이 연구는 부분적으로 국립 과학 재단 (NSF 1,227,359)는 각각 국립 과학 재단 (EPS-1003907), 국립 과학 재단 (1007978) 후원 WVU 사전 사무실 및 WVU PSCoR에 의해 자금 WVU EPSCoR 프로그램에 의해 지원되었다. 미세 작업은 WVU 공유 연구 시설 (클린 룸 설비)과 웨스트 버지니아 대학의 칩 연구소 (마이크로 칩 연구소)에 미세 유체 통합 세포 연구로 이루어졌다. 공 촛점 이미징 WVU 현미경 이미징 시설에서 이루어졌다.

1. 포토 레지스트 마스터 금형의 석판 제작 다음 프로토콜은 30 ~ 60 μm의 사이 직경의 미세을 제작하는 과정을 보여줍니다. 작은 직경 (이하 30 μm의), 포토 레지스트의 단일 스핀 코팅 마이크로를 얻으려면이 필요합니다. <ol><li> 사용하기 전에 클린 24 시간에 4 ° C에서 냉장고에서 리플 로우 레지스트를 전송하고 실내 온도를 따뜻하게 할 수 있습니다. </li><li> 실리콘 웨이퍼를 청소하고 탈수 할 수 있도록 150 ° C에서 한 시간 동안 구워. 탈수는 실리콘 기판에 포토 레지스트 접착 도움을 줄 것입니다. </li><li> 스핀 코팅 포토 레지스트의 첫 번째 레이어는 다음과 같은 조리법을 사용하여 : </li></ol><table border="1"><tbody><tr><td> 단계 </td><td> 시간 (초) </td><td> 속도 (rpm) </td><td> 가속 </td></tr><tr><td> 1 </td><td> 2 &lt;/ TD&gt; <td> 300 </td><td> 최고 </td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td> 최고 </td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td> 최고 </td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td> 최고 </td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td> 최고 </td></tr></tbody></table><ol start="4"><li> 그런 다음 90 초 동안 110 ° C의 온도 열판에 웨이퍼를 굽는다. 부드러운 빵을 한 후 포토 레지스트의 두께는 20 ~ 30 μm의 수 있습니다. </li><li> 코트에게 첫 번째 레이어에 사용되는 것과 동일한 조리법에 따라 포토 레지스트의 두 번째 레이어를 회전. </li><li> 부드러운 빵을 90 초 동안 110 ° C의 온도 열판에 배치하여 다시 웨이퍼. 이 부드러운 빵을 한 후 포토 레지스트의 두께는 40 ~ 60 μm의 수 있습니다. </li><li> photor를 노출하여 긍정적 인 마스터 패턴을 생성영화 마스크를 통해 14,500 엠제이 / cm 2의 노광량으로 UV 빛을 ESIST. </li><li> 탈 (DI) 물 (1시 2분 (V / V))의 개발을 희석. 패턴이 완전히 개발 될 때까지 용액에 반복적으로 웨이퍼를 씻어. 다음 DI 물과 웨이퍼를 세척하고 질소 가스를 사용하여 말립니다. </li><li> 리플 로우 : 4 분, 용매 증발을 방지하기 위해 유리 페트리 접시 덮개를위한 120 ° C의 온도 열판에 배치 웨이퍼. 열판에서 웨이퍼를 제거하고 실온까지 냉각 할 수 있습니다. 리플 로우 후의 레지스트 두께는 50 μm의 수 있습니다. </li></ol> 2. PDMS 마이크로 채널 네트워크 소프트 리소그래피 제작 <ol><li> 10:1 중량 비율 (기본 : 경화제)에서 폴리 디메틸 실록산 (PDMS) 솔루션을 준비하고 행성 원심 믹서를 사용하여 철저하게 섞는다. </li><li> 리플 로우 포토 레지스트 마스터 금형에 PDMS 솔루션을 캐스팅합니다. 가스를 15 분 동안 데시 케이 터에서 주조 PDMS를 놓고. 필요한 경우 남아있는 거품을 제거하기 위해 질소 가스를 사용합니다. </li><li> 그것을 치료 할 수 있도록 3 시간 동안 60 ° C의 온도에서 오븐에 PDMS를 굽는다. 그런 다음 마스터 금형 치료 PDMS 층을 제거합니다. </li><li> 채널 네트워크에 구멍을 펀치로 유입 / 유출 구멍을 만들 날카롭게 주먹을 사용합니다. 질소 가스를 사용하여 PDMS의 표면을 청소합니다. </li><li> 4.5 × 10 -2 Torr의 및 3.5 피트 / 분의 산소 유량의 압력에서 플라즈마 클리너 내부에 30 초 동안 산소 플라즈마 두 PDMS 레이어를 처리합니다. 그런 다음 광학 현미경으로 수동으로 PDMS의 표면을 맞 춥니 다. 물 한 방울 정렬의 더 나은 제어에 필요한 경우에 사용합니다. </li><li> 60 ° C 영구적 인 결합을 달성하기 위해 30 분 동안 오븐에서 장치를 굽는다. </li></ol> 3. 칩 HUVEC 문화 및 시드 <ol><li> 문화는 L-글루타민과 문화 매체와 HUVECs 10 % 태아 소 혈청 (FBS)와 보충. 일에 대한2와 5 사이에 전자 실험 구절을 사용 하였다. </li><li> HUVECs가 합류되면, 37 ℃에서 2-6 분 동안 세포를 계산하고 최초의 HEPES이 (HEPES-BSS) 생리 식염수를 버퍼로 세포를 세척하여 그들을 수확 후 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)과 부화와 세포 치료 트립신이 완료된 후, 트립신을 중화 솔루션과 트립신 / EDTA (에틸렌 다이아 민 테트라 초산)을 중화. 원심 분리를 수집하기 위해 8 % 덱스 트란과 문화 매체에있는 세포를 일시 중지합니다. 덱스 트란은 더 나은 세포 파종 및 첨부 돕기 위해 중간 점도를 높이기 위해 사용되었다. </li><li> 5 4.5 × 10 -2 Torr의의 작동 압력 분 및 3.5의 산소 유량 피트 3 / 분 산소 플라즈마 장치를 취급합니다. 다음 DI 워터로 장치를로드하고 멸균 층류 생물 안전 후드에서 8 시간 동안 UV 빛으로 취급합니다. </li><li> 세포가 준비되어 하루 전에 1X 인산염 장치를 세척 피브로넥틴 (100 ㎍ / ㎖, DIL와 코트 다음 (1X PBS) 완충 식염수1X PBS로 uted), 4 ° C에서 하룻밤 냉장고에 품어. </li><li> 피브로넥틴 코팅 후 1X PBS 다음 배지와 부하 장치를 씻는다. 15 분 37 ° C의 온도에서 장치를 품어. </li><li> 3-4 X 10 6 세포 / ML의 세포 농도와 8 % 덱스 트란 문화 매체에 HUVEC 세포를로드합니다. 장치의 하나 입구에서 세포의 20 μL 방울을 배치하고 미세 유체 채널에 세포를 소개를 기울이십시오. 15-20 분 후 세포는 채널의 측면 벽에 부착하기 시작합니다. 장치에게 전지보다 균일 한 분포를 생성 할 때마다 15 분을 돌립니다. 필요한 경우, 추가 하중을 수행 할 수 있습니다. </li></ol> 4. 장기 관류 설정 정적 문화의 5-6 시간 후, 첨부 HUVECs 완전히 확산하기 시작합니다. 10 μL / 시간의 지속적인 흐름으로 원격 제어 주사기 펌프 시스템을 사용하여 관류를 설정합니다. 관류는 F를 조정할 수 있습니다또는 더 높은 유량,는 2 주 4 일 사이 기간 동안 지속될 수있다. 5. 세포 염색 및 현미경 특성 <ol><li> 세포가 장치 안에 합류에 도달하면, 첫째, 철저하게 매체를 제거 1X PBS로 장치를 세척한다. 다음 희석제 (4 μL-1 ML)로 희석 붉은 염료로 장치를로드합니다. 셀 로딩 절차와 비슷 염료를 넣습니다. 상온에서 5 분 동안 어둠 속에서 장치를 배양 한 후 염색을 중지 배지로 장치를 세척한다. 염료의 긴 배양은 세포 독성과 disadhesion가 발생할 수 있습니다. </li><li> 1X PBS (ML 당 2 방울)에 희석 파란색 염료로 장치를로드합니다. 상온에서 5 분 동안 어둠 속에서 부화 후 철저하게 1X PBS로 장치를 세척한다. </li><li> 거꾸로 광학 현미경으로 세포 염색을 검사합니다. 염색이 좋으면 (1X PBS로 희석 3.5 % 파라 포름 알데히드) 고정 매체로 마이크로를로드 한 후 잠수함중간과 완전히 알루미늄 호일로 커버를 고정에있는 장치. 4의 온도에 냉장고에 장치를 보관 ° C 건조하고 photobleaching에에서 장치를 방지 할 수 있습니다. 고정 장치는 이제 공 촛점 현미경 스캐닝 레이저로 할 수있는 공 촛점 이미징을위한 준비가되어 있습니다. </li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

<html> 1. 마스터 금형 제작 혈관의 형태 학적위한 설계 원칙 중 하나는 네트워크를 통해 혈관 직경의 분포는 최소의 에너지 고려에 의해 지배되는 진술 머레이의 법칙 16로 알려져 있습니다. 또한 분기점에서 부모 혈관의 직경 큐브 딸 혈관의 직경 큐브의 합에 해당된다는 진술 ( <img alt="식 1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/50771eq1.jpg" src="/files/ftp_upload/50771/50771e...

미세 혈관은 혈액 순환 시스템의 일부로서, 혈액과 조직 사이의 상호 작용을 중재 신진 대사 활동을 지원, 조직의 미세 환경을 정의하고, 많은 건강과 병적 상태에 중요한 역할을한다. 체외에서 기능 미세 혈관의 재현부는 복잡한 혈관 현상의 연구 플랫폼을 제공 할 수 있습니다. 그러나, 내피 세포 마이 그 레이션 분석, 내피 관 형성 분석과 쥐 및 쥐 대동맥 링 분석 등의 생체 미세 혈관 분석, 종래의 입체 (3D) 형상과 연속 흐름 제어에 대해 생체 내 미세 혈관의를 다시 할 수 없습니다 1-8. 동물 모델과 같은 각막 신생 혈관 분석, 병아리 융모 막 혈관 분석 및 마트 리젤 플러그 분석 등의 생체 분석,에서를 사용하여 미세 혈관의 연구는 더 많은 시간이 소요되는 비용 높은 관찰과 quantifications에 대해 도전하고 있습니다윤리적 문제 1, 9-13 올립니다. 하지 않았을 등 쉽게 엄격하게 제어 생물학적 조건과 역동적 인 유체 환경과 같은 연구 14, 동물 생체에 관련된 높은 실험 비용과 복잡성을 축소 및 단축하면서 micromanufacturing 및 미세 유체 칩 기술의 발전은 생명 과학에 대한 통찰력의 다양한 활성화 기존의 거시적 기술로 할 수 있었다. 여기, 우리는 endothelialized를 구성하는 방법을 제시 마이크로 - 온 - 칩 생체 내 미세 혈관에서의 3D 형상을 모방하고 포토 리소그래피 리플 로우 레지스트 기술, 소프트 리소그래피, 그리고 미세 유체의 조합을 사용하여 제어 지속적인 혈류의 흐름에서 실행됩니다.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

생체 내 동물 연구는 더 많은 시간이 소요되는, 비싼, 그리고 관찰과 정량화가 매우 어려운됩니다 때문에 노력은 미세 혈관의 연구를위한 생체 분석의 개발에 초점을 맞추고있다. 세 가지 차원 (3D) 형상에 대해 생체 내 미세 혈관의 대표 및 지속적인 유체 흐름을 제공 할 때 그러나, 생체 미세 혈관 분석에서 기존의 제한이 있습니다. 포토 리소그래피 리플 로우 레지스트 기술, 소프트 리소그래피, 그리고 미세 유체의 조합을 사용하여, 우리는 멀티 깊이 원형 단면 endothelialized 개발 한 제어 연속 관류에 따라 생체 내 미세 혈관에서의 3D 형상을 모방하고 실행 마이크로 - 온 - 칩 흐름. 긍정적 인 리플 로우 레지스트는 반원형 단면 미세 네트워크와 마스터 몰드를 제조하는 데 사용되었다. REPL 개의 폴리 디메틸 실록산 (PDMS) 마이크로의 정렬 및 본딩마스터 금형 icated, 원통형 마이크​​로 네트워크를 만들었습니다. 마이크로의 직경은 잘 제어 할 수 있습니다. 또한, 칩 내부에 씨앗을 품고 기본 인간 제대 정맥 내피 세포 (HUVECs)는 세포 4 일 이주 사이의 기간 동안 지속적인 제어 관류에 따라 마이크로의 내부 표면을 지어 것으로 나타났다.

멀티 깊이 마이크로 네트워크를 제조하는 우리의 접근 방식은 미세 크로스 섹션에게 15 반올림하는 생체 미세 혈관에서의 복잡한 3D 형상을 모방. 또한, 부모 분기 채널의 직경과 딸 채널은 대략 전체 채널 저항이 낮고 유속이 네트워크 16-18 전역에 균일 있도록 필요한 수준에서의 유체 흐름을 유지하기 위해 머레이의 법칙을 준수하십시오. 반원형 포토 레지스트 마스...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

마이크로 - 온 - 칩 플랫폼은 포토 리소그래피 리플 로우 레지스트 기술, 소프트 리소그래피, 그리고 미세 유체의 조합에 의해 개발되었다. endothelialized 마이크로 플랫폼은 생체 내 미세 혈관에서의 3 차원 (3D) 구조를 모방 제어 지속적인 혈류의 흐름에서 실행하고, 고품질의 실시간 영상을 허용하고 미세 혈관 연구에 적용 할 수 있습니다.

멀티 깊이 원형 단면 Endothelialized 마이크로 채널 - 온 - 칩의 개발을위한 절차

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

procedure-for-development-multi-depth-circular-cross-sectional

Research

JoVE Journal

Bioengineering

13.8K 조회수.  West Virginia University.  마이크로 - 온 - 칩 플랫폼은 포토 리소그래피 리플 로우 레지스트 기술, 소프트 리소그래피, 그리고 미세 유체의 조합에 의해 개발되었다. endothelialized 마이크로 플랫폼은 생체 내 미세 혈관에서의 3 차원 (3D) 구조를 모방 제어 지속적인 혈류의 흐름에서 실행하고, 고품질의 실시간 영상을 허용하고 미세 혈관 연구에 적용 할 수 있습니다. 

비디오: Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

룽 공학을위한 절차

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

연구

생체공학

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Hematologic 질환의 Microvascular 상호 작용 유학을위한 Endothelialized Microfluidics

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

퇴행성 관상 재관류에 의한 돼지 심장의 Decellularization 절차

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

바이오 필름 및 서식지의 이질성의 공동 개발을 특성화하기위한 방법

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

고체 지질 나노 입자 (SLNs) 세포 내 타겟팅을위한 응용 프로그램

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

비행 시간 형 질량 분석과 2D 가스 크로마토 그래피를 사용하여 조류의 열수 액화에서 수성 분수의 질적 특성

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

인간의 장 점막의 다세포 3 차원의 Organotypic 모델의 개발은 미세 중력 하에서 성장

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

마이크로 웰 어레이 플랫폼 내 미생물 공동체 개발의 구성 및 추적

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

목재 접착제의 응집력 개발을 위한 표준 시험 방법 ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

현탁액에서 금 나노 입자의 크기 조정을위한 비대칭 유동 필드 흐름 분획

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...