저자는 다음 자금 출처 인정: 심혈 관 연구에 대 한 마술을 문제 기금.

1입니다. Cardiomyocytes의 준비
<ol><li>앞에서 설명한17지하실 멤브레인 매트릭스와 문화 인간 유도 만능 줄기 세포 (hiPSCs)와 6 잘 플레이트 코트.</li>
	<li>앞에서 설명한 방법18을 사용 하 여 hiPSC CMs로 hiPSCs를 구분 합니다.</li>
	<li>16-18 d 후 차별화에서 일시 중지는 cardiomyocytes 인산 염 버퍼 식 염 수 (PBS) x 1의 2 mL와 함께 각 잘 헹 궈 서 칼슘 또는 마그네슘, 인큐베이션 1 mL/트립 신 또는 셀 분리 시 약 (참조 테이블의의 뒤에 없이 재료) 실 온에서 5 분.</li>
	<li>트립 신 또는 셀 분리 시 중화 ( 재료의 표참조) 로스웰 파크 기념 연구소 (RPMI) 셀 미디어 B-27 (RPMI/B-27 셀 미디어)와 보완의 동일한 볼륨을 사용 하 여. 위쪽 및 아래쪽 준수 셀을 풀고 플라스틱.</li>
	<li>10 mL 혈 청 학적인 피 펫과 정지 cardiomyocytes 수집 하 고 50 mL 원뿔 튜브에 그들을 전송.</li>
	<li>세포 현 탁 액 셀 펠 렛을 얻기 위해 상 온에서 5 분 동안 250 x g 에서 원심</li>
	<li>RPMI/B-27 셀 미디어 10 mL에 펠 릿을 resuspend.</li>
	<li>20 µ L 세포 현 탁 액의 0.4 %Trypan 블루 솔루션의 동일한 금액으로 결합 하 고 부드럽게 혼합.</li>
	<li>수동 hemocytometer를 사용 하 여 계산 하 고 세포 현 탁 액의 농도 세포 생존 능력을 구하십시오.</li>
</ol>2입니다. 섬유 아 세포의 준비
<ol><li>앞에서 설명한16인간의 심장 섬유 아 세포 (HCF) (성인 심 실 타입) 셀 라인의 문화를 시작 합니다.</li>
	<li>트립 신 또는 셀 분리 시의 적절 한 금액으로 그들을 배양 하 여는 HCFs 일시 중단 ( 재료의 표참조) 실내 온도16시 5 분. T175 플라스 크에 10 mL의 트립 신 또는 셀 분리 시 약 사용 되었다.</li>
	<li>트립 신 또는 셀 분리 시 중화 ( 재료의 표참조) 다음 50 mL 원뿔 튜브에 샘플을 전송 매체의 동일한 볼륨을 사용 하 여, 고를 실 온에서 5 분 동안 250 x g 에 세포 현 탁 액을 원심을 펠 릿입니다.</li>
	<li>섬유 아 세포 성장 매체의 10 mL에 펠 릿을 resuspend ( 재료의 표참조).</li>
	<li>20 µ L HCF 세포 현 탁 액의 0.4 %trypan 블루 솔루션의 동일한 금액으로 결합 하 고 부드럽게 혼합.</li>
	<li>자동된 셀 카운터 또는 수동 hemocytometer를 사용 하 여 계산 하 고 새로운 세포 현 탁 액의 농도 세포 생존 능력을 구하십시오.</li>
</ol>3입니다. 내 피 세포의 준비
<ol><li>앞에서 설명한17인간 탯 줄 정 맥 내 피 세포 (HUVEC) 라인의 문화를 시작 합니다. 트립 신 또는 셀 분리 시의 적절 한 금액으로 그들을 배양 하 여는 HUVECs를 일시 중단 ( 재료의 표참조) 실내 온도17시 3 분. 트립 신 또는 셀 분리 시 중화 ( 재료의 표참조) 다음 50 mL 원뿔 튜브에 전송 매체의 동일한 볼륨을 사용 하 여, 및 펠 릿을 실 온에서 5 분 동안 250 x g 에 세포 현 탁 액을 원심. 참고: T175 플라스 크에 10 mL의 트립 신 또는 셀 분리 시 약 사용 되었다.</li>
	<li>내 피 세포 성장 매체의 10 mL에 펠 릿을 resuspend ( 재료의 표참조).</li>
	<li>HUVEC 서 스 펜 션의 20 µ L를 결합 하 여 0.4 %Trypan 블루 솔루션의 동일한 금액으로 그것을 얼룩 하 고 부드럽게 혼합.</li>
	<li>자동된 셀 카운터 또는 수동 hemocytometer를 사용 하 여 계산 하 고 세포 현 탁 액의 농도 세포 생존 능력을 구하십시오.</li>
</ol>4입니다. 드롭 Spheroids 매달려의 창조
<ol><li>교수형 장소 살 균 6 잘 플레이트에 (각각 350 µ m의 직경) 850 micropores를 포함 하는 드롭 장치.</li>
	<li>위에서 설명한 세 가지 종류의 세포를 분리: hiPSC CMs, HCFs, 및 HUVECs. ML 당 2,475,000 세포의 농도에 RPMI/B-27 셀 미디어 70 %iPSC-CMs, 15% HCFs 50 mL 원뿔 튜브에서 15 %HUVECs 비율에 그들을 결합.</li>
	<li>드롭 시스템 (350 µ m의 작은 직경) 6 잘 플레이트에 매달려 매우 낮은 첨부 파일의 각 음에 세포 현 탁 액 (mL 당 2,475,000 세포)의 4 mL를 분배.</li>
	<li>Spheroids는 교수형에 세포 현 탁 액을 분배의 12 h 이내 저절로 형성 드롭 장치. 37 ° C, 5% CO2, 그리고 95% 습도에서 72 h (3 d)의 총에 대 한 문화는 spheroids의 성숙에 대 한 계속.</li>
	<li>문화의 72 h 후 수확 교수형의 바닥에 숨 구멍을 통해 spheroids 매체와 접시에 장치를 배치 하 고 소용돌이 그것 부드럽게 교수형에서 spheroids를 해제 하 여 시스템을 드롭 드롭 장치.</li>
</ol>5. 3 차원 패치 창조 소설 그물 형을 사용 하 여
<ol><li>기지, 평방 바닥판, 바닥 그물, 및 10 층된 측면 그물은 소독 집게의 쌍의 원조와 함께 소설 몰드를 만드는 조립 첫째, 스택 및 기지, 바닥판 평방, 정렬 하단 코너 게시물을 사용 하 여 net. 그런 다음 스택과 고급 스테인레스 스틸 접지 단자의 그물을 만드는 방향으로 번갈아에 10 쪽 그물 레이어.</li>
	<li>플러시 PBS 또는 매체를 표면 장력을 감소, 다음, 전송 충전 베이스에 조립된 형 기본 작성.</li>
	<li>PBS와 같은 방법에 있는 매체 그물 금형 캐비티에 젖은. 천천히 원하는 크기의 전제 된 구멍에는 spheroids를 입력 (2 x 2 x 1 mm, 4 x 4 x 1 m m, 또는 6 x 6 x 1 m m). 참고: 셀 집계에 꽉 연결 하 고 정적 유지 구멍에 spheroids의 예상된 하는 볼륨의 120% 공급 있는지 확인 합니다.</li>
	<li>Net 상단과 상단 스퀘어 플레이트 코너 게시물에 어셈블합니다 및 stoppers 및 지주 튜브는 spheroids의 유실을 방지 하기 위해 보안.</li>
	<li>6 잘 플레이트 RPMI/B-27 셀 미디어 또는 전체 조립된 형을 충당 하기 위해 충분 한 매체와 멸 균 용기에 6 mL와 채워진된 그물 형 시스템 전송.</li>
	<li>3000-4000 x g에 7-10 일 동안 스윙 통에 채워진된 금형 시스템 6 잘 플레이트를 품 어. 참고:는 인큐베이션 기간 심장 패치의 크기에 의해 결정 됩니다. 큰 패치, 보육 시간 심장 패치 성숙에 대 한 증가 합니다.</li>
</ol>6. 소설 그물 형에서 패치 제거
<ol><li>미디어에서 금형 시스템을 제거 하 고 소독된 처리 매트 살 균 10 cm 접시에에 놓습니다.</li>
	<li>지주 관, stoppers, 상단 사각 접시 및 최고 그물을 제거 합니다. 신중 하 게 살 균 집게의 쌍을 가진 계층된 측면 그물 하나에 의해 밖으로 슬라이드. Decannulation, 후 그대로 심장 패치 하단의 그물 위에 얻을 수 있다.</li>
	<li>소독 집게를 사용 하 여 패치 순 하단을 선택 하 고 하단의 net RPMI/B27 미디어의 5 mL와 함께 35 mm 접시에 전송. 부드럽게 천천히 접시, 또는 살 균 세포 스 크레이 퍼와 함께 그물을 소용돌이 의해 그물 아래에서 패치를 풉니다. 그물 아래에서 초연, 후 심장 조직 RPMI/B27 미디어 양식 수 있습니다.</li>
	<li>35 mm 접시에 부동성 3 차원 심장 패치를 품 어 계속. 금형에서 제거 후 빠르면 24 시간 기능 동기 박동을 관찰할 수 있습니다.</li>
	<li>그물 형에서 패치를 제거한 후 모양을 무결성 및 시간 동기 박동 증가의 강도 유지는 관찰 합니다. 참고: 3 차원 그물 형 기반 심장 패치 전시 decannulation 후 구성 요소 cardiospheres의 전기 통합. 우리는 60에서 80 분당 60 일 이상 유지 하는 구타 주파수를 관찰.</li>
</ol>

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+Cardiac+Tissue+Exhibiting+Mechanical+Integration+of+Spheroids+Using+3D+Bioprinting.">Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting.</a> Journal of Visualized Experiments. 125 (e55438), (2017).</li><li>Tan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+number+per+spheroid+and+electrical+conductivity+of+nanowires+influence+the+function+of+silicon+nanowired+human+cardiac+spheroids.">Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.</a> Acta Biomaterialia. 51, 495-504 (2017).</li><li>Karra, R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redirecting+cardiac+growth+mechanisms+for+therapeutic+regeneration.">Redirecting cardiac growth mechanisms for therapeutic regeneration.</a> Journal of Clinical Investigation. 127 (2), 427-436 (2017).</li><li>Bartholoma, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+reaggregates+of+embryonic+cardiomyocytes:+a+potential+model+system+for+monitoring+effects+of+bioactive+agents.">Three-dimensional in vitro reaggregates of embryonic cardiomyocytes: a potential model system for monitoring effects of bioactive agents.</a> Journal of Biomolecular Screening. 10 (8), 814-822 (2005).</li></ol>

저자는 공개 없다.

이 방법의 중요성의 재현성 및 결과 다층 심장 조직의 효율성에 있다. 심장 조직 공학 분야에서 현재 목표 중 하나는 치는, 다층, 고 기능 3 차원 심장 패치를 생성 하는 방법을 식별 하입니다. 우리는 spheroids 소설 그물 형으로 cardiomyocytes, 내 피 세포, 섬유 아 세포의 구성의 직접 수동 시드 다층된 심장 조직 만들기의 효율적이 고 재현 가능한 방법 보고. 이 방법에 사용 되는 그물 형 6 x 6 x 1 m m 2 x 2 ...

현재 심장 조직 공학의 목표는 대체 또는 손상 된 심근 조직1의 기능과 구조를 복구 하는 치료를 개발 하는 것입니다. 기본 심장 조직의 중요 한 수축 및 electrophysiological 속성을 전시 하는 3 차원 심장 조직 모델을 만드는 방법 급속 하 게 확대 되어 있다2,3. 다양 한 전략을 탐험 하 고 연구4,5에서 사용 되었습니다. 이 방법에서에서 범위 특정 합성 및 천연 생리 활성 hydrogels, 젤라틴, 콜라겐, 섬유 소, 그리고 펩 티 드6, 같은 사용 하 여 바이오 잉크 증 착 기술2 및 bioprinting 기술7.
그것은 비 자유로운 방법 외국 비 계 소재8통합의 단점 없이 소재 기반 방법으로 유사한 조직 생산할 수 표시 되었습니다. 오 렌 Caspi 외. 셀의 다양 한 종류의 매우 vascularized 인간의 조작된 심장 조직9의 세대 수는 설명 했다. 턱 외. spheroids에서 심장 패치 생성을 위한 3 차원 인쇄 방법을 개발. 결과 패치 cardiomyocytes, 섬유 아 세포, 내 피 세포는 70:15:15 비율10에 구성 됩니다. Spheroids 하 여 효과적인 "빌딩 블록" 비 계 없는 심장 조직 창조의 그들은 저 산소 증에 대 한 저항으로 이식11,12에 대 한 충분 한 기계적 무결성을가지고 표시 되었습니다. 이전 학문은 회전 타원 체 생성에 대 한 여러 가지 제작 방법을 설명 했다, 교수형의 사용을 포함 하 여 방법, 스피너 플라스 크13, 미세 시스템14및 비 점착 문화 표면 코팅 또는 코팅 agarose 마이크로 금형15. 이 프로토콜을 사용 하 여 교수형 드롭 장치, spheroids의 수백을 한 번에 집 광을 위한 micropores를 포함.
이 연구는 소설 평방 금형 캐비티에는 spheroids를 수동으로 시드 및 성숙에 대 한 통에 조직 배양 포함 심장 조직 창조에 대 한 효율적인 비 계 없는 방법을 선물 한다. 일반적인 정적 문화 조건 하에서 산소 보급 조직 구조, 중앙 괴 사 발생의 외부 측면에 제한 됩니다. 그러나, 그물 형 금형에 시드 모든 spheroids 영양분과 산소의 증가 보급에 대 한 수 있도록 지속적인 유체 운동, 미디어에 몰두. 또한,이 형 기반 방법 수동 최소의 노력으로 다른 크기의 조직 패치의 동시 생성 가능 하며 결과 조직 형에서 쉽게 제거 될 수 있습니다. 이 새로운 메서드는 비 계, 다층 심장 패치의 효율적이 고 재현 가능한 창조에 대 한 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS-3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

이 프로토콜 고 추가 비 계 자료 없이 3 차원 (3 차원) 심장 조직을 만들 쉬운 그물 형 기반 방법에 설명 합니다. 인간 유도 만능 줄기 세포 유래 cardiomyocytes (iPSC-CMs), 인간의 심장 섬유 아 세포 (HCFs), 그리고 인간의 탯 줄 정 맥 내 피 세포 (HUVECs)는 격리 하 고 70 %iPSC-CMs, 15 %HCFs, 및 15 %HUVECs 세포 현 탁 액을 생성 하는 데 사용. 그들은 spheroids의 수백을 한 번에 집 광을 위한 micropores를 포함 하는 매우 낮은 첨부 파일 "걸기 하락" 시스템에서 공동 경작. 셀 집계 하 고 자발적으로 공동 문화의 3 일 후 구타 spheroids를 형성 한다. spheroids 수확, 새로운 금형 캐비티에 시드 고 인큐베이터에 통에 경작. spheroids가 성숙한 기능 조직 시드 후 약 7 일. 만족 스러운 구조적 무결성 및 동기 박동 동작 융합된 spheroids 결과 다층된 조직에 의하여 이루어져 있다. 이 새로운 메서드는 나중에 심장 마비의 치료에 대 한 설계 조직 만들기를 재현 하 고 비용 효율적인 방법으로 유망 가능성이 있다.

우리의 실험에 우리는 70 %iPSC-CMs, 15% HCFs RPMI/B-27 셀 미디어 mL 당 2,475,000 세포의 농도에 15 %HUVECs 세포 현 탁 액을 활용. 세포 현 탁 액을 만든 후 우리는 프로토콜의 4.3 단계에서 설명한 대로 드롭 시스템에 매달려 매우 낮은 첨부 파일의 각 음에 세포 현 탁 액 4 mL 적절. 교수형의 사용 문화 37 ° C, 5% CO2, 그리고 95% 습도에서 3 일 후에 spheroids를 치고 수백의 자발적인 형...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

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

비 계-무료 3 차원 심장 조직 창조의 그물 형 기반 방법

이 프로토콜 만족 구조적 무결성 및 동기 박동 동작 3 차원 비 계 없는 심장 조직을 만들 순 형 기반 방법을 설명 합니다.

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

a-net-mold-based-method-scaffold-free-three-dimensional-cardiac

Research

JoVE Journal

Bioengineering

8.8K 조회수.  The First Hospital of Jilin University. 이 프로토콜 만족 구조적 무결성 및 동기 박동 동작 3 차원 비 계 없는 심장 조직을 만들 순 형 기반 방법을 설명 합니다.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

입체 광학 해상도 Photoacoustic 현미경

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

연구

생체공학

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

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 ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

서 주 신장 조직 세그먼트와 소설 비 계 없는 건설 기술 관

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...