저자는 DMF 장치와 DMF 허브의 설계 및 개발에 대한 그들의 공헌에 대한 로널드 F. Renzi의 마이클 S. BARTSCH 감사합니다. 이 연구는 완전히 디아 국립 연구소의 연구실 감독 연구 개발 프로그램에 의해 지원되었다. 샌디 계약 DE-AC04-94AL85000에서 에너지의 국립 핵 보안 관리의 미국학과에 대한 Sandia 공사, 록히드 마틴사의 자회사에 의해 관리 및 운영 멀티 프로그램 실험실이다.

이 일반적인 프로토콜은 연구의 폭 넓은 다양한 플랫폼의 응용 프로그램을 지원하도록 설계되어,이 보고서에서 설명하는 대표적인 연구로 특정 측면은 괄호 밖으로 분리되어 그림 2 프로토콜을 사용하여 수행 대표적인 연구 결과를 보여줍니다.. 프로토콜에서 모든 &quot;지시&quot;명령은 미리 정해진 스크립트를 사용하여 자동화되어 있습니다. 2 단계는 1 단계 (단계 1.7 및 1.8시 등)와 병렬로 수행 할 수도 있습니다, 그 단계 2.1은 종종 무시됩니다 (패턴 / 코팅 DMF 장치 플레이트를 세척하고 재사용 할 수 있기 때문에, 단계 5.2 참조) . 1. 세포 관류 챔버를 조립하고 채우기 <ol><li> 코트 살균 (멸균) microcapillaries (융합 된 실리카 15cm 길이; 679 μm의 외경, 534 μm의 ID)의 내부 표면 살균 피브로넥틴 용액 (50 ㎍ / ㎖의 30 μl를) 그릴 주사기 펌프를 지시하여 피브로넥틴과 함께, 각에microcapillary,, (37 2 시간 동안 ° C) 잠복기 솔루션을 철회 할 수있는 주사기 펌프를 지시하고, microcapillaries 건조 (6 시간 실온 (RT))시키는. </li><li> CapTite 피팅 (6 재관류 챔버, 8 포트 주사기 펌프)를 사용하여 폴리 카보 네이트 튜브 (500 μm의 외경, 100 μm의 ID) 및 멀티 포트 주사기 펌프 튜브에 각 피브로넥틴 코팅 microcapillary (재관류 챔버)를 연결합니다. </li><li> 테이프를 사용하여 원하는 온도로 설정 열 블록 (37 ° C) 각 재관류 챔버의 본체를 고정합니다. </li><li> 저수지의 재관류 챔버의 열린 끝 (물론 microcentifuge 튜브 또는의 microtiter 플레이트) 신선한 성장 매체에 정지를 포함하는 세포 (P388D.1 쥐 대 식세포) (RPMI 1640, 10 % FBS, 500 단위 / ML 페니실린, 500 μg 빠져 / 원하는 농도 (10 6 세포 / ML)에서 ML 스트렙토 마이신). </li><li> 충분히 AI 다음 각 재관류 챔버에 세포를 (10 μL, 10 4 세포) 그릴 주사기 펌프를 지시R은 열 블록에 고정 섹션에 액체 플러그 (~ 4.5 cm)를 이동합니다. </li><li> 세포 관류 실의 fibronectin의 코팅 내부 표면 (- 2 시간 37 ° C 1)을 준수 할 수 있습니다. 한편, 세포 저수지에서 신선한 성장 매체를 포함하는 새로운 저수지 재관류 챔버의 열린 끝을 전송할 수 있습니다. </li><li> 준수 기간 후에, 낭비 새로운 배지 (10 μL)을 철회하고, 준수를 통해 새로운 액체 플러그 (새로운 배지)를 위치하기에 충분한 공기를 따라 액체 플러그 (에어컨 미디어 및 무소속의 세포)를 보낼 주사기 펌프를 지시 세포. </li><li> 세포 인구가 (- ~ 1,000 세포 / 챔버의 마이크로 스케일의 문화 생성 인구의 24 시간 16) 충분히 평형 및 확장 될 때까지 일정한 간격 (매 2 시간)에서 매체 교환 (즉, 단계를 반복 1.7) 수행을 계속합니다. </li></ol> 2. DMF 장치를 조작하고 허브 아키텍처를 조립 <ol><l난&gt;으로 십육 인듐 주석 산화물 (ITO) 포토 리소그래피와 에칭 전극 (물방울 작동의 드라이버), 5 파릴 렌-C의 μm의 50와 코트 DMF 장치의 바닥 판을, 이전에 14,29 설명한 패턴 테플론 AF의 뉴 멕시코. 위와 같이, 코팅 테플론 AF로 패터닝되지 않은 ITO 유리 기판을 DMF 장치의 상단 덮개를 형성한다. </li><li> 이 간격, 작동 전극의 크기 (2.5 mm 2)와 함께 물방울 볼륨 (2.5 μL를 정의 금속 압축 프레임을 사용하여 판 사이 400 μm의 간격을 유지 오목과 폴리머 캐스트 14,30으로 DMF 플레이트를 고정 전극 당). DMF 어셈블리가 단순한 수단 (24)에 의해 수행 할 수 있습니다. </li><li> 삽입 테프론 코팅 전송 microcapillaries (360 μm의 외경, 100 μm의 아이디, 3.5-4.0 ㎝ 길이) DMF 플레이트 사이의 공간에, 그 동족 작동 전극의 가장자리로 확장하는 각각의 위치. </li><li> 반대 EN에각 전송 microcapillary 부착 CapTite 피팅 라. </li><li> ITO 전극에 전압을 공급하는 전원 커넥터를 갖습니다. 전극 활성 시퀀스는 미리 정해진 스크립트를 실행하는 컴퓨터 제어 전자 인터페이스를 사용하여 자동화되어 있습니다. </li><li> 옵션 : 물방울의 추적을 용이하게하기 위해 전하 결합 소자 (CCD) 카메라 (DCR-HC96 (소니, 일본))가 장착 된 현미경의 번역 단계 (SZ-6145TR (올림푸스, 일본))에 조립 DMF 허브로 이동 . 31 </li></ol> 3. DMF 허브로 재관류 챔버를 연결하고 세포 집단을 자극 <ol><li> 중간 저수지에서 재관류 챔버의 열린 끝을 (단계 1.6 참조)를 제거하고 DMF 허브의 전송 microcapillaries (단계 2.4 참조)의 끝 부분에 부착 된 CapTite 피팅에 삽입. </li><li> 낭비 재관류 챔버의 액체 플러그 (에어컨 미디어)를 보낼 주사기 펌프를 지시합니다. </li><li> 이달온 - 보드 저수지에서 (E. 대장균은 0.1 % 플루로 닉 F127 W / V 신선한 배지에서 100 ㎍ / ㎖에서 BioParticles) 자극을 그리고 각 전송 ​​microcapillary에 적절한 크기 (10 μL)의 비말을 제공하기 위해 DMF 허브를 ruct . 온 - 보드 저수지는 원통 모양 구획 (1.5 × 1.5 cm 각)으로 구성되어 있으며 튜브를 통해 DMF 허브에 연결되어 있습니다. </li><li> 자극이 전달 microcapillaries에 연결 그려 재관류 챔버의 세포 집단에 플러그를 배치하기에 충분한 공기를 따라 주사기 펌프를 지시합니다. </li><li> 자극과 세포를 (- 4 시간 37 ° C 1) 품어. 선택 사항 : 정기적으로 신선한 자극 (즉, 단계를 반복 3.2-3.4)에 대한 환율. </li></ol> 4. 자극을 종료 및 분석을위한 세포 용 해물을 복구 <ol><li> 옵션 : 포스트 자극 기간이 필요한 경우 (즉, 단계를 반복합니다 3.2 새로운 매체에 대한 자극 함유 액체 플러그를 교환 - 3.4) 자극에 대한 새로운 매체를 대체하고, 배양하고 필요에 따라 교환을 계속한다. </li><li> 환율 세척 버퍼 (전주곡 직접 용해 모듈의 버퍼 B) (즉, 단계를 반복 3.2-3.4, 자극에 대한 세척 버퍼를 대체)에 대한 재관류 챔버의 액체 플러그, 그리고 (5 분)이 필요 품어. </li><li> 교환 용해 용액의 액체 플러그 (버퍼 세척) (전주곡 직접 용해 모듈의 버퍼 0.1 % 플루로 닉은 F127 W / V와 함께) (즉, 단계를 반복 3.2-3.4, 자극에 대한 용해 용액을 대체) 및 배양이 필요 (5 분) . </li><li> DMF 허브에 액체 플러그 (세포 용 해물)를 보낼 주사기 펌프를 지시합니다. </li><li> DMF 허브를 분해, DMF 장치 (이것은 물방울 혼합을 초래할 수 있으므로, 옆 판을 기울 않도록주의를 사용하여)의 상판을 제거하고 오프 플랫폼 분석을 위해 Pipetman이나 주사기를 사용하여 해물 (유전자 발현 프로파일을 수집 qPCR의를 통해). </li></ol> 5. 클린 플랫폼 하드웨어다시 사용 <ol><li> RT에서 10 분 동안 10 %의 표백제에 전송 microcapillaries 및 CapTite 피팅 (물에 v / v)의 몰두, 질소 가스를 이용하여 증류수, 드라이 잘 헹구십시오. </li><li> RT에서 10 분 동안 10 %의 표백제 DMF 장치 플레이트를 담가, 이소프로판올 잘 씻어은 탈 이온수 다음, 건조하여 질소 가스를 10 분 동안 160 ° C에서 베이킹에 의해 따랐다. </li><li> 주사기 펌프의 폴리 카보 네이트 튜브 및 DMF 디바이스의 폴리머 캐스트 및 압축 프레임, 세척하지 않고 다시 사용할 수 있습니다. 재관류 챔버 microcapillaries은 각 실험 후 삭제됩니다. </li></ol>

<ol>
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(33), e1603 (2009).</li><li>Thaitrong, 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=Quality+Control+of+Next+Generation+Sequencing+Library+through+an+Integrative+Digital+Microfluidic+Platform.">Quality Control of Next Generation Sequencing Library through an Integrative Digital Microfluidic Platform.</a> , (2012).</li><li>Shin, Y. J., Lee, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+vision+for+digital+microfluidics.">Machine vision for digital microfluidics.</a> Review of Scientific Instruments. 81, (2010).</li><li>Thornton, M., Eward, K. L., Helmstetter, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+minimally+disturbed+synchronous+cultures+of+hematopoietic+cells.">Production of minimally disturbed synchronous cultures of hematopoietic cells.</a> BioTechniques. 32, 1098-1105 (2002).</li><li>Huang, X., Dai, W., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforced+adhesion+of+hematopoietic+cells+to+culture+dish+induces+endomitosis+and+polyploidy.">Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy.</a> Cell Cycle. 4, 801-805 (2005).</li><li>Raje, M., 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=Charged+nylon+membrane+substrate+for+convenient+and+versatile+high+resolution+microscopic+analysis+of+Escherichia+coli+&amp;+mammalian+cells+in+suspension+culture.">Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli &amp; mammalian cells in suspension culture.</a> Cytotechnology. 51, 111-117 (2006).</li><li>Chatterjee, D., Hetayothin, B., Wheeler, A. R., King, D. J., Garrell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+microfluidics+with+nonaqueous+solvents+and+solutions.">Droplet-based microfluidics with nonaqueous solvents and solutions.</a> Lab Chip. 6, 199-206 (2006).</li><li>Perroud, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic-based+cell+sorting+of+Francisella+tularensis+infected+macrophages+using+optical+forces.">Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces.</a> Analytical Chemistry. 80, 6365-6372 (2008).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+A+revolutionary+tool+for+transcriptomics.">RNA-Seq: A revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10, 57-63 (2009).</li><li>Malone, J. H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, M., 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=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, 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=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., 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=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

우리는 마이크로 규모의 세포 배양과 자극 실험을위한 간단하고 다양한 자동화 플랫폼을 개발했습니다. 문화 크기는 더 작은 직경의 microcapillaries의 사용을 통해 줄일 수 있으며, 플랫폼은 우리가 작은 문화 볼륨 및 세포 인구 (- - 20 μL, 100 2000 세포 챔버 당 1 개)를 사용하여 작업 할 수 있습니다. 이러한 규모에서 작업 루틴 연구의 비용을 절감하고 귀중한 시약 및 / 또는 세포의 사용?...

(체외 분석) 문화 유지 세포의 연구는 복잡한 생물학적 시스템과 인간의 건강을 지배하는 기본 원리와 분자 메커니즘에 대한 귀중한 통찰력을 제공하고 있습니다. 문화, 자극, 그리고 페트리 요리의 microtiter 플레이트를 활용 분석을위한 세포의 수집을위한 기존의 방법은 밀리미터 규모의 문화 볼륨의 셀 (≥ 10 5) (≥ 0.1 ml)에 많은 인구 연구를 위해 설계되었습니다. 그러나, 세포의 제한된 양을 사용할 수있는 많은 경우 (예 : 일차 전지), 또는 세포의 작은 집단 (예를 들면 인구를 통해 휴대 전화의 변동성을 줄이기 위해) 바람직한, 또는 필요한 시약 얻기 어렵다 또는 엄청나게 비싼 (예 : 정제 된 세포 분비 인자). 이러한 문제를 성공적으로 배양 크기를 아래로 조정에 의해 해결 될 수있는 모든 소비를 줄이는 이점이 있습니다시약은 체외 분석 1,2에 필요합니다. 불행하게도, 기존의 장비와 방법은 마이크로 스케일의 문화와 3-11이 너무 복잡하고 대부분의 실험실에 의해 일상적인 사용을위한 전문화되어 현재 사용할 수있는 미세 유체 기반의 자동화 된 시스템의 정확하고 재현성 조작을 지원하지 않습니다. 마이크로 스케일 볼륨에서 (1 - 20 μL) -이 보고서에서, 우리는 자동으로 문화, 자극, 세포의 작은 인구 (2,000 세포 100)의 복구를위한 간단하고 다양한 기술 플랫폼의 조립 및 사용을 설명합니다. 피브로넥틴 코팅 microcapillaries는 ( &quot;세포 관류 실&quot;모듈) 마이크로 스케일 문화의 확립, 유지 보수 및 자극에 대한 사이트 역할의 집합, 그리고 디지털 마이크로 유체 (DMF : 플랫폼 아키텍처 (그림 1) 모듈 식 디자인 &quot;전송&quot;microcapillaries ( &quot;중앙 허브&quot;모듈) 14,15 노선 세포 장착) 12,13 장치과하고 재관류 챔버에서 시약. DMF는 사용자가 개별적으로 동시에 여러 방울을 해결하고 장치 하드웨어를 변경하지 않고 조작을 (예 : 재구성 샘플 처리 기차)를 변경하거나 다시 주문 할 수 있습니다. 그 엄청난 유연성 세포 배양 16, 17, 효소 분석 18,19, 면역 20,21, DNA 분석 22,23, 단백질 처리, 24,25 등 다양한 애플리케이션의 핵심 기술로 최근의 출현에 분명하다 임상 검체 처리. 26,27 우리의 중앙 허브는 DMF 장치에 내재 된 유연성을 활용, 더욱 전문화 된 주변 장치의 조작의 일부를 (예를 들어, 세포 배양) 수행 할 기회를 제공 microcapillary 인터페이스의 추가를 통해 향상 오히려 DMF 장치 자체에보다 모듈. 이런 방식으로 처리 열차의 구획화는 괞 찮아의 설계를 단순화TForm 클래스 아키텍처 (모든 처리 단계를 수행 할 수 DMF 장치를 구축 할 필요 없음) 새로운 함수로의 진화를 촉진는 (단순히 필요에 따라 새로운 주변 장치 모듈을 통합)가 필요합니다. 중앙 허브에서 세포 및 시약의 수송은 DMF 장치 13,28 내에 전극을 순차적으로 활성화에 의해 생성 된 힘 전기 습윤에 의해 구동됩니다;에서 전송로 및 재관류 챔버 내에서 고정밀 주사기에 의해 생성 된 압력 변화에 의해 제공됩니다 펌프. 이러한 유체 움직임의 모든 간단한 전자 인터페이스를 통해 제어되고 미리 정해진 스크립트를 사용하여 자동화되어 있습니다. 대표적인 예로서, 우리는 박테리아의 반칙으로 (그림 2)에 따라 면역 세포에 이끌려 전사 반응의 연구를위한 플랫폼을 사용하는 방법을 보여줍니다. 플랫폼에서 이러한 실험을 수행하는 것은 우리가 세포의 작은 숫자 (experimen 당 ~ 1,000 작동 할 수있게탈 조건), 실험 - 투 - 실험 다양성, 보존 시약 및 노동 손에 다시 직접을 최소화합니다. 그것이 부여하는 장점뿐만 아니라 접근성과 다양성을 감안할 때,이 플랫폼은 실험실과 응용 프로그램의 다양한 사용을 발견하고 제한된 수량에서 사용할 수있는 세포와 자극의 분석을 용이하게 특히 유용합니다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

문화의 세포 (체외 분석)의 연구는 복잡한 생물학적 시스템에 중요한 통찰력을 제공하고 있습니다. 생체 분석을위한 기존의 방법과 장비가 잘 밀리미터 규모의 볼륨 (≥ 0.1 ml)에 세포 (≥ 10 5) 다수의 연구 적합합니다. 그러나, 관심 및 / 또는 그들의 문화, 자극, 또는 처리에 필요한 시약 세포의 소비를 줄이기 위해 문화 크기를 확장 할 필요하거나 바람직되는 많은 경우가 있습니다. 불행하게도, 기존의 접근 방식은 마이크로 스케일 문화의 정확하고 재현성 조작을 지원하지 않으며, 현재 사용 가능한 미세 유체 기반의 자동화 된 시스템이 너무 복잡하고 대부분의 실험실에 의해 일상적인 사용을위한 전문입니다. 마이크로 스케일의 볼륨 -이 문제를 해결하기 위해, 우리는 자동화 된 문화, 자극, 세포의 작은 인구 (2,000 세포 100)의 복구를 위해 간단하고 다양한 기술 플랫폼을 개발했습니다S (1 - 20 μL). 이 플랫폼은 마이크로 스케일 문화를 확립, 유지 및 자극하는 내 fibronectin의 코팅 microcapillaries ( &quot;세포 관류 실&quot;)의 집합으로 구성되어 &quot;전송&quot;microcapillaries ( &quot;중앙 허브에 장착 된 디지털 마이크로 유체 (DMF) 장치 &quot;)하고 재관류 챔버에서 루트 세포 및 시약되는, 힘의 재관류 챔버와 중앙 허브 사이의 자료 전송 고정밀 주사기 펌프, 어떤, 재료의 전송 제어 기능을 제공하는 전자 인터페이스입니다 조정과 미리 정해진 스크립트를 통해 자동화. 예를 들어, 우리는 박테리아와 도전에 면역 세포에 이끌려 전사 반응의 연구를 촉진하는 플랫폼을 사용했다. 플랫폼의 사용은 우리가, 세포 및 시약의 소비를 줄이는 실험 - 투 - 실험 변동을 최소화 할 수있게하고, 다시 직접 손에 노동. 그것이 부여하는 장점뿐만 아니라 접근성과 다양성, 오 부여UR 플랫폼은 실험실과 응용 프로그램의 다양한 사용을 발견하고 제한된 수량에서 사용할 수있는 세포와 자극의 분석을 용이하게 특히 유용합니다.

자동화 플랫폼의 데모로, 우리는 박테리아 도전 면역 세포의 작은 인구는 마이크로 규모의 문화에서 성장했다하는 연구를 수행하는 데 사용되며 (그림 2 프로 염증 반응의 오프 플랫폼 분석을 위해 용해 ). 여섯 세포 관류 챔버의 각 성장 매체의 10 μL에 현탁 10 4 면역 세포 (P388D.1 생쥐 대 식세포) 씨앗을 품고 있었다. 준수 기간 (37 ℃에서 2 시간 동안 C)와 ...

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A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

우리는 마이크로 스케일 세포 자극 실험에 자동 세포 배양 및 심문 플랫폼을 개발했습니다. 플랫폼은 양성 세포의 작은 인구를 자극 분자 분석을위한 해물을 복구하는 간단한 다목적 및 정밀한 컨트롤을 제공합니다. 플랫폼은 물론 귀중한 세포 및 / 또는 시약을 사용하는 연구에 적합합니다.

마이크로 스케일 세포 자극 실험을위한 다목적 자동화 플랫폼

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K 조회수.  Sandia National Laboratories. 우리는 마이크로 스케일 세포 자극 실험에 자동 세포 배양 및 심문 플랫폼을 개발했습니다. 플랫폼은 양성 세포의 작은 인구를 자극 분자 분석을위한 해물을 복구하는 간단한 다목적 및 정밀한 컨트롤을 제공합니다. 플랫폼은 물론 귀중한 세포 및 / 또는 시약을 사용하는 연구에 적합합니다.

비디오: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT : 바이오 매스의 효소 발견과 칵테일 최적화를위한 자동 플랫폼

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

연구

생체공학

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

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

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

세포와 조직 Mechanobiology의 응용 프로그램에 대한 새로운 스트레칭 플랫폼

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

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

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

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

높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

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

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

계층화 된 알긴산를 구축 : 이기종 세포 인구의 공동 문화를위한 플랫폼

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

세포 분화 및 견인 힘의 상관 관계 분석을위한 높은 처리량 세포 마이크로 어레이 플랫폼

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

패터닝 단백질과 세포의 다목적 방법

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Semiflexible 고분자 공부를 다양 한 도구로 DNA 나노튜브

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

장기간 무세포 유전자 발현의 전도를 위한 다층 미세유체 플랫폼

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

동적 압축으로 연골 세포 자극을 위한 미세 유체 플랫폼

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...