This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

이 프로토콜은 동물 연구 윤리위원회의 LUMC 지침을 따른다. 악기 1. 준비, 문화 매체, 및 MTCS 참고 : 층류 후드에있는 모든 준비를 수행합니다. MTCS 관류 실, 버블 트랩 및 스탠드 리버 등. 2010 년 (11)에 설명되어 있습니다. <ol><li> 70 % 에탄올로 포셉 및 마이크로 가위를 소독. 멸균 타이로드의 버퍼 (130 mM의 염화나트륨, 5.4 밀리미터의 KCl, 6.0 mM의 헤 페스, 1.2 mM의 황산, 1.2 21G 바늘 5 ML의 주사기를 준비 mM의 KH 2 PO 4, 20 mM의 포도당, 1.5 mM의 염화칼슘 2 · 2H 2 O, 의 pH = 7.2). 무균의 KCl 용액 (100 mM의)와 21G 바늘 5 ML의 주사기를 준비합니다. </li><li> 65 심장 당 매체 ml의 준비 : (A /; 페니실린의 100 단위, 100 스트렙토 마이신의 μg의, 및 암포 테리 신 B의 0.25 UG / ㎖) DMEM 10 % 태아 송아지 혈청, 안티 곰팡이 / 항생제로 보충을, 인슐린 Transferrin-셀레늄 (ITS / ml의 인슐린 10 μg의 5.5 μg의 / ㎖ 트랜스페린, 6.7 NG / ㎖ 셀렌 산 나트륨) 및 필터 소독. </li><li> 70 % 에탄올 및 종이 타월 건조와 함께 분사하여 관류 챔버와 버블 트랩을 소독. 초기 관류 챔버없이 그러나 MTCS (도 1D)을 조립한다. 스탠드에 거품 트랩을 배치합니다. 쉬운로드 펌프 헤드와 연동 롤러 펌프를 사용합니다. <ol><li> 실리콘 튜브는 저수지 (50 ML 튜브) 및 펌프, 펌프와 버블 트랩 버블 트랩과 저수지 사이의 연결 (그림 1D 참조)를 만들어 사용. 매체는 가스 투과성 실리콘 호스 (1m)과 가능한 한 짧게 인큐베이터 외부 튜브를 통해 가스 배양기에서 가스와 평형에 도달 할 수 있도록 충분한 시간 동안 인큐베이터 안에 튜브를 만든다. </li></ol></li><li> (적절한 순환을 허용 / 1 ml의 주위 분의 흐름 속도를) 펌프를 켜, 70 % 에탄올로 50 ML 튜브를 입력하고 에탄올 CIR하자모든 튜브를 살균 30 분 동안 culate. 탱크를 비우고 시스템에서 에탄올을 펌핑하여 에탄올을 제거합니다. </li><li> 멸균 증류수로 50 ML 튜브를 입력 펌프를 켜고 30 분 동안 물 순환 에탄올 나머지 없애 보자. 탱크를 비우고 시스템에서 물을 펌핑하여 물을 제거합니다. </li><li> 매체의 45 mL를 50 ml의 튜브를 작성, 인큐베이터에있는 스탠드를 넣어. 펌프 (1 ml / 분의 주위의 흐름 속도)를 켜고 모든 튜브를 채우기 위해 매체를 순환, 모든 기포를 제거하고 배지는 적어도 1 시간 동안 배양기에서 가스 조성에 적응하도록 허용한다. 표준 조직 배양 조건 (5 % CO 2, 37 ℃로)에서 인큐베이터를 유지한다. 기포가 튜브에 존재하지 않는 것을 확인합니다. </li></ol> 마우스 심장의 2. 분리 <ol><li> 헤파린 500 단위와 마우스를 주입한다. 이는 응고로부터 혈액을 방지하고 (B)의 제거를 허용 할 것이다마음에서들 (100d). 10 분 후, 정밀 증발기를 이용하여 4 % 이소 플루 란 유도 챔버에 마우스를 마취. 마우스가 발가락 핀치에 응답하지 않는 경우 마취는 충분하다. </li><li> 해부 보드에 마우스를 전송하고, 동축 회로에 접속 보호구 통해 마취를 유지한다. 70 % 알코올로 마우스의 모피를 소독. 가위 복강을 열고 사용하면 대정맥과 다이어프램을 노출. </li><li> 심장을 노출 마지막에서 첫 번째 갈비뼈에 시작 횡 절개를하고 마우스의 머리 흉부 케이지를 반영합니다. 마우스가 더 이상 숨을 쉬지으로 마취를 중지합니다. 심장 박동을 관찰한다. </li><li> (다이아 프램을 통해) 흉강으로 복부에서 하대 정맥에 살균 타이로드의 버퍼를 포함하는 5 ML의 주사기에 부착 된 21G 바늘을 삽입합니다. 혈액이 바늘 삽입의 대정맥에 꼬리를 종료 할 수 있는지 확인합니다. 제 관류전자 타이로드의 버퍼 부드럽게과 대정맥에 일정한 압력으로 심장이 부분적으로 붉은 색을 잃을 때까지. 참고 : 심장은 심장에서 혈액의 제거를 허용 치는 남아있다. </li><li> 무균의 KCl 솔루션을 포함하는 5 ML의 주사기에 부착 된 21G 바늘과 바늘을 교체합니다. 조심스럽게 심장이 박동을 멈출 때까지 대정맥에의 KCl 용액을 관류하고 바늘을 제거합니다. 참고 : KCl을 솔루션은 관상 시스템의 쉽게 관류 바늘의 삽입 및 관류를 허용 편안한 확장기의 마음을 유지합니다. </li><li> 약간 곡선 집게를 사용하여 마음을 들어 올려 사용하는 가위는 심장 손상과 심장에 부착하는 근위부 동맥과 정맥을 주변 조직에서 무료로 심장을 해부 그러나 둡니다. 얼음처럼 차가운 PBS는 항진균제 / 항생제 (/)로 보충이 포함 된 15 ML 튜브에 마음을 전송합니다. 최대 3 시간 동안 얼음처럼 차가운 PBS의 마음을 보관합니다. </li></ol> 3. Cannulatio관류 상공 회의소에서 마우스 마음의 N <ol><li> 층류 후드의 모든 절차를 수행합니다. 10cm 페트리 접시에 15 ML 튜브에서 격리하는 마음을 전송하고 추가 PBS는 A / 보충. </li><li> 해부 현미경, 모든 비 심장 조직을 제거하는 미세 가위와 집게를 사용하지만 상행 대동맥을 보존 마음에 팔 머리 동맥과 폐동맥 혈관, 2mm의 기부와 최소한 분기에. 좌심실 내강 (일반적으로 2mm)에 대한 액세스를 만드는 마음의 정점의 끝을 잘라. 해부 현미경 흐름 후드에서 재관류 챔버를 놓습니다. </li><li> 실리콘 튜브를 사용하여 삽입 바늘 매체 5 ML의 주사기를 연결합니다. 20 ㎖ 매체와 관류 챔버를 입력하고 관류 챔버에서 2 무딘 바늘 (그림 1) 사이에 회전 무대에서 마음을했습니다. 심장이 앞에 위치하도록 회전 스테이지의 높이를 조절바늘. </li></ol> 승모판을 배양 4. 결찰 (그림 1 참조) <ol><li> 봉합사 (실크 7.0)과 대동맥을 결찰. 봉합 좌심방을 결찰. 좌심방에 폐정맥을 통해 (그림 1. 1 NR) 바늘을 삽입하고 좌심방에서 해당 항목에 봉합 근위부와 결찰. 이 승모판 막에 손상을 하듯이 바늘이 좌심방으로 너무 멀리 있지 않은지 확인하십시오. </li><li> 좌심실로 직선 운동으로 바늘 (그림 1 nr.2)를 삽입합니다. 50 ㎖ 튜브에 관류 챔버로부터 전송 매체. 생체 접착제와 심근에 바늘을 밀봉합니다. </li><li> 접착제가 건조 후, 신중하게 nr.1 바늘과 누출이 있는지 확인하기 위해 매체 채워진 주사기를 연결하여 마음에 매체를 주입. 바늘 nr.2 마지막 남은 혈액이 심장에서 관류되어에서 해당 매체 종료를 확인합니다. </li></ol> 5. 리대동맥 판막을 배양 gation (그림 1 참조) <ol><li> 대동맥의 바늘 (그림 1 nr.1)를 삽입하고 봉합 결찰. 이 대동맥 판막을 손상하는 것처럼 바늘이 대동맥으로 너무 멀리 있지 않은지 확인하십시오. 좌심실로 직선 운동으로 바늘 (그림 1 nr.2)를 삽입합니다. 50 ㎖ 튜브에 관류 챔버로부터 전송 매체. 생체 접착제와 심근에 바늘을 밀봉합니다. </li><li> 접착제가 건조 후, 신중하게 nr.2 바늘과 누출이 있는지 확인하기 위해 매체 채워진 주사기를 연결하여 마음에 매체를 주입. 바늘 nr.1 마지막 남은 혈액이 심장에서 관류되어에서 해당 매체 종료를 확인합니다. </li></ol> 스탠드에 6. 관류 상공 회의소 <ol><li> 매체를 옮겨 20 mL를 관류 챔버를 입력합니다. 챔버에 가스켓 및 뚜껑을 놓고 세탁기와 나사로 조입니다. </li><li> incuba에서 조립 MTCS 제거터. 스탠드에 재관류 챔버를 연결합니다. 튜브를 연결합니다 (그림 1D 참조). 인큐베이터 (5 % CO 2, 37 ℃로)에 스탠드를 놓습니다. 펌프에 튜브를 연결합니다. 600 μL / 분 주위에 흐름 속도와 펌프의 전원을 켭니다. </li><li> 저장 및 / 또는 기포 트랩 매질의 레벨이 수신 매체 방울의 하강에 의해 흐름의 존재의 시각화를 허용하는지 확인. 배양 후, 심장, 시스템으로부터 제거 고정 조직 학적 검사를 위해 처리된다. </li></ol>

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The authors declare no conflicts of interest.

심장 마우스 밸브를 배양에서 중요한 단계는 흐름의 적절한 방향을 보장하기 위해 마우스에서 심장 절제 밸브에 수직 바늘의 생존과 라이 게이션을 위해 가능한 한 짧은 관류 챔버에서 결찰 사이의 시간을 포함 . 또한, 매체없이 관류 챔버에서 결찰 후 흐름을 확인하는 것은 적절한 삽입 및 바늘의 결찰을 보장합니다. 그것은 멸균 문화를 유지하고 잠재적으로 흐름을 방해 마음에 갇혀 얻을 수있?...

심장 판막 질환은 서구 세계에서 이환율과 사망률의 주요 원인이다 그 유병률은 연령에 따라 증가하며 75년 인구의 10 % 이상 및 1 이전에 영향을 미친다. 마음의 전신 부분의 밸브는, 대동맥 및 승모판 밸브는 주로 영향을받습니다. 심장 판막 질환 (2)의 기계적 특성 변화를 초래 밸브의 고도로 조직화 구조의 손실을 특징으로한다. 구조적 완전성은 밸브의 기능에 따라서 중요하다. 밸브의 전단지는 판막 간질 세포 (VIC), 판막 내피 세포 (VEC), 고도로 계층화 된 패턴 3,4으로 구성되어 세포 외 매트릭스로 구성되어있다. VIC를은 전자 재료 합성, 분해 및 조직에 대한 책임이 있습니다. 요인은 혈액, 내장 컴퓨터에서 나오는 또는 그 기능을 조율 VIC를에 ECM 행위에 거주. 또한,기계적 힘은 층류 또는 진동형 전단 응력, VIC를도 5의 동작에 영향을주는 압축 또는 인장 응력을 초래 심장주기 동안 전단에 작용. 밸브의 구조를 조절하는 방법을 이해하기 위해서는, 먼저 VIC를가 심장 사이클 동안 경험 한 자극의 다양한 세트에 응답하는 방법을 이해하여야한다. 시험 관내 연구 판막 세포의 특성 및 능력에 대한 매우 유익한왔다. 시험관 내에서 이들 세포의 반응은, 그러나 항상 정확하게 생체 반응 6을 흉내낼 수있다 예를 들면, 자극에 VIC의 반응의 EC의 존재 및 ECM의 조성 5에 따라 달라진다. 더욱이, 자극 판막 세포의 반응은 전단 7에 특정 위치에 달려있다. 생화학 적 자극에 더하여, 판막 셀의 동작은 O 작용 기계적 힘에 의해 결정된다N 밸브 (8). 밸브의 각 지역은 혈역학 적 스트레스의 고유의 세트 실시한다. 현재 생체 모델은 기계적인 힘이 판막의 구조 (5)의 중요한 결정 요인임을 보여 주었다 있지만, 관련 메커니즘은 아직 불분명하다. 생체 내 모델은 판막 개발 9,10의 기초가되는 분자 메커니즘에 대한 통찰력을 제공하고 있지만, 성인 판막 생물학에 대한 통찰력은 여전히 애매하다. 따라서, 생체 외 유동 모델되는 심장 밸브는 11 시간의 연장 된 기간 동안 심장에 자연스러운 위치에서 배양 될 수 개발되었다. 이는 밸브가 구성 천연 유지와 VIC를 최대한 자연스러운 VIC를 자극에 반응을 생체 내와 같은 환경을 경험할 수있는 장점을 갖는다. 또한, 마음에 그들의 자연적인 위치에있는 밸브의 문화는 각 쓰는 용이관련 혈역학 적 스트레스에 판막 지역. 즉,이 생체 모델에서, 소형 조직 배양 시스템 (MTCS)는, 밸브는 심장 밸브 리모델링에서의 역할의 조사를 허용하는 다른 생화학 적 및 혈역학 적 자극에 노출 될 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

대동맥 판막 (그림 2) 또는 승모판 막 (11)은 최소 3 일 동안 배양 할 수있다. (대동맥판과 승모판 대한 확장기의 총수에 대한 수축기 위치를 나타낸다)가 개방 위치에서 배양함으로써, 판막 세포 생존 유지. TUNEL 양성 세포 (도 2H, I) 또는 절단 된 카스파 제 -3 발현의 부재에 의해 결정된 어떠한 세포 사멸이 관찰되지 않는 (도시되지 리버 외., 2010 (11)). (...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

소형 조직 배양 시스템에서 마우스 심장 밸브를 배양

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.7K 조회수.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

비디오: Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Proteolytically 분해 3 차원 시스템에서 난소 대과의 캡슐화 및 문화에 대한 방법

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

연구

생체공학

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

심장 조직 공학을위한 모세관 력 리소그래피

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

콘트라스트 계산하기 관류 고정 시편의 단층 촬영을 이용하여 인간의 심장 정맥 시스템의 해부학 적 재건

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

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 ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

비 계-무료 3 차원 심장 조직 창조의 그물 형 기반 방법

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...