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 histolog...

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 border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">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 ( <strong clas...

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

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

세포 네트워크의 창조를위한 플라즈마 리소그래피 표면 Patterning

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

연구

생체공학

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized는 조직 고유의 하이드로 겔 Bioink를 사용하여 구축

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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. ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

젤라틴 메타크릴 하이드로겔 기반 바이오잉크의 3D 바이오프린팅 프로토콜

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

다층 하이드로겔 바이오잉크를 3차원 바이오프린팅에 사용하여 균일한 세포 분배

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

내피 네트워크에 3D 다세포 유방 스페로이드의 직접 생체 인쇄

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

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 ...

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 ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

신경과 같은 조직을 위한 Murine 피질 성상 세포의 3D 생물 인쇄

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...