The authors thank Bernd Krostitz for technical assistance, Radoslaw Kedzierski for initial contributions to the project, Erik Wade for proofreading and giving helpful comments, and Prof. Heike Walles for providing the bioreactor and sharing her valuable experience with organ decellularization. We are also thankful for funding of the project and publication by the Berlin University of Technology.

참고 : RL은 Landesamt 대 아퍼 싶게 Soziales (LaGeSo)에서 장기의 외식에 대한 윤리적 승인을 획득했습니다. 간은 스터 쥐에서 외식되었다. 대정맥과 간 포털 정맥은 22 G 정맥에 삽관 하였다. 외식 간의 탈세 포화하는 방법은 이전에 설명되었다. 1 % 나트륨 데 옥시 콜레이트와 쥐 간 과도한 관류에 의해 수득 된 4,5- 여기서 사용 세포 외 매트릭스. 쥐 간의 세포 외 기질 (ECM) 1. Recellularization <ol><li> 간 세포주 인 HepG2의 확장 <ol><li> 배양 된 로스웰 파크 메모리얼 연구소 (RPMI) 10 % 소 태아 혈청 및 2 mM 글루타민 및 페니실린 및 스트렙토 마이신이 mM의 각각의 보충 1640의 간암 세포주 인 HepG2. </li><li> T175 병 1.5 × 10 7 세포를 시드 및 37 ℃, 5 % CO 2에서 세포 성장 </sub&gt;. 37 ° C에서 5 분 동안 트립신에 의해 사일 후 수확 세포, 5 % CO 2. </li><li> 300 XG에서 세포를 스핀 다운 4 ml의 PBS에서 그들을 재현 탁하고 현미경 아래 노이 바 우어 챔버와 세포를 계산합니다. 하나 T175 병은 약 4.5 × 10 7 세포를 얻을 것입니다. 참고 : 문화 ECM 당 6 × 10 8 인 HepG2 세포 (4-5 × 10 8 세포 각각 10 × 175cm 2 배양 플라스크)와 쥐의 간 ECM의 recellularization 충분한 세포 수를 포함해야합니다. </li></ol></li></ol><img alt="그림 1" src="/files/ftp_upload/54633/54633fig1.jpg" /> 그림 1. 생물 반응기 시스템. A)이 생물 반응기 시스템은 주문 제작했다. 그것은 37 ° C와 5 % CO 2의 장기 모델을 유지합니다. 연동 펌프의 유량 간은 성장 챔버 내에 배치된다) B. 개별적으로 조절하고 연결할 수있다연동 펌프, 중간 저장, 버블 트랩 및 압력 센서로 구성 관류 회로에 에디션. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54633/54633fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol start="2"><li> 전자 재료의 Recellularization <ol><li> 간 관류 챔버, 관류 시스템, 매체의 저장 및 버블 트랩을 포함, 생물 반응기 시스템을 설정합니다. 생물 반응기 시스템 (121 ° C, 15 분) 소독. </li><li> 연동 펌프와 생물 반응기 시스템을 연결하고 인큐베이터가 적절한 조건을 제공하는 배치 (37 ° C, 5 % CO 2). 생물 반응기 시스템 (그림 1)의 간 관류 챔버에서 decellularized 쥐의 간 비계 8 놓습니다. </li><li> 관류 시스템에 관 클립을 사용하여 유관 포털 정맥과 대정맥을 연결합니다. (1.1에서 설명) 150 ml의 RPMI 배지로 발판을 평형/ 분 1.25 mL의 유속으로 5 D 이상. </li><li> 미디어 회로로부터 간 발판을 분리하고 5 ML의 주사기를 사용하여 포털 정맥 (5 ㎖)에 3 × 10 8 인 HepG2 세포와 지지체를 접종, 공기 방울 형성을 방지하고 세포가 그들을 배양하여 ECM을 다시 채울 수 있도록 펌프와 발판에 1 시간은 꺼져. </li><li> 점차적으로 10 분 동안 125 ml / 분으로 시작 펌프를 조정함으로써, 유량을 증가; 20 분 동안 250 ml / 분 최종적 3.75 ㎖ / 분으로 30 분간. </li><li> 반복은 6 × 10 (8)의 총 세포 수에 도달 1.2.4-1.2.5 단계를 반복합니다. </li><li> / 분 3.75 ml의 유속으로 바이오 리액터 시스템을 실행. 문화 이주에 대한 recellularized 쥐의 간. 참고 : 새로운 배지 (50 ㎖) 매일과 매체의 3 분의 1을 교체합니다. 이러한 락 테이트 탈수소 효소 활성 매체 샘플의 pH 및 글루코스 및 락 테이트의 농도 등의 생체 변수를 측정하기 위하여 배양액 샘플. </li></ol></li></ol> Recellularized 쥐 간 2. 형질 도입 <ol start="1"><li> AAV 벡터의 대규모 생산 <ol><li> 전술 한 바와 같이 생산 정제 및 AAV 벡터를 정량화 : 11 <ol><li> 간략하게, 롤러 병에서 AAV 벡터를 생성하고 iodixanol 구배 원심 분리에 의해이를 정제. PD10 겔 여과 컬럼을 통해 여과하여 잔류 iodixanol를 제거합니다. 표준으로 게놈 AAV의 DNA를 사용하여 qPCR에 의한 AAV 벡터의 농도를 결정합니다. 참고 : 본 연구에 사용 된 자기 보완, pseudotyped AAV2 / 6 벡터 전달 효율 및 내생 적 발현 유전자의 최저 대한 shRNA를 발현 카세트 (인간의 시클로 필린의 B (hCycB), 그림 2)을 보여주기 위해 기자로 EmGFP 인코딩 . 혈청 형 6 (간 모델 당 2.7 × 10 13 AAV 벡터)의 pseudotyped scAAV 벡터의 충분한 양을 확인합니다. </li></ol></li></ol></li></ol><img alt="그림 2" src="/files/ftp_upload/54633/54633fig2.jpg" /> 본 연구에 사용 된 자기 보완, pseudotyped AAV2 / 6 벡터 그림 2.지도. 게놈은 AAV2의 반전 터미널 반복 (ITR) 구성 및 제어 아래의 shRNA뿐만 아니라 CMV 프로모터의 제어하에 EmGFP를 인코딩 U6 프로모터의. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54633/54633fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <ol start="2"><li> 간 모델의 형질 도입 <ol><li> PBS의 각각의 양을 첨가하여 5 ㎖ 2.7 × 10 13 벡터 게놈의 최종 농도로 AAV 벡터 용액을 조정한다. </li><li> 정맥에서 튜브를 제거하여 미디어 회로로부터 간을 분리합니다. 포털 정맥의 정맥에 5 ML의 주사기를 연결하고 전체 AAV 벡터 용액 (5 ml)에 주입. 참고 : OptionallY는 간 걸쳐 AAV 벡터 용액의 분배를 수행하는 페놀 레드 (5 μg의 / ㎖)을 추가한다. </li><li> 펌핑없이 1 시간 동안 품어. 서서히 10 분 / 125 ㎖로 분부터 유량을 증가; 2.5 ㎖ / 20 분, 30 분 3.75은 ㎖ / 분 분간. 문화 육일 동안 recellularized 쥐의 간. 주 : 매체의 3 분의 1을 교체 1.2.7에 따라 주에 주어진 </li></ol></li></ol> Recellularized 형질 쥐 간 3. 평가 <ol start="1"><li> 헤 마톡 실린 및 에오신 (HE) 염색 및 면역 조직 화학적 분석 <ol><li> 메스를 사용하여 각 간 로브에서 샘플 (0.5 × 0.5 × 1.5 cm)를 가져 가라. </li><li> 4 % 파라 포름 알데히드 (PFA), 4 ℃에서 1.5 시간 동안 + 4 % 수 크로스 용액 (3.1.1부터) 샘플을 인큐베이션. (주의 :.. PFA는 항상 흄 후드 내에서 PFA를 유지하고 적절한 보호 복을 착용 독성 및 발암) 세척 PBS로 3 회 (세척 단계 당 1 분) 8 %에 품어4 ℃에서 자당 하룻밤. </li><li> 플라스틱 cryomolds에 매체를 고정 부어 기포의 매체 무료 고정에 샘플을 배치합니다. 샘플이 잘 덮여 때까지 고정 매체를 추가합니다. 스토어 더 사용할 때까지 -80 ° C에서 샘플을 포함. </li><li> cryotome (12) 냉동 섹션 (10 μm의)를 준비합니다. 헤 마톡 실린 + 에오신 염색 8 recellularization을 평가합니다. (: EmGFP 여기) 유전자-의 이익에 대한 면역 조직 화학 염색에 의해 전달 효율을 평가합니다. </li></ol></li><li> 분자 생물학적 시료 채취 <ol><li> 생검 펀치 (직경 4mm)와 각 간 로브 샘플. EmGFP의 유전자 발현의 평가를위한 제조업체의 지침에 따라 전체 RNA, DNA와 단백질을 분리, 8을 노크 hCycB. </li></ol></li></ol>

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The authors have nothing to disclose.

여기에 설명 된 재구성 차원 간은 인간화 시스템에서 바이러스 벡터를 연구하는 모델을 제공합니다. 인간의 간세포 암 세포주와 쥐의 간의 ECM의 다시 채우기가 큰 바이오 의약품의 연구를 허용하는 혈관 시스템을 생성합니다. 이러한 결과는 개념 증명 재구성 간 모델을 효율적으로 바이러스 벡터로 형질 도입 될 수있다 제공한다. 여기에 도시 된 실험에서 간 각자 로브는 AAV 벡터에 의해 형질 도입 하였다. 그러나, 몇 가지 예비 실험에서, 단일 엽 세포 증식을하지 않았다. 이 혈관 폐색 시스템에서 세포 파편 또는 다른 구성 요소를 방지하는 것이 중요하다. 모든 돌출부가 관류 될 수 있는지 여부를 시험하기 위해, 페놀 레드 등의 무독성 염료 간 모델을 플러싱 할 수있다. 또 다른 중요한 문제는 멸균 간 골격을 유지하고있다. 에탄올 또는 항생제 치료는 disadv 동안antageous 혈관 시스템, γ-방사선의 세포 외 기질의 조사가 혈관을 보존 시료를 멸균. recellularization 절차 및 시간 다시 채우기 위해 사용되는 세포의 수는 재현 가능한 결과를 얻기 위해 조정되어야 할 수 있도록 또한, 이식 된 간 크기가 다르다. 본 연구에 사용 된 쥐 간은 비교적 큰 셀 테스트 시약 (예컨대, AAV 벡터)의 대용량을 필요로한다. 또한, 다시 채우기 절차는 2 주 이상 걸렸다. 이것은 합리적인 노력을 함께 할 수있는 복제의 수를 제한합니다. 현재는보다 적은 세포 적은 테스트 시약의 사용을 허용 쥐 간 부피의 약 오분있는 마우스 간을위한 모델을 설정한다. 세포 수의 비례 미세화는 타당하지만, 정확한 양은 작고에 결정되어야그녀의 실험. 본 모델의 또 다른 단점은 간세포 인 HepG2 세포주를 사용하는 것이다. 실험은 생리 학적 관련성이 모델을 제공 유도 다 능성 줄기 세포로부터 분화 된 간세포의 사용을 개발하는 것이 계속된다. 또한, 간은 간세포, 예를 들면, 쿠퍼 세포 및 sinosoids 외에도 여러 종류의 세포로 구성된다. 우리는 ECM이 여러 세포 유형으로 recellularized 때 다른 세포 유형의 자연 환경을 다시 채워야한다고 가정합니다. 차원 간 모델은 몇 가지 장점을 결합한다. 생체 내 모델에서 기존의 가장 큰 단점은 동물 생리학은 인간의 생리 실질적으로 차이가 있다는 것입니다. 인간 환자의 치료 독성 부작용 때문에 미결 남아있다. 이러한 단점은 더 가깝게 후 생물학 반영 인간 세포 간 차원 모델을 재구성함으로써 극복 될 수있다남자 환자. 간 모델의 두 번째 장점은 동물 복지에 기여한다. 동물 성분이 재구성 실험 요구되지만 잉여 동물 즉 즉, 추가적인 동물은 필요하지 않다, 다른 동물 실험을 위해 희생되었다 사용될 수있는 바와 같이, 방법은 여전히 3R 원칙 (여분 환원, 정제)의 목적을 따른다 접근 방식은 완전히 자주 생체 실험과 연관된 동물의 고통을 피할 수 있습니다. 구 상체는 3D 시스템에서 세포 과정을 연구하기위한 대체 수단이다. 많은 생물학적 물질이 구조의 내측 부분에 깊이 침투하지 않도록하지만, 타원체는 혈관 아니다. 이러한 문제는 혈관 간 3D 모델로 극복되었다. 가 유전자 치료에 가장 유망한 후보 중에서 때문에 여기에 설명 된 실험에서 AAV 벡터는 조사했다응용 프로그램. 수많은 유전자 치료 접근법 간염 바이러스 또는 알파 -1- 항 트립신 결핍증 감염의 치료를 위해, 예를 들면, 간을 표적 겨냥은 3D 간암이 AAV 벡터를 개발하는 과정에서 사용될 수있다. 또한, 물론, 다른 hepatotropic 바이러스 벡터, 예를 들어, 아데노 바이러스 벡터의 조사에 적합하다. 또한, 이러한 간염 B 또는 C 바이러스 감염성 간염 바이러스를 연구하기 위해 사용될 수있다. 또, 예를 들면, 바이러스의 새로운 전략을 설계하는데 이용 될 수있다. 또한, 3D 모델 기관의 암을 치료 및 독성 학적 연구를 수행하기 위해 새로운 세포 증식 치료법을 개발하는 유망한 도구를 나타낸다. 긴 안목에서 인공 간은 이식 등 재생 의학에 이용 될 수있다. 함께, 차원 간 모델은 감염 생물학에 응용 프로그램 및 생물 의학 연구의 다른 분야의 넓은 범위를 제공합니다.

Most current biomedical research relies on one of two approaches, either two-dimensional (2D) cell culture experiments or animal models, which are three-dimensional (3D) by their very nature. However, these approaches have some severe drawbacks. Cells grown in 2D culture have been shown to differ in gene expression patterns and cell physiology from those cultivated under 3D conditions.1 Animal models, in addition to being associated with ethical concerns, often do not model human physiology well. Although the lack of obvious toxic effects of a compound must be confirmed in animal models prior to the first dosing in humans, multiple cases have been documented in which severe, sometimes fatal, adverse effects have occurred in clinical trials.2
To overcome these shortcomings, humanized 3D ex vivo organ models have become important research tools. When cultivated under suitable conditions, cells self-assemble into 3D structures known as spheroids. However, these spheroids lack a vascular system, which limits the distribution of small molecular compounds, large biologics and viral vectors alike. For example, adenoviral vectors only transduced the outer cell layers of spheroids prepared from human glioblastomas.3 A solution to this problem is the use of an organ model containing a vascular system. To this end, the organ of interest can be explanted from an animal, and the animal cells can be replaced by human cells. Various methods for decellularization of animal livers by treatment with detergents or sodium cholate have been described.4-6 The resulting extracellular matrix (ECM) harbors cytokines and growth factors which regulate various cellular processes.7 It can be used as a scaffold for recellularization with human cells to obtain a functional organ model.
In a recent study, we used a humanized 3D liver model to study distribution and transgene expression of an adeno-associated virus (AAV) vector.8 AAV vectors belong to the most promising viral vectors for gene therapeutic applications.9 The first, and to date only, approved gene therapeutic intervention in the Western world uses an AAV vector for the transfer of lipoprotein lipase.10

<table border="0" cellpadding="0" cellspacing="0" width="735">
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			<td height="20" style="height:20px;width:216px;">Incubator</td>
			<td style="width:233px;">Fraunhofer</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peristaltic Pump</td>
			<td>Fraunhofer</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flange with groove</td>
			<td>Duran</td>
			<td>2439454</td>
			<td>modified by gaffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Ring Transparent</td>
			<td>Duran</td>
			<td>2922551</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quick Release Clamp</td>
			<td>Duran</td>
			<td>2907151</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flat Flange Lid</td>
			<td>Duran</td>
			<td>2429857</td>
			<td>modified by gaffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw thread Tube</td>
			<td>Duran</td>
			<td>2483802</td>
			<td>modified by gaffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw thread Tube</td>
			<td>Duran</td>
			<td>2483602</td>
			<td>modified by gaffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone sealing Ring</td>
			<td>Duran</td>
			<td>2862012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw Cap</td>
			<td>Duran</td>
			<td>2924013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw Cap</td>
			<td>Duran</td>
			<td>2924008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw Cap with aperture</td>
			<td>Duran</td>
			<td>2922709</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screw Cap with aperture</td>
			<td>Duran</td>
			<td>2922705</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter&#160;</td>
			<td>Sarstedt</td>
			<td>831,826,001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Tubing</td>
			<td>VWR</td>
			<td>228-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube connector</td>
			<td>Ismatec</td>
			<td>ISM556A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biocompatible Tubing</td>
			<td>Ismatec</td>
			<td>SC0736</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">T175 culture flasks</td>
			<td style="width:233px;">Greiner bio-one</td>
			<td style="width:119px;">660 160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>BioWest SAS (Th. Geyer)</td>
			<td>L0501-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glutamine</td>
			<td>BioWest SAS (Th. Geyer)</td>
			<td>X0551-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>BioWest SAS (Th. Geyer)</td>
			<td>L0940-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/ streptomycin</td>
			<td>BioWest SAS (Th. Geyer)</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fetal calf serum</td>
			<td style="width:233px;">cc pro</td>
			<td style="width:119px;">S-10-M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Tissue-Tek O.C.T.</td>
			<td style="width:233px;">Weckert-Labortechnik</td>
			<td style="width:119px;">600001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HepG2</td>
			<td style="width:233px;">DSMZ</td>
			<td style="width:119px;">ACC 180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryomold 15x15x5mm</td>
			<td style="width:233px;">Sakura</td>
			<td style="width:119px;">4566</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Biopsy punch 4mm</td>
			<td style="width:233px;">pfm medical</td>
			<td style="width:119px;">48401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Nucleospin miRNA</td>
			<td style="width:233px;">Macherey &#38; Nagel</td>
			<td style="width:119px;">740971.10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Nucleospin RNA/DNA Buffer Set</td>
			<td style="width:233px;">Macherey &#38; Nagel</td>
			<td style="width:119px;">740944</td>
			<td></td>
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study of viral vectors in a three-dimensional liver model repopulated with the human hepatocellular carcinoma cell line hepg2

이 프로토콜은 3 차원 (3D)의 발생 생체 간 모델 및 바이러스 벡터 시스템의 연구 및 개발에의 응용을 설명한다. 이 모델은 인간 간세포 세포주와 decellularized 래트 간 세포 외 기질을 다시 채우기에 의해 얻어진다. 이 모델은 살아있는 동물에 유해한 실험을 대체하는 혈관 3D 전지 시스템 연구를 허용한다. 또 다른 장점은 동물 모델보다 인간 생리에 가까운 모델의 인간화 특성이다. 본 연구는 아데노 - 관련 바이러스 (AAV 벡터) 유래의 바이러스 벡터와이 간 모델의 전달을 입증한다. 미디어와 3D 간 모델을 공급하는 관류 회로는 벡터를 적용 할 수있는 쉬운 방법을 제공합니다. 시스템 간 주요 대사 파라미터의 모니터링을 허용한다. 최종 분석을 위해, 조직 샘플은 recellulariza의 범위를 결정하기 위해 수행 될 수있다조직 학적 기술에 의해 기. 전달 된 유전자의 바이러스 벡터와 표현의 분포를 정량적 PCR (qPCR에), 웨스턴 블롯 및 면역 조직 화학 염색에 의해 분석 될 수있다. 기초 연구 및 유전자 치료 응용 개발 벡터 모델의 다수의 애플리케이션은 신규 한 항 바이러스 치료법의 개발, 암 연구 및 바이러스 벡터에 관한 연구 및 잠재적 부작용을 포함하여 구상 할 수있다.

recellularization 정도의 평가를 위해, 크라이 섹션은 각각 recellularized 간 모델의 각 로브로부터 제조 하였다. 그런 다음 섹션을 헤 마톡 실린 및 에오신 염색으로 분석 하였다. 도 3에서 알 수있는 바와 같이, TLM1, TLM2로 표시된 세 간 모델은 각 로브는 (recellularized하지만 형질 도입되지 않은 제어 간 모델) Ctrl 키 (간 모델 일 +2를 형질) 인 HepG2 세포의 증식을 하였다. 이 간세포 암 세포주는 15 세의 아르헨티나 소년의 종양 조직에서 설립되었다. 간 모델의 일부 영역을보다 집중적으로 다른 사람보다 recellularized했다. 이러한 차이에 대한 이유가 결정될 남아있다. <img alt="그림 3" src="/files/ftp_upload/54633/54633fig3.jpg" /> 그림 3. 헤 마톡 실린과 세 가지의 각 엽의 에오신 염색 간 모델을 분석 TLM1 + 2 :. 형질 간모델 1, 2; recellularized하지만 형질 도입되지 않은 Ctrl 키 : 컨트롤 간 모델입니다. 스케일 바 : 200 μm의. (참고 허가를 재 인쇄. 8) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54633/54633fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 다음 단계에서, 간 모델의 전달을 평가 하였다. 이를 위해, DNA를 간이 모델의 각각의 펀치 생검 28로부터 제조하고, 벡터 역가 qPCR에 의해 정량화 하였다. 평균 셀 (VG / 셀) 당 55에서 90 내재화 벡터 게놈 두 형질 간 모델에서 측정되었다. 2D 세포 배양 실험에서 30 VG / 셀 강한 유전자 발현 및 RNAi를 매개 된 침묵 충분 보여준다. EmGFP의 생산은 RT-PCR 및 웨스턴 블롯으로 분석 하였다. 사실, 리포터의 발현은 각각의 mRNA와 단백질 수준에서 생검 80-90 %가 검출되었다.도 8은 전달 효율의 포괄적 인 사진을 얻기 위해 극저온 섹션 immunohistologically 분석 하였다. 도 4에서 알 수있는 바와 같이, DAPI 염색으로 가시화로 성공적 recellularized 된 간 모델의 영역도 EmGFP 발현의 면역 조직 화학적 분석에서 강한 형광 신호를 주었다. 예상대로되지 AAV 벡터로 처리 제어 간 모델은 임의 EmGFP 발현을 나타내지 않았다. <img alt="그림 4" src="/files/ftp_upload/54633/54633fig4.jpg" /> EmGFP 표현의 그림 4. 면역 조직 화학적 분석 간 모델을 DAPI 염색 (파란색)에 의해 가시화했다 다시 채워 세포.; EmGFP 발현은 면역 화학적 염색 (적색)에 의해 가시화되었다. TLM1 + 2 : 형질 간 모델 (1)과 (2); recellularized하지만 형질 도입되지 않은 Ctrl 키 : 컨트롤 간 모델입니다. 스케일 바 : 200 μm의. (허가를 재 인쇄참고 문헌에서. 8) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54633/54633fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 최종 테스트에서 hCycB의 AAV 매개 된 녹다운을 QRT-PCR로 분석 하였다. hCycB의 최저 70 ~ 90 % 사이 인 것으로 밝혀 두 형질 간 모델의 모든 돌출부 (도 5)에 대하여 평균 하였다. <img alt="그림 4" src="/files/ftp_upload/54633/54633fig5.jpg" /> hCycB의 그림 AAV-형질 3D 간 모델 hCycB 5. RNAi를 매개 최저. 음소거는 QRT-PCR에 의해 결정되었다. 평균값과 표준 편차 (SD)를 비 형질 제어의 평균 값 (각각 TLM1 2) 각각의 형질 3D 간 모델의 모든 샘플에 대해 계산 된 정규화 된 (Ctrl 키.). (R의 허가를 다시 인쇄EF. 8) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54633/54633fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2 at JoVE.com

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

인간의 간세포 암 세포주 인 HepG2와 3 차원 간 모델 다시 채워에서 바이러스 성 벡터의 연구

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.0K 조회수.  Institute of Biotechnology, Berlin University of Technology. The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

비디오: Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

콜론-26 암 종양 베어링 암 악액질의 연구를위한 모델로 마우스를

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

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A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

포탈 정맥 주입 모델 간 전이 유방암의 공부를

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

머리와 목 편평 ​​세포 암에서 신경초 침략을위한 모델

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

위쪽으로 이동: 간단 하 고 유연한 생체 외에서 3 차원 침공 분석 실험 프로토콜

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

아데노 관련 바이러스-Cas9 시스템을 사용하여 유전자 조작 된 뮤린 머리와 목 암 세포라인의 시험관 내 설립

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

간세포 암종 환자를 위한 경질 접근 화학색전성

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

튜닝 가능한 바이오 물속성과 3D 환경에서 종양-스트로마 상호 작용을 연구하는 간암을 위한 생체 모방 모형

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

대장암 줄기세포를 연구하는 스페로이드의 3차원 모델

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

제브라피쉬에서 비알코올성 지방간 질환 관련 간세포암의 초기 진행 중 간 미세환경 분석

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

간세포 암에서 종양-기질 상호 작용을 조사하는 3차원 스페로이드 모델

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...