CHO-P 세포 박사 바바라 Furie (베스 이스라엘 디코 니스 메디컬 센터, 하버드 의과 대학)에서 일종의 선물이었다. 이 작품은 JMK에이 작품은 또한 JMK에 Movember - 전립선 암 재단 챌린지 상에 의해 부분적으로 지원되었다 건강 보조금 HL095722의 국립 연구소에 의해 지원되었다

1. 세포 배양 <ol><li> 인간 골수성 백혈병 (HL-60) 세포 <ol><li> 75cm 문화 HL-60 세포를 20 % (V / V) 소 태아 혈청 (FBS), 1 % (V / V) L-글루타민 및 1 보충 Iscove의 수정 된 둘 베코의 중간 (IMDM), 15 ㎖ 2 플라스크 % (V / V) 페니실린 - 스트렙토 마이신을. </li><li> 세포 현탁액의 볼륨의 절반을 흡입하고 완전한 IMDM 미디어로 교체하여 3 일마다 미디어를 변경합니다. </li><li> 카르복시 디 아세테이트를 들어, 숙신 에스테르 (CFSE) 염색, 원심 분리기 HL-60 세포 현탁액 (400 XG, 5 분), 1 μM의 CFSE 용액에 resuspend (데워진 PBS에서 준비) 및 37 ℃에서 15 분 동안 품어 그런 다음 30 분 동안 신선한 데워진 배지에서 원심 분리기 세포, 기음 뜨는에 resuspend 세포. PBS로 세포를 세척 한 후 (P-SEL 코팅 표면에 CFSE 염색 HL-60 세포의 대표 이미지 그림 1B 참조) 롤링 실험을 위해 사용합니다. </li></ol></li></ol><ol start="2"><li> 폐의 미세 혈관 내피 세포 (LMVECs) <ol><li> 코트 100mm 페트리 0.1 % 젤라틴 용액 (PBS에서의 v / V)와 함께 요리와는 적어도 30 분 동안 37 ° C에서 알을 품다. </li><li> 전체 내피 성장 매체의 젤라틴 코팅 100mm 배양 접시에 문화 LMVECs은 (내피 기초 매체-2 (EBM-2)), 특정 성장 보조 장비 보충은) 시약을 참조하십시오. 미디어에게 80~90% 합류에 도달하면 매일과 하위 문화의 세포를 변경합니다. </li><li> 하위 문화, PBS로 세포를 씻어 준 후 분리 세포를 1X 트립신-EDTA 4 ㎖로 3 분 동안 37 ° C에서 완전 EBM-2 미디어의 동일한 볼륨에 중화. 15 ML 튜브 및 centrifug에 세포 현탁액을 전송E (400 XG, 5 분). 원심 분리 후, 완전한 내피 미디어 1 ML에서 펠렛을 재현 탁하고 혈구와 세포를 계산합니다. 이 모든 실험을위한 통로 (7)에 따라 만 세포를 자신의 형태와 기능 사용에 영향을 미치는 등, 이상하지 통로 세포를 수행합니다. </li></ol></li></ol><ol start="3"><li> 중국어 햄스터 난소-P-셀렉틴 (CHO-P) 세포 <ol><li> 안정적으로 인간의 P-SEL을 표현하는 형질 전환 된 CHO 세포이다 CHO-P 세포, 공동 작업자 (베스 이스라엘 디코 니스 메디컬 센터, 하버드 의대) (11, 12)에 의해 제공되었다. </li><li> F-12 미디어 25 ㎖에있는 T175의 CM 문화 CHO-P는 세포 2 플라스크. </li><li> 계대를 들어, 4 ~ 5 초 동안 PBS 10 ㎖로 세포를 세척 한 다음 전체 미디어에서 중화 한 다음 37 ℃에서 3 분에 대한 1X 트립신-EDTA (에틸렌 다이아 민 테트라 초산) 10 ㎖에를 Trypsinize. </li><li> 원심 분리 세포 현탁액 (400 XG, 5 분), 정중, 상등액을 흡인 전체 미디어 1 ㎖ 중에 세포 펠렛을 재현 탁과 함께 세포를 세어혈구. </li></ol></li></ol> 2. 통합 다 잘 플레이트 미세 유체 시스템의 작동 <ol><li> 모든 장비가 제대로 연결되어 있는지 확인하고, 다른 모듈을 켜 : 컴퓨터, 컨트롤러, 거꾸로 현미경과 CCD 카메라. </li><li> 이미징 소프트웨어를 열고, 멀티 웰 플레이트 모듈과 영상 모듈이 화면에 표시되어 있는지 확인하십시오. </li><li> (컨트롤러에 연결) 증기 트랩에 튜브를 연결하고 또한 그들에게 압력 인터페이스에 연결합니다. </li><li> 플레이트 히터 / 어댑터 멀티 웰 플레이트를 놓습니다. 우물 (아래에 설명)에 시약을 추가하고 접시 위쪽에 인터페이스를 연결합니다. 자동화 된 단계에 이미징 플레이트를 놓습니다. </li><li> 인터페이스는 플레이트의 상부에 부착하고 멀티 웰 플레이트를 이용하여 쉽게 제어 할 정의 유속 미세 유체 채널을 통해 유체를 구동 웰의 상부에 컨트롤러로부터 공기압을 적용수동 모드에서 모듈 화면. </li><li> 웰 사이에 관찰 영역에 걸쳐 채널 흐름 시약. 미세 유체 채널의 크기는 70 μm의 높이 350 ㎛의 넓​​은 x입니다. 선형 채널의 길이가 1mm이고 채널의 바닥은 브라이트, 위상, 및 공 초점 형광 현미경과 호환 180 μM의 유리 커버 슬립을 포함한다. </li><li> CCD 카메라 (스트림 획득, 11 프레임 / 초)를 사용하여 영상을 획득하고 호환 가능한 소프트웨어를 통해 분석 할 수 있습니다. </li></ol> 3. 단백질 기질 또는 세포 단층과 미세 유체 채널의 코팅 <ol><li> 피브로넥틴 또는 P-/E-selectin과 미세 유체 채널을 코팅 <ol><li> PBS에서 20 ㎍ / ml의 피브로넥틴 용액 1 ㎖를 준비합니다. 코팅되어야하는 채널들의 수 (채널 당 피브로넥틴 25-50 μl를 사용)에 기초하여 부피를 바꾼다. </li><li> 물론 각각의 입구에 피브로넥틴 솔루션의 25 ~ 50 μl를 추가합니다. 2 DYN / ㎠의 전단력 적용5 분 채널을 perfuse하는 데 필요한 권한입니다. 액체가 아니라 콘센트에 나타나는의 구슬을 유의하시기 바랍니다. 실온에서 30 ~ 45 분 알을 품다 </li><li> 우물에서 솔루션 (채널을 공급하는 중간 원에서 직접 흡인하지 않습니다) 1,13 대기음. 콘센트도에 PBS의 200 ~ 500 μl를 추가하고 5 분 동안 2 DYN / ㎠의 전단 흐름을 적용하여 PBS와 채널을 씻어. 채널은 정상적으로 피브로넥틴 및 사용 준비로 코팅된다. </li><li> 표면 코팅을 할 수 있도록 37 ° C에서 1 시간 배양과, 상기 한 바와 같이 P 또는 E-SEL, PBS에서 원하는 사람의 재조합 단백질의 5 ㎍ / ㎖의 용액을 제조하고, 코트 채널 코트. </li></ol></li><li> 미세 유체 채널 내부 CHO-P 또는 LMVEC 단층의 생성 <ol><li> 부드럽게 전체 미디어와 원심 분리기의 2 배 용량 (400 XG에서 5 분)를 사용하여 소멸, 3 분 동안 배양 접시에서 세포를 Trypsinize. 전체 미디어와 원심 분리기 10 ㎖ (400 XG에서 5 분)로 재현 탁 셀 전압 강하N. </li><li> 현탁액으로 세포 농도를 결정하기 위하여 세포를 카운트. 채널 내부 합류 LMVEC 단층의 형성을 보장 15-20000000 세포 / ml로 세포 농도를 가지고. 합류 CHO-P 세포 단일 층의 경우, 50-60000000 세포 / ml를 사용합니다. 각 채널에 대한 세포 현탁액의 25 ~ 50 μl를 사용합니다 - 따라서 실험에 사용되는 결정 초기 셀 수. </li><li> 잘 입구에 적절한 농도로 세포 현탁액의 25 ~ 50 μl를 추가합니다. 현미경의 무대에서 접시를 놓고 세포가 전체 채널을 작성 화면에서 관찰하고 흐름을 중단 할 때까지 채널 (2 DYN / ㎠)로 세포를 소개합니다. </li><li> 전체 LMVEC 또는 CHO 미디어 중 하나를 200 ㎕ 씩 출구와 입구를 모두 입력합니다. 세포가 정착하자 인큐베이터에서 3 시간 동안 준수 (37 ° C에서 5 % CO 2). </li><li> 3 시간 배양 한 후, 무소속 제거하기 위해 전체 미디어 (2 DYN / ㎠, 10 ~ 15 분)와 채널을 씻어세포. 세포는 이제 완전히 합류 나타납니다과 채널의 사용 준비가 완료됩니다. 초기 셀 시딩 밀도에 따라, 안정화 시간을 추가로 2 ~ 3 시간은 세포 표면의 전체 범위를 확인해야 할 수 있습니다. </li></ol></li></ol> 4. LMVEC 프로 염증 활성화 및 P-/E-selectin의 항체 차단 <ol><li> LMVEC 기초 미디어에서 TNF-α 용액 (10 NG / ML)를 준비합니다. </li><li> , 채널에 LMVEC의 염증 활성을 유도 아니라 입구에 TNF-α 용액 100 μl를 추가하고 5 분 동안 2 DYN / ㎠의 전단 흐름을 적용하여 채널에 솔루션을 소개합니다. 제어 채널 (활성화되지 않은되는 EC)는, 흡입구 LMVEC 기저 매체의 100 μl를 잘 추가 채널 (5 분간 2 DYN / cm 2)로 소개한다. 채널은 지금 롤링 분석을위한 준비가되어 있습니다. </li><li> P-SEL 및 LMVECs 및 CHO-P 세포에 E-SEL을 차단하기 위해, P-SEL (복제 AK4, 바에서 5 ㎍ / ㎖의 중화 소개샐 미디어) 또는 E-SEL (복제 P2H3, 기저 미디어 5 ㎍ / ㎖)을 채널로 항체 및 37 ℃에서 1 시간 동안 배양 다음으로, (5 분 2 DYN / ㎠) 기저 매체와 채널을 씻어. 채널은 지금 롤링 분석을위한 준비가되어 있습니다. </li></ol> 5. 기판 / 세포 단층 코팅 미세 유체 채널에서 HL-60 롤링 분석 <ol><li> 조심스럽게 채널이 제대로 (세포와 코팅의 경우, 완전히 합류 세포 단층이 관찰되어야한다) 코팅되어 있음을 확인하기 위해 현미경으로 채널을 검사합니다. </li><li> 롤링 실험, 원심 분리기 HL-60 세포 현탁액 (400 XG에서 5 분)에 대한 HL-60 세포 현탁액을 준비하고 기저 매체와 한 번 씻어. 5,000,000 세포 / ml로 HL-60 세포 현탁액을 생성하기 위해 (칼슘 2 + 2 + 및 Mg를 함유 기저 매체) IMDM에서 세포에 resuspend 횟수. 롤링 분석을 수행하기 위해 각 채널에 대한 세포 현탁액의 25 ~ 50 μl를 사용합니다. </li><li> 셀 suspe의 25 ~ 50 μl를 추가합니다nsion 현미경 단계에 온도 조절 플레이트 홀더 (37 ° C) 및 장소 안에 잘 장소 접시를 콘센트에. 다음으로, (세포가 입구에 콘센트에서 흐르는 15 초 이내에 관찰되어야한다) 2 DYN / ㎠의 전단력을 적용하여 채널에 세포를 소개합니다. </li><li> 전단 응력의 ​​함수로서 압연 응답을 검사하려면, (5 DYN까지 0.25부터 서서히 전단력을 증가 DYN / cm 2 0.25 전단을 줄이고 원하는 각 전단에 ( &quot;스트림 취득&quot;기능을 사용) 20 ~ 30 초의 영상을 취득 / cm 2. 그것은) 높은 가위를 사용하는 것도 가능하다. </li><li> CCD 카메라 (스트림 획득, 11 프레임 / 초)를 사용하여 영상을 획득하고 호환 가능한 소프트웨어를 통해 경로를 회전하고 회전 속도를 분석 할 수 있습니다. </li></ol> 6. 표면 분자의 발현을 감지하는 유동 세포 계측법 <ol><li> 트립신 처리 후, 원하는 세포 유형 (HL-60,-CHO의 (1-2 × 105 세포 / 샘플을 사용) 세포 현탁액을 준비PBS에서 P 또는 LMVECs) (- / -), 2 % FBS가 보충. 두 번 세포를 세척하고 50 μL (같은 버퍼를 사용하여)에 샘플 볼륨을 가져온다. </li><li> 20 분 4 ° C에서 원하는 형광 - 복합 AB (자세한 내용은 첨부 표 참조) (알루미늄 호일로 커버) 각 샘플을 품어. </li><li> 두 번 (같은 버퍼) 세포를 세척하고 200 μL에 스테인드 세포 현탁액의 최종 볼륨을 가져옵니다. 표면 분자의 발현을 검출하는 유동 세포 계측기를 사용하여 샘​​플을 분석한다. </li></ol>

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A model for an antibody-based microfluidic screening system.</a> Microelectronic Eng. 98, 668-671 (2012).</li><li>Perozziello, G., 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+devices+modulate+tumor+cell+line+susceptibility+to+NK+cell+recognition.">Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.</a> Small. 8, 2886-2894 (2012).</li><li>Perozziello, G., 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+biofunctionalisation+protocols+to+form+multivalent+interactions+for+cell+rolling+and+phenotype+modification+investigations.">Microfluidic biofunctionalisation protocols to form multivalent interactions for cell rolling and phenotype modification investigations.</a> Electrophoresis. , (2013).</li><li>Simone, G., 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+facile+in+situ+microfluidic+method+for+creating+multivalent+surfaces:+toward+functional+glycomics.">A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics.</a> Lab Chip. 12, 1500-1507 (2012).</li><li>Sarkar, 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=Engineered+cell+homing.">Engineered cell homing.</a> Blood. 118, e184-e191 (2011).</li><li>Cheng, Z., 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=Targeted+Migration+of+Mesenchymal+Stem+Cells+Modified+With+CXCR4+Gene+to+Infarcted+Myocardium+Improves+Cardiac+Performance.">Targeted Migration of Mesenchymal Stem Cells Modified With CXCR4 Gene to Infarcted Myocardium Improves Cardiac Performance.</a> Mol. 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저자는 이익의 갈등을 선언하지 않습니다.

외인성 세포 기반 치료의 성공적인 번역의 주요 과제 중 하나는 효율적으로 높은 생착 효율 3 부상과 염증의 사이트에 세포를 제공 할 수 없다는 것입니다. 셀룰라 롤링 결국 조직 (5)에 내피 통해 확고한 부착 및 이주에 이르는, 혈관의 벽에 세포의 감속을 용이 셀 원점 복귀하는 과정에서 중요한 단계를 나타낸다. 후보 세포 유형에 대한 압연 공정의 더 나은 이해는 세포 호밍을 ?...

세포 기반 치료의 성공적인 임상 번역의 주요 과제 중 하나는 비효율적 인 배달 또는 원하는 사이트 3,4에 체계적으로 주입 된 세포를 대상으로합니다. 따라서, 휴대폰 호밍을 개선하기위한 방법 일정한 검색이있어, 특히 세포 치료를 개선하기위한 전략으로, 압연 전지. 혈관에 롤링 세포는 고전적인 질병 사이트 5 모집 백혈구에 대해 정의 된 세포 유도 폭포,에서 중요한 단계입니다. 이 단계는 내피 셀렉틴 사이 특정 상호 작용에 의해 지배된다, 즉, P-및 E-셀렉틴 (P-및 E-SEL), 백혈구 5,6의 표면에 자신의 카운터 리간드. 더 나은 이해와 셀 원점 복귀의 효율 향상, 특히 압연 단계는 세포 기반 치료를 개선하기 위해 새로운 플랫폼에 대한 탐구에 매우 중요합니다. 지금까지이 두 평면 플랫폼을 포함, 평행 판의 흐름 챔버 (PPFCs)를 사용하여 달성되었다세포 현탁액을 주사기 7,8, 9 펌프 사용하여 관류 통해 상부 플레이트에있는 유입과 유출 포트와 그들 사이 가스켓와 에스. 바닥 판의 표면은 중요한 세포 단층 / 기질로 코팅 될 수 있고, 전단 유동하 관류 세포와 표면 사이의 상호 작용은 다음 7을 탐구이다. 그러나 PPFC 거품 형성, 누출, 주요 단점을 제시 제대로 제어 흐름과 낮은 처리량, 시약 소모 및 매우 지루한 방법입니다. 전통적인 PPFC에 대한 대안 기술은 낮은 시약 소비 1,10으로, 정확하고, 컴퓨터로 제어되는 전단 흐름에서 세포 분석의 높은 처리 성능 (PPFCs보다 최대 10 배 이상)을 허용, 멀티 웰 플레이트 미세 유체 시스템입니다. 셀 롤링 실험은 USI를 세포 단일 층 또는 설계 기판 코팅 및 이미지가 될 수는 미세 유체 채널 내에서 수행된다NG 롤링 특성을 가진 현미경은 쉽게 적절한 소프트웨어를 사용하여 분석 하였다. 이 연구에서, 우리는 인간 골수성 백혈병 다른 표면에 (HL-60) 세포의 회전 특성을 연구하여이 멀티 웰 플레이트 미세 유체 시스템의 기능을 보여줍니다. HL-60은 P-및 E-SEL뿐만 아니라 다른 롤링 수용체를 발현하는 세포의 단층을 분석 하였다 같은 기판 상에 롤링. 또한, 항체 (AB)에 그 표면에 HL-60의 회전 운동을 중재의 특정 셀렉틴의 직접 참여를 설명하는 데 사용되었다 블로킹. 롤링 실험은 같은 회전 속도, 회전 세포 수 및 압연 경로 속성으로 키 롤링 매개 변수의 효율적인 분석을 허용하는, 최소한의 시약 / 셀 소비, 안정적인 전단 흐름에 따라, 용량 증가 하였다.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Human Lung Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2527</td>
			<td></td>
		</tr>
		<tr>
			<td>P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)</td>
			<td>Kind gift by Dr. Barbara Furie11,12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 Cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium Supplements</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 MV SingleQuots</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM - Iscove&#39;s Modified Dulbecco&#39;s Medium 1x</td>
			<td>Gibco</td>
			<td>12440</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12 (1x) Nutrient Mixture (Ham)</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (L/G) 200 mM</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>Sa550</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>BD Falcon</td>
			<td>BD-353003</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml polypropylene conical tubes)</td>
			<td>MedSupply Partners</td>
			<td>TC1500</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution (2%)</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS (without calcium chloride and magnesium chloride)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution (10x)</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Anti-hE-Selectin/CD62E</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hP-Selectin</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG&#173;1 &#954; Isotype Control</td>
			<td>BD Bioscience</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SLeX /CD15s Ab, Clone: 5F18</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgM-FITC Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC2859</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD162, Clone: KPL-1</td>
			<td>BD Pharmingen</td>
			<td>556055</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1 k Isotype Control</td>
			<td>BD Pharmingen</td>
			<td>550617</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-Selectin Ab (AK4)</td>
			<td>Santa Cruz</td>
			<td>SC19996</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-Selectin Ab, Clone P2H3</td>
			<td>Millipore</td>
			<td>MAB2150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC3877</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human P-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>137-PS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human E-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>724-ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human, Plasma</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Bioflux 1000</td>
			<td>Fluxion Biosciences</td>
			<td>Bioflux Montage was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlux 48-well plates</td>
			<td>Fluxion Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>CFlow Plus was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-S</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic analysis of in vitro cell rolling using a multi-well plate microfluidic system

세포 기반 치료를위한 주요 과제는 전신 정맥 내 또는 동맥 내 주입 다음 그 조직에 고효율로 생존 세포의 대량을 대상으로 할 수 없다는 것이다. 따라서, 휴대폰 호밍을 증가하는 것은 현재 세포 치료를 개선하기위한 전략으로 연구된다. 혈관 내피 세포에 압은 셀 원점 복귀하는 과정에서 중요한 단계이며 평행 평판 유동 챔버 (PPFC)를 이용하여 시험관 프로빙 할 수있다. 그러나,이 저조한 제어 흐름 조건, 매우 지루한, 낮은 처리량 분석이다. 대신, 우리는 정밀하게 제어, 생리학 관련 전단 흐름 1,2에서 높은 처리량 세포 롤링 특성의 연구를 가능하게하는 멀티 웰 플레이트 미세 유체 시스템을 사용했다. 이 논문에서, 우리는 P-및 E-셀렉틴 코팅 표면뿐만 아니라 세포 단층 코팅 표면에 HL-60 (인간 골수성 백혈병) 세포의 회전 특성이 readil 할 수있는 방법을 보여y를 조사했다. 더 염증성 조건을 시뮬레이트하도록, 미세 유체 채널 표면이어서 상당히 동적 조건하에 HL-60 세포와의 상호 작용을 증가 종양 괴사 인자-α (TNF-α)로 활성화 된 내피 세포 (EC에)으로 코팅 하였다. 향상된 처리량과 같은 속도를 압연과 경로를 압연과 같은 매개 변수의 신속한 분석을 허용 통합 된 다중 매개 변수 소프트웨어 분석 플랫폼은 체외 세포 롤링 특성을 평가하기위한 중요한 장점이다. 세포 압연 및 원점 복귀에 영향을 미칠 수 있도록 설계 엔지니어링 방식의 신속하고 정확한 분석을 허용,이 플랫폼은 사전 외인성 세포 기반 치료를하는 데 도움이 될 수 있습니다.

HL-60 세포가 피브로넥틴에 P-및 E-셀렉틴 표면에 롤 아니지만 그들은 롤링 리간드 P-SEL 당 단백질 리간드-1 (PSGL-1) 시알 릴 루이스 X (SLEX)을 포함하여 원점 복귀 리간드의 다양한 5,14 (그림 1A 표현으로 HL-60 세포는 황금 표준 &quot;롤러&quot;로 간주됩니다 ). 과 특정 상호 작용을 매개하는 테트라 당 SLEX위한 발판으로 표면 단백질 PSGL-1의 역할, P-및 E-SEL, 염증 5...

Watch this Scientific Journal Video about Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System at JoVE.com

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

이 연구는 크게 생리학 관련 전단 흐름에 따라 세포 롤링 연구의 처리량을 증가, 멀티 웰 플레이트 미세 유체 시스템을 사용했다. 캐스케이드 및 환자 세포의 외인성 개체군의 전신 전달 다음 세포 호밍의 중요성을 원점 다단계 셀에서 셀 압연의 중요성을 감안할 때,이 시스템은 세포 기반 치료를 개선하기위한 스크리닝 플랫폼으로 잠재력을 제공한다.

의 체계적인 분석<em&gt; 체외</em멀티 웰 플레이트 미세 유체 시스템을 사용하여&gt; 셀 롤링

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

systematic-analysis-vitro-cell-rolling-using-multi-well-plate

Research

JoVE Journal

Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

11.9K 조회수.  Brigham and Women's Hospital.  이 연구는 크게 생리학 관련 전단 흐름에 따라 세포 롤링 연구의 처리량을 증가, 멀티 웰 플레이트 미세 유체 시스템을 사용했다. 캐스케이드 및 환자 세포의 외인성 개체군의 전신 전달 다음 세포 호밍의 중요성을 원점 다단계 셀에서 셀 압연의 중요성을 감안할 때,이 시스템은 세포 기반 치료를 개선하기위한 스크리닝 플랫폼으로 잠재력을 제공한다. 

비디오: Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

종양 Microenvironment를 재현을위한 마이크로 유체 장치<em&gt; 체외에서</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

연구

생체공학

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

비대칭 수용체 패턴에 셀 롤링의 탄도를 공부

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

높은 처리량의 사용<em&gt; 체외</em&gt; 미세 유체 시스템 구강 다중 종의 바이오 필름을 개발하는

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

다 잘 판에 대 한 시스템을 삽입 하는 결단 력과 침투성 막에 3 차원 조직 모델에서 보급의 시뮬레이션 하는 방법

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

자동된 슬라이드 스캔 및 Widefield 하이 콘텐츠 분석 시스템을 사용 하 여 붙일 표시 된 조직에서 시장 세분화

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

가 여러 명의 병렬 접시 흐름 챔버를 사용 하 여 스트레스에 노출 된 내 피 세포의 유전자 표정 분석

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

라이브 셀 분석의 전단 응력에 녹 농 균 등 처리량 미세 자동 시스템을 사용 하 여

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

미세 유체 칩으로 결정적인 방식으로 단일 셀 기반 공동 배양 구축

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...