This work was supported by The British Heart Foundation and The Oak Foundation.

실험 동물의 사용 및 관리에 대한 기관위원회에 의해 승인 된 프로토콜에 따라 모든 동물 실험을 수행합니다. 문화 미디어 1. 준비 <ol><li> F-12K 보통, 10 % 소 태아 혈청 (FBS) 및 100 U / ml의 페니실린과 스트렙토 마이신 : 인간 섬유 아 세포주 CCL-153 배지 만들기. </li><li> PiPSC 생성을위한 프로그램을 다시 미디어를 확인 : 마네 둘 베코의 수정 독수리의 매체 (DMEM) 20 % 녹아웃 혈청 교체, 0.1 MM의 β-머 캅토 에탄올, 0.1 mM의 최소 필수 중간 (MEM) 비 필수 아미노산, 10 NG / ml의 염기성 섬유 아세포 성장 인자를 포함하는 2 (bFGF를-2) 및 100 U / ml의 페니실린과 스트렙토 마이신. 미디어를 사용하기 전에 bFGF를-2를 갓마다 추가합니다. </li><li> 평활근 세포를 유도 분화 매체 (DM)를 확인 (SMC) 분화 : 10 % FBS, 0.1 밀리미터 β 머 캅토 에탄올, 100 U가 / ㎖ 및 스트렙토 마이신 25 NG / ml의 혈소판 유래 성장 인자를 포함하는 미디어-MEM (α)(PDGF-ββ). </li><li> 확인 내피 분화 미디어 내피 세포 (EC)의 분화를 유도하는 (EC-DM) : 내피 성장 미디어 -2- (EGM-2) 50 NG를 함유 배지 / ㎖ 혈관 내피 성장 인자 (VEGF) 및 100 U / ml의 페니실린과 스트렙토 마이신. </li></ol> 부분적으로 유도 다 능성 줄기 세포로 재 프로그램 2. 인간 섬유 아세포 (PiPSC) <ol><li> 배양액을 0.04 % 젤라틴 용액 도포 플라스크에 배양 인간 섬유 아세포 세포주 CCL-153. </li><li> 통로 셀 (1)의 비율로 3 일마다 6. PiPSC 발생의 최적의 효율성을 위해 통로 (9) 전에 세포를 사용합니다. 90 % 컨 플루 - 세포는 80에 도달하면 형질 전환을위한 준비가되어 있습니다. </li><li> (4) 재 프로그래밍 인자를 함유하는 폴리 시스 트론 플라스미드 pCAG2LMKOSimO 선형화 옥타 - 결합 전사 인자 4 (인 Oct4)를 SOX2, 크 룹펠 형상 인자 4 (KLF4) 및 C-MYC (pCAG-OSKM). 37 ° C에서 3 시간 동안 PvuI 제한 효소 5 유닛 플라스미드 5 μg의 다이제스트. 정화제조 업체의 프로토콜에 따라 상용 키트와 함께 플라스미드. </li><li> 트랜 제조 업체의 프로토콜에 따라 인간 피부 섬유 아세포 (NHDF) nucleofection 키트와 함께 전기에 의한 선형 pCAG-OSKM 플라스미드 4 μg의 2 × 10 (6) 인간 섬유 아세포. 부드럽게 미리 예열 재 프로그래밍 매체 5 mL를 함유하는 미리 0.04 % 젤라틴 코팅 된 T25 플라스크에 형질 감염된 세포를 시드. </li><li> 24 시간 후, 형질 세포의 순수한 인구를 선택하는 25 ㎍ / ml의 네오 마이신의 보충제 매체 변경합니다. </li><li> 4 일까지 매일 매체를 변경합니다. 참고 : 인간 섬유 아세포는 재 프로그래밍 4 일 후에 PiPSC된다. </li></ol> 내피 세포 및 평활근 세포에 PiPSC 3. 차별화 <ol><li> 4 일 동안 DM을 포함하는 콜라겐 IV의 사전 코팅 요리에 씨 PiPSC는 SMCs로 분화를 유도합니다. 4 일까지 매일 매체를 변경합니다. </li><li> 콜라겐 IV의 사전 코팅 요리 C에 씨앗 PiPSC육일에 대한 내피-DM을 ontaining은 내피 세포로 분화를 유도합니다. 6 일까지 매일 매체를 변경합니다. </li></ol> 4. 탈세 포화 대동맥 이식 준비 참고 : 모든 솔루션과 장비는 멸균해야한다. <ol><li> 자궁 경부 전위로 마우스를 희생하고 해부 현미경 부정사 위치에 마우스를 고정합니다. </li><li> 흉강을 엽니 다 흉골을 통해 흉곽 주위를 잘라. 대동맥을 노출 심장, 폐, 식도를 제거합니다. 부드럽게 집게로 사망시 대동맥의 지방을 제거합니다. </li><li> 가위로 앞쪽 끝에서 대동맥을 잘라 부드럽게 무딘 집게로 끝을 잡고. 무딘 절개에 의한 뒤에 척추 열에서 대동맥를 분리합니다. 조심스럽게 바이폴라 electrocoagulator와 결찰에 의해 대동맥에서 모든 분기 동맥을 닫고 가위로 결찰의 끝에서 절단. </li><li> 3m로 대동맥 루멘를 세척하기 위해 5 ML의 주사기를 사용하여혈전의 형성을 방지하기 위해 100 U 헤파린을 함유하는 식염수 L. </li><li> 신장으로 가지 전에 대동맥의 뒤쪽 끝을 잘라. 대동맥의 무결성을 유지하면 전체 절차를 수행하는 동안 매우 중요합니다. </li><li> 인산 완충 식염수 (PBS)에 희석 0.075 % 소듐 도데 실 설페이트 (SDS) 용액 5 ㎖와 대동맥 루멘 플러시. </li><li> 10cm 페트리 접시에 0.075 % SDS 솔루션의 흉부 대동맥을 적 십니다. 실온에서 150 rpm에서 2 시간 동안 진탕에 페트리 접시를 놓습니다. </li><li> PBS의 5 ml의 대동맥 루멘을 플래시합니다. </li><li> 10cm 페트리 접시에 PBS의 대동맥을 적 십니다. 실온에서 150 rpm에서 2 시간 동안 진탕에 페트리 접시를 놓습니다. PBS마다 20 분을 새로 고칩니다. </li><li> PBS의 5 ml의 대동맥 루멘을 플래시합니다. </li><li> 최대 일주일 동안 4 ° C에서 PBS에서 탈세 포화 대동맥 이식을 유지합니다. </li></ol> PiPSC 및 생물 반응기 컨디셔닝 5. 듀얼 시드 참고 : 모든 솔루션을s와 장비는 멸균해야한다. 조직 문화 후드에서 모든 작업을 수행합니다. <ol><li> 그림 1에 도시 된 바와 같이. 생물 반응기의 흐름 회로를 조립 튜브와 함께 배양 챔버, 미디어 저수지, 연동 펌프 및 규정 준수 챔버를 연결합니다. 순환에 필요한 매체의 최소한의 부피는 40 ml의입니다. </li><li> 전제 조건 10cm 페트리 접시에 DM 매체 대동맥 담가 배지와 탈세 포화 된 용기. 실온에서 50 rpm으로 1 시간 동안 진탕에 페트리 접시를 놓습니다. </li><li> 현미경으로 탈세 포화 된 선박의 양쪽에 1cm 길이 나일론 튜브 (OD 0.9 mm, ID 0.75 mm)를 삽입합니다. 선박과 8-0 실크 봉합사와 튜브를 묶어. </li><li> 챔버 벽으로부터의 입구 및 출구 포트 (1/32 &quot;튜브)와 나일론 튜브를 연결하여 배양 챔버에서 탈세 포화 된 대동맥 이식술을 조립한다. 매체의 5mls 각 시간 배양 챔버를 유지한다. </li><li> 추가 예열로를 Trypsinize PiPSC트립신은 배양 접시를 커버하고 부드럽게 요리 10 배 바위. 트립신을 취소하고 2 인큐베이터에서 접시를 떠나 - 3 분. 접시에 예열 문화 매체를 추가하고 세포와 매체를 혼합한다. </li><li> 혈구를 사용하여 세포 수를 계산합니다. 원심 분리기 300 x g에서 5 분 동안 5 × 105 세포를 함유하는 각각의 세포 현탁액의 두 분액. 완전히 뜨는을 대기음. </li><li> 완전히 상층 액을 흡입 한 후, DM의 50 μL에 주사위 세포 펠렛 1 펠렛을 재현 탁. 탈세 포화 혈관 내강에이 세포 현탁액을 주입한다. </li><li> 이어서, 마트 리겔 100 ㎕의 주사위에 다른 세포 펠렛을 재현 탁. 조심스럽게 탈세 포화 대동맥 이식 상 혼합물을 피펫. </li><li> 혼합물이 용기 외부 표면을 감싸는 고르게 겔 상태로 전환 때까지 15 분 - 10 기다립니다. DM으로 배양 챔버를 입력합니다. </li><li> 37 ° C에서 5 % CO 2 배양기에서 설정 전체 생물 반응기를 배치. 수동으로 처음 2 시간에서 탈세 포화 이식을 종축 모든 반 시간 약 90 ° 회전합니다. </li><li> 세포 부착 가능하도록 12 시간 동안 정적 문화를 유지합니다. </li><li> 평활근 세포의 분화를 유도하는 연동 펌프로 루멘을 통해 DM을 제공한다. 5 ml / 분 및 24 시간에 걸쳐 20 ㎖ / 분으로 단계적으로 증가시 초기 유속을 시작합니다. </li><li> 24 시간 동안 20 ㎖ / 분에서 매체 순환 유량을 유지. </li><li> 매체 흐름을 중지하고 인큐베이터에서 설정 생물 반응기를 이동합니다. </li><li> 다시 씨앗 PiPSC와 이식의 루멘. 5.6 -를 Trypsinize는, 단계 5.5로 계산 원심 분리기 1 × 6 PiPSC. 50 NG / ㎖ VEGF를 함유 EGM-2 배지에 50 μl의 세포를 재현 탁. 그래프트의 루멘으로 세포 혼합액을 주입한다. </li><li> 내피 세포의 분화를 유도하기 위해 50 NG / ml의 VEGF를 포함 EGM-2 배지에 배양 챔버 및 전체 순환 매체를 변경합니다. </li><li> 토륨 설정을 다시 생물 반응기를 이동전자 인큐베이터. 수동으로 이식을 처음 2 시간 90 ° 30 분마다 회전 한 다음 12 시간 동안 정적 문화를 유지. </li><li> 5 ㎖ / 분, 35 ㎖ / 분으로 단계적으로 상승에 순환류 시작. 오일에 35 ㎖의 유속 / 분 유지. 매일 순환 챔버 매체 변경합니다. </li><li> 마우스에 더 이식에 대한 설계 이식을 수확. </li></ol> 마우스로 두 번 시드 PiPSC 이식 6. 손톱 <ol><li> 사용 NOD.CB17-Prkdc의 SCID / 혈관 이식받는 사람으로 NcrCrl 수컷 마우스. 35g, 그리고 좋은 건강 상태를 유지하고 있습니다 - 항상 마우스는 약 10 주입니다 약 25의 무게를 확인합니다. </li><li> 복강 Hypnorm (25 ㎎ / kg) 및 Hypnovel (25 ㎎ / kg)의 조합을 투여함으로써받는 마우스를 마취. 마취 동안 건조를 방지하기 위해 눈에 바셀린 안과 연고를 적용합니다. 마우스는 편안한 근육을 가지고 꾸준히 호흡하도록함으로써 효율적인 마취를 확인합니다. <ol><li> 목 면도 및 확장에 누운 위치에 마우스를 고정합니다. 마우스 호흡기가 선명합니다 있는지 확인하기 위해 마취제와 함께 아트로핀 황산 (1.7 ㎎ / ㎏)을 관리합니다. </li></ol></li><li> 마우스의 흉골에 하악에서 중간 선 절개를 준비합니다. 5 ~ 10 배 증폭과 해부 현미경, 측면 오른쪽 침샘을 들어 올려 오른쪽 경동맥을 노출 할 권리 cleidomastoid 근육을 제거합니다. </li><li> 조심스럽게 기단을 향해 선단에서 오른쪽 경동맥을 동원에 부착 된 조직을 제거합니다. 8-0 실크 봉합사로 두 번 경동맥 중간 부분을 결찰하고 두 관계 사이 해부. </li><li> 각 용기의 끝에서 멸균 나일론 튜브로 만든 커프를 통과 microhemostat 클램프 각 끝을 고정합니다. </li><li> 한 끝에 봉합 넥타이를 제거하고 헤파린의 드롭 (100 U / ml)에 적용됩니다. 직후 번째의 선단부를 반전전자 동맥 전체 커프 몸을 커버하고 팔목에 8-0 실크 봉합사로 반전 용기를 해결합니다. <ol><li> 동맥의 다른 부분에 동일한 절차를 반복한다. 식염수로 세척 동맥이 혈전을 제거합니다. </li></ol></li><li> 동맥 커프 통해 용기의 탈세 포화 된 단부 슬라이딩 8-0 실크 봉합사로 고정하여 경동맥 양단 탈세 포화 용기 그래프트 임플란트. 이식의 맥동을 평가하기 전에를 종료에서 혈관 클램프를 제거합니다. </li><li> 원래 위치로 오른쪽 침샘을 놓습니다. 6-0 polyglactin 봉합사를 사용하여 중단 된 봉합 수술 위치에 피부를 닫습니다. </li><li> 절차가 완료된 후에는 의식이 재개 될 때까지 지속적으로, 마우스의 활력 징후를 관찰한다. 자궁 경부 전위 수확 용기 이식에 의해 24 시간 이상 추가 분석을 위해 삼주를받는 사람 쥐를 희생. </li></ol>

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저자는 충돌하는 금전적 이해 관계가 없습니다.

현재 프로토콜은 기능적 조직 공학 혈관 PiPSC 인간 섬유 아세포를 사용하여 생성 될 수있는 사운드, 빠르고, 단순하고 재현성 효율적인 전략을 나타낸다. 이 기술은 가까운 미래의 재생 의학, 조직 공학 및 잠재적 환자 맞춤형 세포 치료를위한 유용한 도구를 나타냅니다. 프로토콜의 효능을 보장하는 중요한 단계는 혈관 생물 반응기에서 PiPSC의 준비, 멸균 완전히 탈세 포화 대동맥 이식, 발판에 PiP...

혈관 도관의 건설은 심혈관 질환으로 인한 손상 및 손상된 혈관의 수술 수리에 대한 기본적인 전략이다. , 동종 이식,자가 조직 (심낭 또는 복재 정맥)과 이종 이식 1, 현재까지 수술에 사용 된 이식 재료는 생체 적합성 합성 고분자 ([고어 텍스의 ePTFE] 또는 폴리에틸렌 테레 프탈레이트 [쿠론] 폴리 테트라 플루오로 에틸렌 [테플론, 확장 된 폴리 테트라 플루오로 에틸렌)을 포함한다. 인공 이식 (예를 들어 고어 텍스 쿠론)이 가장 일반적으로 사용되는 반면, 이러한 재료 가능성 협착증, 칼슘 증착, 혈전 색전 감염을 포함 수많은 장단기 합병증을 일으킨다. 와 현재 생물학적 이식 환자는 혈전 색전증 감소하지만, 그들은 여전히 석회화 열화이 때문에 차 이식 실패 및 단축 내구성으로 제한을 발생합니다. 따라서, 수술 t을 크게 향상에도 불구하고년, 연구자와 임상의를 통해 echniques 여전히 혈관 질환을위한 이상적인 도관을 식별의 필요성 부담한다. 더 최근에, 혈관 조직 공학의 연구 분야는 세포를 성공적 래프팅 1 기능성 용기 전형 생체 모방 환경을 만들 목적으로, 생분해 성 지지체에 혼입되어있는 개념을 생성하고있다. 기본적으로 혈관 구조의 성공은 세 가지 필수 구성 요소에 따라 달라집니다; 즉, 발판, 내피 세포 내층 및 평활근 세포 층, 네이티브 맥관계에 필적 기계적 특성을 제공하기 위해 적절한 세포 외 기질을 함유하는 지지체, 및 / 조절을 개시에 필요한 세포 / 분자 시그널링을 포함 셀 수리. 장기 이식 개통과 신 조직의 지속적인 개발, 제 비계의 효과적인 세포 파종에 크게 의존ereby 매우 중요한 세포 유형의 결정을 렌더링. 몇몇 보고서 소경 도관 3-6 개발 성숙한 내피 다양한 소스로부터 평활근 세포의 사용을 입증한다. 유망하지만, 충분한자가 혈관 내피 부족 성숙한 평활근 세포가 상당한 부담으로 작용 얻었다. 최근에, 다양한 소스로부터의 줄기 세포는 혈관 조직 공학 어플리케이션에 이용되어왔다. 실제로, 배아 줄기 세포 (7)를 포함한 줄기 세포 다양한 종류의 유도 된 다 능성 줄기 세포 (iPSCs) 8,9, PiPSC 10,11, 골수 유래 단핵구 (12), 중간 엽 줄기 세포 (13), 혈관 내피 전구 세포 및 성숙한 혈관벽 줄기 세포 유래 한 항원 SCA (-1) + (14, 15)이 정의 된 모든 미디어에 응답 내피 기능 중 하나 또는 평활근 세포로 분화 할 수있는 것으로 입증되었다 줄기 / 선조 세포 및배양 조건. 또한, 줄기 세포의 무제한 자기 갱신 용량은 그들 만 성숙한 내피 성장 정지 및 노화를 진행하기 전에 한정된 횟수 위해 나눌 수 평활근 세포와 달리 더 나은 후보를 만든다. 그 래프팅은 생체 적합성, 생체 역학적 특성 및 생분해 속도 등 여러 가지 요인에 따라 달라위한 골격 물질의 선택은 성공적인 조직 공학적 용기를 생성한다. 기본적으로, 물질은 생분해해야 이식을위한 발판을 만드는 데 사용하고 불필요한받는 사람 면역 반응을 탑재되지 않습니다. 또한, 세포 부착과 이후의 생존에 적합한 기공 및 미세을 포함해야합니다. 현재까지, 혈관 조직 공학에서 사용되는 지지체 가장 일반적인 재료는 폴리 글리콜 산, 폴리 락트산, 폴리 카프로 락톤 ε (16)의 중합체를 포함한다. 최근 탈세 포화 된 생물학적 물질은 할또한 일부 성공을 적용. 여러 실험실자가 세포와 탈세 포화 인간, 개 또는 돼지의 혈관을 뿌리는 것은 응고 내막 증식증 17-19 저항 생물학적 이식을 한 것으로 나타났습니다. 혈관 조직 공학의 다른 전략은 비계 지원 (20), (21)없이 예를 들어, 세포 외 기질 단백질 기반의 혈관 이식, 섬유소 젤 (13)에 파종 세포 생성 세포 시트를 포함한다. 현재의 프로토콜 기능 내피 세포와 평활근 세포, 중증 합병 면역 결핍증에 조직 공학 혈관의 기능 PiPSC 유래 혈관 세포를 항구 탈세 포화 된 혈관 지지체로 구성된 생물 반응기 및 이식의 세대 (SCID에 인간 PiPSC의 분화를 보여줍니다 ) 마우스. PiPSC이 세포는 쥐에서 종양을 형성 윤리적 제기하지 않기 때문에 혈관 이식의 조직 공학에 사용할 최적의 세포 유형이며,동종 면역 반응. 더욱이, 우리는 핍 내피 세포 및 핍 - 평활근 세포를 생성하기위한 전략이 효율적인 10,11 재현임을 보여 주었다. 그 후, 우리는 따라서 이식과 생존 효능을 향상 밀접하게 네이티브 용기 내에 존재하는 기질 단백질을 모방하는 PiPSC 유래 혈관 세포의 파종을위한 탈세 포화 된 선박을 설계​​했습니다. 또한, 종래 PiPSC 뿌리기에 용기의 탈세 포화가 대 식세포 등의 면역 세포 유형에 의해 마운​​트 염증 반응의 발생을 방지한다. 더 중요한 것은,이 프로토콜은 단지 인간 번역에 대한 혈관 도관 유망 생성하는 방법을 나타내지 않는, 또한 연구 및 마우스 모델을 통해 혈관 조직 재생을 제어하는​​ 분자 메커니즘을 이해 귀중한 수단을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

PiPSC의 성공적인 세대는 4 전사 인자, 인 Oct4, SOX2, KLF4 및 C-MYC (OSKM)를 운반하는 선형화 된 pCAG2LMKOSimO 플라스미드와 인간의 섬유 아세포를 nucleofecting 후 사일 확인되었다. PiPSC 섬유 아세포 (그림 2A)에 비해 현저하게 별개의 표현형을 표시의 mRNA (그림 2B)와 단백질 (그림 2C) 레벨 10에서 4 재 프로그래밍 요소를 표명했다. PiPSC 기반 혈관 이식의 효능은 모두...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

여기, 우리는 두 번 시드를 부분적으로 유도 만능 줄기 세포로 쥐에 이식을위한 기능적인 조직 설계 혈관 이식을 생성하는 프로토콜을 제시 (PiPSC은) - 탈세 포화 선박 발판 생물 반응기에서 유래 내피 세포 - 평활근 세포 및 PiPSC 산출했다.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

11.9K 조회수.  King's College London BHF Centre.  여기, 우리는 두 번 시드를 부분적으로 유도 만능 줄기 세포로 쥐에 이식을위한 기능적인 조직 설계 혈관 이식을 생성하는 프로토콜을 제시 (PiPSC은) - 탈세 포화 선박 발판 생물 반응기에서 유래 내피 세포 - 평활근 세포 및 PiPSC 산출했다. 

비디오: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

트라이 전단지 폴리머 밸브의 상대 유체 역학 평가를위한 프로토콜

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

연구

생체공학

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

결합 표면의 접목 및 Postmodification 과정에 의한 빛의 응답 멤브레인의 제조

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

티슈 엔지니어링 혈관 이식 및 이후의 이식을위한 수술 기법<em&gt; 생체</em&gt; 모니터링

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

결합 된 3 차원 조직은 엔지니어링<em&gt; 체외</em&gt; /<em&gt; 인 실리코</em특정 돌연변이 배경에서 약물의 효과를 예측&gt; 폐 종양 모델

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

의 응용 프로그램<em&gt; 생체</em&gt; 조직 평가를위한 쥐 앞정 전방의 기능 테스트는 골격근 수리 엔지니어링

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

큰 힘줄 결함을 복구하는 조직 엔지니어링 콜라겐 이식과 새로운 테노르래피 봉합사 기술

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

시드 및 마우스 모델에서 생 합성 조직 설계 Tracheal 이식의 이식

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...