1. MEA 준비 <ol><li> 이전 18 설명 또는 멀티 채널 시스템과 같은 MEA 제조 업체의 공급 업체에서 직접 구입으로 electroporation 실험에 사용 MEAs는 두 표준 석판 술 기술을 사용하여 제조 될 수있다 <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> 및 알파 MED 과학 주식회사 <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)가</a> . 우리 실험에 사용 된 MEAs는 고체 전자 연구 (CSSER)의 센터에서 관리 애리조나 주립 대학의 클린 룸 시설에서 - 집에서 제조되었습니다. 우리 실험에 사용 된 MEAs가 잘 1cm 내경 유리 내의 50-200 μm의 전극 직경 크기 4 배열하여 8 32 인듐 틴 산화물 전극했다. 유리는 잘 MEA를 사용 polydimethylsiloxane (PDMS)에 접합되었다. </li><li> 셀의 이전 압력솥에 MEA를 검사eeding. 그들은 MEA의 무결성을 손상하지 않는 한, 이러한 70 %의 에탄올에 몸을 담글 수로 살균의 다른 방법, 다른 곳에서 설명 된 바와 같이 UV 치료 및 온수 치료도 사용할 수 있습니다. </li><li> 층류 흐름 후드에 autoclaved MEA를 전송하고 표준 멸균 폴리스티렌 페트리 접시에 배치합니다. 이전 MEA에 셀을 퍼 뜨리고하기 20 분에 UV 빛을 설정하여 판상 흐름 후드를 소독. MEA는 UV 빛 흐름 후드가 UV 조명되는 동안 멸균 알루미늄 호일로 MEA를 포함하는 페트리 접시를 커버에 민감합니다. </li></ol> 2. MEA에 심는 세포 <ol><li> 트립신 / EDTA (에틸렌 다이아 민 테트라 초산) 및 도금 1 ML 셀 미디어에서 100-200 셀 / μl의 농도로 높이를 사용하여 점착성의 포유류 세포에서 실행중인 셀 줄에서 수확 세포. 우리 실험에서는 배양 NIH 3T3 섬유 아세포 세포 라인, HELA 세포 라인과 MEA에 대한 기본 대뇌 피질과 hippocampal 뉴런에 성공했습니다. </li><li&gt; MEA의 세포와 미디어 20-30 μl 방울을 넣으십시오. 보육에 MEA와 페트리 접시를 전송합니다. 이 세포 드롭 크기가 MEA의 모든 전극을 커버 할 수있을만큼 큰 수 있도록하는 것이 중요합니다. </li><li> 세포가 층류 후드에 보육에서 MEA와 페트리 접시를 전송하고 부드럽게 잘 MEA에 사전 예열 미디어 400-500 μl (37 ° C)을 추가 퍼 뜨리고 후 2 시간. 그 세포가 여전히 부착 고르게 MEA 표면에 확산되도록하기 위해 현미경으로 확인합니다. 보육에 MEA을 다시 전송합니다. 미디어를 추가하기 전에 2 시간의 대기 기간은 세포가 MEA 표면을 준수 할 충분한 시간을 보장합니다. 너무 일찍 셀을 퍼 뜨리고 후 세포 미디어의 추가는 세포를 씻어 수 있습니다. 세포가 MEA에 잘 준수하지 않을 경우, 그 표면은 세포 접착을 향상시키기 위해 세포 부착 요인 등 aspolylysine, fibronectin과 laminin으로 처리 할 수​​ 있습니다. 우리 HELA 세포 실험에서 우리는 일반적으로 fibronecti과 MEA 표면 처리1-2 시간을위한 N (10 μg / ML). </li><li> 셀을 퍼 뜨리고 후 8-12 시간은 모든 죽은 세포를 제거하는 PBS로 1-2 세차를 수행합니다. 필요에 따라 셀 매체는 모든 24-48 시간을 대체 할 수 있습니다. 전형적인 실험에서 세포는 시딩 후 보통 transfection 24-48 시간에 대한 준비가되어 있습니다. electroporation 효율의 일관성을 유지하려면 그것은 문화가 electroporation 전에 합류 최소한 80 % 될 때까지 기다리는 것이 좋습니다. </li></ol> 3. siRNA의 특정 사이트 Transfection <ol><li> 핵산 솔루션을 준비합니다. siRNA의 transfection를 들어, 200-400 μl의 최종 볼륨을 달성하기 위해 얼음 차가운 electroporation 버퍼에 1-2 μM의 siRNA의 electroporation 솔루션을 준비합니다. 이 동영상 글에서 우리는 siRNA의 알렉사 488 복합 불가능한 시퀀스를 HELA 세포의 transfection을 보여줍니다. </li><li> 보육의 무균 층류 후드에 셀로 MEA를 전송합니다. MEA에서 세포 미디어를 제거하고 PBS로 세척을 수행합니다. 이후 나는의 200-400 μl를 추가합니다잘에 CE 추운 1 μM의 siRNA의 electroporation 솔루션입니다. </li><li> 대상 전극 (전기 펄스의 최적의 매개 변수를 결정하기위한 4 단계로 참조)에 양극 평방 전기 펄스를 적용 할 수 있습니다. 우리 실험에서는 전기 펄스와 MEA 본드 패드와 접촉을 할 듀얼 인라인 패키지 (DIP) 16 핀 클립을 생성하는 실용적인 악기주세요 2414A 파형 발생기를 사용했습니다. 파형 발생기는 각 전극의 선택성을 제공 할 수 demultiplexor 인쇄 회로 기판을 통해 DIP 클립에 연결 할 수 있습니다. 참조 전극은 세포 미디어에서 스틸 음극을 배치합니다. 미디어의 강 참조 전극을 낮추는 동안은 MEA 표면과 접촉하지 않도록 매우 중요합니다. 접촉되면, 그것은 세포와 MEA에 손상을 일으킬 수 있습니다. 그것은 세포 위의 1 mm의 위치에 대한 참조 전극을 낮출 것을 권장합니다. 1mm의 높이 스페이서는 심판의 위치에서 도움을 잘에 배치 할 수 있습니다erence 전극. 외부 참조 전극을 사용하는 다른 접근 방식은 음극 - 양극 쌍으로 주변 전극을 사용하는 것입니다. </li><li> 바로 MEA의 대상 전극에 전기 펄스를 적용한 후에, 보육에 MEA을 다시 전송합니다. 5 분의 잠복기 후, 부드럽게 사전 예열 (37 ° C) 셀 미디어와 electroporation 버퍼의 75 %를 교체하십시오. 그 후, 형광 이미징은 세포에 휘황 태그 siRNA의 전달을 확인 할 수 있습니다. </li></ol> 4. Electroporation 매개 변수 최적화 <ol><li> 펄스 진폭, 펄스 기간, 그리고 펄스의 수의 값의 범위에 대한 electroporation, 디자인 electroporation 실험을위한 최적의 전기 펄스 매개 변수를 결정합니다. 헬러 세포와 우리의 경험에서 우리는 관찰 그 미를 성공적으로 electroporation으로 이어 100 μm의 전극 크기에 대한 단일 전압 3 V 9 V에서 진폭의 펄스 및 밀리 초 (1 ~ 10) 밀리 초까지 기간nimal 세포의 죽음. 그것은 하나의 매개 변수가 한 번에 다양 할 수 있으며 다른 사람들이 지속적으로 유지하고있는 것으로 추천합니다. 펄스 매개 변수 각각에 대해 electroporation 효율을 quantitate하기 위해, 우리는 단계 4.2-4.5에서 설명 propidium 요오드화물 (PI), 셀 impermeant을 염색이 얼룩 핵산 물질, 라이브 분석 기반의 방법론을 사용합니다. </li><li> 1 단계와 2 단계에 설명 된 지침에 따라 MEA에 세포 배양을 설정합니다. </li><li> 이전 electroporation은 얼음 차가운 electroporation 버퍼에서 30 μg / ML PI 솔루션의 300-500 μl와 calcien는 PBS의 솔루션을 오전 4 μm의 400 μl를 준비합니다. </li><li> 보육의 층류 후드에 MEA를 전송합니다. MEA에서 세포 미디어를 제거하고 PBS로 세척을 수행합니다. 이후 PI과 함께 얼음 차가운 electroporation 버퍼의 300-500 μl를 추가합니다. </li><li> 대상 전극 (elec을 생성하고 적용하는 우리의 실험에 사용 된 장비에 원하는 펄스 진폭 및 기간과 전기 펄스를 적용MEA에 tric 펄스 단계 3.3)에 설명되어 있습니다. 참조 전극의 경우도 잘 MEA의 electroporation 버퍼에 강 전극을 낮추거나 참조로 인근에 전극을 사용합니다. 선택적으로 다른 전극으로 다양한 진폭 또는 기간의 펄스를 적용하여 여러 실험 같은 장치에서 수행 할 수 있습니다. 예를 들어, 32 전극 MEA에, 8 실험은 8 그룹, 4 전극으로 구성된 각 분할에 의해 32 전극을 수행 할 수 있습니다. 8 그룹의 각 다음 컨트롤로 사용할 수 있습니다 0.5 V 단위와 한 그룹에서 3 V-6 V 범위에서 다양한 진폭과 지속 시간 5 밀리 초의 단일 직사각형 전압 펄스에 의해 자극 될 수 있습니다. </li><li> 즉시 신청 후 전압 펄스가 5 분의 보육에 MEA를 전송합니다. 그 후, 부드럽게 잘 MEA의 electroporation 버퍼의 75 %를 제거하고 따뜻한 휴대 미디어로 교체. 보육에 MEA을 다시 전송합니다. </li><li> 2-4 시간의 잠복기 후4 μm의 300-500 μl 잘 MEA의 셀 미디어를 대체이 Calcein PBS의 솔루션입니다. 또 다른 30 ​​분을 위해 보육에 MEA을 다시 전송합니다. </li><li> Calcein AM과 세포 배양 후, PBS있는 두 개의 세차 후 PBS로 잘 MEA에 솔루션을 Calcein 오전 바꿉니다. 그리고 Calcein AM (여기 / 방출 - 490 nm/520 NM) - PI (535 nm/617 nm의 여기 / 방출)에 대한 필터를 광학 현미경으로 사용하는 세포의 형광 이미지를 얻을 수 있습니다. electroporation에 의한 PI의 이해는,에 세포가 빨간색 형광 및 Calcein AM 녹색 형광을 살아있는 세포의 원인이됩니다. 관찰 할 수 있습니다 세 가지 시나리오)) 살아 세포 electroporated하고 살아 (적색과 녹색을 모두 형광 것), 2) 세포 1아르하지만 electroporated (단지 녹색 형광합니다), 3하지 electroporation에 의한 죽은 세포는 (단지 빨간색 형광됩니다) . 세포 생존 능력은 전극에 살아 세포의 총 수의 퍼센트 electroporation의 EFF로 계산iciency는 PI / siRNA와로드 셀의 총 수의 비율로 계산되었다. </li></ol> 5. 대표 결과 그림 1은 PI와 헬러 세포의 특정 사이트 로딩을 보여줍니다. 이 전극에서만 세포가 PI (그림 1B)의 이해를 보여 관찰 할 수 있습니다. 라이브 검정이 게시물 electroporation은 세포 (그림 1A)의 생존 능력을 평가하는 데 사용 된 수행했습니다. 그림 1에 표시된 예를 들어, electroporation의 효율성과 세포 생존은 각각 81.8 %와 96.1 %입니다. 높은 세포 생존 능력과 electroporation 효율은 electroporation 펄스 매개 변수를 최적화하여 달성 될 수있다. 최적화 된 electroporation 펄스에 대한 휘황 태그 siRNA (8 V, 1 밀리 초)와 헬러 세포의 특정 사이트 transfection은 그림 2에 표시됩니다. 8 V, 1 밀리 초에서 siRNA의 electroporation 효율 ± 74.4이 발견 16.0 %와 한세포 생존 능력은 87.9 ± 8.4 % (평균으로 표시 모든 데이터가 ± 표준 편차, N = 5)으로 발견되었다. <img alt="그림 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> 그림 200 μm 전극 (6 V, 5 밀리 초)에 HELA 세포의 PI 1. 사이트 별 제공. A) Calcien 기반으로 실시간 분석은 2 시간 게시물 electroporation을 수행. 살아 셀은 녹색 형광으로 표시됩니다. B) 레드 형광이 전극에서 세포 PI의 이해를 보여줍니다. C) A와 B 흰색 화살표 중 겹쳐 이미지는 electroporated과 살아있는 세포를 나타냅니다. 검은 색 화살표는 죽은 세포를 나타냅니다. A, B 및 C의 흰색 원은 전극의 개요를 나타냅니다. <img alt="그림 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> 그림 2. 휘황 태그 scrambl와 헬러 세포의 TransfectionsiRNA의 전자 배열. A) 100 μm 전극에 HELA 세포의 이미지 알렉사 488 태그 siRNA (8 V, 1 밀리 초)와 transfected. B) 세포 핵의 이미지는 DAPI 물들. 흰색 원은 전극의 가장자리를 표시합니다.

<ol>
	<li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Caplen, N. J., Parrish, S., Imani, F., Fire, A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+gene+expression+by+small+double-stranded+RNAs+in+invertebrate+and+vertebrate+systems.">Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.</a> Proceedings of the National Academy of Sciences. 98, 9742 (2001).</li><li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550 (2002).</li><li>Lee, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+small+interfering+RNAs+targeted+against+HIV-1+rev+transcripts+in+human+cells.">Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.</a> Nat. Biotechnol. 20, 500-505 (2002).</li><li>Miyagishi, M., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U6+promoter-driven+siRNAs+with+four+uridine+3'+overhangs+efficiently+suppress+targeted+gene+expression+in+mammalian+cells.">U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.</a> Nat. Biotechnol. 20, 497-500 (2002).</li><li>Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+expression+of+small+interfering+RNA+in+human+cells.">Effective expression of small interfering RNA in human cells.</a> Nat. Biotechnol. 20, 505-508 (2002).</li><li>Xia, H., Mao, Q., Paulson, H. L., Davidson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+gene+silencing+in+vitro+and+in+vivo.">siRNA-mediated gene silencing in vitro and in vivo.</a> Nat. Biotechnol. 20, 1006-1010 (2002).</li><li>Chu, G., Hayakawa, H., Berg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+for+the+efficient+transfection+of+mammalian+cells+with+DNA.">Electroporation for the efficient transfection of mammalian cells with DNA.</a> Nucleic Acids Res. 15, 1311 (1987).</li><li>Felgner, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proceedings of the National Academy of Sciences. 84, 7413 (1987).</li><li>Raptis, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=applications+of+electroporation+of+adherent+cells+in+situ,+on+a+partly+conductive+slide.">applications of electroporation of adherent cells in situ, on a partly conductive slide.</a> Mol. Biotechnol. 4, 129-138 (1995).</li><li>Jordan, M., Schallhorn, A., Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+mammalian+cells:+optimization+of+critical+parameters+affecting+calcium-phosphate+precipitate+formation.">Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.</a> Nucleic Acids Res. 24, 596-601 (1996).</li><li>Gao, Y., Liu, X. L., Li, X. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+on+siRNA+delivery+with+nonviral+carriers.">Research progress on siRNA delivery with nonviral carriers.</a> International Journal of Nanomedicine. 6, 1017 (2011).</li><li>Ziauddin, J., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays+of+cells+expressing+defined+cDNAs.">Microarrays of cells expressing defined cDNAs.</a> Nature. 411, 107-110 (2001).</li><li>Yamauchi, F., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+gene+transfer+by+electroporation+into+adherent+cells+on+plasmid+DNA-loaded+electrodes.">Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.</a> Nucleic Acids Res. 32, e187 (2004).</li><li>Yamauchi, F., Kato, K., Iwata, H. <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+Assembly+of+Poly+(ethyleneimine)+and+Plasmid+DNA+onto+Transparent+Indium?+Tin+Oxide+Electrodes+for+Temporally+and+Spatially+Specific+Gene+Transfer.">Layer-by-Layer Assembly of Poly (ethyleneimine) and Plasmid DNA onto Transparent Indium? Tin Oxide Electrodes for Temporally and Spatially Specific Gene Transfer.</a> Langmuir. 21, 8360-8367 (2005).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsystem+for+transfection+of+exogenous+molecules+with+spatio-temporal+control+into+adherent+cells.">Microsystem for transfection of exogenous molecules with spatio-temporal control into adherent cells.</a> Biosensors and Bioelectronics. 22, 863-870 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-chip+for+spatially+controlled+transfection+of+nucleic+acid+payloads+into+cells+in+a+culture.">Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture.</a> Lab on a Chip. 7, 1004-1011 (2007).</li><li>Jain, T., Muthuswamy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+array+(MEA)+platform+for+targeted+neuronal+transfection+and+recording.">Microelectrode array (MEA) platform for targeted neuronal transfection and recording.</a> Biomedical Engineering, IEEE Transactions on. 55, 827-832 (2008).</li><li>Jain, T., McBride, R., Head, S., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+parallel+introduction+of+nucleic+acids+into+mammalian+cells+grown+in+microwell+arrays.">Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays.</a> Lab on a Chip. 9, 3557-3566 (2009).</li><li>Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+high-throughput+electroporation+microchip+applicable+for+siRNA+delivery.">An efficient and high-throughput electroporation microchip applicable for siRNA delivery.</a> Lab Chip. 11, 163-172 (2010).</li><li>Li, Z., Wei, Z., Li, X., Du, Q., Liang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+high-throughput+electroporation+chips.">Efficient and high-throughput electroporation chips.</a> , (2011).</li><li>Jain, T., Papas, A., Jadhav, A., McBride, R., Saez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+surface-bound+siRNAs+in+microwell+arrays.">In situ electroporation of surface-bound siRNAs in microwell arrays.</a> Lab Chip. 12, 939-947 (2012).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Electroporationfor+Gene+Transfer+In+Vivo.">Single-Cell Electroporationfor Gene Transfer In Vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Akaneya, Y., Jiang, B., Tsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+gene+silencing+by+local+electroporation+in+targeting+brain+region.">RNAi-induced gene silencing by local electroporation in targeting brain region.</a> J. Neurophysiol. 93, 594 (2005).</li><li>Lee, W. G., Demirci, U., 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=Microscale+electroporation:+challenges+and+perspectives+for+clinical+applications.">Microscale electroporation: challenges and perspectives for clinical applications.</a> Integr. Biol. 1, 242-251 (2009).</li><li>Boukany, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanochannel+electroporation+delivers+precise+amounts+of+biomolecules+into+living+cells.">Nanochannel electroporation delivers precise amounts of biomolecules into living cells.</a> Nature Nanotechnology. 6, 747-754 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 동영상 글에서 우리는 siRNA의 스크램블 순서와 헬러 세포의 특정 사이트 transfection을위한 MEA의 사용을 보여줍니다. 이 기술의 장점 중 하나는 기본 세포 라인을 포함한 여러 세포 라인에의 적용이다. 우리 연구실은 이전에 E18 일 늙은 쥐와 스크램블 siRNA 시퀀스와 플라스미드 GFP 18, 19와 NIH-3T3 세포에서 기본 hippocampal neuronal 문화의 특정 사이트 transfection이 기술의 사용을 보여주었습니다....

<table border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 세포 미디어 : 고급 가상 L-글루타민 200 MM 페니실린 / 스트렙토 마이신 태아 소 혈청 </td><td> Gibco / Invitrogen Himedia 연구소 / VWR Lonza 그룹 (주) Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> 세포 미디어 구성 : 사전 가상의 2% FBS, 2% L-글루타민과 2% Pennstrep </td></tr><tr><td> 트립신 EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Mediatech 주식회사 </td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech 주식회사 </td><td> 21-040 - CV </td><td></td></tr><tr><td> 태그 알렉사 488과 rhodamine는 sequ 스크램블줬어 용 siRNA </td><td> Qiagen 주식회사 </td><td> 1027292 </td><td></td></tr><tr><td> Electroporation 버퍼 </td><td> Biorad 연구소 </td><td> 165-2677 </td><td></td></tr><tr><td> 파형 발생기 </td><td> 사실적인 </td><td> 2414A </td><td> 원하는 펄스를 제공 할 수있는 파형 / 펄스 발생기 사용할 수 있습니다. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

eukaryotes 및 siRNA와 shRNA 등의 RNAi 요원의 후속 개발에 RNAi 경로의 발견, 기능 유전체학 및 치료학에 대한 특정 유전자에게 1-8 입을위한 강력한 방법을 달성했습니다. RNAi 기반 연구에 참여하고있는 주요 과제는 대상 셀에 대한 RNAi 요원의 전달입니다. 이러한 대량 electroporation 및 화학 transfection 방법 등의 전통 비 바이러스 성 전달 기술은 종종 배달을 통해 필요한 공간 컨트롤을 결여하고 9-12 가난한 transfection 효율 여유. 이러한 양이온 지질, 양이온 폴리머와 나노 입자 등의 화학 transfection 방법의 최근 발전은 매우 향상된 transfection 효율 13 결과했습니다. 그러나 이러한 기술은 아직 상당히 높은 처리량 기술, 단일 세포 연구 및 세포 세포의 상호 작용 조사를 소형화 혜택을 누릴 수 있습니다 전달 이상 정확한 공간 제어를 제공하기 위해 실패합니다. NT는 &quot;유전자 전달에&gt; 최근 기술 진보. 자기편 세포의 높은 처리량 transfection 14-23, microscale electroporation을 사용하는 대부분을 사용하도록 설정 한 Microscale electroporation은 (최대 단일 셀에) 전달 이상 정확한 spatio - 시간적 제어를 제공하며, 표시되었습니다 또한 높은 효율성 19 24-26을. 달성하기 위해, electroporation 기반의 접근 방식은 화학 기반의 transfection 방법에 필요한 siRNA와 DNA 단지와 부화의 연장 기간을 (일반적으로 4 시간)이 필요하고 벌거 벗은 siRNA와 DNA의 직접 항목으로 이어지지 않는 세포 세포질에 분자. 결과 유전자 발현으로는 transfection 27 후 육시간 초부터 달성 될 수있다. 우리 연구소는 이전에 다음 ID로 자기편 포유류의 세포 배양 17-19에서 특정 사이트 transfection에 대한 microelectrode 배열의 사용 (MEA)를 보여주었습니다. MEA 기반의 접근 방식에서, 유전자 페이로드의 전송이 지역화 된 마이크로 스케일 electroporati를 통해 이루어집니다세포의 있습니다. 선택한 전극에 전기 펄스의 응용 프로그램은 자극 전극의 지역에 존재하는 세포의 electroporation로 연결 지역 전기장을 생성합니다. 마이크로 전극의 독립적 인 제어 transfection 이상의 공간과 측두엽 제어 기능을 제공하고 또한 실험 처리량을 증가와 문화 - 투 - 문화 다양성을 감소 같은 문화에 수행 할 여러 transfection 기반의 실험을 할 수 있습니다. 여기 우리는 실험 설정 및 electroporation을 사용하여 휘황 태그 불가능한 시퀀스 siRNA와 자기편 헬러 전지의 대상 transfection에 대한 프로토콜을 설명합니다. 동일한 프로토콜은 또한 플라스미드 벡터의 transfection에 사용하실 수 있습니다. 또한 여기에 기술 된 프로토콜은 쉽게 약간의 수정을 포유류의 세포 라인의 다양한 확장 할 수 있습니다. 사전 정의 및 사용자 정의 모두 전극 패턴과 MEAs의 상용 여부는이 기술 접근​​ 할 수 t를기본 세포 배양 장비와 O 대부분의 연구 실험실.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

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

microelectrode 배열 (MEA)를 사용하여 자기편 포유류의 세포 배양에 siRNA의 스크램블 시퀀스의 특정 사이트 transfection에 대한 기사 내용은 프로토콜.

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

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

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

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

13.4K 조회수.  Arizona State University. microelectrode 배열 (MEA)를 사용하여 자기편 포유류의 세포 배양에 siRNA의 스크램블 시퀀스의 특정 사이트 transfection에 대한 기사 내용은 프로토콜.

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

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

표피 성장 인자를 사용하여 인간의 췌장 암 세포의 유전자 전달 및 Transfection은 다행 기반 엔지니어링된 Nanovectors를 수용체 타겟

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

연구

생체공학

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

사이트 특정 박테리아 염색체 공학 : ΦC31 Integrase 중재 카세트 거래소 (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

단일 합성 Curli 오페론을 생성하여 공학 점착성의 박테리아를

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

단일 세포 및 3D 감금 단일 배아 주문 : 하이 콘텐츠 스크리닝을위한 새로운 장치

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

siRNA를 치료제의 전달을위한 다공성 실리콘 미세 입자

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

양이온과 음이온 리포좀을 사용 CNS에 치료의 siRNA 배달

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 보완 방법을 사용 하 여 태 반 대상 약물 전달의 안전과 효율성의 종합 평가

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

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

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

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

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

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

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

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

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

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

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

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

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

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

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

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

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