우리는 기꺼이이 프로젝트에 사용 된 표본 공급 Olbrich 정원의 존 Wirth 감사 하 고 싶습니다. 이 작품은 국립 심장, 폐, 혈액 연구소에 의해 부분에서 지원 (G.R.G.에 R01HL115282) 국립 과학 재단 (J.R.G와 G.R.G.를 DGE1144804), 위스콘신 대학 외과 및 동문 기금 (H.D.L.). 이 작품 또한 일부 환경 보호 기구 (스타 그랜트 no. 83573701)에서 건강의 국가 학회 (R01HL093282-01A1 및 UH3TR000506), 그리고 국립 과학 재단 (IGERT DGE1144804)에 의해 지원 되었다.

1. 식물 조직 세제 기반 접근을 사용 하 여 decellularization
<ol><li>
신선 또는 냉동 F. hispida, 잎 샘플을 사용 합니다. -20 ° C 냉동 고 (최대 1 년) 나중에 사용에 대 한 저장소에서 사용 되지 않는 신선한 샘플을 동결. 참고: 거의 모든 원하는 식물의 줄기 나 잎 조직을 사용 합니다. 확장된 저장 시간은 조직에 손상을 일으킬 수 있습니다.<ol>
<li>샘플의 용도 기준으로 하 여 처리 하는 샘플의 형태와 크기를 결정 (즉, 샘플 스트립으로 잘라 기계적 테스트 응용 프로그램에 대 한 적합, 한편 8 m m 디스크 샘플 다 잘 자란 응용 프로그램에 유용 ). 잘라 잎 8 m m 디스크 날카로운, 깨끗 한 생 검 펀치 실내 온도 (20-25 ° C) 이온된 H2o. 잠수 하는 동안 참고: 전체 잎과 줄기에는이 프로토콜을 사용할 수 있습니다. 그러나, 작은 샘플은 빨리 decellularize 것입니다.</li>
		<li>실내 온도 (20-25 ° C) 이온된 H2O 세척 및 그들을 녹여 낮은 속도 설정 흔들 플레이트 세트에 5-10 분에 대 한 샘플을 품 어. 충분히 이온된 h 조2O를 사용 하 여 모든 샘플은 철저 하 게 침수 되도록.</li>
	</ol>
</li>
	<li>
10% (w/v) 나트륨 라우릴 황산 염 (SDS) 이온된 H2o. 장소에서에서의 솔루션 (유리 또는 플라스틱 접시 이상적 이다) 적합 한 컨테이너에 샘플을 준비 하 고 SDS 솔루션 샘플 표지를 추가. 손상을 방지 하기 위해 샘플에 낮은 속도를 흔들 플레이트 세트에 실내 온도 (20-25 ° C)에서 5 일에 대 한 샘플을 품 어. 참고:이 decellularization 과정을 감속 하 고 SDS에 의해 고르지 치료 이어질 수 컨테이너를 혼잡 하지 않습니다. 이 단계 동안 샘플 갈색 색조를 취득 해야 합니다.<ol>
<li>5 일 후 바꿉니다 SDS 솔루션 이온된 H2o. 품 어떤 잔여 SDS 솔루션을 철저 하 게 씻어을 흔들어 접시에 추가 10-15 분에 대 한 샘플을.</li>
	</ol>
</li>
	<li>10% (v/v) 표 백제 솔루션에서 1% (v/v) 비 이온 계면 활성 제를 준비 합니다. 500 mL 솔루션, 백제의 50 mL와 5 mL의 비 이온 계면 활성 제를 혼합 후 이온 H2o. Submerge 445 mL을 추가 갓된 솔루션에서 샘플링 합니다. 참고: 비 이온 계면 활성 제/표 백제 솔루션 없는 긴 수명, 따라서, 준비의 48 h 사용할 수 있습니다.</li>
	<li>
샘플은 완전히 지워질 때까지 비 이온 계면 활성 제/표 백제 솔루션 마다 24 h를 교체 (시각적 비교를 위해 그림 1A 참조). 다음 씻어 초과 비 이온 계면 활성 제/표 백제 솔루션을 2 분 동안 흔들어 접시에 이온된 수에 샘플을 품 어. 흔들어 접시 샘플을 손상을 방지 하기 위해 낮은 설정으로 설정 합니다. 참고: 샘플 사용의 선택을 취소 하는 데 필요한 시간 따라 달라 집니다, 유형 및 식물의 종. 전체 decellularization의이 샘플의 완전 한 변색 (철저 한 청소를 보장 하기 위해 DNA 정량화를 수행).<ol>
<li>년까지 그들을 저장 하는 샘플을 lyophilize (플래시 동결 액체 질소를 사용 하 여 선호,-80 ° C에서 샘플을 동결 하 셔도 됩니다) 실내 온도 (20-25 ° C) 낮은 습도에 그들을 저장 하 고.</li>
		<li>Tris HCl (10 m m, pH 8.5)에서 샘플을 다시 구성 하기 충분히 코트 샘플을 사용 하 여. 혈 청 무료 미디어 사용 (린스, 표준 24 잘 접시에서 당즉 사용 150-300 µ L) 하기 전에 micropipette를 사용 하 여 부드럽게 2-3 번 샘플 린스. 참고: Tris HCl 버퍼 및 혈 청 무료 미디어 (즉 DMEM, 그러나 어떤 기본적인 세포 배양 매체를 대체할 수) 낮추는 이온 H2O 혼자 치료 보다 SDS 처리 샘플의 세포 생존 능력에 대 한 영향에 더 효과적입니다.</li>
	</ol>
</li>
</ol>2. 세제 무료 Decellularization 접근을 사용 하 여 샘플의 준비
참고:이 절차의 초기 단계 일치 단계 1.1-1.1.2 (위 참조).
<ol><li>5% (v/v) 표 백제 (NaClO)와 3% (w/v) 나트륨 중 탄산염 (NaHCO3) 솔루션을 준비 합니다. F. hispida 잎 샘플 잘라 뜨거운 접시에 감동 하면서 8 m m 디스크에 대 한 60-70 ° C에 증기 두건에서 솔루션을 따뜻한. 참고: 중 탄산 나트륨 탄산 나트륨 (Na2CO3), 또는 수산화 나트륨 (NaOH)로 교체하실 수 있습니다. 원하는 온도 크게 (90 ° C로 실 온)에서 변화 하 고 사용 하는 샘플의 속성에 따라 조정 되어야 한다.</li>
	<li>솔루션에는 원하는 온도 도달 하면, 일단 샘플 잠수함 하 고 그들을 손상 되지 않도록 교 반 속도 줄일 수 있습니다. 후 샘플 지워집니다 가시 (참조 그림 1B 시각적 비교를 위해), 목욕에서 신중 하 게 제거. 이온된 H2O 초과 표 백제 솔루션을 제거 하려면 1-2 분에 한 번 샘플을 품 어. 참고: 샘플을 취소 하는 데 필요한 시간이 달라질 수 있습니다. 예를 들어 전체 잎 또는 줄기 완전히 취소 하 고 열 탕에 시간이 걸릴 수 있습니다와 같은 컷된 파 슬 리 10-15 분, 두꺼운 또는 더 큰 샘플 동안에 지울 수 있습니다.</li>
	<li>샘플을 lyophilize 하 고 실내 온도 (20-25 ° C) 낮은 습도에 그들을 저장 합니다.</li>
</ol>

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저자는 공개 없다.

여기, decellularize 식물 조직에 두 가지 방법은 설명 합니다. 여기, 결과 나온 프로토콜 가능성이 있다는 제안25, 이전 연구 결과 함께 결합의 광범위에 적용 가능한 종 식물, 줄기와 잎에서 수행할 수 있습니다. 이 절차는 간단 하 고 공장 decellularization 대부분 실험실에서 실행 될 수 있다 그래서 전문된 장비를 필요 하지 않습니다. 그것은 주목할 만한 그 decellularization, 후에 건설 기?...

조직 생활을 만드는 1980 년대에 등장 하는 조직 공학 대체, 그리고 잠재적으로 주소 중요 한 장기 및 조직 부족1. 한 전략은 자극 하 고 누락 된 조직이 나 장기를 재생성 하기 위해 시체를 안내 건설 기계를 사용 하 고. 첨단 3 차원 인쇄 생산 독특한 물리적 특성으로 건설 기계와 같은 접근을 제조, 비록 달성 물리적 및 생물학적 속성의 다양 한 범위와 건설 기계를 제조 하는 능력 유지 도전2 , 3. 또한, 기능 혈관 네트워크의 부족으로 이러한 기술을 제한 되어 3 차원 조직 재생에. Decellularized 인간과 동물 조직으로 건설 기계를 사용 하 여가 문제4,5,,67우회 주 었 있다. 그러나, 높은 비용, 일괄 배치 변화, 및 제한 된 가용성의 광범위 한 사용을 제한할 수 있습니다 decellularized 동물 투어8. 또한 환자에 게 잠재적인 질병 전송 및 일부 decellularized 포유류 조직9면역학 반응에 대 한 우려가 있다.
셀 루 로스, 식물 및 세균성 소스에서 파생 된 다양 한 응용 프로그램에 대 한 바이오 재생 의학에서 생성 하 광범위 하 게 사용 되었습니다. 몇 가지 예는 다음과 같습니다:10,11, 연골12,,1314 및 상처 치유15뼈. 그들은 내 구성과 저항 포유류 세포에 의해 분해 되 고 하는 룰의 구성 되어 건설 기계 추가 혜택이 있다. 이것이 사실은 포유류 세포는 셀 룰 로스 분자를 분해 하는 데 필요한 효소를 생산 하지 않습니다. 비교에서는, 건설 기계는 세포 외 기질, 콜라겐 등에서 고분자를 사용 하 여, 쉽게16 를 세분화 생산과 장기 응용 프로그램에 적합 하지 않을 수 있습니다. 콜라겐 건설 기계 화학 cross-linking에 의해 안정 될 수 있다. 그러나, 건설 기계17의 생체 적합성에 영향을 주는 cross-linkers의 고유한 독성 때문 교환이 이다. 반대로, 셀 루 로스 통하지 포유류 세포18,,1920효소 저하 때문에 시간의 연장된 기간에 대 한 이식의 사이트에 남아 있을 가능성이 있다. 이 가수분해 전처리를 통해 저하의 속도 cellulases21장비의 공동 배달을 조정 하 여 변경할 수 있습니다. Decellularized 플랜트-파생 셀 룰 로스 건설 기계에서 vivo에서 의 생체 적합성도 쥐22연구에서 입증 되었습니다.
진화의 년의 수백만의 수백을 통해 식물의 구조와 구성의 유체 수송 및 유지의 효율성을 증가 하 정제는. 공장 혈관 혈관은 작은 혈관, 머 레이 법23에 따르면 포유류 맥 관 구조에 유사한으로 분기 하 여 유압 저항을 최소화 합니다. Decellularization, 후 식물의 용기 및 상호 연결 된 숨 구멍의 복잡 한 네트워크 유지 됩니다. 쉽게 사용할 수 있는 고유 식물 종의 수많은 고려 건설 기계 공장에서 파생 된 현재 조직 공학24,25건설 기계에 영향을 미치는 디자인 한계를 극복 하기 위해 가능성이 있다. 예를 들어, Modulevsky 그 외 여러분 시연 신생 및 셀 마이그레이션 decellularized 애플 조직 마우스22의 뒤에 피하 이식 했다 때 발생. 마찬가지로, Gershlak 외. 내 피 세포 decellularized 잎24의 맥 관 구조 내에서 성장 될 수 있었다 보여주었다. 별도 실험에서 Gershlak 그 외 여러분 또한 cardiomyocytes 잎의 표면에 성장 수와24계약 수를 보여줄 수 있었다.
식물은 또한 현재까지 개발 된 가장 진보 된 제조 기법으로도 달성 하기 어려운 거시적인 규모에 복잡 한 조직 세포에서 포함 됩니다. 식물 조직의 복잡 한 계층 구조 디자인은 그들이 그들의 성분26의 합계 보다 더 강한 합니다. 식물 줄기 등 견고 하 고 힘든 구성에서 배열 하는 다른 기계적 특성의 과다 보유와 같은 훨씬 더 유연 하 고 유연한 것27를 나뭇잎. 잎 크기 면에서 종에 따라 다릅니다, 그리고 모양, 강도, vascularization, 정도 휴식 및 화란의 다른 학위를가지고 있습니다. 전반적으로, 이러한 식물 속성 decellularized 식물 독특하고 대단히 기능적인 의료 기기, 건설 기계 엔지니어링 조직 등으로 사용할 수 있습니다 것이 좋습니다.
이 프로토콜 decellularize 식물 조직에 두 가지 방법에 초점을 맞추고와 같은 고 조직 공학에서 공중 발판으로 사용 하기 위해 유래한 다. 첫 번째 방법은 DNA와 포유류 decellularize 조직6,22,25 식물에 널리 사용 되는 기술에서 적응 되어 세포 물질을 제거 하는 일련의를 사용 하는 세제 기반 기술 ,,2829,30. 두 번째 방법은 세제 무료 이며 일반적으로 나뭇잎31의 부드러운 조직을 제거 하는 데 사용 하는 "skeletonization" 프로토콜에서 적응 됩니다. 이전 작업 그 주변의 부드러운 조직31는 맥 관 구조 분리를 촉진 표 백제와 나트륨 중 탄산염 해결책에서 잎을 끓인 것을 보여주었다. 이 기술은 17번째 및 18번째 세기 Albertus Seba32 및33에드워드 패 리 쉬 작품 등에서 실시 하는 실험을 다시 인용 될 수 있다. 이러한 실험을 중심으로 잎과 과일, 같은 공장 문제를 떠나 장기간 물에 잠긴 (달 주) 시간과 수 있도록 자연스럽 게 멀리 부패 부드러운 조직. 여기 "skeletonization" 접근은 셀룰러 잔류물을 제거 하 고 부드러운 조직 구조를 크게 방해 하지 않도록 낮은 온도에서 더 이상 보육 시간 등 온화한 조건, 사용에 적응 됩니다. 여기 자세한 실험에 대 한 3 개의 식물 종류 사용 되었다: 무화과나무 hispida, Pachira aquatica 및 필리핀의 종. DNA 정량화, 기계적 테스트, 그리고 두 방법 모두에서 세포 대사 활동에 미치는 영향의 결과 설명 합니다.

<table><tbody><tr><td>Sodium dodecyl sulfate</td><td>Sigma Life Science</td><td>75746-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>MP Biomedicals, LLC</td><td>807426</td><td>Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td></tr><tr><td>Concentrated bleach (8.25% sodium hypochlorite)</td><td>Clorox</td><td>Item #: 31009</td><td>Standard concentrated bleach.</td></tr><tr><td>Sodium bicarbonate</td><td>Acros Organics</td><td>217120010</td><td>Can be substituted with sodium hydroxide or sodium carbonate.</td></tr><tr><td>8 mm Biopunch</td><td>HealthLink</td><td>15111-80</td><td>Cuts samples that fit well in 24 well plate</td></tr><tr><td>Belly Dancer-Shake table</td><td>Stovall Life Sciences</td><td>BDRAA115S</td><td>Use low speeds to not damage tissues. Can use any model/brand of shake table.</td></tr><tr><td>Isotemp hot/stir plate</td><td>Fisher Scientific</td><td></td><td>Can use any style/brand of hot/stir plate.</td></tr><tr><td>Beaker</td><td>Any</td><td></td><td>Can use any size beaker as long as it will fit your samples and not overcrowd them.</td></tr><tr><td>Tris Hydrochloride</td><td>Fisher Scientific</td><td>BP153-500</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT50003PC</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA assay</td><td>Life Technologies</td><td>P11496</td><td>Can use any dsDNA quantification mehtod on hand.</td></tr></tbody></table>

two methods for decellularization of plant tissues for tissue engineering applications

헌, 합성, 및 동물 파생 된 이식 조직 교체에 대 한 건설 기계로 현재 사용 되는 낮은 가용성, 불 쌍 한 생체 적합성 및 비용 제한이 있습니다. 식물 조직 그들이 유일 하 게 적합 상호 다공성, 기존 혈관 네트워크, 그리고 다양 한 기계 건설 기계, 높은 표면적, 우수한 물 수송 및 보존, 등으로 사용 하는 유리한 특성이 있다 속성입니다. 조직 공학 응용 프로그램에 대 한 식물 decellularization의 2 개의 성공적인 방법이 여기 설명 되어 있습니다. 첫 번째 방법은 포유류 조직 취소 하는 데 사용 하는 이전 설립된 방법과 비슷합니다 세포 물질을 제거 하는 세제 목욕을 기반으로 합니다. 두 번째 잎 맥 관 구조를 격리 하 고 온수 표 백제 및 잎과 줄기를 소금 목욕의 사용을 포함 하는 프로토콜에서 적응 하는 세제 무료 방법입니다. 두 방법 모두 유사한 기계적 특성 및 낮은 세포 대사 충격, 따라서 더 나은 그들의 의도 된 응용 프로그램을 적합 한 프로토콜을 선택 하려면 사용자를 허용 건설 기계를 얻을.

두 방법 모두 굴복 건설 기계 세포 배양 및 조직 엔지니어링 응용 프로그램에 적합 했다. 그림 1 세제 기반 메서드와 세제 없는 메서드에 대 한 컷된 샘플 (8 m m 직경)에 그대로 잎을 사용 하 여 decellularization 프로세스의 일반적인 워크플로 보여 줍니다. 무화과나무 hispida 조직 두 방법 모두 다음의 성공적인 decellularization 나왔고 명확 하 고 그대?...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

여기에 우리가 현재, 및 대비 2 프로토콜 decellularize 식물 조직 하는 데 사용: 세제 기반 접근 및 세제 무료 접근. 두 방법 모두 다음 조직 공학 응용 프로그램에 대 한 건설 기계로 활용 될 수 있습니다 사용, 식물 조직의 세포 외 매트릭스 뒤에 남겨두고.

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

two-methods-for-decellularization-plant-tissues-for-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.5K 조회수.  University of Wisconsin-Madison. 여기에 우리가 현재, 및 대비 2 프로토콜 decellularize 식물 조직 하는 데 사용: 세제 기반 접근 및 세제 무료 접근. 두 방법 모두 다음 조직 공학 응용 프로그램에 대 한 건설 기계로 활용 될 수 있습니다 사용, 식물 조직의 세포 외 매트릭스 뒤에 남겨두고.

비디오: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

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

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

자연 무 세포 창자 매트릭스의 생산을위한 탈세 포화 방법론

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

시험관 연구에 대 한 3D 같은 플랫폼으로 태아와 성인 심장 조직 Explants의 비교 Decellularization

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

세포 배양을 위한 태아 마우스 골격근의 3D 탈세포화 매트릭스 준비

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

발생학

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularization 및 Recellularization 방법론을 인간의 Saphenous 정 맥에 대 한

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

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 ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

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 ...

뼈 조직 공학을 위한 Cell-seeded Decellularized Apple-derived Skaffolds 분석

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

저자 스포트라이트: 뼈 조직 공학을 위한 Apple 유래 셀룰로오스 지지체 사용에 대한 통찰력 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.