이 작품은 프랑스 문화원 퀴리, 파리,에 의해 설립되었다. 기계 들것 데미안 CUVELIER (문화원 퀴리)에 의해 디자인되고 GREM에서 제조 된 (MECANIQUE-grem.com). PDMS의 패터닝 아마르 Azioune (보르도 II 대학)에 의해 개발되었다.

1. PDMS의 보호 <ol><li> PDMS의 조각을 잘라 약은 전 장에서 X 20mm 35mm (예를 들어, GelPak, 재료의 표에 나와있는대로)가. </li><li> 플라스틱의 상단과 하단에 보호 층을 제거 (필요한 경우)와 페트리 접시를 (처리 문화를 셀 수 없습니다) 플라스틱 PDMS를 배치 핀셋을 사용합니다. </li><li> / 분 (30) 진동에서 회 전자에 5 분 동안 70 % 에탄올로 PDMS를 씻으십시오. </li><li> 거기에 공기를 유입하여 표면을 건조시킵니다. </li><li> 약 5 ㎝의 UV 전구에서 거리에서 5 분 깊은 UV (λ = 180 nm의)와 조명 (램프 참고 용 재료 장을보고, 매개 변수는 다른 램프에 따라 다를 것이다). </li><li> 각각의 조각을 PDMS를위한 EDC / NHS 용액 200 μL (용액의 반응성이 시간 만에 붕괴 때문에 그냥 사용하기 전에 준비해야합니다)를 준비합니다. pH가 6.0에서 0.05 M MES + 0.5 M NaCl을 버퍼 1 ㎖를 들어, 설포-NHS의 11.5 밀리그램과 EDC의 19.2 밀리그램을 추가합니다. </li><li> 이동조명되지 않은 페트리 접시의 뚜껑에 페트리 접시에서 PDMS의 장. 이는 PDMS의 주변은 매우 소수성 보장하고 다음 단계를 용이하게한다. </li><li> EDC / NHS 솔루션을 추가하고 실온에서 15 분 동안 품어. </li><li> 물과 EDC / NHS를 씻어. </li><li> PLL-g-PEG 용액 (HEPES 10 mM의 pH를 8.6에서 0.5 ㎎ / ㎖)를 첨가하고 3 시간에 하룻밤 실온에서 품어. </li><li> 물을 PLL-g-PEG를 씻어. (PLL-g-PEG 기능화) 비활성화 된 PDMS는 4 ℃에서 며칠 동안 저장 될 수있다 </li></ol> 2. PDMS의 패터닝 <ol><li> PDMS 시트를 가지고 패터닝 microfeatures 베어링 합성 석영 포토 마스크에 배치 (그림 1 참조). PLL-g-PEG는 포토 마스크의 크롬 측에 직면 베어링 PDMS면을 넣습니다. </li><li> 약 5cm의 UV 전구로부터의 거리에 포토 마스크를 통해 12 분 동안 밝히는.</li><li> 마스크 + PDMS에 물을 천천히 마스크 오프 PDMS 껍질. </li><li> 실온에서 1 시간 동안의 pH 8.6에서의 HEPES 버퍼에서 50 ㎎ / ㎖에서 피브로넥틴 용액으로 배양한다. 그것은 다른 사람에게 ECM 단백질을 사용하는 것이 가능하지만, 이러한 프로토콜이 테스트되지 않았다. </li><li> PBS로 씻어. </li></ol> 3. 장치 설치 <ol><li> 스트레칭 장치 (문제 해결을위한 토론 참조)에 이전에 비활성화 된 PDMS를 탑재합니다. <ol><li> 들것의 고정 부분에 PDMS의 한 쪽을 연결합니다. </li><li> PDMS 시트의 너무 많은 클램핑하지 않고 들것의 모바일 부분에 다른 쪽을 고정합니다. </li></ol></li><li> 두꺼운 PDMS 찌르기에 PDMS의 사각형 (22mm X 19mm)을 잘라 (그림 2 참조) 세포와 미디어를 유지하는 풀 (또는 프레임)을 부여하기 위해 내부에 또 다른 사각형을 잘라. </li><li> 이 풀을 PDMS에서 실리콘 그리스를 추가하고 의대를 만들 PDMS 시트의 상단에 배치IUM 풀을 유지. 그리스 스트레칭 동안 PDMS 시트에 풀의 활공을 허용합니다. </li></ol> 4. 셀 패터닝 <ol><li> 센과 50 % 자랄 문화 플라스크에서 세포를 분리. </li><li> 세포를 카운트하고 200,000 세포 / ml의 농도로 재현 탁 그들을. </li><li> 풀의 세포 현탁액 1 ㎖ (자세한 내용에 대한 설명을 참조)를 추가합니다. </li><li> 세포 (RPE-1 세포를 10 분 및 HeLa 세포를 20 분 소요되며, 세포 유형에 따라) 10-30 분 동안 패턴에 바인딩하자. </li><li> 부드럽게 평형 배지 (배양기에서 매체를 평형)와 부동 세포를 세척하십시오. </li><li> 세포가 패턴에 몇 시간 동안 전파하자 (RPE-1은 최소 2 시간이 필요합니다, 헬라 세포는 3 시간이 필요합니다). </li><li> PDMS 나누기 경우 증발 매체의 누설을 방지하기 위해 풀의 상단에 커버 슬립을 추가합니다. </li><li> 거꾸로 현미경에 장치를 넣어 영상을 시작합니다. 기름 침수 산부인과의 사용을 피하십시오이 때문에 문제를 중심으로 작동하지 않습니다으로 jectives. </li></ol> 5. 스트레칭 <ol><li> X-,-y 및 z 축 (주로 X)의 무대 위치를 보정하는 동안 마이크로 미터 나사를 돌립니다. 스테이지 위치는 PDMS의 확폭 및 포커스의 손실을 상쇄하도록 보정 될 필요가있다. (토론 단락 &quot;스트레치하는 동안&quot;참조). </li></ol>

<ol>
	<li>Vogel, V., Sheetz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+force+and+geometry+sensing+regulate+cell+functions.">Local force and geometry sensing regulate cell functions.</a> Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).</li><li>Terenna, C. R., 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=Physical+mechanisms+redirecting+cell+polarity+and+cell+shape+in+fission+yeast.">Physical mechanisms redirecting cell polarity and cell shape in fission yeast.</a> Curr. Biol. 18 (22), 1748-1753 (2008).</li><li>Guillot, C., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+epithelial+tissue+homeostasis+and+morphogenesis.">Mechanics of epithelial tissue homeostasis and morphogenesis.</a> Science. 340 (6137), 1185-1189 (2013).</li><li>Bosveld, F., 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=Mechanical+control+of+morphogenesis+by+Fat/Dachsous/Four-jointed+planar+cell+polarity+pathway.">Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.</a> Science. 336 (6082), 724-727 (2012).</li><li>Farhadifar, R., Roper, J. C., Aigouy, B., Eaton, S., Julicher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+cell+mechanics,+cell-cell+interactions,+and+proliferation+on+epithelial+packing.">The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing.</a> Curr. Biol. 17, 2095-2104 (2007).</li><li>Rauzi, M., Verant, P., Lecuit, T., Lenne, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+anisotropy+of+cortical+forces+orienting+Drosophila+tissue+morphogenesis.">Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.</a> Nat. Cell Biol. 10, 1401-1410 (2008).</li><li>Mitrossilis, D., 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=Real-time+single-cell+response+to+stiffness.">Real-time single-cell response to stiffness.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (38), 16518-16523 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchison, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 12, 1783-1790 (2007).</li><li>Fink, J., 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=External+forces+control+mitotic+spindle+positioning.">External forces control mitotic spindle positioning.</a> Nat. Cell. Biol. 13 (7), 771-778 (2011).</li><li>Azioune, A., Storch, M., Bornens, M., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+process+for+single+cell+micro-patterning.">Simple and rapid process for single cell micro-patterning.</a> Lab Chip. 9 (11), 1640-1642 (2009).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+A+direct+printing+protocol+using+deep+Uvs.">Protein micropatterns: A direct printing protocol using deep Uvs.</a> Methods Cell Biol. 97, 133-146 (2010).</li><li>Azioune, A., 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=Robust+method+for+high-throughput+surface+patterning+of+deformable+substrates.">Robust method for high-throughput surface patterning of deformable substrates.</a> Langmuir. 27 (12), 7349-7352 (2011).</li><li>Carpi, N., Piel, M., Azioun, A., Cuvelier, D., Fink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+on+silicon+elastomer+(PDMS)+with+deep+UVs.">Micropatterning on silicon elastomer (PDMS) with deep UVs.</a> Protoc. Exch. , (2011).</li><li>Sinha, B., 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=Cells+respond+to+mechanical+stress+by+rapid+disassembly+of+caveolae.">Cells respond to mechanical stress by rapid disassembly of caveolae.</a> Cell. 144 (3), 402-413 (2011).</li></ol>

저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

이 기술은 여러 번 사용하고 철저하게 테스트되어 있지만, 실패한 실험으로 이어질 수있는 몇 가지 중요한 단계가 있습니다. PDMS 소개 : 이 작품의 경우, GelPak, 시판 얇은 PDMS 시트를 사용 하였다. 또는 PDMS 시트는 PDMS 믹스에서 직접 캐스팅 할 수 있습니다. 더 재현하고, 사용자 정의 만든 PDMS에 비해 휴식 덜하기 때문에 우리는 GelPak를 사용하는 것이 좋습니?...

고등 생물의 조직을 구성하는 세포는 기계적인 긴장과 외부 환경이나 주변의 세포 1, 2에서 하나 오는 스트레칭 힘이 적용됩니다. 세포에 적응하고 조직의 무결성을 유지하기 위해 이들의 힘에 저항한다. 이러한 힘은 개발 3,4 동안 조직의 형태 형성에 중요하다. 배양 된 세포에 기계적인 힘을 적용하는 것은 조직에 일어날 일에 모방 할 수있는 방법이지만, 세포의 모양과 세포의 변형 5,6의 양적 및 독립 제어와. 이를 위해 여러 가지 기술들이 사용될 수있다. 하나는 (전체 세포 또는 그 일부)를, 예를 들어 AFM 또는 파생 7,8 또는 세포가 성장 기판을 스트레칭을 사용하여 셀을 누를 수 있습니다. 이 문서에서 설명하는 방법은 세포로 도금 평면 기판을 스트레칭하는 방법을 보여줍니다. 이 기술은 원래 평가하기 위해 개발 된 힘의 역할은 m에 작용itotic 포유 동물 세포 9. 유사 분열 세포가 수축 섬유를 통해 기판에 연결 상태를 유지하고 막을 스트레칭 차례로 분열 스핀들의 회전을 유발하는 섬유에 힘을 발휘. 접착제 미세 패턴을 결합하고 스트레칭의 관심은 힘과 개별 셀의 모양을 독립적으로 제어를 달성하는 것입니다. 그것은 축 스트레칭이인가 된 상태에서, 완벽하게 등방성 둥근 모양으로 난형 세포를 스트레칭 예를 들어 수 있습니다. 세포는 미세 패턴에 엮어되지 않으면 축성 대부분의 세포는 스트레치 축과 정렬 된 장축을 갖는 세포 연신율 결과 스트레칭. 이것은 장축 정렬과 셀에 적용된 스트레칭 효과의 영향을 분리하기 위하여 다음 어렵다. 이 디바이스는 시간이 오래 경과 형광 현미경을 포함한 모든 살아있는 세포 이미징에 적합하고, 약은 실험 기간 동안 추가 할 수 있습니다. 깊은 UV를의 미세 방법 10Azioune에 상세하게 설명했다 등. PDMS 11 패터닝는 설명했다 Azioune 등. 12 프로토콜을 스트레칭 선물 카르 피 등. (13)의 비디오 버전입니다

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelPak</td>
			<td style="width:71px;">GelPak</td>
			<td style="width:119px;">PF-60-X4</td>
			<td style="width:167px;">Different thickness/stickiness are available. One alternative could be to cast your PDMS yourself.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Silicon grease</td>
			<td style="width:71px;">GE Bayer Silicones</td>
			<td style="width:119px;">Baysilone-Paste</td>
			<td>This one is biocompatible</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stretching device</td>
			<td style="width:71px;">GREM m&#233;canique</td>
			<td style="width:119px;">Stretcher 2011</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">EDC (N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">3450</td>
			<td>Stable 6 months at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NHS (N-Hydroxysulfosuccinimide sodium salt)</td>
			<td style="width:71px;">Sigma</td>
			<td align="right" style="width:119px;">56485</td>
			<td>Protect from humidity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pll-g-peg (PLL(20)-g[3.5]-PEG(2) 20 mg)</td>
			<td style="width:71px;">SurfaceSolutions (Zurich)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Synthetic Quartz photomask</td>
			<td style="width:71px;">Toppan</td>
			<td style="width:119px;"></td>
			<td>Take standard binary photomask in Quartz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretching micropatterned cells on a pdms membrane

기계의 힘은 세포에 작용 및 / 또는 조직은 다수의 프로세스에 중요한 역할을한다. 우리는 폴리 디메틸 실록산 영상과 호환 (PDMS) 막에 도금 세포 스트레칭 장치를 개발했습니다. 이 기술은 재현하고 다양한입니다. PDMS 막을 구체적인 형상으로 세포 또는 조직을 한정하기 위해 마이크로 패턴 될 수있다. 첫 번째 단계는 딥 UV 기술로 PDMS 막 상에 미세 패턴을 인쇄하는 것이다. PDMS 막이어서 기계적 들것에 장착된다. 챔버는 스트레치 동안 활공 수 있도록 생체 적합성 그리스와 멤브레인의 상단에 바인딩됩니다. 세포를 시딩하고 미세 패턴에 몇 시간 동안 확산 할 수있다. 샘플은 마이크로 미터 나사를 이용하여 여러 번 연신 및 연신 될 수있다. 그것은 (30 % 정도)의 전체 범위에 스트레치를 적용하는 분 미만 소요됩니다. 여기에 제시된 기술에 필요한 동력 장치를 포함하지 않습니다신속 및 / 또는 컴퓨터 제어 연신 반복 스트레치 사이클 pplying 있지만이 구현 될 수있다. 세포 스트레칭이나 조직 세포의 힘, 기계적인 스트레스 나 조직의 형태 형성에 대한 세포 반응에 관련된 질문에 관심이있을 수 있습니다. 이 비디오 프리젠 테이션은 단순 해 보이는 이런 유형의 실험을 수행 할 때 발생할 수있는 일반적인 문제를 방지하는 방법을 보여줍니다.

<html> 이 비디오 프로토콜에서 제시된 방법은 포유 동물 세포의 유사 분열 후퇴 섬유에의 힘의인가를 허용했다. 실제로, 세포 분열 동안, 유사 분열 단계에서, 포유류 세포는 구형 형상을 가지고, 기판에 부착 된 막에 의해 둘러싸인 얇은 액틴 케이블 뒤에 남겨 후퇴. 이 케이블 (수축 섬유) 부문에 가기 전에 셀 구조의 메모리입니다. PDMS 박막의 포토 마스크를 통해 깊은 UV와 미세 패턴 제작 (?...

Watch this Scientific Journal Video about Stretching Micropatterned Cells on a PDMS Membrane at JoVE.com

Stretching Micropatterned Cells on a PDMS Membrane

이 원고는 단방향 스트레칭을 사용하여 부착 세포 나 조직에 힘을 적용하거나 해제하는 방법을 제시한다.

PDMS 막에 마이크로 패턴 세포를 스트레칭

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

stretching-micropatterned-cells-on-a-pdms-membrane

Research

JoVE Journal

Bioengineering

15.2K 조회수.  Institut Curie.  이 원고는 단방향 스트레칭을 사용하여 부착 세포 나 조직에 힘을 적용하거나 해제하는 방법을 제시한다. 

비디오: Stretching Micropatterned Cells on a PDMS Membrane

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned 표면은 암세포와 Hyaluronic 산성 상호 작용을 연구하기 위해

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

연구

생체공학

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Micropatterned 세포를 사용하여 약물의 영향 향상된 시각화 및 정량 분석

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules. By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

상수 강제 축 방향 광학 족집게로 DNA의 짧은 시퀀스를 스트레칭

Single-molecule techniques for stretching DNA of contour lengths  less than a kilobase are fraught with experimental difficulties. However, many ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

세포와 조직 Mechanobiology의 응용 프로그램에 대한 새로운 스트레칭 플랫폼

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

발광 공명 에너지는 포유 동물 세포에 표현 막 단백질의 구조 변화를 연구에 전송

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

폴리 (ε 카프로 락톤) 전기 방사 원사에 인간 중간 엽 줄기 세포의 첨부 파일을 최적화

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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. ...

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.

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 ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

미세 패턴 매크로 스케일 3D 셀룰러 아키텍처 모듈 형 하이드로 겔 시트의 건설

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

폴리 디메틸 실록산 (PDMS) 공동 축류 초점 장치를 사용하여 이중 에멀젼 생성

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...