We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

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The authors have nothing to disclose.

여기서 우리는 만성 혈관 질환 경로 1,23,24의 확장 된 실험 시간은 전형적인 수 있도록 이전에 개발 된 vMTF 기술을 기반으로 구축하는 프로토콜을 제시한다. 이를 위해, 우리는 이전 MTF 수축력 실험에 사용하기위한 개선 된 혈관 조직 생존력과 엔지니어링 동맥 박편을 수득 미세 증착 기술을 이용하여 PDMS 기판 (11)의 장기간의 작용을 제공하는 것으로 나타났다 genipin를 미세 패턴. 매케인 등. 관련 MTF 모델 25 몇 주 동안 설계 심장 조직의 확장 된 문화에 대한 대안 micromolded 젤라틴 하이드로 겔 기판을 개발했다. 이 프로토콜은 성공적으로 2 주에 걸쳐 동맥 라멜라 구조 (그림 2)과 기능 (그림 3) 모방 vMTFs을 얻었다. 제시된 프로토콜을 성공적으로 완료하여 원하는 해상도를 얻을 수 있지만질병 관련 시간 코스에 대한 확장 된 문화 시간의 ULT (예를 들어, 최대 14 일 26 지속 뇌 혈관 경련의 병리학 적 효과), 일반적으로 다음과 같은 몇 가지 함정이 발생한다. 장치에 지사의 일부 또는 전체 차단 될 수 있습니다 해로운 손상에 PDMS 미세 유체 장치 결과의 반복 사용. 따라서, 새로운 디바이스는 각각의 실험을 위해 사용되어야한다. 또 다른 문제는, 일반적인하지는 않지만, 조직의 예상치와 일치 박리입니다. 이 사건은 발생 자연에서 무작위 드문 것 같았다. 관찰에 의해, 조직은 얇은 막으로 분리하기 전에 arch- 또는 루멘 같은 구조를 형성한다. 우리는 또한 미세 접촉 프린팅 기술을 사용하여 제작 된 조직에서이 동작을 관찰했다. 따라서, 우리는이 문제가 오버 파종 또는 표현형 비정상적인 행동 결과 배양 된 혈관 평활근 세포의 스위치가 아니라 여기에 제시된 제조 방법의 직접적인 결과 중 하나의 결과로 생각합니다. 현재 마이크로 유체IC 설계 선형 흐름 패턴을 필요로하며, 같은 하나의 주 배향 방향과 조직에 한정된다. 이러한 심실 심근 구조 (27)의 &quot;벽돌 벽&quot;패턴으로 더 복잡한 패턴을 필요로하는 조직에 응용 프로그램은보다 정교한 미세 유체 설계가 필요합니다. 우리는 다른 세포 유형으로 genipin 수정 기판을 접종하지 않았습니다. 그러나 Genchi 외. genipin 수정 PDMS 11 등방성 골격 근육의 생존 능력을 확장했다. 따라서, 우리는이 프로토콜의 최소한의 수정 버전이 기관 - 온 - 어 - 칩 기술의 미래 발전에 다른 기관 시스템에 광범위하게 적용이 있는지 확신. 이전의 방법 (8)에 기초 vMTFs위한 응력 계산 방법은 박막의 조직에서의 상대적으로 수축 및 대응 절단 길이에 의해 제한된다. 너무 오래 잘라 경우, 영화 자체에 컬합니다. 너무 짧게 잘라 경우, 필름은되지 않습니다차. 어느 경우는 제한을 모델에 따라 수축 특성의 적절한 분석을 방지 할 수 있습니다. 적절한 절단 길이는 반복 실험을 통해 정제해야한다. 아가 왈 등으로하는 레이저 조각 시스템의 구현은,., 반복성과 MTF (28)의 절단 품질을 개선 할 수있다. 더 단단히 통제되는 자 급식 실험 시스템에서 네이티브 조직의 구조와 기능을 요점을 되풀이하는 능력은 생물 공학 분야에서의 키 추적이다. 이 추구는 기본적인 생리 학적 및 기관계 29,30의 병리학 동작의 이해를 증진, 여러 시험 관내 조직 모방 다기능 아직 단순화 된 모델 기관 온어 칩의 개발을 유도 하였다. PDMS 기판 상 genipin 증착을 사용하여, 2 주간에 걸쳐 연장 된 세포 생존 및 혈관 평활근 기능의 유지를 증명 하였다. 이것은 이전 제조 TECHN 비해 상당한 개선이다배양 19 7 일, 및보다 강력한 동맥 온칩 방법 개발에 도움이있다 - 조직 및 4 후의 세포 죽음의 박리를 초래할 수 iques. 때문에 대부분의 혈관 질환의 만성적 인 특성으로,이 사전은 특정 혈관 병변에 관련된 수축 메커니즘으로 미래 연구의 다양한 프레임 워크를 제공합니다.

이러한 뇌 혈관 경련, 2, 3, 고혈압, 동맥 경화증 및 4와 같은 혈관 질환은 서서히 발전 전형적으로 사실상 만성적이며, 혈관 평활근 세포 (혈관 평활근 세포)로 역기능 힘 발생을 수반한다. 우리의 생체 내 모델보다 실험 조건의 미세한 제어 체외 방법에서 사용이 느리게 진행 혈관 장애를 연구하는 것을 목표로하고 있습니다. 시험관의 기능 수축력을 측정하는 우리는 이전에 개발 한 혈관 근육 박막 (vMTFs)은 심장 혈관 조직 (5)를 설계하지만,이 방법은 상대적으로 단기 연구에 제한되어있다. 여기서는 장기 측정 이전 vMTF 기술을 확장 기판 개질 기술을 제시한다. 혈관 내피 세포는 전체 기능에도 중요하지만, 설계 라멜라 동맥 혈관의 변화를 평가하는데 유용한 모델 시스템을 제공하는질병의 진행 동안 수축. 기능적인 혈관 질환 조직 모델 구조 및 동맥 라멜라의 기능을 모두 엔지니어, 용기의 기본 수축 부, 고 충실도로 효과적으로 요약되어야한다. 동맥 라멜라 6 엘라스틴의 시트로 구분 수축 혈관 평활근 세포의 동심, 원주 방향으로 정렬 시트입니다. 폴리 디메틸 실록산 (PDMS) 기판 상에 세포 외 기질 (ECM) 단백질의 마이크로 콘택트 프린팅 이전 심혈관 조직 5,7-10 정렬 모방 조직 조직 유도 신호를 제공하기 위해 사용되어왔다. 그러나 조직은 만성 연구에서 자신의 적용을 제한, 문화 3-4 일 후에 무결성을 잃을 수 미세 접촉 인쇄를 사용하여 패턴. 이 프로토콜은 새로운 미세 증착 기술로 기존 미세 접촉 인쇄 기술로 대체함으로써이 문제에 대한 해결책을 제공한다. genipin와 f와 Genchi 등. 수정 된 PDMS 기판운드, 문화 (11)에 1 개월 심근 세포의 생존을 연장. 여기서는 PDMS에 패터닝 된 혈관 평활근 세포의 배양을 확장하는 유사한 접근법을 사용한다. Genipin, 치자 나무 열매의 천연 가수 유도체는 유사한 가교제 및 조직 복구 12,13 및 ECM 수식 (14)의 분야에서 생체 재료로서의 사용이 증가에 비해 인해 비교적 낮은 독성 기판 변형위한 바람직한 후보 15. 이 프로토콜에서, 피브로넥틴은 이전 미세 접촉 인쇄 방법에서와 같이, 셀 안내 큐로서 이용되고; 그러나 genipin 피브로넥틴은 패터닝 이전에 PDMS 기판 상에 증착된다. 세포가 패턴 매트릭스 저하로 따라서, 첨부 된 혈관 평활근 세포에서 새로 합성 된 ECM은 genipin 코팅 된 PDMS 기판에 바인딩 할 수 있습니다. 이 프로토콜은 두 단계 genipin과 ECM 증착을위한 미세 유체 전달 장치를 이용한다. 미세 유체 장치 모방 microco의 디자인이전의 연구 (16)에서 설계 동맥 박편에 사용 ntact 인쇄 패턴. 따라서,이 프로토콜이 성공적으로 구조 및 생체 동맥 박편의 수축 기능에 고도로 정렬 요점을 되풀이 동맥 라멜라 모방을 수득 할 것으로 예상. 우리는 또한 genipin 시험관 혈관 질환 모델에서 장기에 적합한 기판 수정 화합물 확인하기 위해 조직의 수축력을 평가.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Coverslip staining rack</td>
			<td>Electron Microscopy Sciences</td>
			<td><a href="http://www.emsdiasum.com/">www.emsdiasum.com/</a></td>
			<td>72239-04</td>
			<td>Alternative coverslip rack may be used</td>
		</tr>
		<tr>
			<td>Microscope cover glass - 25 mm</td>
			<td>Fisher Scientific, Inc.</td>
			<td><a href="http://www.fishersci.com/">www.fishersci.com</a></td>
			<td>12-545-102</td>
			<td>Alternative brand and size may be used; Microscope slides may also be substituted as substrate base</td>
		</tr>
		<tr>
			<td>Poly(N-iso-propylacrylamide) (PIPAAm)</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td>#21458</td>
			<td>Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation</td>
		</tr>
		<tr>
			<td>1-butanol</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td align="right">360465</td>
			<td>Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 &#176;C, avoid heating; alternative product may be used</td>
		</tr>
		<tr>
			<td>Spincoater</td>
			<td>Specialty Coating Systems, Inc.</td>
			<td><a href="http://www.scscoatings.com/">www.scscoatings.com</a></td>
			<td></td>
			<td>SCS G3P8 Model; Alternative brand and/or model may be used</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives (Dow Corning)</td>
			<td><a href="http://www.ellsworth.com/">www.ellsworth.com</a></td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Alternative distributor may be used</td>
		</tr>
		<tr>
			<td>Fluorescent microbeads</td>
			<td>Polysciences, Inc.</td>
			<td><a href="http://www.polysciences.com/">www.polysciences.com/</a></td>
			<td align="right">17151</td>
			<td>Alternative brand and/or larger size may be used</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Wafer World, Inc.</td>
			<td><a href="http://www.waferworld.com/">www.waferworld.com</a></td>
			<td align="right">2398</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Photoresist&#160;</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 3025 allows 20-25-&#181;m feature height</td>
		</tr>
		<tr>
			<td>Contact mask aligner</td>
			<td>Suss MicroTec</td>
			<td><a href="http://www.suss.com/">www.suss.com</a></td>
			<td></td>
			<td>MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure</td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem Corp.</td>
			<td><a href="http://www.microchem.com/">www.microchem.com</a></td>
			<td></td>
			<td>SU-8 Developer; Hazard: flammable</td>
		</tr>
		<tr>
			<td>Tridecafluro-trichlorosilane</td>
			<td>UCT Specialties, Inc.</td>
			<td><a href="http://www.unitedchem.com/">www.unitedchem.com</a></td>
			<td>T2492</td>
			<td>Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. )</td>
		</tr>
		<tr>
			<td>Surgical biopsy punch</td>
			<td>Integra LifeSciences Corp.</td>
			<td><a href="http://www.miltex.com/">www.miltex.com</a></td>
			<td>33-31AA-P/25</td>
			<td>Alternative brand and/or size may be used</td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Cayman Chemical</td>
			<td><a href="http://www.caymanchem.com/">www.caymanchem.com</a></td>
			<td align="right">10010622</td>
			<td>Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>Mediatech, Inc.</td>
			<td><a href="http://www.cellgro.com/">www.cellgro.com</a></td>
			<td>21-031-CV</td>
			<td>Alternative brand may be used</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning, Inc.</td>
			<td><a href="http://www.corning.com/">www.corning.com</a></td>
			<td align="right">356008</td>
			<td>Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Inc.</td>
			<td><a href="http://www.lifetechnologies.com/">www.lifetechnologies.com</a></td>
			<td>15140-122</td>
			<td>Alternative brand and/or size may be used, as long as concentration is the same</td>
		</tr>
		<tr>
			<td>Umbillical artery smooth muscle cells</td>
			<td>Lonza</td>
			<td><a href="http://www.lonza.com/">www.lonza.com</a></td>
			<td>CC-2579</td>
			<td>Alternative cell types may be used for alternative applications. Media should be modified accordingly</td>
		</tr>
		<tr>
			<td>Tyrode&#39;s solution components</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>various</td>
			<td>Alternative brand may be used for mixing solution</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Zeiss</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
			<td>4350020000000000</td>
			<td>SteREOLumar V12; Alternative brand/type of stereomicroscope may be used</td>
		</tr>
		<tr>
			<td>Temperature-controlled platform</td>
			<td>Warner Instruments</td>
			<td><a href="http://www.warneronline.com/">www.warneronline.com</a></td>
			<td>641659; 640352; 641922</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>E7764-50UG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
		<tr>
			<td>HA-1077</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a></td>
			<td>H139-10MG</td>
			<td>Alternative amount may be purchased, as long as treatment concentration is maintained</td>
		</tr>
	</tbody>
</table>

microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

이 작품의 주요 목표는 소수성 PDMS 기판에 미세 패턴 혈관 평활근 세포의 생존을 연장했다. 이 패턴 genipin 및 PDMS에 피브로넥틴 (그림 1) 입금 미세 유체 전달 시스템을 통합하여 수행되었다. 미세 유체 전달을 사용하여 ECM 단백질의 침착 genipin 및 피브로넥틴 (그림 1D)의 선 사이의 벌거 벗은 PDMS와 채널 패턴의 높은 충실도 전송을 얻었다. 이전의 마이크로 콘택트 인쇄 법 5,10,16 유사한 동맥의 라멜라 구조를 생체 모방 부착 세포 (도 1E) 형태 합류 단층 (도 2). 이러한 조직은 그의 응력 vMTF 기술을 사용하여 측정 하였다 (도 3)에 응답하여, 수축성 구조체를 수득 하였다. 이주의 과정을 통해 조직의 생존 능력의 질적 평가는 genipin - 후 변형에 최소한의 악화를 보였다에드 기판 (그림 2A). 조직 합류 및 정렬 2 주 (그림 2B, 2C 및 2D) 이상 유지되었다. 두 가지 주요 스트레스 값은 모든 vMTF 계산 하였다 :. 1) 기초 톤, 2) 유도 수축 (그림 3C 및 3D)이 기저 톤 평형 비자극 혈관 평활근 세포에 의해 유지되는 스트레스입니다. 수축력이 유도 된 엔도 텔린 1 자극에 의​​해 유도 된 부가적인 응력이다. 기저 톤 유도 수축성 모두 (도 3E 및 3F)에 걸쳐 혈관 활성 조직을 보여주는 2 주 시간 걸쳐 일관된 동작을 나타내었다. 혈청 결핍 혈관 평활근 세포 증식을 19 않기 때문에 분석의 끝에 수축성 조직에 약간의 감소는, 조직을 구성하는 세포의 감소 된 수의 직접적인 결과이다. 배지에 혈청의 최소 기저 수준의 첨가는 향후 연구에서이 결과를 완화 할 수있다. <imgALT = &quot;그림 2&quot;SRC = &quot;/ 파일 / ftp_upload / 52971 / 52971fig2.jpg&quot;/&gt; 그림 2. 조직이 성공적으로 Genipin 수정 기판에 2 주 동안 모방 생체 동맥 강철 구조 유력한 남아 이주의 과정을 통해 희생 시점에서 조직의 (A) 대표 위상차 이미지 (스케일 바 : 200 μm의).. F-액틴 필라멘트, 파란색 : 핵 (세포의 존재를 확립하는 것으로), 스케일 바 혈청 기아 (녹색 후 (B) 1 일 고정 genipin 수정 기판에 제작 된 조직의 대표 면역 이미지, 4 일 및 10 일째 .. - 7) (D) 조직 정렬 F - 굴지 방향 순서 매개 변수 (OOP에 의해 측정) (22) (오차 막대 표준 오차, N = 3 : F - 굴지 범위 (오차 막대에 의해 측정 된 100 ㎛) (C) 비율 합류 : 표준 오차, N = 3-7). © 안압 PublishiNG. 재현 및 / 또는 허가를 수정했습니다. 판권 소유. 모든 권리 보유. (19)는 <a href="https://www.jove.com/files/ftp_upload/52971/52971fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52971/52971fig3.jpg" /> 그림 3. 조직 수축성은 2 주에 걸쳐 유지된다. (A) 대표 vMTF 절단 방식. (B) 컷 vMTF의 개략도. 32 ° C 이하로 냉각되면, PIPAAm 용해는 vMTF 해제. 현재 셀 층에서 스트레스는 구부리 수동 PDMS 층됩니다. 돌출 길이의 측정이 Grosberg의 방법에 따른 곡률 반경으로 변환 할 수 등. (8)의 곡률 반경은 조직의 평균 단면 응력을 계산하는 데 사용된다. (C) 순차 송신 LIG대표 수축력 분석의 HT 이미지 (스케일 바 : 1mm). 조직은 평형에 도달 완전한 휴식을 할 수 있도록 엔도 텔린-1, 다음 HA-1077 치료로 자극한다. 아래 :. 수축력 분석을위한 분석의 과정에서 이상적인 조직의 사이드 뷰의 도식 (D) 대표 스트레스 곡선. 두 가지 주요 스트레스 값이 계산됩니다. 기저 톤 평형 응력 상태와 이완 응력 상태 사이의 차이이다. 유도 수축은 엔도 텔린 -1 자극 상태로 평형 상태에서 응력의 변화이다 (E) 기저 톤 (오차 막대 : 표준 오차, N = 5-12) (F) 유도 수축 (오차 막대 :.. 표준 오차, N = 5-12). © 안압 출판. 재현 및 / 또는 허가를 수정했습니다. 모든 권리 보유. (19) <a href="https://www.jove.com/files/ftp_upload/52971/52971fig3large.jpg" target="_blank">의 더 큰 버전을 보려면 여기를 클릭하십시오</a>이 그림.

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

미세 패턴 혈관 근육 박막의 확장 문화 미세 유체 Genipin 증착 기술

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

microfluidic-genipin-deposition-technique-for-extended-culture

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.6K 조회수.  University of Minnesota. We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

비디오: Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

동적 마스크 프로젝션 석판술를 사용하여 신경 문화 시스템의 Micropatterned Hydrogels의 제작

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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생체공학

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biomarker 디스커버리를위한 Mesoporous 실리카 박막의 낮은 분자량의 단백질 농축

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

미세 유체 기술은 셀 변형능을 검사 할

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

티슈 엔지니어링 혈관 이식 및 이후의 이식을위한 수술 기법<em&gt; 생체</em&gt; 모니터링

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

에 Microengineered 인간의 장내 융모와 생활 마이크로 바이 옴의 공동 문화 창자 - 온 - 어 - 칩 미세 유체 장치

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

혈관 유래 미세 유체 네트워크의 이미지 유도, 레이저 기반의 제조

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

판막 다층 미세 유체 장치에 대한 여러 단계의 변수 높이 포토 리소그래피

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...