Name: easy and accurate mechano-profiling on micropost arrays
Uploaded: 2015-11-17T15:00:00
Description: Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

This work was supported by the Committee Technology and Innovation (CTI) Switzerland grant 14796-PFLS-LSCTI. MicroPost arrays were generously provided by MicroDuits GmbH.

Micropost 배열 1. 배양 세포 참고 : 모든 단계는 불임을 보장하기 위해 생물 안전 캐비닛에서 수행해야합니다. 여기에 지정된 볼륨은 T25 세포 배양 플라스크에 대한 것입니다. 세포 배양 배지 및 세포 레시피 파종 밀도 골암 세포주 HuO9 및 M132에 최적화되어있다. <ol><li> micropost 배열에 파종을위한 세포 배양을 준비합니다. <ol><li> 표준 광학 현미경에 배양 플라스크를 배치하여 ?...

<ol>
	<li>Sharma, R. I., Snedeker, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+biomechanical+gradients+for+directed+bone+marrow+stromal+cell+differentiation+toward+tendon+and+bone.">Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone.</a> Biomaterials. 31 (30), 7695-7704 (2010).</li><li>Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+biophysics+of+cancer+cells.">Biomechanics and biophysics of cancer cells.</a> Acta Biomater. 3 (4), 413-438 (2007).</li><li>Bartalena, G., 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=A+novel+method+for+assessing+adherent+single-cell+stiffness+in+tension:+design+and+testing+of+a+substrate-based+live+cell+functional+imaging+device.">A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device.</a> Biomed Microdevices. 13 (2), 291-301 (2011).</li><li>Tan, J. L., 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+lying+on+a+bed+of+microneedles:+An+approach+to+isolate+mechanical+force.">Cells lying on a bed of microneedles: An approach to isolate mechanical force.</a> PNAS. 100 (4), 1484-1489 (2003).</li><li>Akiyama, Y., Hoshino, T., Iwabuchi, K., Morishima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Room+Temperature+Operable+Autonomously+Moving+Bio-Microrobot+Powered+by+Insect+Dorsal+Vessel+Tissue.">Room Temperature Operable Autonomously Moving Bio-Microrobot Powered by Insect Dorsal Vessel Tissue.</a> PloS ONE. 7 (7), (2012).</li><li>Biais, N., Ladoux, B., Higashi, D., So, M., 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=Cooperative+retraction+of+bundled+type+IV+pili+enables+nanonewton+force+generation.">Cooperative retraction of bundled type IV pili enables nanonewton force generation.</a> Plos Biology. 6 (4), 907-913 (2008).</li><li>Wuang, S. C., Ladoux, B., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Chemo-Mechanical+Effects+of+an+Anti-Cancer+Drug+Emodin+on+Breast+Cancer+Cells.">Probing the Chemo-Mechanical Effects of an Anti-Cancer Drug Emodin on Breast Cancer Cells.</a> Cell Mol Bioeng. 4 (3), 466-475 (2011).</li><li>Schoen, I., Hu, W., Klotzsch, E., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Cellular+Traction+Forces+by+Micropillar+Arrays:+Contribution+of+Substrate+Warping+to+Pillar+Deflection.">Probing Cellular Traction Forces by Micropillar Arrays: Contribution of Substrate Warping to Pillar Deflection.</a> Nano Lett. 10 (5), 1823-1830 (2010).</li><li>Lemmon, C. 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=Shear+Force+at+the+Cell-Matrix+Interface:+Enhanced+Analysis+for+Microfabricated+Post+Array+Detectors.">Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors.</a> Mech Chem Biosyst. 2 (1), 1-16 (2005).</li><li>Lam, R. H. W., Weng, S. N., Lu, W., Fu, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+subcellular+measurement+of+cell+stiffness+using+a+microengineered+stretchable+micropost+array+membrane.">Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane.</a> Integr Biol-UK. 4 (10), 1289-1298 (2012).</li><li>Roure, O., 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=Force+mapping+in+epithelial+cell+migration.">Force mapping in epithelial cell migration.</a> PNAS. 102 (39), 14122-14122 (2005).</li><li>Papenburg, B. J., Rodrigues, E. D., Wessling, M., Stamatialis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+role+of+material+surface+topography+and+wettability+on+cell-material+interactions.">Insights into the role of material surface topography and wettability on cell-material interactions.</a> Soft Matter. 6 (18), 4377-4388 (2010).</li><li>Badique, 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=Directing+nuclear+deformation+on+micropillared+surfaces+by+substrate+geometry+and+cytoskeleton+organization.">Directing nuclear deformation on micropillared surfaces by substrate geometry and cytoskeleton organization.</a> Biomaterials. 34 (12), 2991-3001 (2013).</li><li>Sniadecki, N. 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=Magnetic+microposts+as+an+approach+to+apply+forces+to+living+cells.">Magnetic microposts as an approach to apply forces to living cells.</a> PNAS. 104 (37), 14553-14558 (2007).</li><li>Weng, R. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silicone-based+stretchable+micropost+array+membrane+for+monitoring+live-cell+subcellular+cytoskeletal+response.">A silicone-based stretchable micropost array membrane for monitoring live-cell subcellular cytoskeletal response.</a> Lab Chip. 12, 731-740 (2012).</li><li>Desai, R. A., Yang, M. T., Sniadecki, N. J., Legant, W. R., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Post-Array-Detectors+(mPADs):+an+Approach+to+Isolate+Mechanical+Forces.">Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces.</a> J Vis Exp. (8), e311 (2007).</li><li>Yang, M. T., Fu, J. P., Wang, Y. K., Desai, R. A., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+stem+cell+mechanobiology+on+microfabricated+elastomeric+substrates+with+geometrically+modulated+rigidity.">Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity.</a> Nat Protoc. 6 (2), 187-213 (2011).</li><li>Sniadecki, N. J., Han, S. J., Ting, L. H., Feghhi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+in+Cell+Biology.">Micropatterning in Cell Biology.</a> Methods in Cell Biol. 121, 61-73 (2014).</li></ol>

이 작품은 실질적으로 진입 기술 및 실제적인 장벽을 낮춤으로써 견인 힘 현미경 분야의 발전을 추구한다. 이 장벽은 양측에서 온다. 가장 먼저 재현성 제조 및 실험적 micropost 배열을 의뢰하기 위해 극복해야 할 많은 비 사소한 기술적 문제가 있습니다. 전형적인 실험이 수백 또는 세포의 수천의 분석이 필요할 수 있습니다 염두에두고 - 둘째, 마찬가지로 적지 않은 단일 셀 힘의 신뢰할 수있는 반...

- 기계에 민감한 세포 기반 분석은 역학 세포 생물학에서 재생할 수있는 중심적인 역할을 반영하는 다양한 애플리케이션으로, 부착 세포를 조사 할 수 있습니다. 이 애플리케이션은 세포 내 프로세스 또는 전체 셀 동작을 구동하는 기본 메커니즘에 초점을 맞추고있다. 한편, 예컨대 세포 외 매트릭스 조성물 또는 매트릭스 강성 같은 외부 환경 요인 극적 셀의 기계적 및 생물학적 반응에 영향을 미칠 수있다. (1) 같은 약학 적 활성 화합물의 많은 클래스의 사용 후에 관측 될 수 있고, 효과가있는 아르 종종 세포 배양 모델을 이용하여 특징으로한다. (2) 등의 골격 구조 및 기능의 변화와 관련된 세포 표현형으로 표시 변경을 유도 할 수 자발 또는 실험적으로 유도 된 유전자 돌연변이에 의한 것과 같은 다른 한편 유전자형 특성에서. (3)이 예는 많은 수의 몇 가지주제에있는 세포의 기계적 표현형 관련이며,이 모든 유용하게 micropost 배열로 조사되었다. 이 글을 쓰는 시점에서, 약 200 기사 세포 micropost 상호 작용을 설명하는 발표되었다. 이 작품은 micropost 편향 원칙의 이론적 인 측면뿐만 아니라 제조에 실제적인 지침을 설명합니다. 세포와 유연한 micropost 배열의 상호 작용을 설명하는 첫 번째 기사는 연속 부드러운 기판을 탄 등 nanonewton 규모의 세포 수축력을 추정하는 데 사용되는 고전적인 견인 힘 현미경 (TFM)과는 대조적으로 2003 년 4 탄 및 동료에 의해 ​​출판되었다. 실리콘 엘라스토머로 이루어지는 다수의 근접하게 이격 된 수직 빔을 사용하는 방법을 설명했다. 이 기술의 주요 장점은 두 가지 주요 기능에서 등장. 유지하면서 제 휴대 명백 기판의 강성을 변경하기 위해 하나의 유일한 micropost 치수를 변경해야달리 일정하므로, 표면 토폴로지 및 화학의 차이를 피하는 기판 조성물. 둘째 microposts는 이산 개별 초점 유착 정도의 힘과 공간 해상도로 분석 할 수 있습니다 및 표준 TFM에 의해 유사한 분석에 고유 한 분석 문제를 줄일 수있는 개별 스프링과 같은 역할을합니다. 오늘 micropost 배열에 대한 응용 프로그램의 범위는 크게 몇 하나의 셀에 대한 힘의 단지 매핑을 초과합니다. 예를 들어, 아키야마는 곤충 근육 탑재 자율 마이크로 로봇을 개발하기 위해서는, micropost 어레이 액츄에이터 방 애벌레로부터 격리 등쪽 혈관 조직의 사용을보고한다. (5) 그러나 microposts 대부분의 게시 된 응용 프로그램은 감염이나 암과 같은 질환의 연구에 초점을 맞추고있다. 예를 들어, micropost 어레이는 나이 세리아 gonorrh 번들의 타입 IV의 필리의 구동력 발생을 연구하기 위해 사용되어왔다. 신호 캐스케이드 감염 향상과 연관된 6 기타 세포 골격 타겟팅 약학 화합물로 처리 유방암 세포를 연구 microposts를 사용한 콜로니 OEA. 7 micropost 편향 종종 셀을 가정 단부 하중 고전 캔틸레버 빔 이론을 이용하여 설명하는 매우 micropost의 선단에 부착. 편향 δ 원인이 여기에 적용되는 힘 (F)은 micropost의 &quot;휨 강성&quot;K에 의존에 의해 계산된다 : <img alt="식 (1)" src="/files/ftp_upload/53350/53350eq1.jpg" /> (1) E, I, L 및 인 영률 각각 관성 빔 길이의 면적 모멘트. 기판 휨이 교류로 촬영되지 않습니다 그러나,이 방정식의 결과는 빔 전단 이후 작업뿐만 아니라 굴곡의 힘의 근사치를 제공카운트. microposts 전형적 폴리 디메틸 실록산 (PDMS)와 같은 부드러운 재료로 제조되는 것을 고려할 때 이러한 요소가 포함될 필요가 실리콘 고무 기반. . Schoen 외는 micropost (L / D) 및 해당 중합체의 포아송 비 (V)의 종횡비에 기초하여 이러한 교정 인자가 있다는 것을 증명 8 그것은로 주어진다. : <img alt="식 (2)" src="/files/ftp_upload/53350/53350eq2.jpg" /> (2) T 기울기는 (V) 동일한 문서에서 발견 될 수있는 피팅 파라미터 = 1.3를 포함 틸팅 계수되면서 : <img alt="식 (3)" src="/files/ftp_upload/53350/53350eq3.jpg" /> (3) 즉 micropost 보정의 강성 K CORR은 순수한 굽힘 강성 K = K 벤드의 생성물 및 보정 계수 CORR 의미주어진: <img alt="식 (4)" src="/files/ftp_upload/53350/53350eq4.jpg" /> (4) 따라서, 셀 력 계산 이제 판독 식 (1)의보다 정교한 변형을 사용하여 수행한다 : <img alt="식 (5)" src="/files/ftp_upload/53350/53350eq5.jpg" /> (5) 보정의 영향은 즉시 micropost 치수에 대한 일반적인 값을 사용으로 더 분명해진다. 예를 들면, 원형 단면과 PDMS 계 실리콘 고무로 만들어진 5 ㎛의 직경 15 마이크론 길이 micropost는 0.77의 보정 계수에 이르게하기 때문에 보정 전 계산은 23 %가 소비 된 전지의 힘을 과대 평가한다. 이것은 훨씬 더 심각한 작은 종횡비 microposts에 대한된다. 전통적으로, micropost 이미지 분석은 이상적인 빔 굽힘 이론에 근거하고있다. 2005 년 MICR의 사용을 개척 한 그룹opost 어레이 micropost 분석에 적합한 이미지 분석 소프트웨어를 발표 9 소프트웨어는 소프트웨어 라이센스가 필요하고 사용자가 각각의 위치에 대한 세 개의 이미지를 취한다.; 전송 모드와 스테인드 세포에 형광 모드에서 또 하나의 micropost의 상단과 하단면에서 한 각. 각각의 상부 및 하부 위치를 비교 한 후 소프트웨어가 힘 벡터 필드를 결정하고, 포스트 당 힘과 같은 관련 파라미터를 계산 micropost. 기타 소프트웨어 패키지는 존재와 그 분석의 원칙은 간단하게 그들을 설명하는 해당 기사에 언급되어 있지만, 이러한 분석 소프트웨어 패키지는 일반적으로 공개적으로 사용할 수 없습니다. (10, 11) 셀 매핑 힘을 위해 설계 micropost 어레이는 모든 이웃 microposts 사이의 등거리 간격 장점이 후자의 직교 micropost 레이아웃 또는 육각형 하나에 되로 분류 될 수있다. 일반적인 microposts 하원형 단면을 적이 그들의 사이즈 타원 또는 사각형 단면 microposts 또한보고되었다 그러나, 길이가 50 ㎛ 인 내지 1.0 μm의 직경이 10 ㎛ 내지 2. (4)를 배열한다. 12,13 micropost 재료로서 PDMS 기반 실리콘의 혼합물의 혼합물을 사용하는 나노 입자를 추가로 허용한다. 코발트 나노로드를 추가 예를 들어 micropost의 자기 활성화를 가능하게하고, 따라서 잠재적 인 실험 디자인에 자유의 또 다른 학위를 제공합니다. 14 대부분의 그룹은 커버 유리와 같은 또는 페트리 접시 내부 평면 딱딱한 기판 상에 자신의 micropost 배열을 생산하고 있습니다. 그러나 맨과 동료들은 최근에 신축 막에 형성된 micropost 배열을보고했다. 15 세포 수축성의 관점에서 라이브 셀 세포 내 동적 응답을 공부하는 동안이 부착 세포에 세포 스트레칭 힘의 응용 프로그램을 할 수 있습니다. 널리 사용되는 가장 맛 음료SU8 포토 레지스트를 이용하여 실리콘 웨이퍼 위에 마이크로 구조를 생성하는 데 사용되는 간단한 표준 클린 룸 공정에서 Sniadecki 동료. 16-18의 통찰력 프로토콜에 기재된 바와 같이 micropost 어레이 제작 오리스 공정은 소프트 리소그래피에 기초한다. 이것은 실리콘 고무 몰드로 이송 구조 위에 캐스팅 된 상기 복사 프로세스가 이어진다. 제 2 단계에서, 이들 몰드 선택된 기판 위에 실리콘 고무를 사용하여 초기 미세 구조를 복제하기 위해 사용된다. 그러나 microposts위한 제조 공정을 확립 그들의 애플리케이션과 관련된 서적의 큰 증가에도 불구하고 마이크로 공학 전문가에도 상당한 시간이 소요; 허용 가능한 품질 레벨을 산출하기 위해 특정 테스트 환경과 micropost 레이아웃 최적화 및 적응을 필요로하는 많은 공정 단계가있다. 상업 micropost 배열은 준비-T에 사용할 수 있습니다지속적으로 높은 품질의 오 사용 ( &quot;기성&quot;) 형식입니다. 따라서 그들은 현장 생산에 필요한 복잡하고 긴 제조 공정에 대한 대안이다. 이 논문에서 시판 micropost 어레이는 단일 시야 현미경 이미지를 사용하여 셀룰러 힘을 매핑하는데 사용 하였다. 더 중요한이 문서에서는이 원고에 대한 보충 자료 등을 다운로드 할 수 있습니다 MechProfiler라는 이름의 완전한 기능을 오픈 소스 소프트웨어를 설명합니다. 소프트웨어 적극적 유지 버전도 http://www.orthobiomech.ethz.ch에서 찾을 수있다. &quot;기성품&quot;분석과 호환 오픈 소스 분석 소프트웨어의 조합은 현저히 TFM 정확한 실험을 달성하기 엔트리 장애물을 낮춘다. 클린 룸 시설이나 소프트웨어 개발 전문 중 하나에 대한 액세스 권한이없는 연구원은 성공적으로 세포의 힘을 분석 할 수 있습니다. 이 메케에 초점을 사용자로 하여금ensitivity 분석 출력보다는 기술 자체, 및 더 넓은 지역에 사용할 견인력을 측정한다. 또한, 이것은 micropost 어레이 전자동 스크리닝 향해 방법을 포장하는 중요한 단계이다. MechProfiler 분석 소프트웨어는 파일 형식 TIFF, PNG, BMP와 JPG의 이미지를 처리​​합니다. 이미지는 형광, 위상차 또는 밝은 장광 현미경을 사용하여 수행 될 수있다. 독립 실행 형 프로그램은 (이용 가능한 그림 12) 무료 matlab에 컴파일러 런타임과 함께 실행하고 기본 알고리즘은 약 1 분에 하나 또는 여러 개의 세포와 이미지를 처리하는 사용자 수 있도록 효율적인 이미지 처리를 위해 수 있습니다. 또한, 이들 세포 중 살아있는되거나 &quot;고정&quot;. MechProfiler 소프트웨어 크게 상업용 품질 micropost 어레이의 재현성에 의존하여 데이터 분석 처리량을 증가시킬 수있다, 구체적으로는, 기본 및 #8220은 비 - 편향 &quot;배열의 각 포스트의 위치가 이상적 그리드에 대해 추정 될 수있다 (본 연구에 사용 된 배열에서 그리드 제조 편차를 100nm 이하였다). 단 하나의 관심 영역에 작물, 분석 용 이미지 파일의 선택이 열리면 세포에 의해 덮여 게시물을 정의되거나 또는 폐기해야하는 사후 위치를 결정하고, 편향 / 이상적 그리드에 대해 힘을 산출하고, 최종적으로 표준 Office 스프레드 시트에 포함, 수출 가능성을 가진 모든 세포 관련 데이터를 저장합니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>T25 cell culture flasks</td>
			<td>Nunc</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS-buffer</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium DMEM/F12</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS South America</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100x</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge vials</td>
			<td>Sarstedt</td>
			<td>62.554.502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropost array&#160;</td>
			<td rowspan="2">MicroDuits</td>
			<td rowspan="2">MPA-col1/FN</td>
			<td>Micropost dimensions:</td>
		</tr>
		<tr>
			<td>pre-coated with Collagen I/Fibronectin</td>
			<td>&#216;= 6.4 &#181;m, l=18.2 &#181;m; grid=&#160;13 &#181;m, kcorr= 2.87 nN/&#181;m</td>
		</tr>
		<tr>
			<td>Ethanol abs p.A.</td>
			<td>Merck</td>
			<td>100.983</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Nunc</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered 10%</td>
			<td>Sigma</td>
			<td>HT5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Blue G-250</td>
			<td>Sigma</td>
			<td>27815</td>
			<td>Coomassie blue</td>
		</tr>
		<tr>
			<td>Methanol ACS p.A.</td>
			<td>Merck</td>
			<td>1.06009</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>WillCo Wells BV</td>
			<td>GWSb-3522</td>
			<td>35 mm diameter, aperture 22&#160;mm</td>
		</tr>
		<tr>
			<td>T-100 Eclipse</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>D3-L3</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Camera controler</td>
		</tr>
		<tr>
			<td>DS Fi2</td>
			<td>Nikon</td>
			<td></td>
			<td>Camera</td>
		</tr>
	</tbody>
</table>

easy and accurate mechano-profiling on micropost arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

설명 된 기술의 주요 장점은 일상적인 실험실 작업에 신속하고 효율적인 통합을위한 단순하고 잠재력에있다. 오픈 소스 소프트웨어와 결합 고품질 상용 센서 어레이의 조합하고자 달리 이미지 분석 소프트웨어 개발의 지식 크린룸 시설 및 심층 액세스해야 - 기계 감도에 대한 정보를 제공한다. (1)가 제공 방법의 흐름을 도시한다. 그것은 micropost 배열에 세포를 준비 시드...

Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

Easy and Accurate Mechano-profiling on Micropost Arrays

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Micropost 배열에 쉽고 정확한 - 기계 프로파일

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

easy-and-accurate-mechano-profiling-on-micropost-arrays

Research

JoVE Journal

Bioengineering

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

비대칭 수용체 패턴에 셀 롤링의 탄도를 공부

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

연구

생체공학

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Microelectrode 배열을 사용하여 siRNA와 자기편 세포의 높은 효율, 특정 사이트 Transfection (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

무 세포 생물학 밀봉 Femtoliter 상공 회의소 배열

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

지원되는 지질 이중층에 리간드 나노 클러스터의 배열

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode 배열 마이크로 신경 통신 및 Axonal 신호 전파 공부에 대 한 인터페이스

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

재발 동작 전위 기록을 위한 멀티웰 마이크로 전극 어레이의 인간 iPSC 유래 심근구 네트워크

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

생체 내 신경 녹음을 위한 Ti3C2 MXene 마이크로 전극 어레이 의 제조

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

하위 구조 분석기: 형광 현미경 이미지에서 세포 체의 신속한 탐색 및 정확한 분석을 위한 사용자 친화적인 워크플로우

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

절개된 임플란트 조직의 면역 기능 분석을 위한 고차원 유세포 분석

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

인간 iPSC 유래 심근세포, 심장 섬유아세포 및 내피 세포를 사용하여 3D 심장 미세 조직 배열의 제조

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...