 간 매트릭스는 인간 간 세포 증식을 할 수있다. 이 인간화 마이크로 간 질환 (예., 선천성 대사 질환, 바이러스 성 질환 또는 악성 종양) 또는 전임상 제약 테스트 3에 대한 연구를위한 생체 모델이 될 수 있습니다. 쥐의 간 관류 탈세 포화에 대한 몇 가지 다른 프로토콜은 이미 4-13 발표되었다. 모든 프로토콜에서, decellular화는 유관 간문맥 통해 알칼리 이온 성 또는 비이 온성 세제의 관류에 의해 달성 하였다. 우리가 아는 한, 우리는 문맥 및 / 또는 쥐 간 동맥 (14)를 통해 선택적 관류에 의해 쥐의 간 탈세 포화를보고 첫 번째 그룹이었다. 간에서 서로 다른 혈관 시스템의 선택적 관류 활성화 셀룰러 다시 채우기에 중요한 역할을 할 수 있고, 또한, 더 나은 결과 탈세 포화를 가능하게하고있다. 여기에서 설명 된 연구에서, 간은 진동 압력 조건 하에서 관류있게 주문품 독점 관류 장치로 관류 하였다. 이러한 압력 조건은 간 생리적 호흡기 의존 관류 모방 : 시츄, 간은 호흡 운동시 간 혈류에 직접적인 영향을 다이어프램의 접합부 아래에 건다. 영감은 특히 최적화, 다이어프램의 저하 및 간 압박에 이르게간세포 정맥 유출 반면 만료 간 상승 및 포털 정맥 유입 15을 최적화하기 복강 내 압력의 저하로 연결. 우리의 목표는 진동 압력 조건은 복강 내 조건 생체를 모방하여 쥐의 간 관류 탈세 포화의 동질성에 영향이 있는지 여부를 알아보고자 하였다. 탈세 포화 과정의 균질성 관류 탈세 포화의 과소 평가 요소 일 수있다. 알려진 모든 에이전트는 ECM에 간 탈세 포화 원인 변경에 사용됩니다. 다른 영역이 이미 완전히 탈세 포화되는 반면 저조한 관류 영역의 세포는 ECM 내에 남아있다. 남아있는 세포를 용해, 관류 시간 또는 압력은 잘 관류 부위에 더 많은 변경을 일으키는 상승되어야한다. 따라서, 탈세 포화에 대한 세제는 기관 내에서 균일하게 분산되어야한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
		</tr>
		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
		</tr>
		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
		</tr>
		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
		</tr>
		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
		</tr>
		<tr>
			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
		</tr>
		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
		</tr>
		<tr>
			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
		</tr>
		<tr>
			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
		</tr>
		<tr>
			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
		</tr>
		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
		</tr>
		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for decellularization of rat livers in an oscillating-pressure perfusion device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

균질성 따라서 다른 탈세 포화 프로토콜의 효과를 육안 관찰, 조직 학적 분석, 탈세 포화 간 매트릭스 내의 나머지 DNA 함량 분석에 의해 평가 하였다. 또한, 부식 주조 탈세 포화 후 간 손상의 microanatomy을 시각화 하였다. Macroscopy 탈세 포화 동안, 간 세포는 콘텐츠의 제거를 나타내는, 루슨트된다. 문맥을 통해 관류 된 간을 탈세 포화 동안 및 (흰색 화살표...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

진동 압 관류 장치에서 쥐 간을의 탈세 포화 절차

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K 조회수.  Charité – Universitätsmedizin Berlin. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

비디오: Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

전체 간은의 Decellularization 및 Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

연구

생체공학

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

퇴행성 관상 재관류에 의한 돼지 심장의 Decellularization 절차

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

당뇨병 쥐 모델에서 신경 전도 대 한 초음파 도구 차단

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Foodborne 병원 체를 검출 하는 전기 화학 DNA 바이오 센서의 개발

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Decellularization 식물 조직의 조직 공학 응용 프로그램에 대 한 두 가지 방법

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Vivo에서 부분 간 엔지니어링을에서 위한 쥐를 기초로 새로운 수술 기법

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

거꾸로 방향 가압된 주머니 안에 전체 인간의 마음의 decellularization

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

고처리량 미세 유체 장치를 사용하여 동적 환경 조건의 생성

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

맞춤형 관류 생물 반응기에서 돼지 혈관 플랩의 조달 및 관류-탈세포화

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

혈관화된 복합 동종 이식을 위한 생체 내 관류 기반 생물반응기를 사용한 쥐 뒷다리의 조달 및 탈세포화

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...