이 작품은 로우 랜드 주니어 펠로우 프로그램에 의해 지원됩니다. 크리스 스톡스 주문 제작, 컴퓨터를 이용한 압력 제어 설정, 박사 다이앤 Schaak의 개발에 그의 도움을 : 저자는 하버드 로우 랜드 연구소의 과학자들과 직원에게 감사의 말씀을드립니다 생물학적 시료 처리에 대한 그녀의 입력, 윈필드 힐에 대한 전기 설치, 박사 Alavaro 산체스 개발 압력 설정에 필요한 기계 배관 부품 가공에 대한 흐름 cytometer, 스콧 Bevis, 케니 스펜서 돈 로저스에 대한 액세스 권한을 부여합니다. 미세 유체 주인은 하버드 대학 나노 시스템을위한 센터 (CNS)에서 제작 하였다.

1 셀 준비 <ol><li> 접시 1 × 10 10 % (v / v) 소 태아 혈청과 1로 보충 Leibovitz의 L-15 중간에 조직 배양 용 T75 플라스크 당 10 ㎖의 부피 전이성 유방암 세포주 MDA-MB-231의 5 세포 / ml % 페니실린 - 스트렙토 마이신. </li><li> 0 % CO 2 환경과 37 ° C에서 가습 배양기에서 MDA-MB-231 세포를 품어. </li><li> 2 분 동안 0.25 % 트립신-EDTA로 세포를 처리함으로써 이일 파종 후 실험 수확 세포 및 성장 배지 8 ㎖에 첨가함으로써 트립신의 활성 효소를 불 활성화. </li><li> 5 × 105 세포 / ml의 최종 농도 (칼슘 및 마그네슘 2 +없이 DPBS, 1X) 둘 베코 인산염 완충 식염수를 200 × g 및 재현 탁에서 5 분 동안 원심 분리하여 세포 펠렛. </li></ol> 2 장치 설계 및 제작 참고 : 마스크, 마스터 몰드 날조와마이크로 채널 클로징 처리는 폴리 디메틸 실록산 (PDMS) 미세 주조법이 일반 실험실 벤치 탑상에서 수행 될 수있는 동안 클린 룸 안에서 실시하여야한다. <ol><li> AutoCAD를 그림 1에 나타낸 바와 같이 미세 유체 장치를 설계합니다. 이 장치는 (L = 7mm W = 40 μm의, 그리고 H = 70 μm의) 여러 주입 포트, 거친 필터와 두 개의 평행 한 직선 직사각형 관성 중심 채널 입구로 구성되어야합니다. 개별 직선 채널은 알루미늄 전극 구멍을 통해이와 떨어져 800 μm의 배치 5 일렉트로 챔버 (W C = 400 μm의)로 구성되어 있습니다. 두 횡 방향으로 인접하는 일렉트로 챔버 음극 용 구멍을 공유한다. 두 개의 직선 채널은 일렉트로 지역의 하류에있는 콘센트에 병합합니다. </li><li> LinkCAD를 사용하여 GDSII 파일에 마이크로 패턴 CAD 파일을 변환합니다. 빈 5 &quot;× 5&quot;포토 마스크에 미세 패턴을 작성레이저 마스크 라이터. 제조 업체의 프로토콜에 따라 마스크를 개발한다. 참고 : 마스크 개발 프로세스는 포토 레지스트 현상, 크롬 에칭 및 레지스트 제거를 포함한다. 대안 적으로, 마이크로 패턴을 고해상도 투명도에 인쇄 (&gt; 20,000 DPI)를 타사 메이커로부터 구입하고, 포토 마스크로 사용될 수있다. 포토 마스크에 최종 마이크로 기능은 네가티브 포토 레지스트의 극성이 일치하도록 투명해야한다. </li><li> 70 μM의 최종 두께를 갖도록 2000 rpm에서 4 인치 실리콘 웨이퍼 상에 네거티브 포토 레지스트를 스핀 코트 표준 소프트 리소그래피 기술 (23)을 사용하여 장치 설계의 주형을 제조. </li><li> 95 ° C에서 20 분 동안 네가티브 포토 레지스트와 웨이퍼를 프리 베이킹. </li><li> 웨이퍼와 마스크를 연락 마스크 얼 라이너를 사용하여 80 초 동안 UV 소스 (17.4 엠제이 / cm 2)에 노출. </li><li> SU-8 개발에 4 분 동안 노출 된 웨이퍼를 개발한다. </li><li> 측정표면 프로파일로 미터를 사용하여 개발 된 포토 레지스트 두께. </li><li> 제 (실리콘 엘라스토머 키트) 경화 엘라스토머 1 비율 및 PDMS 복제본을 생성하는 주형에 부어 혼합물 : 엄격 10 섞는다. 30 분 동안 주조 엘라스토머를 탈기하고 2 시간 동안 70 ° C의 오븐에서 탈기 엘라스토머 주형을 경화. </li><li> 핀 바이스를 사용하여 입구, 출구 및 전극 삽입을위한 금형 및 펀치 구멍에서 마이크로 패턴과 경화 된 PDMS 복제를 박리. NOTE : 핀 바이스의 통상 절삭 날 직경 단단히 대신 PEEK 튜빙 (32 분의 OD &quot;) 및 알루미늄 전극 (0.029의 OD&quot;)를 유지하기위한 충분한 0.76 mm이어야한다. </li><li> 각각 75 W, 500 mTorr로의 고주파 전력 및 산소 분압에서 7 초간 산소 플라즈마 클리너로 처리 된 유리 슬라이드에 결합 PDMS 복제본으로 둘러싸인 미세 유체 채널을 생성. </li></ol> 3 흐름 실험 <ol><li> 삽입알루미늄 전극 (0.029 &quot;OD) 및 출구 PEEK 튜빙 (32 분&quot;미세 구멍을 통해 지정된 장소에 OD)는 (그림 3B 참조). </li><li> 알루미늄 전극에 고전압 짧은 구형파 펄스를 발생하기위한 18 담당 전기 기기를 연결 PDMS 몰드에 흐르는 용액과 접촉하는 (도 3c 참조). 참고 :이 장비는 펄스 발생기 및 자체 내장 된 고전압 앰프로 구성한다 (18). </li><li> (도 3a 참조) 세포 및 생체 분자와 네 개의 50 ml의 원심 개별적 DPBS를 함유하는 튜브, 용액을 준비하고, 공기 유량 제어 시스템에 연결된 각각의 바이알 홀더에 각각 끼운다. 18 </li><li> 미세 유체 장치의 각 입구 구멍에 유리 병 홀더에서 유입 PEEK 튜브를 연결합니다. </li><li> 저런 전기를 갖기 위해 100 V로, 구형파 펄스 V의 크기를 설정LD 강도, E = V / L 전자, 전기 천공 챔버 걸쳐 0.7 kV로 / cm 동등. NOTE : 여기서, L 전자는 양극과 음극이 흐르는 용액과 접촉하는 두 점 사이의 거리 (도 1 참조). </li><li> 40 PSI에 압력 조절기를 설정합니다. NOTE :. 단일 수동 조절 질소원 균일 모든 샘플 튜브를 가압하기 위해 사용되며, 고속 매니 폴드 적시 맞춤식 LabVIEW 소프트웨어를 사용하여 각각의 용액 포트를 활성화하기 위해 이용되는 18 </li><li> 밸브 제어의 경우, 밸브 러너 레이블 주문 제작 LabVIEW 소프트웨어를 열고 운영한다라는 제목의 드롭 다운 메뉴에서 실행을 클릭합니다. </li><li> 해당하는 각 밸브 아이콘을 클릭하여 밸브를 켭니다. 참고 :이 활성화 될 때 밸브의 아이콘이 녹색으로 변합니다. 활성화 된 아이콘을 클릭하면 비활성화하고 밸브를 닫습니다. 닫힌 밸브 아이콘이 회색으로 설정해야합니다. </li><li> (DPBS 저수지에 밸브를 열고즉., 1.5 분 동안 안정한 세포 트래핑 와류 발생에 필요한 주요 흐름 속도로 세정액). </li><li> 30 초 (즉, 셀 - 포착 공정)를위한 전기 천공 챔버에서 트랩 세포에 세포 용액으로 세척 용액으로부터 활성 용액 포트 스위치. </li><li> 용액 스위칭 단계 동안 파쇄 흐름을 보장하기 위해 셀 - 포착 단계 이전에 10 초 동안 동시에 장치를 통해 모두 세탁 셀 솔루션을 흐른다. 참고 :이 간단한 공동 흐름 단계는 각 솔루션 전환 단계를 반복해야합니다. </li><li> 세척 포트 켜고 비 오염 포집 세포를 제거하기 위해 20 초 동안 세척 장치. </li><li> 관심 장치로 (3000 다 중성 및 음이온 덱스 트란 분자 또는 1.5 밀리그램 / 70000 다 중성 덱스 트란 분자 ml의 0.2 ㎎ / ㎖)의 제 분자를 함유하는 용액을 주입한다. </li><li> 즉시 주사 후 5 쇼트 펄스 (Δt를 2 초 간격으로 30 밀리 초 =) 적용분자 용액 및 오실로스코프를 사용하여 실시간으로 적용되는 전기 펄스의 크기 및 지속 기간을 모니터링한다. </li><li> 3.10만큼 추가 분자의 수와 같은 단계를 반복 배달 할 수 있습니다. 각 분자 납기 후 분자 용액에 100 초 동안 세포를 배양한다 (100)의 과잉 물질을 제거하기위한 세척 용액으로 세척 장치. </li><li> 5 이하 PSI 작동 압력을 낮춤으로써 하류 분석 용 96 - 웰 플레이트에서 세포를 릴리스. 참고 : 100 셀 솔루션의 약 100 ㎕의 각 릴리스에서 수집됩니다. </li><li> 실험은 유동 세포 계측법을위한 충분한 세포를 수집하는 3.16 세 시간을 통해 3.9 단계를 실행합니다. </li><li> 228 × g에서 5 분 동안 처리 된 세포를 포함하는 96 - 웰 플레이트를 원심 분리기. </li><li> 초과 형광 분자를 포함하는 상층 액을 제거합니다. </li><li> 유동 세포 계측법 분석을 위해 DPBS 세포를 재현 탁. </li><li> 분자 U에 대한 흐름 cytometer에 로드셀효율성 분석 ptake. </li></ol>

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M., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vortex-assisted+DNA+delivery.">Vortex-assisted DNA delivery.</a> Lab Chip. 10, 2057-2061 (2010).</li><li>Jia, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonviral+minicircle+vector+for+deriving+human+iPS+cells.">A nonviral minicircle vector for deriving human iPS cells.</a> Nature Methods. 7, U146-U197 (2010).</li><li>Dunbar, 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=Gene+transfer+to+hematopoietic+stem+cells+Implications+for+gene+therapy+of+human+disease.">Gene transfer to hematopoietic stem cells Implications for gene therapy of human disease.</a> Annual Review of Medicine. 47, 11-20 (1996).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Angew Chem-Int Edit. 37, 551-575 (1998).</li><li>Graziadei, L., Burfeind, P., Barsagi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+Unlabeled+Proteins+into+Living+Cells+by+Electroporation+and+Isolation+of+Viable+Protein-Loaded+Cells+Using+Dextran+Fluorescein+Isothiocyanate+as+a+Marker+for+Protein-Uptake.">Introduction of Unlabeled Proteins into Living Cells by Electroporation and Isolation of Viable Protein-Loaded Cells Using Dextran Fluorescein Isothiocyanate as a Marker for Protein-Uptake.</a> Anal Biochem. 194, 198-203 (1991).</li><li>Dimitrov, D. S., Sowers, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Electroporation-Fast+Molecular-Exchange+by+Electroosmosis.">Membrane Electroporation-Fast Molecular-Exchange by Electroosmosis.</a> Biochimica Et Biophysica Acta. 1022, 381-392 (1990).</li><li>Sukharev, S. I., Klenchin, V. A., Serov, S. M., Chernomordik, L. V., Chizmadzhev Yu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+electrophoretic+DNA+transfer+into+cells.+The+effect+of+DNA+interaction+with+electropores.">Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.</a> Biophysical Journal. 63, 1320-1327 (1992).</li><li>Glogauer, M., McCulloch, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+large+molecules+into+viable+fibroblasts+by+electroporation+Optimization+of+loading+and+identification+of+labeled+cellular+compartments.">Introduction of large molecules into viable fibroblasts by electroporation Optimization of loading and identification of labeled cellular compartments.</a> Experimental Cell Research. 200, 227-234 (1992).</li><li>Verspohl, E. J., KaiserlingBuddemeier, I., Wienecke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+specific+antibodies+into+electropermeabilized+cells+is+a+valuable+tool+for+eliminating+specific+cell+functions.">Introducing specific antibodies into electropermeabilized cells is a valuable tool for eliminating specific cell functions.</a> Cell Biochemistry and Function. 15, 127-134 (1997).</li></ol>

저자가 공개하는 게 없다.

처리량 및 다중 분자 전달 효율의 새로운 병렬화 일렉트로 플랫폼, 10 배 향상된 기능을 통해 이전에 개발 된 단일 챔버 시스템이 제공하는 모든 장점에 부가하여 달성되었다. 18 이전 가능한 장점은의 (ⅰ) 전처리 정제를 포함 생존 능력 향상을위한 균일 한 크기 분포, (ⅱ) 정확한 분자량 및 개별 투여 량 조절, 및 (ⅲ) 낮은 동작 전류와 표적 세포. 그들은 분자량, 공액 형광 유형 및 전하의 ...

최근, 세포 분자의 세포 내 전달을 용이하게하는 전기 펄스의 사용은 포유 동물 세포를 조작 매력적인 수단이되었다. 일 또한 일렉트로이라고하는이 과정은, 가역적으로 액세스하는 본래 세포막 투과성 분자를 허용 세포막을 permeabilizes 세포의 세포 적 환경이다. 거의 모든 분자 일렉트로를 이용한 세포의 모든 종류의 막으로 임시 만들어진 기공을 통해 세포질 내로 도입 될 수 있기 때문에, 기술이보고 된 것으로, 더욱 재현성 보편적으로 적용하고, 바이러스 - 매개 화학을 포함하는 다른 방법에 비해보다 효율적 세포 생존은 그대로 유지하면서, 광학 접근법. 2-3이 기술은, 형광 물질을 도입하는 약물 4 5 핵산 6-7을 이용하고있다. 이러한 이점을 감안할 때, 일렉트로는 일반적인 노동으로 채택되었다DNA 형질 atory위한 기술, 생체 내 유전자 치료 8 세포 접종 연구. 성공적인 일렉트로 필요한 전계 강도가 밀접 셀의 직경과 상관하기 때문에 종래의 전기 천공 시스템은 동시에 대형화와 이질 샘플 실용적인 효율 및 생존 가능성을 달성하는 것은 여전히​​ 그러나 어렵다. 더욱이, 이러한 시스템은 다수의 분자 양의 정밀한 제어가 벌크 스토캐스틱 분자 전달 프로세스에 대한 의존도로 인해 전달되는 것을 허용하지 않는다. 9는 이러한 문제를 해결하기 위해, 많은 그룹은, 저급 CORPORATION에서 전압의 이점을 제공하고, 미세 유체 일렉트로 플랫폼 개발 더 형질 감염 효율, 세포 사망의 큰 감소, 및 복합 분자를 제공하는 능력. 10-13 이러한 장점은 그 전극 피치 마이크로 전기 천공 시스템의 작은 풋 프린트에 의해 가능하게되었다길이 극적 성공적인 전달을 위해 필요한 전압을 감소시키는, 서브 밀리미터이다. 더욱이, 이들 마이크로 일렉트로 시스템은 전달 효율을 향상하면서 감소 된 세포 사망을 산출, 생성 된 열을 방출 빠르게 균일 한 전계 분포를 달성 할 수있다. 추가적으로 이러한 마이크로 투명 재료 사용률 프롬프트 파라미터 변경에 대한 전기 천공 과정 시츄 관찰에 있습니다. 2,12을 그러나 정확한 투여 량 제어 및 연구 및 치료 적 응용, 6 신흥 필요한 분자 - 및 세포 - 의존 파라미터 제어를, 14-16는 여전히 해결되지 못한 상태로 남아있다. 이 작업은 순차적으로 표적 세포의 미리 선택된 동일한 집단에 여러 분자를 전달할 수있는 미세 유체 와류를 이용한 전기 천공 시스템을 제공한다. 균일 한 크기 분포를 가진 세포는 이전에보고 된 실리콘을 사용하여 이전에 전기 천공 법 (electroporation)에 격리되어제 &#39;선택적 트래핑 메커니즘. 17-18 균일 한 크기 분포, 적은 편차 일렉트로 효율 및 달성 하였다 주어진 전계 강도 당 강화 가능성을 갖는 바이. 19 또한, 연속 마이크로 와류를 사용하여 포획 세포를 교반을 통해 분자의 균일 한 전달 허용 결과와 일치하여 전체 세포질은, 이전에,이 시스템은 일반적으로 생물학적 인 응용에 이용 분자의 넓은 범위에 적용될 수 있음을 입증하기 위해. 다른 와류 이용한 일렉트로 플랫폼을 사용하여 (20)을보고로 전달 된 분자량 광범위한 고분자 전이성 유방암 세포. 또한, 실시간 프로세스 모니터링의 도움으로,이 작품은 주로 확산 매개. 14 대 전기 매개되고, electrporation 동안 분자 전달의 메커니즘에 관한 오랜 논쟁에 종지부를 찍을 더 많은 증거를 제공 </sup&gt; 다른 전기 천공 시스템과는 달리,이 플랫폼 고유 일렉트로의 정확한 멀티 분자 전달, 고분자 전달 효율, 최소의 세포 사망, 크기 및 전달 분자의 전하의 넓은 범위뿐만 아니라 실시간 시각화 결합 된 장점을 제공한다 과정. 이러한 기능을 감안할 때, 개발 된 일렉트로 시스템은 일렉트로 분자 전달 메커니즘에 대한 깊이있는 이해를 필요로하는 세포 리 프로그래밍 연구, 6,14,21-22 약물 전달 응용 프로그램 10,19 및 애플리케이션을위한 다양한 도구로 실제적인 가능성이있다.

<table><tbody><tr><td>MDA-MB-231 cancer cell line</td><td>American Type Culture Collection (ATCC)</td><td>HTB-26</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 Medium</td><td>Cellgro, Mediatech, Inc.</td><td>10-045-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Cellgro, Mediatech, Inc.</td><td>21-030</td><td></td></tr><tr><td>Trypsin</td><td>Gibco, Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Flow Cytometer easyCyte HT</td><td>Millipore</td><td>0500-4008</td><td></td></tr><tr><td>Oxygen Plasma Cleaner</td><td>Technics Micro-RIE</td><td></td><td></td></tr><tr><td>Dektak 6M surface profiler</td><td>Veeco</td><td></td><td></td></tr><tr><td>KMPR 1050</td><td>Microchem</td><td></td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Compressed Nitrogen gas</td><td>Airgas</td><td>NI 300</td><td></td></tr><tr><td>High Pressure Regulator</td><td>McMaster-Carr</td><td>6162K22</td><td></td></tr><tr><td>Downstream regulator</td><td>McMaster-Carr</td><td>4000K563</td><td></td></tr><tr><td>High-speed 3/2way-8 valve manifold</td><td>Festo</td><td></td><td></td></tr><tr><td>Inline Check Valve</td><td>Idex Health and Science</td><td>CV3320</td><td></td></tr><tr><td>5/32&quot; OD x 3/32&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-156F-0</td><td></td></tr><tr><td>1/4&quot; OD X 0.17&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-250PB-4</td><td></td></tr><tr><td>1/16&quot; PEEK tubings</td><td>Festo</td><td>P1533</td><td></td></tr><tr><td>1/32&quot; PEEK tubings</td><td>Idex Health and Science</td><td>P1569</td><td></td></tr><tr><td>PEEK tubing unions</td><td>Idex Health and Science</td><td>P881</td><td></td></tr><tr><td>Pulse Generator</td><td>HP</td><td>8110A</td><td></td></tr><tr><td>Aluminum Wire</td><td>Bob Martin Company</td><td>6061 ALUM</td><td></td></tr><tr><td>Oscilloscope</td><td>Agilent</td><td>DSO3062A</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>VWR</td><td>21008-216</td><td></td></tr><tr><td>T75 culture flask</td><td>VWR</td><td>82050-862</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 3,000 MW, Anionic</td><td>Gibco, Life Technologies</td><td>D3307</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>D1819</td><td></td></tr><tr><td>Dextran, Texas Red, 3,000 MW, Neutral</td><td>Gibco, Life Technologies</td><td>D3329</td><td></td></tr></tbody></table>

microscale vortex-assisted electroporator for sequential molecular delivery

물리적으로 세포에 비 - 투과 외인성 분자 프로브를 도입하기위한 아주 강력한 기술이기 때문에 전기 천공은, 과거 몇 년 동안 증가 주목을 받아왔다. 이 작업은 정밀한 분자 종속 매개 변수 컨트롤과 함께 포유 동물 세포에 여러 분자 전달을 수행 할 수있는 마이크로 유체 일렉트로 플랫폼을보고한다. 균일 한 크기 분포와 세포를 분리하기위한 시스템의 능력은 주어진 전계 강도 당 전기 천공 효율 적은 편차를 허용; 따라서 샘플 생존력을 향상. 또한, 그 공정 시각화 기능 효율 향상을위한 반응계에서 프롬프트 분자 전달 파라미터 조정을 허용 실시간 형광 분자 흡수 과정의 관찰을 허용한다. 보고 된 플랫폼의 광대 한 능력을 보여주기 위해, 서로 다른 크기와 전기 요금과 거대 분자 (예를 들면, 덱스 트란은 3000 및 70000 다의 MW로)했다테스트 된 모든 분자 높은 전달 효율 (&gt; 70 %)로 전이성 유방암 세포로 전달. 개발 플랫폼은 온 - 칩 기술은 일렉트로 유리할 수있다 연구 분야의 확장에 사용 가능성을 증명했다.

개발 병렬 미세 electroporator 전이성 유방암 살아있는 세포로 다양한 크기 및 전하와 거대 분자를 전달. 성공적인 전달 분자는 질적 인 시츄 electroporation하여 선회 세포의 형광 강도의 변화를 모니터링함으로써 결정과 유세포 분석을 통해 정량 측정에 의해 확인되었다. 4A가 처리 된 세포의 90 %가 중성 70000 다 덱스 트란 통풍 관 것을 보여준다. 통계적인 분석을 위해, ?...

Watch this Scientific Journal Video about Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery at JoVE.com

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

미세 유체 와류를 이용한 일렉트로 플랫폼은 정확하고 독립적 인 투여 량 제어와 동일한 세포 집단에 여러 분자의 순차적 전달을 위해 개발되었다. 시스템의 크기에 기초하여 목표 셀 정제 단계 이전 일렉트로 분자 전달 효율 및 처리 된 세포의 생존 능력을 향상시키는 데 도움.

마이크로 소용돌이를 이용한 순차 분자 전달을위한 Electroporator을

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

microscale-vortex-assisted-electroporator-for-sequential-molecular

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Bioengineering

8.6K 조회수.  Harvard University.  미세 유체 와류를 이용한 일렉트로 플랫폼은 정확하고 독립적 인 투여 량 제어와 동일한 세포 집단에 여러 분자의 순차적 전달을 위해 개발되었다. 시스템의 크기에 기초하여 목표 셀 정제 단계 이전 일렉트로 분자 전달 효율 및 처리 된 세포의 생존 능력을 향상시키는 데 도움. 

비디오: Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

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공학

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

음향 나노 디스펜싱 기술을 사용한 고처리량 DNA 플라스미드 멀티플렉싱 및 트랜스펙션

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

유전학

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

Microfluidic 장치에 수동 펌핑을위한 고속 비말 기반 전달 시스템

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

생물학

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

단일 셀 게놈 시퀀싱 초고 처리량 미세 플랫폼

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

생체공학

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

드롭릿 기반 미세 접근 및 단일 가닥 DNA Amplicons 위한 스피어 PCR 증폭

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

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 ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Craniofacial Mesenchyme의 Electroporation

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

전기 - 무료, 선형 핵산 및 단백질 분리

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

연속 흐름의 제조 및 작동, 투과화 감지 기능이 있는 미세 전기 천공 시스템

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

인간 소변에서 지속적인 세포외 소포 분리를 위한 미세유체 장치

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

저자 스포트라이트: Bioreactor Integration을 위한 비대칭 전계 흐름 분획

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

분기된 A4F 미세유체 장치를 사용한 대용량 시료에서 세포외 소포의 단일 단계 분리

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...