저자는 교수 박사 S.E.R. Hovius, 박사 M.A.M. Mureau 및 모든 외과 피하 지방이 많은 직물의 컬렉션에 대 한 성형 수술의 부의 감사. P. Sukho 연구의 행위 동안에 네덜란드 친목 프로그램 (NFP-12/435)에서 교부 금에 의해 지원 됩니다. 사원 Bastiaansen Jenniskens 네덜란드 관절염 재단 (LP11)에서 교부 금에 의해 지원 됩니다.

복 부 피하 지방 조직 승인 의료 윤리 위원회 (#MEC-2014-092), 에라스무스 엠씨 대학 의료 센터, 로테르 담, 네덜란드의 인간 기증자 로부터 얻은 것입니다. 모든 동물 실험 윤리 위원회의 동물 실험, 에라스무스 엠씨 대학 의료 센터, 로테르 담, 네덜란드 (133-14-01)에 의해 승인 되었다.
1. 인간의 ASCs 격리와 문화
<ol><li>1g의 지방 조직에 대 한 절연 매체의 3 mL를 준비 합니다. 혼합 낮은 포도 당 Dulbecco의 수정이 글의 중간 (LG-DMEM) 10 g/L 소 혈 청 알 부 민 (BSA)와 콜라 1 g/L 입력 1.</li>
	<li>인간의 피하 복 부 지방 조직 해 부 (n = 8, 모든 여성, 평균 나이 40 ± 9 살) 살 균 생물 SafetyCabinet의 메 마른 외과 블레이드 크기 10, Adson 브라운 조직 집게와 Metzenbaum가 위를 사용 하 여 작은 조각 (0.5 cm).</li>
	<li>메 마른 유리 미디어 저장 병에 해 부 조직을 저장 합니다. 해 부 조직 총 양의 무게 고 준비 절연 매체 병 당 절연 매체의 150 mL와 함께 지방 조직 (50 g)와 소화를 시작 합니다. 프로토콜 롤러 믹서에 하룻밤 해 부 지방 조직 및 4 ° C에서 절연 매체 병을 저장 하 여 여기 일시 중지 될 수 있습니다. 그런 다음 다음 단계를 계속 하기 전에 15 분 동안 실내 온도 25 ° C에 다음 날 병 prewarm. 참고: 복 부 피하 지방 조직 기증자 승인 에라스무스 MC 의료 윤리 위원회 (# 멕-200)의 규범에 따라 유 방 재건 수술에서 남은 자료로 얻어 졌다: "적절 한 보조 사용 인간의 조직"의 (< http://www.federa.org>). 남은 지방 조직만 기증자 않았다 하지 탈퇴 보조 사용에서 사용 되었다.</li>
	<li>다이제스트 150 rpm에서 1 h 동안 37 ° C 배양 기 통에에서 멸 균 유리 미디어 저장 병에 해 부 지방 조직.</li>
	<li>50 mL 튜브로 소화 솔루션을 분할 하 고 튜브 390 x g에서 10 분 원심.</li>
	<li>문화 매체를 준비: LG DMEM 10% 태아 둔감 한 혈 청 (FBS), 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B. 보충</li>
	<li>원심 분리, 다음은 상쾌한을 제거 하 고 배양 (LG-DMEM 10% 태아 둔감 한 혈 청 (FBS), 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B 보충) 20 mL에 셀 펠 릿 resuspend. 390 x g에 5 분 동안 다시 세포를 원심 고는 상쾌한을 제거 합니다.</li>
	<li>문화 매체 (LG-DMEM 10 %FBS, 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B 보충)의 10 mL와 함께 셀 펠 릿을 resuspend 하 고 세포 현 탁 액 100 µ m 필터를 통해 필터링.</li>
	<li>세포 현 탁 액의 50 µ L을 (적혈구 세포)에 대 한 3% 초 산/메 틸 렌 블루 솔루션의 50 µ L을 추가 하 고 솔루션을 섞어 10 µ L를 사용 하 여 셀을 계산. 세포는 hemocytometer와 계산을 수행 합니다. 다음 문화 매체 (LG-DMEM 30 %FBS, 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B 보완)에 40, 000 셀/c m2 의 조밀도에 세포 격판덮개.</li>
	<li>24 시간 5% CO2 와 다습 한 대기권에서 37 ° C에서 세포를 품 어.</li>
	<li>갓 부 화, 워시와 따뜻한 (37 ℃) 성 세포와 식 염 수 (PBS) 세포 파편을 제거 하 고 10 %FBS 문화 매체와 미디어를 대체 버퍼링을 다음 추가 ascorbic 산-2-인산 염 (25 µ g/mL)와 인간의 재조합 섬유 아 세포 성장 요인 2 (FGF2, 1 ng/mL).</li>
	<li>Subculture ASCs 표준 0.25% trypsin EDTA 솔루션 (3 mL T175 플라스 크에 대 한)를 사용 하 여 90% 합류에. 3-5 분 후 중화 문화 매체 (LG-DMEM 10 %FBS, 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B 보충)의 10 mL와 0.25% trypsin EDTA 솔루션. 참고: 일반적인 ASC 표면 마커 및 트라이-혈통 차별화 분석 수행 이전 그리고 발표 ASCs이 프로토콜 표시 ASC 특정 특성21,22를 사용 하 여 격리를 사용 하 여 cytometry 분석 흐름.</li>
	<li>T175 플라스 크에 대 한 문화 매체 (LG DMEM 보충 10 %FBS, 50 µ g/mL gentamicin 및 1.5 µ g/mL ampothericin B)의 10 mL로 세포 세척 하 고 원심 250 x g. 제거에서 8 분에 대 한 셀은 상쾌한 문화 매체의 1 mL에 셀 resuspend. hemocytometer 셀을 계산 합니다.</li>
	<li>8, 000 셀/c m2의 조밀도에 T175 문화 플라스 크에 세포 격판덮개</li>
	<li>더 사용 하기 전에 문화 매체에서 10% 디 메 틸 sulfoxide (DMSO)와 액체 질소에 나머지 ASCs를 고정.</li>
</ol>2. ASC 시트 준비
<ol><li>FBS의 1 mL와 함께 온도 응답 문화 요리 (3.5 c m 직경)를 미리 coat 다음 셀 뿌리기 전에 적어도 30 분 동안 37 ° C에서 인큐베이터에서 요리를 놓습니다.</li>
	<li>3-5 분 (3 mL T175 문화 플라스 크에 대 한) 표준 0.25% trypsin EDTA 용액을 사용 하 여 ASCs trypsinize Trypsinization 후 어떤 셀 수 풀 경우 계산 전에 100 µ m 필터를 통해 셀을 필터링 합니다.</li>
	<li>트립 신 솔루션을 무력화 LG DMEM 10% 보충 FBS (T175 플라스 크에 10 mL) 250 x g. 8 분 대 세포 현 탁 액 분리기는 상쾌한 제거와 LG DMEM 10% 보충의 1 mL에 셀 resuspend FBS.</li>
	<li>FBS와 온도 응답 문화 요리 코팅, 후 37 ° C에서 지구 온난화 접시에 인큐베이터에서 요리를 이동 하 고 접시에서 FBS를 제거 합니다.</li>
	<li>씨앗 ASCs 400000 셀/cm2 (총, 3.52 × 106 세포 배양 접시 당 2 mL에 희석).</li>
	<li>신중 하 게 가능한 균등 하 게 요리를 부드럽게 스윙 하 여 세포를 배포 합니다.</li>
	<li>5% CO2와 다습 한 대기권에서 37 ° C에서 48 h에 대 한 문화 ASC 시트. 참고: 문을 여는 인큐베이터의 온도 강하를 ASC 시트 형성을 방해할 수 있습니다 또는 구성 된 ASC 시트의 조기 분리를 방지 하기 위해 ASCs 시드 후 최소화 하십시오.</li>
</ol>3. 부분 Colectomy 및 대 장 문 합
<ol><li>유발 하 고 남성 Wistar 쥐 (250-350 g 사이 무게) 1 L/min isoflurane의 유지 (1.5-5%)/oxygen 흡입 buprenorphine 0.05 mg/kg을 피하 주사 하 고.</li>
	<li>일단 동물 일반 마 취, 마 취 깊이 반사 테스트를 사용 하 여 평가 합니다. 건조를 방지 하기 위해 눈에 비타민 A 포함 눈 연 고를 적용 합니다. 마 취는 수술에 대 한 충분 한 것으로 간주, aseptically 머리를 면도 하 고 외과 영역 70% 에탄올으로 두 번 살포 하 여 복 부를 준비 합니다. 살 균 종이 커튼으로 복 부 드러 워 진. 무 균 기술을 사용 하 고 전체 수술 중 살 균 분야를 유지 합니다.</li>
	<li>메 마른 외과 블레이드 크기 10으로 5 cm의 중간 선 복 부 절 개를 만들고 Metzenbaum가 위 절 개를 확장. 확인 하 고 콜론 exteriorize 염 분 moistened gauzes와 복 부 구멍의 나머지에서 결 팩. 오른쪽, 중간, 식별 하 고 퉁 명 스럽게 Halsted 모기를 사용 하 여 각 선박 주위 해 부는 mesentery에서 산 통 동맥을 왼쪽, 고 흡수 비 꼰 실크 4/0 각 개별 선박 선.</li>
	<li>맹 하 aborally 1.0 cm 사이 꼬리 mesenteric 동맥 위에 0.5 cm 저 승 세그먼트를 격리 합니다. 건강 한 콜론 Metzenbaum가 위를 사용 하 여 통해 transect. 저 승 세그먼트의 절제술, 후 두 긴 면봉, 원심 결 통해 트랜스 anally 그리고 근 위 결 장 끝에 도입 하 여 콜론의 인접 하 고 원심 끝을 함께 가져.</li>
	<li>수술 현미경을 사용 하 여, 한 레이어 반전 5 중단된 봉합 (비 흡수 monofilament 폴 리아 미드 8/0)를 사용 하 여 봉합에 의해 부족 하 게 sutured 엔드-투-엔드 문 합을 만듭니다. 결 장 벽의 모든 레이어를 통해 중단된 봉합 놓고 노트 extraluminally 위치.</li>
	<li>컨트롤 또는 ASC 시트 그룹으로 무작위로 쥐를 나눕니다.</li>
</ol>4. ASC 시트 이식
<ol><li>40 분 비보에 이식, ASC 시트 분리를 촉진 하기 전에 실내 온도에 아래로 냉각 하기 위하여 문화 요리를 하실 수 있습니다. 참고:는 초연 후 약 2 cm 직경에서 축소 ASC 시트. ASC 시트 생존 분리20후 최소한 3-6 h에 대 한 높은 남아 있습니다.</li>
	<li>문화 매체와 혈 청 1 mL와 함께 그것은 lg 전자 DMEM 무료 교체를 제거 합니다.</li>
	<li>부드럽게 atraumatic 집게와 ASC 시트의 테두리를 잡아 고 anastomotic 라인 위에 ASC 시트의 요리 측을 두십시오.</li>
	<li>신중 하 게 ASC에 밖으로 스트레칭 약 0.25 cm 위와 아래 anastomotic 라인 atraumatic 집게 랩 콜론 주위 시트를 사용 하 여 시트. 랩 등 측면 주위 ASC 시트까지 콜론을 들어올립니다. 참고: ASC 시트는 anastomotic 라인을 자발적으로 준수, 조직 접착제를 사용 하는 필요 또는 다른 소재. 제어 그룹 추가 치료에 영향을 받지 않습니다.</li>
	<li>목화 면봉을 제거 하 고 장갑과 악기를 변경. 복 부에 콜론을 바꿉니다. 흡수 되기 쉬운 봉합을 사용 하 여 실행 한 레이어와 닫기 복 부-교육 알바 꼰 polyglycolic 산 5/0. 봉합은 subcutis와 함께 흡수 봉합을 사용 하 여 실행의 한 계층에서 커티스 꼰 polyglycolic 산 5/0.</li>
</ol>5. 수술 후 평가 및 후속
<ol><li>즉시 5 mL 따뜻한 식 염 수의 피하 주사로 동물 postoperatively rehydrate. 체온을 유지 하 고 동물을 sternal recumbency를 유지 하기 위해 충분 한 의식 회복 될 때까지 생체 신호를 모니터링 하는 열 램프 아래에서 동물을 놓습니다. 참고: 수술을 받은 했다 동물 했다 완전히 복구 될 때까지 다른 동물의 회사에 반환 되지 않습니다. 복구, 후 물에 자유 접근 및 일반 쥐 차 수 있도록. 수술 후 통증 완화를 위해 관리 0.05 mg/kg buprenorphine 피하 모든 6-8 h 3 일 동안. 이 연구에서 언제 든 지 아무 항생제 관리 되었다.</li>
	<li>웰빙 무게의 일일 임상 평가 얻을. 매우 낮은 웰빙 점수 또는 심한 체중 감소, 동물 마 취 아래 심장 찔린 통해 exsanguination에 의해 안락사 해야 한다.</li>
	<li>수술 후 당일 또는 하루 3 7 anesthetize 1 L/min isoflurane의와 다시 쥐 (1.5-5%)/oxygen 흡입 buprenorphine, 없이 U 자 모양의 절 개를 다시 개복 술을 수행 하 고. 복 부 복 막 염만, 섬유 유착, 그리고 농의 존재의 표시를 확인 하십시오. 유착의 심각도 점수와 복 부와 anastomotic 지역에서 농 양.</li>
	<li>문 합을 포함 하 여 콜론의 닫힌된 세그먼트에 공기 insufflation에 의해 anastomotic 파열 압력을 결정 합니다. 첫 번째 공기 파열 압력으로 누설 하는 때 공기 압력과 파열의 장소를 기록 합니다.</li>
	<li>각 희생된 동물에서 콜론을 제거 하 고-장 문 합을 포함 하는-콜론의 컷된 세그먼트 버퍼링 4% 포름알데히드, 파라핀 포함 및 콜론 4 μ m 두꺼운 부분으로 절단에 의해 해결.</li>
	<li>심장 찔린 통해 exsanguination에 의해 직접 문 합 세그먼트의 컬렉션 후 마 취 동안 동물을 안락사. 호흡 운동과 심장 박동의 부재와 함께 동물 죽음을 확인 합니다.</li>
	<li>되며 & 오신 빨강, Picro 시리우스 등 조직화 학적인 stainings 및 인간 미토 콘 드리 아, 쥐 내 피 세포 (CD34 안티) 및 (CD3 +, CD163 +) 면역 체계의 세포에 대하여 항 체와 immunohistochemical stainings 수행 합니다.</li>
	<li>컴퓨터 분석에 대 한 슬라이드를 디지털화 하 고 동물 그룹을 비교.</li>
</ol>

<ol>
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Cell Harvesting by Temperature Reduction Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf">https://tools.thermofisher.com/content/sfs/brochures/AppNote-Nunc-UpCell-N20230.pdf</a> (2008)</li><li>Wu, Z., 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=Colorectal+anastomotic+leakage+caused+by+insufficient+suturing+after+partial+colectomy:+a+new+experimental+model.">Colorectal anastomotic leakage caused by insufficient suturing after partial colectomy: a new experimental model.</a> Surg Infect (Larchmt). 15 (6), 733-738 (2014).</li><li>Wu, Z., 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=Reducing+colorectal+anastomotic+leakage+with+tissue+adhesive+in+experimental+inflammatory+bowel+disease.">Reducing colorectal anastomotic leakage with tissue adhesive in experimental inflammatory bowel disease.</a> Inflamm Bowel Dis. 21 (5), 1038-1046 (2015).</li><li>Wu, Z., 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=Reducing+anastomotic+leakage+by+reinforcement+of+colorectal+anastomosis+with+cyanoacrylate+glue.">Reducing anastomotic leakage by reinforcement of colorectal anastomosis with cyanoacrylate glue.</a> Eur Surg Res. 50 (3-4), 255-261 (2013).</li><li>Aysan, E., Dincel, O., Bektas, H., Alkan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polypropylene+mesh+covered+colonic+anastomosis.+Results+of+a+new+anastomosis+technique.">Polypropylene mesh covered colonic anastomosis. 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저자는 연구 잠재적인 상충으로 해석 될 수 있는 어떤 상업적 또는 금융 관계의 부재에서 실시 되었다 선언 합니다.

Anastomotic 누설은 기본 문 합으로 결 장 절제술을 다음과 같은 가장 심각한 불리 한 이벤트 이다. Anastomotic 장애 및 누설을 방지 하기 위해 최적의 기술을 여전히 부족 한. 다양 한 생체 재료의 로컬 응용 프로그램, 다양 한 결과25,,2627실시 되어 있다. 세포 치료의 목표는 조직 대체 하 여 조직 복구 또는 로컬 치유 통해 paracrine 분 ?...

기본 문 합으로 부분 colectomy는 대 장 암, 크 론 병, 계실 염1,2등 콜론에 영향을 미치는 많은 질병에 대 한 할 수 있는 일반적으로 수행된 수술입니다. 가장 후 대 장 문 합은 anastomotic 누설3합병증을 겁 나. 비록 anastomotic 누수와 관련 된 몇 가지 위험 요소 확인 되었습니다, 누설을 방지 하기 위한 솔루션 알4,5남아 있습니다.
지방 조직 유래 stromal 세포 (ASCs) 염증 및 영양 특성6,7, 이러한 세포 재생 요법8에 대 한 후보를 유망 하 게 연결 되어 있습니다. 조직 치유를 촉진 하는 ASCs의 효과 심장 근육, 피부, 식도9,,1011,,1213등 다양 한 조직에 표시 했다. 그러나, 장 치유를 촉진 하는 ASCs 사용 하 여에 몇 가지 보고 있다. ASC 코팅 biosutures 통해 실험 대 장 anastomoses에 ASCs의 지방 이식 또는 ASCs serosal 주사14 치유 하거나 개선 보였다 anastomotic 더 유리한도 불구 하 고 anastomotic 누설을 방지 하지 않았다 치유15.
정지에서 ASCs의 지방 이식 함께 또는 생체 부족 세포 보존 또는 이식된 세포11의 부적 절 한 배포에 연관 될 수도 있습니다. 셀 시트 기술16,17 ASC 배달18,19의 혁신적인 방법을 제공합니다. 따라서, 이전 연구에 새로운 접근 방식은 어느 ASCs 셀에 시트 실험 대 장 문 합20에 적용 수 있는 제안 했다. 이 연구는 ASC 시트 이식 쥐 모델에서 부분 colectomy 후 대 장 문 합 누출을 감소 시키기에서 성공적 증명. 이 기사 보고 ASC 시트 준비 및 외과 이식 기술 합니다.

<table border="0" cellpadding="0" cellspacing="0" width="689">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">LG DMEM</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">22320022</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Collagenase type I</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">17100-01</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A9418</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal bovine serum</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">10270-106</td>
			<td style="width:167px;">FBS, ASC isolation and culture</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">3% acetic acid with methylene blue</td>
			<td style="width:115px;">Stemcell Technologies</td>
			<td style="width:191px;">7060</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Gentamicin</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15750-037</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ampothericin B&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">15290-018</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Shaker (Gallenkamp Environmental Shaker Model 10X 400)</td>
			<td style="width:115px;">Akribis Scientific</td>
			<td style="width:191px;">F240</td>
			<td style="width:167px;">ASC Isolation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge&#160;</td>
			<td style="width:115px;">Hettichlab&#160;</td>
			<td style="width:191px;">Rotina380</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:216px;">Phosphate buffer saline&#160;</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">14190-094</td>
			<td style="width:167px;">PBS, ASC isolation and culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ascorbic acid-2-phosphate</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">A8960</td>
			<td style="width:167px;">ASC isolation and culture</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Human recombinant fibroblast growth factor 2&#160;</td>
			<td style="width:115px;">AbD Serotec</td>
			<td style="width:191px;">AF-100-15</td>
			<td style="width:167px;">FGF2, ASC isolation and culture</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin EDTA</td>
			<td style="width:115px;">Gibco, Life technologies</td>
			<td style="width:191px;">25200-056</td>
			<td style="width:167px;">ASC culture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:191px;">D2650-5x</td>
			<td style="width:167px;">DMSO, ASC freezing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Thermo-responsive culture dishes</td>
			<td style="width:115px;">Nunc Thermo scientific</td>
			<td style="width:191px;">174904</td>
			<td style="width:167px;">ASC sheet preperation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable braided silk 4/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">21151042</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Non-absorbable monofilament polyamide&#160; 8/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">G1118170</td>
			<td style="width:167px;">surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Absorbable braided polyglycolic acid 5/0</td>
			<td style="width:115px;">B Braun</td>
			<td style="width:191px;">C1048207</td>
			<td style="width:167px;">surgery</td>
		</tr>
	</tbody>
</table>

transplantation of adipose tissue-derived stem cell sheet to reduce leakage after partial colectomy in a rat model

Anastomotic 누설 대 장 수술 후 비참 한 합병증은 이다. 누출 방지에 대 한 현재 메서드는 임상 효능의 다른 수준, 그들은 지금 불완전 한 솔루션까지 있습니다. 줄기 세포 치료 ASC 시트를 사용 하 여이 문제에 대 한 솔루션을 제공할 수 있습니다. ASCs 조직 그들의 영양 및 immunomodulatory 속성 때문에 치유를 승진 시키기를 위한 유망 후보자 이라고 여겨진다. 여기, 우리는 누설을 줄이기 위해 쥐 모델에서 대 장 문 합에 이식 하는 고밀도 ASC 시트를 생산 하는 방법을 제공 합니다. ASCs 셀 시트 쉽게 분리 될 수 있는 온도 응답 문화 요리에 형성. 이식의 날, 5 봉합 대 장 문 합으로 부분 colectomy 수행 되었다. 동물 쥐 당 1 ASC 시트와 함께 즉시 이식 했다. ASC 시트 없이 어떤 접착제, 봉합 사, 또는 어떤 소재 문 합을 자발적으로 준수 됩니다. 동물 그룹 3 ~ 7 일을 희생 했다 postoperatively. 이식된 동물에 비해, anastomotic 농 양 및 누설의 부각 더 높았다 제어 동물. 우리의 모델에서 대 장 문 합 후 ASC 시트 이식 성공적이 고 낮은 누설 율으로 연결 했다.

ASC 시트 문화 및 결 장 절제술 및 문 합 절차를 묘사 하는이 연구의 흐름 차트는 그림 1에 표시 됩니다. 그림 2 는 ASC 시트 현미경 형태학 및 중 고 분리 후 ASC 시트의 거시적인 외관. 그림 3 ASC 시트 분리 및 이식의 다른 단계를 보여 줍니다. 그림 4 는 anastomotic 선 및 누설 방지에 ASC 시?...

Watch this Scientific Journal Video about Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model at JoVE.com

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

쥐 모델에 부분 Colectomy 후 누설을 줄이기 위해 지방 조직 유래 줄기 세포의 이식

준비와 쥐 모델에서 부족 하 게 봉합 대 장 anastomoses에 지방 조직 유래 줄기 세포 (ASC) 시트의 이식 제공 됩니다. 이 새로운 응용 프로그램은 ASC 시트 대 장 문 합 누출을 줄일 수 있습니다 보여줍니다.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

transplantation-adipose-tissue-derived-stem-cell-sheet-to-reduce

Research

JoVE Journal

Medicine

7.5K 조회수.  Utrecht University. 준비와 쥐 모델에서 부족 하 게 봉합 대 장 anastomoses에 지방 조직 유래 줄기 세포 (ASC) 시트의 이식 제공 됩니다. 이 새로운 응용 프로그램은 ASC 시트 대 장 문 합 누출을 줄일 수 있습니다 보여줍니다.

비디오: Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Orthotopic 대동맥 이식 : 만성 Vasculopathy의 개발 연구에 생쥐 모델

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

연구

의학

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 ...

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

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

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

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

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 ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

간 간선 재건과 부분 Orthotopic 간 이식의 쥐 모델에 대한 외과 절차

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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 ...

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 ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

정맥 삽입 위치 모델 : 바이 패스 이식 개통을 연구하기위한 적절한 모델

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

ADSC 매 이식은 확장 식도 내시경 점막하 절개 후 협착을 방지하는

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD.CB17-Prkdcscid/J) mouse. 
In this procedure, two small incisions are made. The first incision is to expose and resect the left lobe of the liver, and another small incision is made to expose the spleen for the intrasplenic transplantation of cells. This procedure does not require any specialized surgical skills, and it can be completed in 5-7 minutes with less stress and pain, faster recovery, and better survival. We have demonstrated the transplantation of hepatocytes isolated from a green fluorescent protein (GFP) expressing mouse (Transgenic C57BL/6-Tg (UBC-GFP) 30Scha/J), as well as hepatocyte like cells of human origin (NeoHep) in partially hepatectomized NOD.SCID mice.

We have described a protocol for performing partial hepatectomy (PHx) and cell transplantation via spleen in NOD.SCID (NOD.CB17-Prkdcscid/J) mice. In this protocol, an incision is made to expose and resect the left lobe of the liver followed by another incision for the intrasplenic transplantation of cells.

끄 덕 임에서 부분 Hepatectomy 후 Hepatocytes의 intrasplenic 이식 SCID 마우스

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

다시는 vivo에서 선박 지향 70% 쥐 부분 간 이식 Arterialized Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...