저자는 국립 과학 재단 (National Science Foundation)을 통해 재료 연구 과학 및 엔지니어링 센터의 일환으로 후원 존스 홉킨스에​​서 표면 분석 연구실의 사용을 인정합니다. LED가 IGERT 트레이와 국립 과학 재단 (National Science Foundation) 대학원 연구원입니다. 이 연구는 부분적으로 NIH 부여 U54CA143868에 의해 지원되었다.

1. Micropatterned 스탬프 제작 표준 석판술 <ol><li> 에탄올과 함께 새로운 실리콘 웨이퍼를 씻어 공기의 흐름과 건조. 웨이퍼를 처리하고 전체 과정에서 표면 결함을 방지하기 위해 집게를 사용하십시오. </li><li> coater를 한 바퀴 돌리고 SU - 2025 부정적인 포토 레지스트와 웨이퍼 표면을 커버하기 위해 웨이퍼를 전송합니다. 포토 레지스트와 웨이퍼의 적어도 80 %를 커버. 3000rpm에서 30 초 뒤에 600rpm에서 10 초 스핀 코트. 포토 레지스트의 photosensitivity으로 인해,이 시점에서 객실 조명을 끄십시오. </li><li> 3 분 95 ° C에서 &quot;부드러운 빵을&quot;에 대한 뜨거운 접시에 코팅된 실리콘 웨이퍼를 포토 레지스트 전송합니다. 핫 플레이트에서 제거하고 1 시간 냉각 수 있습니다. </li><li> 포토 레지스트 덮인 실리콘 웨이퍼 위에 원하는 패턴으로 미리 가공 마스크를 놓고 20 초 동안 자외선 (UV) 조사 (350-450 nm의)에 노출. </li><li> 110 &quot;하드 베이킹&quot;에 철판 ° 육분 위해 C로 전송 실리콘 웨이퍼. </li><li> 불을 켜고 철판에서 웨이퍼를 제거합니다. 독특한 패턴이 시점에서 볼 수 있습니다. </li><li> SU - 8 포토 레지스트 개발로 실리콘 웨이퍼를 주의깊게 린스. 개발자 솔루션 3 rinses 후 에탄올로 린스. 모든 백색 잔류물이 있으면, 포토 레지스트 개발 솔루션과 헹굼 반복하거나 잠수함 웨이퍼를 개발자 솔루션 2 분 헹구세요 에탄올로 다시 진행합니다. 물, 공기 건조와 함께 씻으십시오. </li><li> 믹스 polydimethylsiloxane (PDMS) 10시 1분 무게 비율에 엘라스토머 솔루션과 치료 요원. 공기 방울을 제거하는 5 분간 900rpm에서 원심 분리기. </li><li> 장소는 배양 접시에있는 웨이퍼 패턴과 실리콘 웨이퍼 위에 PDMS 잔. 몇 분 현재 거품, desicator의 장소와 진공이있다면 거품이 사라까지. </li><li> 보완 탄성 스탬프를 형성 실내 온도 (RT)에서 하룻밤 치료. PDMS는 완전히 30 분 60 ° C에서 오븐에서 개최 12시간 후 치유되지 않을 경우. </li><li> 조심스럽게 실리콘 웨이퍼에서 PDMS를 제거합니다. 원하는 크기로 도장을 절단하는 레이저 에지를 사용하십시오. 15 분간 에탄올에 Sonicate. 웨이퍼 새로운 탄성 우표를 만드는 여러 번 이용하실 수 있습니다. </li></ol> 2. HA의 Micropatterning <ol><li> 플라즈마 깨끗한 유리는 5 분 동안 슬라이드. 5 분 챔버와 진공로 모든 슬라이드를 놓습니다. 다음 챔버를 입력 산소의 낮은 흐름을 허용합니다. 5 분에 플라즈마 청소기를 돌립니다. 포셉를 사용하여 배양 접시에있는 플라즈마 청소기와 장소에서 제거합니다. </li><li> 플라즈마 세정 동안 15 분 동안 에탄올의 PDMS 스탬프를 sonicate. </li><li> 15 ML의 원심 분리기 튜브의 95% V / V 에탄올 3 - aminopropyltrimethoxysilane (APTMS)의 신선한 3퍼센트 V / V 솔루션을 준비합니다. silanol를 형성 RT에서 5 분간 반응하도록 허용합니다. </li><li> 플레이스 PDMS의 스핀 coater 척의 패턴까지 우표와 커버 APTMS 솔루션으로 표면을 패턴. 30 초 동안 3500rpm에서 Spincoat. , PDMS 스탬프만을 면을 도장 표면을 만지지 마십시오. </li><li> 패턴 측면의 한쪽 끝을 아래로 향하게 설정 스탬프를하고 1 분 유리 표면과 접촉 완전히되도록 부드럽게 PDMS를 눌러 글라이드 : 플라즈마에 전송 APTMS 솔루션은 유리 표면을 청소. </li><li> PDMS는 부드럽게 도장 제거하고, 패턴 유리 슬라이드 30 분 RT에서 품어 수 있습니다. 에탄올과 공기 건조와 패턴 표면을 씻어. Sonicate PDMS 초과 APTMS을 제거하는 15 분 동안 도장. </li><li> 115에서 1 시간 동안 오븐이나 뜨거운 접시에 열 표면 ° C. 에탄올로 씻어 건조 공기. </li><li> [메톡시 (polyethyleneoxy) 프로필] 톨루엔에 trimethoxysilane - 신선한 폴리에틸렌 글리콜 (PEG) - 실란 20% V / V 2 솔루션을 준비합니다. 이 패턴을 둘러싼 않은 지역 접착제 역할을합니다. </li><li> 75에서 45 분 동안 PEG - 실란 솔루션 유리 페트리 접시와 부화 전송 패턴 유리 슬라이드 뜨거운 접시에 ° C. 톨루엔, 물, 공기와 건조와 린스. </li><li> 생물 학적 안전 캐비닛에 UV 살균 방사선을 사용하여 1 시간 소독. 불임을 유지하기 위해이 시점에서 생물 학적 안전 캐비넷에서 진행합니다. </li><li> 수성 멸균 HA 솔루션을 준비합니다. 1 - 에​​틸 -3 - (3 - 디메틸 아미노) carbodiimide (EDC)의 10mM N - hydroxysuccinimide의 5mM 50μg / ML 플루오레신 - 분류 HA </li><li> 무균 조건에서 패턴 유리 기판에 HA 솔루션을 적용하고 빛으로부터 보호. 패턴 기판은 16-24시간 위해 그대로해야합니다. </li><li> HA 솔루션을 기음과 씻어 인산염 버퍼 호수 (PBS), 1 시간 동안 PBS에서 (BSA) 1% 소 혈청 알부민과 표면을 커버. 표면은 이제 세포 배양을위한 준비가되었습니다. </li></ol> 3. HA Micropatterned 표면에 세포 배양 <ol><li> 확장 MDA - MB - 231 제조 업체의 지시에 따라 인간의 유방암 세포 LS174T 대장암 암종 세포. </li><li> 0.4과 1x10 사이의 밀도와 암 세포의 씨앗 단세포 suspensions <sup18.75 cm 2 유리 면적 당 및 HA에&gt; 6 암세포는 표면 고정화. 5 % CO 2 관리 대기 속에서 humidified 인큐베이터 (37 ° C)에서 알을 품다. 세포 접착은 24 시간 이내에 발생해야하며 거꾸로 가벼운 현미경을 사용하여 볼 수 있습니다 </li><li> 세포 배양 표면은 표준 보육 조건 5 일 이내에 유지 수 있습니다. 매일 미디어를 변경합니다. 세포 형태학 및 성장은 거꾸로 광 현미경을 사용하여 볼 수 있습니다. 추가 셀룰러 assays은 HA의 표면에 반응을 분석하기 위해 적용할 수 있습니다. </li></ol> 4. 대표 결과 : covalently APTMS 패턴 유리 슬라이드에 HA를 무력화하는 데 사용 carbodiimide 화학은 그림 1A에 표시됩니다. EDC는 아민 반응 중간체를 형성하기 위해 HA 카르 복실 그룹과 반응 길이가 0 인 crosslinking 에이전트입니다. 이 중간은 가수 분해에 감염될이므로, NHS는 carbodiimide 반응 효율을 높이는 추가됩니다. HA 솔루션은 APTMS micropatterns, HA 중급 ATPMS의 기본 아민 간의 안정적인 아미드 결합 형태와 상호 작용으로. HA 패턴 표면의 예입니다 (1B - C 그림) 표시됩니다 HA의 패턴 고정을 입증 형광 현미경과 ImageJ 형광 강도 분석을 사용하여 시각. HA micropatterned의 표면 expgenous HA와 암 세포의 상호 작용 연구를 활성화합니다. 두 MDA - MB - 231 인간의 모유와 LS174t 인간의 결장암 세포 라인은 우선적으로 HA micropatterned 영역 (그림 2)을 준수. 스캐닝 전자 현미경을 사용하여 고해상도 이미지는 HA 고정화 표면 (그림 3)과 교양 세포의 상호 작용을 분석하는 데 사용할 수 있습니다. <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="그림 1" /> 그림 1 HA micropatterned 표면 HA 기능성 표면의 발전을 설명하는 (A) 도식, 플루오레신의 (B) 형광 이미지 (녹색) 표시 HA micropatterned 표면. (C) 3D 픽셀 분포지도 이산 HA 패턴을 보여주 ImageJ 소프트웨어를 사용합니다. 스케일 바 = 100μm. Elsevier의 허가를 9 수정. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="그림 2" /> HA micropatterned 표면에 그림 2. 암 세포 접착. 콜론 (A)과 유방 모두 (B) 암종 우선적으로 문화 24 시간과 HA 패치에 준수. Elsevier의 허가를 9 수정. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="그림 3" /> HA와 그림 3. 암 세포 상호 작용. (A) 결장암 세포 낮은 배율 (왼쪽)와 높은 배율 (오른쪽, 왼쪽에있는 사각형)에서 표시 HA 표면에 (A) 교양의 전자 현미경 이미지를 검색 및 유방암 세포 (B)에서 그림 HA 표면에 교양 낮은 배율 (왼쪽)와 높은 배율 (오른쪽, 왼쪽에있는 사각형).

<ol>
	<li>Toole, B. P., Wight, T. N., Tammi, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-cell+interactions+in+cancer+and+vascular+disease.">Hyaluronan-cell interactions in cancer and vascular disease.</a> Journal of Biological Chemistry. 277, 4593-4596 (2002).</li><li>Hamilton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hyaluronan+Receptors+CD44+and+Rhamm+(CD168)+Form+Complexes+with+ERK1,2+That+Sustain+High+Basal+Motility+in+Breast+Cancer+Cells.">The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells.</a> Journal of Biological Chemistry. 282, 16667-16680 (2007).</li><li>G&#246;tte, M., Yip, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparanase,+Hyaluronan,+and+CD44+in+Cancers:+A+Breast+Carcinoma+Perspective.">Heparanase, Hyaluronan, and CD44 in Cancers: A Breast Carcinoma Perspective.</a> Cancer Research. 66, 10233-10237 (2006).</li><li>Lord, M. S., Pasqui, D., Barbucci, R., Milthorpe, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+on+derivatives+of+hyaluronic+acid+and+subsequent+cellular+response.">Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.</a> Journal of Biomedical Materials Research. Part A 91, 635-646 (2009).</li><li>Barbucci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+hyaluronic+acid+by+sulphate+groups+insertion+to+obtain+a+heparin-like+molecule.">Modification of hyaluronic acid by sulphate groups insertion to obtain a heparin-like molecule.</a> Gazz. Chim. Ital. 125, 169-180 (1995).</li><li>Morra, M., Cassinelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fouling+properties+of+polysaccharide-coated+surfaces.">Non-fouling properties of polysaccharide-coated surfaces.</a> Journal of Biomaterials Science, Polymer Edition. 10, 1107-1124 (1999).</li><li>Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+hyaluronic+acid+and+poly-L-lysine+for+patterned+cell+co-cultures.">Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures.</a> Biomaterials. 25, 3583-3592 (2004).</li><li>Ibrahim, S., Joddar, B., Craps, M., Ramamurthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-tethered+model+to+assess+size-specific+effects+of+hyaluronan+(HA)+on+endothelial+cells.">A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.</a> Biomaterials. 28, 825-835 (2007).</li><li>Dickinson, L. E., Ho, C. C., Wang, G. M., Stebe, K. J., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+surfaces+for+high-resolution+analysis+of+cancer+cell+interactions+on+exogenous+hyaluronic+acid.">Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid.</a> Biomaterials. 31, 5472-5478 (2010).</li><li>Gao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+oligosaccharides+promote+excisional+wound+healing+through+enhanced+angiogenesis.">Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.</a> Matrix Biology. 29, 107-116 (2010).</li><li>Slevin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-mediated+angiogenesis+in+vascular+disease:+Uncovering+RHAMM+and+CD44+receptor+signaling+pathways.">Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways.</a> Matrix Biology. 26, 58-68 (2007).</li></ol>

허가 전재 비디오 도식 그림, 저작권 빌리 - VCH Verlag GmbH를 &amp; 주식 KGaA. 디킨슨 LE, Kusuma S, Gerecht S.은 biomaterials를 사용하여 배아 줄기 세포의 분화의 틈새를 재구성. Macromol. Biosci. 2010; 10월 21일. [인쇄 앞서 Epub].

제시 HA의 micropatterning 방법은 외인성 HA와 세포 상호 작용의 연구를 수 있습니다. HA는 암 진행 1 핵심적인 역할을하는 것으로 알려져 있지만 2 차원 패턴 HA 표면에 암 세포의 상호 작용을 조사 제한된 연구가있다. 외인성 HA의 micropatterns에 대한 제어 연구는 암 세포 부착, 성장, 및 마이 그 레이션의 고해상도 시각화를 허용하고 암이 더 기초를 명료하게하다 수 있습니다. <p class="jove_content...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

micropatterned surfaces to study hyaluronic acid interactions with cancer cells

암 침공과 진행은 종양 microenvironment 내에서 성장 요인 / 크린 시토킨 및 세포외 매트릭스 (ECM) 구성 요소에 의해 복잡한 규제하에있는 운동성이있는 세포 표현형을 포함합니다. Hyaluronic 산 (HA)은 강화 침략, 성장 및 angiogenesis 1 종양 진행을 촉진하는 것으로 알려져 하나 stromal ECM 구성 요소입니다. HA의 상호 작용은 세포 표면 수용체 CD44는 따라서 전이성 확산 2-3 증가, 종양 세포 성장, 생존 및 마이 그 레이션을 촉진 이벤트를 신호 유도와 함께. HA는 D - glucuronic 산성 및 DN - acetylglucosamine의 단위를 반복 구성된 음이온, nonsulfated glycosaminoglycan입니다. 반복 disaccharide 단위의 카르 복실와 수산기 그룹의 존재로 인해 고유의 HA는 주로 친수성과 photoreative 고정 4-5에 대한 황산 그룹을 소개 화학 수정 의무가 있습니다. 표면에 HA의 immobilizations 관련된 이전 연구는 6-7 표면에 세포 부착을 제어하기 위해 HA의 bioresistant 행동과 그 sulfated 파생을 활용한다. 이러한 연구의 세포 접착에 우선적으로 비 HA 패턴 영역에서 발생합니다. 외인성 HA와 세포 상호 작용을 분석하기 위해, 우리는 제어 연구 및 HA와 암 세포의 상호 작용 고해상도 시각화를 활성화 패턴 기능화 표면을 개발했습니다. 우리는 유리 표면에 HA의 이산 패턴 영역을 정의하는 microcontact 인쇄 (uCP)을 활용. HA를 무력화하기 위해 carbodiimide 링크 화학을 적용 &quot;tethering&quot;접근 방식은 8 사용되었다. 유리 표면 microcontact aminosilane로 인쇄되었으며 패턴 배열에 HA 고정을 활성화 EDC와 NHS의 최적 비율의 HA 솔루션과 반응. MCP와 carbodiimide 화학을 포함하는 것은 체외 애플 리케이션에 적합한 표면을 생성, 정의 지역 HA의 고정을 활성화. 두 결장암 세포와 유방암 세포는 암시 HA micropatterned 표면과 상호 작용. 암 세포 접착은 48 시간의 확산과 함께 24 시간 이내에 발생했습니다. HA micropatterned 표면을 사용하여, 우리는 그 암 세포 접착은 HA 수용체 CD44을 통해 발생 보여주었다. 또한, HA 패턴의 표면 전자 현미경 (SEM)과 암 세포 접착 라고나할까요 허용된 고해상도 이미징을 검사 및 외인성 HA에 대한 암 세포의 운동성을 분석 HA 패턴에 대한 확산과 호환되었습니다.

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

외인성 hyaluronic의 산성 (HA)과 암 세포의 상호 작용의 고해상도 분석이 가능합니다 새로운 접근 방식이 설명되어 있습니다. 패턴의 표면은 carbodiimide 화학 및 microcontact 인쇄 결합하여 가공하고 있습니다.

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 ...

micropatterned-surfaces-to-study-hyaluronic-acid-interactions-with

Research

JoVE Journal

Bioengineering

15.0K 조회수.  Johns Hopkins University. 외인성 hyaluronic의 산성 (HA)과 암 세포의 상호 작용의 고해상도 분석이 가능합니다 새로운 접근 방식이 설명되어 있습니다. 패턴의 표면은 carbodiimide 화학 및 microcontact 인쇄 결합하여 가공하고 있습니다.

비디오: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

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 ...

연구

생체공학

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Stretching Micropatterned Cells on a PDMS Membrane

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 PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

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 ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

조직 공학 Thermoresponsive 나노 구조 표면의 제조

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

뼈 Morphogenetic 단백질의 2 고체 표면에 화학 클릭 하 여 사이트 감독 동원 정지

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

히 알루 론 산 기반 Hydrogels 세포종 환자에서 파생 된 셀의 3 차원 문화에 대 한

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

인간 유도 만능 줄기 세포 유래 심근 세포에서 단세포 광학 작용 잠재적 측정

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...