This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

EBL 금형에서 PDMS 금형 1. 복제 <ol><li> (29) 실리콘 몰드를 제조 <ol><li> 스핀 코팅은 200 ㎕의 폴리 메틸 메타 크릴 레이트 (PMMA) 1 분 동안 2,500 rpm에서 1 × 1cm의 실리콘 (Si) 기판 상에 용액의 박막을 형성한다. </li><li> 2 분 동안 180 ° C에서 Si 기판의 PMMA 필름을 굽는다. </li><li> 300 μC / ㎠의 면적 도즈 집속 전자 빔을 이용하여 PMMA 필름의 설계 나노 패턴을 작성한다. </li><li> 80 초 동안 개발자의 PMMA의 나노 패턴을 개발합니다. </li><li> 10 kV로, 0.5 mA의 방출 전류는 0.5 Å / sec의 증착 속도의 출력 전압 전자빔 증착기를 이용하여 두께 50 nm의 니켈 층과 PMMA의 나노 패턴을 증착. </li><li> 20 분 동안 80 ° C에서 20 ㎖ 제거제의 PMMA 부 리프트 오프. </li><li> 반응성 이온 에칭 (RIE) Si 기판에 나노 패턴은 원하는 깊이의 실리콘 몰드 도착. 참고 : tetr의 가스 혼합물afluoromethane (CF 4) 150 W 400 W와 RIE 전력의 유도 결합 플라즈마 (ICP) 전원으로 / 산소 (O 2) (90 % / 10 %)이 560 nm의 깊이까지 실리콘 기판을 에칭하기 위해 사용된다. </li></ol></li><li> silanization합니다 실리콘 몰드 <ol><li> 100mm의 PS 페트리 접시에서 유리 커버 슬립하고 실리콘 주형을 넣어 흄 후드에 위치한 유리 데시 케이 터 내에서 전송할. </li><li> 커버 슬립에 10 μL의 1H, 1H, 2H, 2H-Perfluorooctyltrichlorosilane을 삭제합니다. 주의 : 1H는, 1H는, 2H, 2H-Perfluorooctyltrichlorosilane 피부 부식 및 눈에 심한 손상을 일으킬 수 있습니다. 적절한 개인 보호 장비 (PPE)를 착용 할 것. </li><li> 부분적으로 페트리 접시를 커버. </li><li> Si를 몰드의 실란 화를 완료하는 흄 후드에서 5 시간 동안 진공하에 데시 케이 유지. </li></ol></li><li> PDMS 예비 중합체를 준비 <ol><li> 일회용 계량 보트 10 g의 PDMS 수지 1.05 g 경화제를 단다. </li><li> 플라스틱 숟가락을 사용하여 철저하게 PDMS 예비 중합체를 섞는다. </li><li> 맑은 혼합물이 관찰 될 때까지 약 20 분 동안 진공 하에서 플라스틱 건조기에서 PDMS의 예비 중합체를 탈기. </li></ol></li><li> PDMS 몰드 복제 <ol><li> 60mm 페트리 접시에 실란 화시 금형을 넣습니다. </li><li> 페트리 접시의시 금형에 PDMS 예비 중합체를 따르십시오. </li><li> 모든 거품이 사라질 때까지 약 10 분 동안 플라스틱 건조기 및 탈기의 페트리 접시를 놓습니다. </li><li> 열판을 페트리 접시로 이동시켜, 4 시간 동안 70 ° C에서 PDMS의 예비 중합체를 경화. </li><li> 신중 핀셋을 이용하여 Si를 몰드에서 PDMS 몰드를 떼어. 참고 : PDMS 금형은 일주까지 주위 조건에서 저장 될 수있다. 경화 후, PDMS 몰드 (30)의 일부 미가 교 PDMS 수지 분자 잔류 경화제가있다. 저 분자량 분자들은 점차 확산 시간 동안 표면에 축적된다. 이 PDMS 표면 (31)의 지형 및 기계적 특성에 영향을 미친다. DIF융합 1 주일 내 중요하지 않습니다. </li></ol></li></ol> 대형, 단일 금형에 PDMS 금형 2. 바느질 <ol><li> 단계 1.4을 반복하여 여러 PDMS 금형을 준비합니다. 참고 : PDMS 혼합물의 동일한 금액을 동일한 두께의 PDMS 금형을 얻을 때마다 체중. </li><li> 이러한 광학 현미경으로 nanogratings 이방성 PDMS 나노 패턴의 방향을 결정하고, 마커 펜으로 PDMS 몰드의 뒷면에 마크. 참고 : 이러한 나노 기둥으로 등방성 나노 형상의 방향을 표시 할 필요가 없습니다. </li><li> 흄 후드에서 에탄올 Si 기판을 청소하고 압축 공기로 건조. </li><li> 블레이드 각 PDMS 몰드의 패턴 화되지 않은 영역을 낸다. 참고 : 스티치 금형의 둘레에 배치되는 PDMS 금형의 경우, 다른 사람과의 접촉 만이 패턴 화되지 않은 영역이 떨어져 손질해야한다. </li><li> 에 나노 패턴면이 아래로 트리밍 된 PDMS 몰드를 배치미러 Si 기판의면 다음은 가까이 있지만 주변 금형 (들)을 터치하지 다른 금형을 맞 춥니 다. </li><li> PDMS 접착층 준비 <ol><li> 0.5 mm 두께의 층을 형성하기 위해 깨끗한 유리 슬라이드 (2.5 cm × 7.5 cm)에 1g는 PDMS 프리폴리머 (1.05 : 10 PDMS 수지와 경화제의 비) 탈기 전송. </li><li> 3-5 분 동안 핫 플레이트상에서 100 ℃에서 PDMS 층 굽는다. 레이어를 터치 레이어가 부분적으로하지만 완치는 것을 보장하기 위해 바늘을 사용합니다. 참고 : 부분적으로 경화 된 PDMS는 경화 PDMS 예비 중합체처럼 흐를 수 없지만 경화 PDMS에 비해 스티커입니다. </li></ol></li><li> 정렬 PDMS 몰드의 뒷면에 PDMS 층을 배치하고 신속하게 조립이 반전하고, 핫 플레이트로 옮긴다. </li><li> PDMS의 접착제 층 및 PDMS 몰드의 배면 사이의 양호한 접촉을 보장하고, 1 시간 동안 100 ° C에서 PDMS를 접착제 층을 경화 조립체의 상부에 금속 블록을 사용 압축력 (5 kPa로)를 적용한다. &lt;BR /&gt; 주 : 조심 조립체의 경사를 방지하기 위해 상기 금속 블록의 위치를 ​​조정한다. </li><li> 금속 블록을 제거하고 실리콘 기판의 단일 스티치 PDMS 몰드를 떼어. </li></ol> PS 기판에 마스터 몰드의 3 세대 주 : 유리 슬라이드에 고정화 스티칭 PDMS 몰드가 PS 판 또는 작동 나노 패턴 기판을 제조 할 수있는에서 PS 박막의 마스터 몰드를 생성하는데 사용될 수있다. <ol><li> 이 PS 판에 마스터 몰드를 생성 <ol><li> 이 PS 판을 준비 <ol><li> 이틀 동안 80 ℃에서 진공 오븐에서 PS 펠렛을 건조. </li><li> 230 ° C에서 프레스 기계를 예열. </li><li> 아래에서 위로 순서대로 알루미늄 판, 폴리 테트라 플루오로 에틸렌 (PTFE) 시트와 알루미늄 스페이서를 조립한다. </li><li> 3cm (L) × 3 cm (W) × 0.3 cm (H)의 사각형 구멍에 알루미늄 스페이서에서로드 3.5 g의 PS 펠릿. 참고 : 스페이서는 약 0입니다.1 PDMS 몰드보다 두꺼운 cm, 따라서 최종 나노 패턴의 PS 기판은 약 0.1 cm의 두께이다. </li><li> 알루미늄 스페이서의 다른 PTFE 시트 다음 다른 알루미늄 판을 놓습니다. </li><li> 프레스 기계 어셈블리를 놓습니다. </li><li> 30 분 동안 230 ° C에서 PS 펠렛을 예열. </li><li> 5 분 동안 어셈블리에 압축 압력 (0.1 MPa의)를 적용합니다. </li><li> 압력을 해제 한 후 조립 0.5 MPa의 압축 압력을 다시 적용합니다. </li><li> 1.5 MPa의 압력의 바람직한까지, 0.5 MPa의 압력 증가 단계를 반복 3.1.1.9 진행한다. </li><li> 프레스 기계의 히터를 끄고 1.5 MPa의 일정한 압력에서 70 ° C 이하로 냉각. </li><li> 어셈블리를 타고 PS 판을 다시 들어가는 습기를 방지하기 위해 80 ℃의 진공 오븐에서 PS 판을 저장한다. </li></ol></li><li> PS 판에 나노 임프린트 스티칭 PDMS 몰드 <ol><li> 알루미늄 스페이서의 PS 판을 놓고3 인치 실리콘 웨이퍼에 설정합니다. 주 : 상기 스페이서의 내부 치수는 PS 판과 동일하다 그래서 PS 판 바로 스페이서 맞는다. </li><li> 30 분 동안 250 ° C에서 열판에 PS 판을 가열한다. </li><li> 용융 PS 판에 나노 패턴면이 아래로 스티치 PDMS 몰드를 넣습니다. 주 : PDMS 몰드의 일측 먼저 PS 판의 표면과 접촉에 배치되고, 다른 쪽의 계면에 기포의 형성을 방지하기 위해 PS 표면과 접촉하여 서서히 저하된다. 주의 : 핫 플레이트의 표면이 뜨겁습니다. 나노 임 프린팅 과정에서 thermogloves을 착용 할 것. </li><li> 스티칭 PDMS 금형의 유리 슬라이드에 알루미늄 판을 놓습니다. </li><li> 알루미늄 판에 금속 블록을 사용하여 압축 압력 (12.5 kPa의)을 적용하고 3 분 동안 기다립니다. 주 : 알루미늄 플레이트가 기울어지지되어 있는지 확인합니다. </li><li> 들어 올려 알루미늄 판에서 금속 블록을 교체하고 난25 kPa의에 압축 압력을 ncrease. </li><li> 압력과 단계를 반복 3.1.2.6는 kPa의 50로 증가. 주의 :이 단계는 PDMS 몰드와 PS 판 사이에 포획 된 공기를 제거하는 것이다. </li><li> 15 분 동안 50 kPa로의 일관된 압력을 받고 240, 250 °의 C 사이의 열판의 온도를 유지한다. </li><li> 열판의 전원을 끄고 전체 설치를 냉각. 주 : 팬은 냉각 과정을 촉진하기 위해 사용될 수있다. </li><li> 의 PS 판에서 스티치 PDMS 몰드 박리 신중 온도가 50 ° C 이하로 된 후, 금속 블록을 제거. 주 : PS 기판 역방향 나노 패턴을 가지고 작업 PDMS 기판을 제조하여 마스터 몰드로서 사용될 수있다. </li></ol></li></ol></li><li> 이 PS 박막에 마스터 몰드를 생성 <ol><li> 에 PS 박막을 준비 <ol><li> 흄 후드에서 10 ml의 톨루엔에 1g의 PS를 녹여. 주의 : 톨루엔은 피부 irritatio가 발생할 수 있습니다n 및 심각한 눈 손상, 그리고 장기간 또는 반복 노출되면 장기에 손상을 줄 수 있습니다. 적절한 개인 보호구를 착용 할 것. </li><li> 1 분 동안 2,500 rpm에서 웨이퍼 2 인에 스핀 코트 1 ML의 PS 솔루션은 ~ 1 μm의 두께 PS 박막을 형성한다. </li><li> 3 일간 흄 후드에서 Si 웨이퍼에 PS 필름을 설정하여, 필름으로부터 톨루엔을 증발시켰다. </li><li> 밤새 80 ℃에서 진공 오븐에서 PS 박막을 어닐링. </li></ol></li><li> 나노 임프린트는 PS 박막의 PDMS 몰드 <ol><li> 열판에 설정되어있는 PS 박막에 나노 형상면이 아래로 스티치 PDMS 몰드를 넣습니다. </li><li> PDMS의 주형의 유리 측에 금속 블록을 사용하여 PDMS 몰드 12 kPa로의 압축 압력을 적용한다. </li><li> 180 ° C에 열판의 온도를 높이고 15 분 정도 유지한다. 주의 : 용융 PS 필름 윤활제로서 기능 할 수있다. 미끄러에서 금속 블록 않도록주의. </li><li> 열판의 전원을 끄고전체 설치를 냉각. 주 : 팬은 냉각 과정을 촉진하기 위해 사용될 수있다. </li><li> 의 PS 필름으로부터 스티치 PDMS 몰드 박리 신중 온도가 50 ° C 이하로 떨어지면 후 금속 블록을 제거. 참고 : 나노 패턴 PS 필름 작업 PDMS 기판을 생산하는 마스터 몰드 될 것입니다. </li></ol></li></ol></li></ol> 세포 행동 4. Nanotopographical 변조 참고 : 대표 nanotopographies 셀 확산의 nanotopographical 변조를 설명하기에 인간의 상피 세포가 배양. <ol><li> 애플리케이션에 따라 3.1 또는 3.2 단계 중 하나에서 생성 된 마스터 주형으로부터 작업 PDMS 기판을 전송. </li><li> 중공 강철 아치 펀치를 이용하여, 특정 멀티 웰 플레이트 (예를 들면, 24- 웰 플레이트)의 구성에 맞도록 디스크에 나노 패턴 PDMS 기판을 절단. </li><li> 오 우물에 PDMS 디스크를 배치 핀셋을 사용하여FA 멀티 웰 플레이트. </li><li> 70 % 에탄올 다음 UV 노출 30 분 동안 각을 이용하여 PDMS 기판을 소독. </li><li> 1 배 멸균 인산 완충 식염수 (PBS)를 세 번으로 PDMS 기판을 세척 할 것. </li><li> 코팅을 실온에서 30 분 동안 세포 외 기질 단백질 (즉, 20 μg의 / ml의 피브로넥틴)와 PDMS 기판. </li><li> 5 분 동안 PDMS 기판을 멸균 PBS로 3 회, 각 린스. </li><li> 10 % 소 태아 혈청 둘 베코 변형 이글 배지에 인간 폐암 세포 A549를 중단하고 혈구 세포를 사용하여 계산. </li><li> 하루 동안 5 % 이산화탄소를 함유하는 가습 분위기하에 37 ℃에서 PDMS 기판 및 배양을 2,000 세포 / ㎠의 밀도로 시딩 세포 접시. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> 4 시간 동안 PBS 중의 4 % 파라 포름 알데히드 및 2 % 글루 타르 알데히드의 혼합물에 세포를 고정 및 CO 2 임계 포를 사용하여 세포를 탈수전자 현미경 관찰 (29)를 스캔 INT 건조기. 주의 : 파라 포름 알데히드와 글루 타르 알데히드는 피부에 심한 화상과 눈에 손상을 줄 수 있습니다. 화학 후드에서 작동하고 적절한 PPE를 착용하십시오. </li></ol>

<ol>
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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=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Gerecht, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Actin+Disrupting+Agents+on+Contact+Guidance+of+Human+Embryonic+Stem+Cells.">The Effect of Actin Disrupting Agents on Contact Guidance of Human Embryonic Stem Cells.</a> Biomaterials. 28 (28), 4068-4077 (2007).</li><li>Bettinger, C. J., Zhang, Z., Gerecht, S., Borenstein, J. T., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+in+vitro+Capillary+Tube+Formation+by+Substrate+Nanotopography.">Enhancement of in vitro Capillary Tube Formation by Substrate Nanotopography.</a> Adv. Mater. 20 (1), 99-103 (2008).</li><li>Thakar, R. G., Ho, F., Huang, N. F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Commun. 307 (4), 883-890 (2003).</li><li>Lee, M. 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=Direct+differentiation+of+human+embryonic+stem+cells+into+selective+neurons+on+nanoscale+ridge/groove+pattern+arrays.">Direct differentiation of human embryonic stem cells into selective neurons on nanoscale ridge/groove pattern arrays.</a> Biomaterials. 31 (15), 4360-4366 (2010).</li><li>Moe, A. A. K., 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=Microarray+with+micro-+and+nano-topographies+enables+identification+of+the+optimal+topography+for+directing+the+differentiation+of+primary+murine+neural+progenitor+cells.">Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.</a> Small. 8 (19), 3050-3061 (2012).</li><li>Dang, J. M., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+induction+of+aligned+mesenchymal+stem+cell+sheets+by+culture+on+thermally+responsive+electrospun+nanofibers.">Myogenic induction of aligned mesenchymal stem cell sheets by culture on thermally responsive electrospun nanofibers.</a> Adv. Mater. 19 (19), 2775-2779 (2007).</li><li>Dasgupta, N., 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=Thermal+co-reduction+approach+to+vary+size+of+silver+nanoparticle:+its+microbial+and+cellular+toxicology.">Thermal co-reduction approach to vary size of silver nanoparticle: its microbial and cellular toxicology.</a> Environ. Sci. Pollut. 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Rev. 105 (4), 1171-1196 (2005).</li><li>Yin, Y., Lu, Y., Gates, B., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-Assisted+Self-Assembly:+A+Practical+Route+to+Complex+Aggregates+of+Monodispersed+Colloids+with+Well-Defined+Sizes,+Shapes,+and+Structures.">Template-Assisted Self-Assembly: A Practical Route to Complex Aggregates of Monodispersed Colloids with Well-Defined Sizes, Shapes, and Structures.</a> J. Am. Chem. 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The authors have nothing to disclose.

우리는 나노 패턴 기판의 넓은 영역을 생성하기 위해, 단순 저렴하면서도 다목적 방법을 제시한다. 충실하게 높은 정의 나노 패턴을 확장하려면 큰 관심은 몇 가지 중요한 단계에 지불해야한다. 첫 번째 다중 PDMS 금형을 트리밍하는 것이다. PDMS의 주형의 패터닝되지 않은 부분을 제거 할 필요가있다. 또한, 주형의 측벽 금형 사이의 갭을 최소화하기 위해 가능한 한 수직으로 완벽 절단한다. 총체적...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>JEOL field emission SEM</td>
			<td>JEOL</td>
			<td>JSM-7600F</td>
			<td>EBL</td>
		</tr>
		<tr>
			<td>E-beam evaporator</td>
			<td>Kurt J. Lesker</td>
			<td>Model: LAB 18 e-beam evaporator</td>
			<td>nickel deposition</td>
		</tr>
		<tr>
			<td>Trion Minilock III ICP/RIE</td>
			<td>Trion technology</td>
			<td>Model: Minilock-phantom III</td>
			<td></td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>PHI Hydraulic Press</td>
			<td>Molde: SQ-230H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>Modle: WS-400A-6NPP-LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 critical dryer</td>
			<td>Tousimis</td>
			<td>Modle: Autosamdri-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>1080</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum plates</td>
			<td>McMaster-carr</td>
			<td>9057K123</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon sheets</td>
			<td>McMaster-carr</td>
			<td>8711K92</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dish</td>
			<td>FALCON</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable weighing boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>Fisher Scientific</td>
			<td>02-913-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Fisher Scientific</td>
			<td>1110049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Ted Pella, inc.</td>
			<td>5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal blocks</td>
			<td>McMaster-carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Brettuns Village Leather Craft Supplies</td>
			<td>Arch punch</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate)</td>
			<td>MicroChem</td>
			<td>495 PMMA A4</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Dow Chemical</td>
			<td>Styron 685D</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td>
			<td>Oakwood Chemical</td>
			<td>7142</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem</td>
			<td>MIBK/IPA at 1: 3 ratio</td>
			<td></td>
		</tr>
		<tr>
			<td>Remover</td>
			<td>MicroChem</td>
			<td>Remover PG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T324-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microsopy Science</td>
			<td>15712-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160;</td>
			<td>Fisher Chemical</td>
			<td>G151-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

<html> 스티치 기법은 높은 정확도와 나노 패턴 기판의 넓은 영역을 생성 할 수있다. 도 1a는 각각, Si 기판 상에 PS 판과 PS 박막 스티칭 PDMS 몰드로부터 전송 된 나노 패턴의 넓은 영역을 표시 1B. 원래 EBL-기록 금형 (그림 1C) 및 기판 (그림 1D)를 작업의 최종 PDMS 사이의 비교는 EBL-기록 된 나노 패턴이 충실하게 작업 기판에 전달...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

셀 동작의 Nanotopographical 변조를위한 나노 패턴 기판 사용 스티치 기법을 확장

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

expanding-nanopatterned-substrates-using-stitch-technique-for

Research

JoVE Journal

Bioengineering

6.5K 조회수.  West Virginia University. A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

비디오: Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

연구

생체공학

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

포토 리소그래피 정의 파릴 렌-C에 대한 세포 패터닝 : 그런가<sub&gt; 2</sub&gt; 기판

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

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 ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

로봇 미세 접촉 인쇄 (R-μCP)를 사용하여 복합 문화 기판 및 순차 친 핵성 치환 제조

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

단일 세포 분비물의 공간 - 시간 영상의 레이블이없는 기술

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

근적외선 Photoimmunotherapy 기술을 사용하여 혼합 된 3D 문화에서 선택적 세포 제거

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Widefield 형광 현미경을 사용 하 여 Mechanobiology 연구에 대 한 부드러운 실리콘 기판의 강성 측정

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinocyte 확산에 2-및 3 차원의 평가 유형 I 교원 질 기판

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

조직 수준 역학을 제어하기 위해 준수 기판에 인간 배아 줄기 세포 식민지의 기하학적 패터닝

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

광학적으로 기화된 퍼플루오로카본 나노방울의 제형 및 음향 변조

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...