저자는 심장 혈관 연구에 대한 기금 모금 및 메릴랜드 줄기 세포 연구 기금 (2016-MSCRFI-2735)과 같은 기금 출처를 인정합니다.

1. 심근 세포의 제조 <ol><li> 생성 및 인간이 유도 한 pluripotent 줄기 세포 (hiPSCs) 10 설명대로 기저막 매트릭스로 코팅 6 잘 접시에. </li><li> 이전에 설명한 방법 11 , 12를 사용하여 hiPSCs를 hiPSC 유래 cardiomyocytes (hiPSC - CMs)로 구분하십시오. </li><li> 분화 후 19 일에, 실온에서 5 분 동안 각 웰에서 0.05 % 트립신 / EDTA 2 mL를 사용하여 심근 세포를 분리한다. </li><li> 세포 해리를 관찰하기 위해 광학 현미경으로 cardiomyocytes를 모니터링합니다. </li><li> 트립신 억제제 0.0125 % 2 mL를 사용하여 트립신을 중화시킨다. </li><li> 분리 된 cardiomyocytes를 풀어 동력 된 피펫 필러를 사용하여 하나의 50 ML 원뿔 튜브로 전송하십시오. </li><li> 펠렛을 얻기 위해 실온에서 250 xg로 5 분 동안 세포 현탁액을 원심 분리하십시오. </li><li> Roswell Park 5 mL에 펠렛을 ResuspendB-27 (RPMI / B-27 세포 배지)가 보충 된 Memorial Institute (RPMI) 세포 배지. </li><li> 세포 배지에 세포 20 μL를 피펫과 0.4 % Trypan 블루 솔루션의 동등한 금액으로 얼룩. </li><li> 자동화 된 세포 카운터 또는 수동 hemocytometer를 사용하여 세포 현탁액의 농도와 세포 생존력을 세고 얻습니다. </li></ol> 2. 섬유 아세포의 제조 <ol><li> 인간의 심장 섬유 아세포 (HCF) (성인 심실 유형) 세포주를 시작하십시오 13 . </li><li> HCF 배양 프로토콜 13 에 따라 항체를 분리한다. </li><li> 세포 배지에 20 μL의 세포를 피펫 팅하고 같은 양의 Trypan Blue 용액 0.4 %로 염색합니다. </li><li> 자동 세포 카운터 또는 수동 hemocytometer를 사용하여 새로운 세포 현탁액의 농도와 세포 생존력을 세고 얻습니다. </li></ol> 3. 내피 세포의 제조 <ol><li> 인간 umb을 시작하십시오.15. 설명 된대로 정맥 내피 세포 (HUVEC) 라인. HUVEC 배양 규약에 따라 항체를 분리하고 분리한다. </li><li> 세포 배지에 세포 20 μL를 피펫과 0.4 % Trypan 블루 솔루션의 동등한 금액으로 얼룩. </li><li> 자동화 된 세포 카운터 또는 수동 hemocytometer를 사용하여 세포 현탁액의 농도와 세포 생존력을 세고 얻습니다. </li></ol> 4. 공동 배양 : <ol><li> RPMI / B - 27 세포 배지에서 3 가지 세포 유형 (hiPSC - CM, HCF 및 HUVEC)을 혼합하여 50 ML 원뿔 튜브에 혼합 세포 솔루션의 재고를 생성, hiPSC - CM : HCF : HUVEC 세포 비율 70:15:15 및 mL 당 165,000 세포의 농도. </li><li> 혼합 채널 솔루션을 다중 채널 피펫을 사용하여 웰 당 200 μL 또는 웰 당 33,000 세포의 초저 애착 96- 웰 U- 바닥 플레이트에 분배하십시오. </li><li> 96 웰 플레이트를 3 일 동안 배양하고 (37 ℃, 5 %의 탄소 다이옥사이드습도 95 %). </li><li> 광학 현미경으로 각 웰의 가운데와 바닥에 혼합 된 세포 다세포 회전 타원체가 있는지 검사합니다. 참고 : 2 차원 현미경 투영에서 이러한 회전 타원체는 원형 모양으로 나타납니다 ( 그림 1 ). Spheroids는 초저 부착 96- 웰 플레이트에 세포를 접종 한 후 24 시간 이내에 형성되어야합니다. 회전 타원체는 96- 웰 플레이트에 세포를 접종 한 후 48 시간 이내에 박동하기 시작해야합니다. 박동하지 않는 회전 타원체는 최종 3D 생체 보정 패치가 작동 할 수 있도록 3D 생체 인쇄에 사용하면 안됩니다. 연결 인자 43 (Cx43) 및 트로포 닌에 대한 회전 타원체의 면역 형광법을 사용하여 회전 타원체 세포 조성 및 품질을 평가할 수 있습니다 15 . </li></ol> 5. 스캐 폴드가없는 심장 조직의 3D Bioprinting <ol><li> 회전 타원체 기반의 3D 바이오 프 린터의 잡지에 회전 타원체가 들어있는 플레이트를 넣으십시오. </li><li> 3D bioprinter를 켜고 3D bioprinting 소프트웨어를 실행하십시오. <ol><li> &quot;Auxillary&quot;, &quot;Operation Preparation&quot;, &quot;Inspect Mode&quot;그리고 &quot;Home Return&quot;을 클릭하십시오. </li><li> &quot;시작&quot;아래의 초록색 아이콘을 클릭하여 회전 타원체 검사를 시작하십시오. </li><li> 화면의 &quot;(2) Dia (μm)&quot;란에 회전 타원체 직경이 표시되는지 확인합니다. </li></ol></li><li> 평균 회전 타원체 직경을 가장 가까운 100 μm로 추정하려면 &quot;초기 설정&quot;을 클릭하고 Stack Z 피치 (μm)에 평균 회전 타원체 직경을 입력하십시오. </li><li> &quot;합계&quot;를 클릭하면 &quot;레이어&quot;아래의 숫자가 자동으로 재조정됩니다. &quot;적용&quot;을 클릭하여 설정을 확인하십시오. </li><li> &quot;Needle Array Map&quot;을 클릭하고 &quot;Layer No.&quot;로 이동하여 3D Bioprinting을위한 원하는 레이어를 선택하고 개별 회전 타원체 위치를 선택하여 화면 왼쪽의 맵에 원하는 3D 생체 설계를 그립니다. 진부한k &quot;적용&quot;을 클릭하여 3D Bioprinting 디자인을 확인하십시오. </li><li> Needle Array 검사를 시작하려면 &quot;Needle Array Check&quot;를 클릭하십시오. 바늘 배열에 초점이 맞지 않으면 0 ~ 500 μm 범위의 값을 &quot;OffsetZ (μm)&quot;에 입력하여 500 μm에서 시작하여 100 μm에서 0 μm까지 줄여서 카메라 초점을 조정합니다. </li><li> &quot;Inspection Parameters&quot;, &quot;Type 1&quot;을 클릭하고 &quot;직경 (μm)&quot;을 원하는 직경 범위로 설정하십시오. &quot;진원도 (%)&quot;및 &quot;매끄러움 (%)&quot;을 원하는 백분율로 설정하십시오. &quot;적용&quot;을 클릭하여 설정을 확인하십시오. 참고 : 여기 회전 타원체 직경은 450 μm에서 550 μm로 설정되었습니다. &quot;진원도 (%)&quot;및 &quot;부드러움 (%)&quot;은 각 회전 타원체에 대한 컴퓨터 계산 평가입니다. 여기서 &quot;진원도 (%)&quot;에 사용 된 설정은 60 %이고 &quot;매끄러움 (%)&quot;은 70 %입니다. </li><li> &quot;Presence&quot;를 클릭하고 &quot;Diameter (μm)&quot;를 동일한 설정으로 업데이트하십시오s를 선택하십시오. &quot;적용&quot;을 클릭하여 설정을 확인하십시오. </li><li> &quot;Stack Mode&quot;를 클릭 한 다음 &quot;Start&quot;아래의 초록색 아이콘을 클릭하면 진공 흡입 및 3D 바늘 어레이의 특정 위치에 회전 타원체를 배치하여 3D 바이오 프린터의 노즐을 사용하여 회전 타원체 픽업을 시작할 수 있습니다. 참고 : 81 개의 회전 타원체 (9 개의 회전 타원체와 9 개의 회전 타원체)로 구성된 심장 패치의 경우 3D 바이오 프린팅은 약 20 ~ 30 분이 소요됩니다. </li><li> bioprinting 후, 무균 포셉을 사용하여 3D bioprinted 패치가 들어있는 바늘 어레이를 들고 RPMI / B-27 세포 배지 150 mL가 들어있는 250 mL 멸균 비이커로 옮깁니다. </li><li> 3 일 (37 ° C, 5 % 이산화탄소, 95 % 습도) 동안 바늘 어레이와 3D bioprinted 패치를 품어. </li></ol> 6. 바늘 어레이 및 패치 성숙에서 3D Bioprinted 패치 제거 <ol><li> 배양 3 일 후, 바늘 어레이의베이스에서 한 손으로 다른 손으로 t 아래 플라스틱 커버에서 다른 손그는 바늘 어레이에서 3D 바이오 프린팅 된 패치를 제거하기 위해 플라스틱 커버를 위로 가볍게 밉니다. 참고 : 필요에 따라 인산염 완충 식염수 (PBS)를 사용한 윤활제를이 단계 직전에 바늘에 적용하여 부드럽고 쉽게 제거 할 수 있습니다. </li><li> 한 쌍의 무균 포셉을 사용하여 3D bioprinted patch가있는 플라스틱 덮개를 들어 올려 RPMI / B-27 세포 배지가 들어있는 35mm 접시에 패치와 플라스틱 덮개를 옮깁니다. </li><li> 핀셋을 플라스틱 커버에 붙이고 RPMI / B-27 셀 미디어에있는 패치로 플라스틱 커버를 부드럽게 흔들어서 패치를 매체에 풀어 놓습니다. </li><li> 광학 현미경으로 3D bioprinted 패치를 검사하여 3D bioprinted 패치가 작동하는지 확인하십시오. </li><li> 3 일 이상 (37 ° C, 5 % 이산화탄소, 95 % 습도) 35mm 접시에 3D bioprinted 패치를 품어 놓습니다. 참고 : 3 일 후, 회전 타원체가 함께 융합되고 구멍이바늘이 있던 패치는 더 이상 보이지 않아야합니다. </li></ol>

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저자는 공개 할 것이 없습니다.

It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histological and mechanical properties.
This approach also allows different types of spheroids to be used to make a patch, as well as the 3D bioprinting of cardiac patches with complex 3D designs. This complexity of the final 3D construct is achievable by 3D bioprinting with precise spheroid positioning, but may be difficult to achieve by gentle rotation of floating spheroids7 or simple molding of spheroids into a cylindrical 3D construct16.
The limitations of this approach are the relatively fixed size of the spheroids between 450 to 550 &#181;m due to the fixed distance of 400 &#181;m between the needles in the needle array, as well as possibly reduced cell viability if the spheroids are too large in diameter. The patches are mechanically fragile immediately after removal from the need array; however, the strength and durability are improved with culture for longer durations of time.
Finally, 3D bioprinting of cardiac patches has promising clinical applications17, in cardiac regeneration18,19 and as 3D models of heart disease. These 3D models can be used for applications in predictive pharmacology/toxicology and development of cell and genome directed therapies. 
In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications in cardiac tissue regeneration and as 3D models of heart disease.

3D bioprinting 1 , 2 , 3 에는 여러 가지 방법이 있습니다. 3D 바이오 프린팅은 잉크젯 바이오 프린팅, 마이크로 압출 바이오 프린팅, 레이저 보조 바이오 프린팅, 방법 조합 또는 새로운 접근법과 같은 예를 사용하여 인쇄 기술 1로 분류됩니다. 3D bioprinting은 scaffold-free 또는 scaffold-dependent 방법으로 분류 할 수 있습니다 4 . 3D bioprinting의 대부분의 방법은 scaffold에 의존합니다. 여기에는 bioinks 5 또는 scaffolds 6 과 같은 생체 재료가 필요합니다. 그러나 발판 의존적 인 3D 바이오 프린팅은 스캐 폴딩 재료의 면역 원성, 독점적 바이오 인크의 비용, 분해 속도가 느린 독성 및 분해 제품의 독성과 같은 많은 문제점과 한계에 직면합니다. Scafspheroids를 사용하는 fold-free cardiac tissue engineering이 시도되고 있으며, 스캐 폴드 의존 조직 공학의 단점을 극복 할 수있는 가능성이있다. 그러나이 논문의 저자들에 의해 인정 된 것처럼, 생물 공정의 과정에서 고정 된 위치에서 회전 타원체를 견고하게 다루고 위치시키는 것은 어려웠다. 3D bioprinting과 회전 타원체 기반 조직 공학을 함께 사용하면 이러한 어려움을 극복 할 수 있습니다. 이 프로토콜에서는 다른 생체 물질을 사용하지 않고 심장 ​​조직의 3D 생체 프린팅을 설명합니다. 단, 회전 타원체 형태의 세포 만 사용하십시오. 스캐 폴드가 필요없는 회전 타원체 기반의 3D 생체 인식 장치 9 는 진공 흡입을 사용하여 개별 회전 타원체를 집어 들고 바늘 배열에 배치 할 수 있습니다. 3D bioprinting에서 바늘 어레이에 회전 타원체를 배치하는 개념은 고대 Japa에서 바늘 배열 ( &quot; kenzan &quot;으로 알려짐)의 사용에서 영감을 얻었습니다.꽃꽂이 , 꽃꽂이의 예술 . 이 시스템은 회전 타원체를 어떤 구성으로도 정확하게 위치시킬 수 있으며 짧은 시간에 개별 회전 타원체가 융합되어 3D 생체 조직을 생성합니다. 따라서이 방법은 회전 타원체를 쉽게 조작 할 수있게 해주 며, 미래의 발판없는 기관 생물 공학에 잠재적 인 영향을 미친다.

<table><tbody><tr><td>Geltrex</td><td>Invitrogen&amp;nbsp;</td><td>A1413202</td><td></td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Thermo Fisher</td><td>15400054</td><td></td></tr><tr><td>Defined Trypsin inhibitor 0.0125%</td><td>Thermo Fisher</td><td>R007100</td><td></td></tr><tr><td>RPMI Cell Media</td><td>Invitrogen</td><td>11875-093</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>B-27 Supplement</td><td>Thermo Fisher</td><td>17504044</td><td>RPMI supplemented with B27 constitutes HIPSC-CM culture media</td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Human cardiac fibroblasts (adult ventricular type)</td><td>Sciencell</td><td>6310</td><td></td></tr><tr><td>Human umbilical vein endothelial cells</td><td>Lonza</td><td>CC-2935</td><td></td></tr><tr><td>PrimeSurface ultra-low attachment 96-well U-bottom plates&amp;nbsp;</td><td>Akita Sumitomo Bakelite Co.</td><td>MS-9096UZ</td><td></td></tr><tr><td>Regenova Bio 3D Printer</td><td>Cyfuse Biomedical K.K.</td><td>N/A</td><td>www.cyfusebio.com/en/</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Troponin T Antibody</td><td>Thermo Fisher</td><td>701620</td><td></td></tr><tr><td>Connexin 43 (Cx43) Antibody</td><td>Chemicon</td><td>MAB3068</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI</td><td>Thermo Fisher</td><td>P36935</td><td></td></tr></tbody></table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

이 프로토콜은 세포만을 사용하여 생체 재료를 사용하지 않고 심장 ​​조직의 3D 생체 인쇄를 설명합니다. Cardiomyocytes, 내피 세포 및 섬유 아 세포를 먼저 분리하고, 원하는 세포 비율로 세고 혼합합니다. 그들은 초저 부착 96- 웰 플레이트에서 개별 웰에서 공동 배양됩니다. 3 일 이내에, 회전 타원체 형태가됩니다. 이 회전 타원체는 진공 흡인을 사용하여 노즐로 집어 들고 3D 바이오 프린터를 사용하여 니들 어레이에 조립됩니다. 회전 타원체는 바늘 어레이에서 융합됩니다. 3 차원 바이오 프린팅한지 3 일 후, 스페 로이드는 손상되지 않은 채로 제거되어 이미 자발적으로 박동하고 있습니다. 3D bioprinted 심장 패치는 구성 요소 spheroids의 기계적 통합을 나타내며 심장 조직 재생 및 심장병의 3D 모델로서 매우 유망합니다.

4.4 단계 (공동 배양)가 끝나면 각 웰의 세포는 초저 부착 96-well U-bottom plate의 바닥에 응집되어 중력에 의해 회전 타원체가 형성됩니다. 이러한 회전 타원체는 hiPSC-CM, HCF 및 HUVEC를 포함하며 광학 현미경으로 육안 검사가 가능하며 2 차원 투영으로 원형으로 나타나야합니다 ( 그림 1 ). 6.3 단계의 끝에서, 3D 생체 보정 심장 패치는 바늘 어레이 ( 그림 2 , 왼쪽)에 의해 생성 된 바늘 구멍 때문에 조직 공극을 포함해야합니다. 이 시점에서, 회전 타원체 사이의 경계는 불분명 해져야하고, 패치는 회전 타원체의 기계적 통합을 보여주는 자발적으로 치닫기 시작해야합니다. 단계 6.5가 끝나면 조직 공극은 주변 조직 ( 그림 2 , 오른쪽)에 의해 채워 져야하고 3D 생체 보정 심장 패치는 계속되어야합니다자발적으로. <img alt="그림 1" class="xfigimg" src="/files/ftp_upload/55438/55438fig1.jpg" /> 그림 1 : 혼합 세포 다세포 회전 타원체의 생성. hiPSC-CM, HCF 및 HUVEC를 70:15:15의 hiPSC-CM : HCF : HUVEC 세포 비율에서 혼합하고, 세포 혼합물을 초저 부착 96- 웰 U- 바닥 플레이트에 분배시켰다. 24 시간 이내에 혼합 된 세포 다세포 spheroids (왼쪽, 오른쪽)가 형성되었다. 스케일 바 = 500 μm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55438/55438fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" class="xfigimg" src="/files/ftp_upload/55438/55438fig2.jpg" /> 그림 2 : 회전 타원체의 기계적 통합을 보여주는 심장 조직의 생성 . 3D 바이오눈에 보이는 바늘 구멍 (왼쪽)이있는 바늘 어레이에서 꺼내 자마자 즉시 인쇄 된 심장 패치. 이때 스 펠로이드 간의 경계는 이미 뚜렷하지 않았고 패치는 이미 자발적으로 뛰기 시작 했으므로 스 펠로이드가 기계적으로 통합되었습니다. 바늘 어레이에서 제거한 지 3 일 후, 바늘 어레이로 인한 조직 공극을 주변 조직 (오른쪽)으로 채우고 패치는 자발적으로 계속 뛰었다. 눈금 막대 = 1mm. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55438/55438fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

이 프로토콜은 생체 재료를 사용하지 않고 심장 ​​조직의 3D bioprinting을 설명합니다. 3D bioprinted 심장 패치는 구성 요소 spheroids의 기계적 통합을 나타내며 심장 조직 재생 및 심장병의 3D 모델로서 매우 유망합니다.

3D Bioprinting을 이용한 Spheroids의 기계적 통합을 보여주는 심장 조직의 생성

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

creation-cardiac-tissue-exhibiting-mechanical-integration-spheroids

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.9K 조회수.  Johns Hopkins Hospital.  이 프로토콜은 생체 재료를 사용하지 않고 심장 ​​조직의 3D bioprinting을 설명합니다. 3D bioprinted 심장 패치는 구성 요소 spheroids의 기계적 통합을 나타내며 심장 조직 재생 및 심장병의 3D 모델로서 매우 유망합니다. 

비디오: Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Microcontact 인쇄를 통해 정렬 기능 심근 조직의 생성

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

연구

생체공학

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

정의 인간 공학 심장 조직의 건설 심장 세포 치료의 메커니즘을 연구하는

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

공학에 대 한 미세 Bioprinting 나가도록 조직 및 Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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. ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

심근 경색의 뮤린 모델에서 3D 생체 인쇄 패치의 이식

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

인간 iPSC 유래 심근세포, 심장 섬유아세포 및 내피 세포를 사용하여 3D 심장 미세 조직 배열의 제조

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

인간 심장 병리학을 연구하기 위하여 체외 생체 공학심장 조직에서와 같이 심장 스페로이드

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

미세 유체 플랫폼 내에서 3D 조직 된 인간 심장 조직 개발

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

수술 전 계획 및 실습 교육에서 응용 프로그램을 갖춘 환자 별 실리콘 심장 모델 생성

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

공학적 심장 조직의 데이터 수집 및 모델 처리량을 개선하기 위한 바이오리액터 설계

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...