Name: human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions
Uploaded: 2015-04-18T14:00:00
Description: Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.

이 프로토콜은 인간의 연구 윤리위원회의 LUMC 및 AMC 지침을 따른다. 1. 수술 및 조직 컬렉션 참고 : Dupuytren의 구축의 수술 적 절제를위한 다양한 기술이 존재하지만, 현재의 황금 표준이 부분 fasciectomy 15입니다. 대부분의 환자는 하루 수술 클리닉에서 처리됩니다. <ol><li> 환자 및 마취의 기본 설정에 환자의 동반 질환에 따라 일반 또는 지역 마취...

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인간 생체 결합 조직 배양에서 가장 중요한 단계는 생존을 보장하기 위해 제거 수술 후의 조직의 즉시 사용이고; 조직은 항상 중간 또는 식염수 용액에 남아 있어야한다; 멸균 문화를 유지; 조직의 횡단면은 최대 200 μm의 두께를 가져야한다; 조직이 매체와 접촉 있지만 완전히 침수하지 않을 경우 체외 배양 시스템의 설정은 최적입니다. 보통은 작은 부피에서 트랜스 웰의 외측 챔버 ...

Dupuytren의 질병 (DD)는 양성 fibroproliferative 질병으로 인해 손의 손바닥에 결절 및 코드의 형성에 손가락의 영구 굴곡이 발생합니다. 질병 확산 북유럽 백인 중에서도 특히 높지만 질환의 근본적인 유전 적 원인은 알려지지 않았다 하나. DD의 주된 특징은, 힘줄 및 손과 손가락의 손바닥의 피부 사이의 공간을 점유 거친 섬유 조직 형성 세포 외 기질 (ECM) 단백질 (예를 들면, 콜라겐)의 과잉 생산, 영구적 미세한 움직임을 방해 한편 (2)의 세. 질병의 재발 한 섬유증, (4)의 기본 원인으로 유전 적 변형을 의미한다. 치료 효과적인 직접 세포 및 분자 수준에서 제어 할 수없는 섬유 성 메커니즘을 타겟팅 할 수 있었다. 섬유증에 우리의 최근 작품은의 발전에 우리를 주도하고있다약물 테스트의 전위와 인간 섬유 성 조직의 단기 배양을 허용 신규 한 3 차원 배양 시스템. 이 시스템은 2D 섬유 아세포 배양 제한하는 접근 방식을 극복하고 섬유증 (5)을 매개로 TGFβ 통로 활성화의 엑손 스키핑에 의해 달성되는 부분 아래로 규제의 역할을 정의하는 데 도움이되었습니다. 우리는 DD 환자의 생체 인간의 절제 표본은 근섬유 사이의 상호 작용과 주변 ECM (5), (6)을 연구하는 문화 방법을 개발했다. DD 결합 조직 섬유화의 연구뿐만 아니라 다른 섬유 성 질환 수술 절제의 조직 병리학 적 분석에 의존 표본, 조직 및 차 문화 또는 셀 정렬 절차의 설립에서 섬유 아 세포의 분리. 그들은 실험에 의한 질병의 특성 또는 치료 개입의 외생 적 조작을 허용하지 않기 때문에 이러한 접근은 매우 정적입니다. 또한, 일차 CELL 문화는 배양 조건에 적응과 유전자 발현 특성도 초기 구절 동안 모든 통로에 본질적으로 생체 내 상황에서 다른 경향 (통로 (3) 사이에 6) 7, 8. 우리는에 폐기물 수술 재료를 유지 관리해야 특정 환자의 특성 연구 및 항 섬유 성 또는 항 염증성 약물 화합물의 스크리닝을 가능 기간에 대한 생체 외 배양 조건. 시스템은 DD 섬유 모세포뿐만 아니라 다른 세포 유형 (9)를 배양 할 때 이전에 관찰 된 바와 같이, 장착시에 조직의 변질을 방지하므로, 매질과 아닌 플라스틱과 조직의 접촉을 허용 니트로 셀룰로오스 막에 기초한다. DD 조직 자체가 단백질의 대량 생산 No를 콜라겐 겔 또는 다른 ECM 단백질 기판은 필요하지 않다. 이 기본 ECM 미세 환경 및 회전율 죄의 유지에 유리하다CE 매트릭스 기판은 조직 구조 및 기능 (10), (11)의 중요한 조절이다. 예를 들어, 피브로넥틴, 라미닌 및 콜라겐과 같은 ECM 단백질, 마찬가지로 상피 세포 (12)에 정점 - 기저 극성 도시 섬유 아세포의 전후 극성, (13)에 영향을 미칠 수있다 . 편광 세포는 세포의 이동 및 유전자 발현, 예를 들어, 막에 α1β1 인테그린 접근성 내가 14 콜라겐 입력 세포 접착에 영향을 미치는 결정 세포 분자의 비대칭 분포를 가지고있다. 이 3D 모델의 주요 목표는 기본 미세을 보존 이었기 때문에, 인공 ECM 매트릭스 기판이 사용되지 않았다. 간단히 : 절제 표본 동등하게 무균 환경에서 절단 및 nitrocellular 세포막에 위치. 주사 투여를 통해 치료가 필요한 경우에 그들이 막 상에 배치 된 후에 조직을 주입된다. 치료는 INJ를 통해 투여 될 필요하지 않은 경우다음의 ection 화합물 (1 % 소 태아 혈청 (FCS), 1 % 페니실린 - 스트렙토 마이신 (P / S)와 함께, 둘 베코 변형 이글 배지 (DMEM)) 배양 배지에 첨가한다. 이전 5 바와 같이 배양, 30 % 수크로오스 용액을 통해 처리 된 티슈는 4 % 파라 포름 알데히드 (PFA)에 고정 된 이후 최대 10 일간,의 OCT 화합물에 매립하고 -80 ℃에서 보관 유지된다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Invitrogen</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td></td>
			<td>high glucose, heat inactivated</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Millicell</td>
			<td>PIHA01250</td>
			<td>0.45um pore size, 12mm diameter</td>
		</tr>
		<tr>
			<td>anti-collagen type I</td>
			<td>Southern Biotech</td>
			<td>1310-08</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- &#945; smooth muscle actin</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-collagen type III</td>
			<td>Southern Biotech</td>
			<td>1330-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor555 Donkey Anti-Mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A-31570</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>TOPRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>methylsalicylate</td>
			<td>Sigma</td>
			<td>M6752</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek OCT</td>
			<td>Sakura</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsccope glass coverslips</td>
			<td>Menzel-Glaser</td>
			<td>BB024060A1</td>
			<td>24x60mm</td>
		</tr>
		<tr>
			<td>Microscope SuperFrost slides</td>
			<td>Menzel-Glaser</td>
			<td>AA00000102E</td>
			<td>76x26mm</td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium</td>
			<td>Vector laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5 II confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Argon-488, 514 nm and HeNe-633 nm laser lines</td>
		</tr>
		<tr>
			<td>Zeiss 710 NLO upright confocal microscope</td>
			<td>Jena, Germany</td>
			<td></td>
			<td>equipped with femtosecond Spectra - Physics Deep See MP laser (Santa Clara, United States) using a Plan -Apochromat 20x/1.0 NA 
			water-immersion objective.</td>
		</tr>
	</tbody>
</table>

human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

생체에있어서, 결합 조직의 3 차원 배양뿐만 아니라 ECM 섬유화 및 DD 조직을 구성하는 다른 세포 성분 및 잠재적으로 다른 유형의 관계를 이해하기 쉽고 견고한 세트 업 시스템이다. 더욱이이 시스템은 신뢰할 수있는 방법은 여러 종류의 세포 및 섬유 성 부하 (20)에 미치는 영향에 대한 화합물의 효과를 테스트 할 수있다. Dupuytren의 섬유증으로부터 유도 수술 폐...

Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren의 질병 (DD)는 손바닥의 fibroproliferative 질환이다. 여기서는 3 차원 (3D) 배양 계에서 배양 DD로부터 표본을 절제 프로토콜을 제시한다. 이러한 단기 배양 시스템은 3 차원 구조의 보존과 섬유 성 조직의 분자 특성을 허용한다.

인간의 Dupuytren의<em&gt; 전의 VIVO</em&gt; 근섬유 아세포의 연구와 세포 외 기질 상호 작용에 대한 문화

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Research

JoVE Journal

Medicine

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

환자 조직 및 유전자 전달의 시험 전의 VIVO 문화

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

연구

의학

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

인간의<em&gt; 전의 VIVO</em&gt; 죽상 경화 모델 병변 생물학을 연구하는

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

인간 복재 정맥 절차<em&gt; 전의 VIVO</em&gt; 관류 및 외부 보강

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

분리, 냉동 보존 및 문화 임상 응용 프로그램에 대한 인간 양막 상피 세포

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

An Ex vivo Model to Study Hormone Action in the Human Breast

An Ex vivo Model to Study Hormone Action in the Human Breast

<em&gt; 생체 내</em&gt; 모델은 인간의 유방암에서 호르몬 행동을 연구하기 위해

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

생체 내 및 생체 외 복막 지방이에 밀키 스팟 구조의 난소 암 전이성 식민지를 연구하는 접근 방식

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Kinomic 프로파일 의약품 응답 테스트를위한 3D 인간의를 Biogel 문화 시스템을 사용 Microtumors 및 환자 유래 아교 모세포종 세포의 생성

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

전략적 내피 세포 관 형성의 분석 : 감소 세포 외 매트릭스 세포 외 기질과 성장 인자를 비교

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...