이 작품은 보건 부여 NIH / NICHD # HD051998 (CAM)을 미국 국립 연구소에 의해 지원되었다.

1. 콜라겐 비드 준비 <ol><li> 이전 3 차원 세포 배양에 대해로드 EVTs에, 하나는 Cytodex - 3 microcarrier 비즈를 준비해야합니다 : <ol><li> 실험에 필요한 Cytodex - 3 구슬의 해당 금액을 달다. 이 프로토콜은 구슬 0.05g이 필요하는, 10ml RCCS 선박에 대한 적응이다. 50ml RCCS 선박 들어, 그에 따라 규모. 50mL autoclavable 원뿔 튜브에 12mL Dulbecco 인산 250 MG Cytodex - 3 구슬을 혼합하면 용액 (DPBS)를 버퍼. 이 금액은 5 RCCS 선박에 대한 충분합니다. </li></ol></li><li> 압력솥 프로세스가 증발하게됩니다대로 적절한 볼륨의 원뿔 튜브에 존재 확인하십시오. 대충 110 10 분 튜브와 압력솥을 캡 ° C. </li><li> 압력솥주기의 완성에있는 원뿔 튜브를 제거하고 Cytodex - 3 비드 솔루션 괜찮아 수 있습니다. </li><li> 원뿔 튜브 룸 온도 냉각 후 1X DP를 사용하여 12.5mL로 전체 볼륨을 가져다 멸균 기술을 사용하여BS. </li><li> 모자와 실온에서 준비 Cytodex - 3 구슬을 저장합니다. 준비 비즈를 사용하기 전에 실내 온도 팽창하고 냉각 허용합니다. Cytodex - 3 구슬 위의 준비 다섯 10mL RCCS 혈관을위한 것입니다. 우리는 콜라겐이 약 한달 후 불안정하므로 상온에서 Cytodex - 3 구슬의 확장 스토리지, 성능 저하가 발생합니다 것이 관찰했습니다. 133-215 μm의 크기에서 Cytodex - 3 구슬이 다​​양합니다. </li></ol> 2. 미디어 준비 <ol><li> 플러스 보조제 및 60% L - 15 Leibovitz의 미디어 (22에서 적응), 40 % 가상 알파 구성되어 GTSF - 2 미디어 (22에서 적응), 최적화된 RCCS의 1L를 준비합니다. 첫째, 함께 보충 가상 알파의 전체 볼륨에서 400mL 준비 : 21.2mM 나트륨 탄산수 소 0.06 %의 펩톤 0.7mM Fructose와 1.4mM 갈락토 오스 5.6mM 포도당 1 % HEPES 1퍼센트 L - 글루타민 0.5 % ITS 10 % FBS </li><li> ITS의 재고 솔루션을 준비하려면, 빙초산 (약 0.05mL)를 추가로 준비 5mL 멸균 산성 H 2 O에 용해. 디졸브로 소용돌이, 45mL 멸균 물을 따르십시오. </li><li> 조직 문화 학년 H 2 O.의 용해에 의한 매체의 600ml 정도로 L - 15 Leibovitz의 분말 달아서 나누다 </li><li> L - 15 Leibovitz의 매체 1L로 세포 배양 매체의 전체 볼륨을 가져와. 필터 소독과 4 ° C 저장을 어둠 속이라서. 사용하는 매체의 각 나누어지는 개별적으로 1퍼센트 페니실린 - 스트렙토 마이신을 추가합니다. </li><li> GTSF - 2 미디어 이외의 대부분의 미디어 공법이 성공적으로 RCCS에서 테스트되었습니다. 개인 실험실 실험이 수행되고 최적의 어떤 매체에 스스로 결정해야합니다. </li></ol> 3. 세포 비드 인큐베이션 <ol><li> ~ 80 % confluency에 EVTs을 전파, trypsinize 및 접수 세포 배양 방법을 사용하여 계산합니다. </li><li> 따뜻한의 4mL에 1x10 6 EVTs을 중지에드 미디어. </li><li> 부드럽게 준비 Cytodex - 3 구슬을 섞는다. 무균 기술을 사용하여 다양한 팁, 10mL 피펫 serological 및 미사용 15mL 원뿔 튜브로 전송을 사용하여 준비 구슬 2.5mL를 제거합니다. 그들은 피펫에 연결과 같은 구슬의 작은 손실이있을 수 있습니다. </li><li> Cytodex - 3 구슬은 튜브의 바닥에 정착하도록 허용합니다. 침전 후 Cytodex - 3 구슬을 방해하지 않고 DPBS의 최상위 층을 제거하는 pipettor을 사용합니다. </li><li> 준비 Cytodex - 3 구슬과 미디어 1x10 6 준비 EVTs를 섞습니다. </li><li> 30 분 상온에서 휴대 비드 혼합를 품어. 주기적으로 부드럽게 섞는다. 37 ° C와 5% CO 2에서 추가로 30 분 알을 품다. 주기적으로 부드럽게 섞는다. </li></ol> 4. RWV를로드 <ol><li> 층류 흐름 캐비닛에서 포장에서 10mL의 RCCS를 제거하고 안정성을위한 살균, 6 - 물론 문화 판에 넣습니다. RC의 표시 그림 1 그림을 참조하십시오CS. </li><li> 포트에서 큰 마개를 제거합니다. </li><li> 예열 미디어와 10mL에 잠복기 휴대 비드 혼합의 전체 볼륨을 가져와. </li><li> 큰 포트를 통해 RCCS에 셀 / 비드 - 혼합을로드합니다. RCCS가에 기울어져 있어야 45 ° 각도 (대형 포트 이상) 잠재적인 기포의 제거에 도움에 로드할 때. 천천히 그리고 꾸준히 혈관을 작성하여 긍정적인 압력 업 빌드를 방지합니다. </li><li> 큰 포트에 마개를 교체합니다. </li><li> 3 - ML 주사기의 피스톤을 제거하고 작은 포트에 빈 주사기를 놓습니다. 각각의 주사기에 미디어의 1 3mL 추가합니다. 천천히 두 밸브를 엽니다. 부드럽게 주사기에 주사기 피스톤을 교체하십시오. 모든 거품이 내부 챔버에서 제거되기 전까지는 한 주사기에서 미디어를 추가합니다. </li><li> RCCS를 들고 거품 확인하기 위해 앞에서 그것을 회전하면서 부드럽게 측면을 누릅니다. 모든 거품이있다면 공기 방울이 형성 O 방해하므로, 당신은 챔버의 그들을 받아야합니다F 셀 집계가와 전단 세력을 소개합니다. 거품이 작은 포트에 따라 때까지 RCCS를 회전하여 거품을 제거합니다. 그런 다음 부드럽게 챔버의 항구 밖으로 거품을 강제로 반대편에있는 주사기 아래로 밀어. </li><li> 한쪽 포트를 닫습니다. 부드럽게 형성에서 거품을 방지 용기로 긍정적인 압력의 작은 금액을 소개하는 다른 주사기 피스톤을 아래로 밀어. 둘째 밸브를 닫습니다. </li><li> 회전자에 RCCS을로드합니다. 37 ° C CO 2 배양기에서 19rpm에서 회전을 시작합니다. </li></ol> 5. 미디어 변경 <ol><li> 매일 이후 다음 첫 사흘 동안 매일 미디어를 변경합니다. </li><li> 날개를 끄고 RCCS를 제거합니다. 일부 흡입을 만드는 각각의 작은 항구에서 주사기를 제거하고 구슬이 큰 포트의 반대를 해결 있도록 각도에 RCCS을 배치 피스톤을 당겨. </li><li> 비즈 모두 해결되면 작은 밸브 중 하나를 열고미디어가 RCCS 및 폐기물 용기 밖으로 흘러 수 있습니다. 이러한 방식으로 미디어의 2 / 3를 제거합니다. 구슬을 방해하지 않도록하고 집계를 폐기하지 않는해야합니다. </li><li> 작은 밸브를 닫고 큰 포트를 엽니다. 다시 큰 포트를 통해 RCCS에 미디어를 추가합니다. 큰 포트에 마개를 교체합니다. </li><li> 4.9 - 4.6 단계를 반복합니다. </li><li> 집계 크기가 증가로서 최적의 속도에서 작은 순환 패턴에서 이상적으로, 항상 정지에서 그들을 유지하기 위해 회전 속도를 증가해야합니다. 집계가 가시 성장 시작되면 일반적으로, 각 수유 후 0.3-0.7 RPM 사이의 속도를 향상시킬 수 있습니다. </li></ol> 6. 수집 세포를 전파 <ol><li> 회전자에서 RCCS를 제거합니다. 각각의 작은 포트에서 주사기를 제거하고 구슬이 큰 포트의 반대를 해결 수 있도록 각도에 RCCS 놓으십시오. </li><li> 비즈 모두 해결되면 작은 밸브 중 하나를 열고 미디어가 흐름 수RCCS 및 폐기물 컨테이너에. 이러한 방식으로 미디어의 1 / 3을 제거합니다. </li><li> 작은 밸브를 닫고 큰 포트를 엽니다. 부드럽게 소용돌이 선박 다시 솔루션에 집계를 해산합니다. 멸균 50ml 원뿔 튜브에 비어있는 액체 혼합물. 수집된 집계 철저하게 집계의 회복을 최대화하기 위해 문화 용기를 씻어 표면에 뜨는 사용하기 전에 원뿔 튜브에 정착하도록 허용합니다. 집계는 같은 유동세포계측법, 침공 assays, immunofluorescence 및 기타로 바로 하류 assays에 사용할 수 있습니다. </li></ol> 7. 대표 결과 Cytodex - 3 구슬에 RCCS 재배 EVT 같은 세포 (SGHPL - 4 trophoblast 세포 라인)의 예제는 그림 2에 표시됩니다. EVT 같은 세포 라인은 떨어져 주요 클러스터에서 확장하고 인접 클러스터에 연결 계획을 표시합니다. 구슬의 대부분이 완전히 전파 세포로 덮여있다. 일단 RCCS 및 괞찮아에서 제거테드는 세포외 기질에 EVT 같은 3 - D 성장 세포가 적극적으로 및 / 또는 마이 그 레이션을 (그림 3) 침공. RT - PCR 데이터는 기존의 세포 배양 monolayers (그림 4) 반대로 3 - D 집계에서 본 MMPs의 증가 표현을 확인합니다. 흥미롭게도, 침략과 연관되지 않은 유전자도 RCCS에 upregulated입니다 (표 1)과 trophoblasts 세포를 침입과 면역 상호 작용 등의 영역을 delineating에 도움이됩니다. <img alt="그림 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> 그림 1. 회전 세포 배양 시스템 (RCCS)의 만화 묘사. <img alt="그림 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> 그림 2. RCCS 5 일 성장 후 대표 골재의 위상 콘트라스트 현미경. 화살표가 침입 세포를 표시하고 *은 Cytodex - 3 microcarrier 비즈를 나타냅니다. <img고도 = &quot;그림 3&quot;SRC = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot;/&gt; 그림 3. RCCS 들어라 집계는 섬유소의 젤에 포함되었습니다. 섬유소 젤을 통해 침공은 (A) 24시간 후 섬유소를 포함 빠르면 준수하고 (B) 48 시간 동안 계속되었다. 화살표가 침입 세포를 표시하고 *은 Cytodex - 3 microcarrier 비즈를 (심판에서 허가, 적응. 7) 나타냅니다. <img alt="그림 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> 그림 4. monolayer와 RCCS의 비교 젤라틴 zymogram는 SGHPL - 4 EVT 같은 세포를 증식. MMP - 2와 MMP - 9의 프로 - 적극적인 형태는 RCCS 성장 trophoblast 세포 (참조. 7, 허가, 적응)에 의해 분비되었다. <img alt="표 1" src="/files/ftp_upload/3367/3367table1.jpg" /> 표 1. RCCS 재배 SGHPL - 4 세포 (참조. 7, 허가, 적응)에서 microarray 결과의 요약. <ol class="lalpha"><li정규 3 - D 유전자의 가치 / 표준 monlayer 유전자 값 : 3 - D 집계에서&gt; 접어 유도는 다음과 같이 계산되었다. </li><li> monolayers의 감지 한계 이하되었습니다 유전자를 나타냅니다. </li></ol>

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Immun. 73, 1129-1129 (2005).</li><li>Myers, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+phenotypic+gap+between+transformed+neuronal+cell+lines+in+culture+and+untransformed+neurons.">Closing the phenotypic gap between transformed neuronal cell lines in culture and untransformed neurons.</a> J. Neurosci. Methods. 174, 31-31 (2008).</li><li>Hjelm, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+three-dimensional+organotypic+human+vaginal+epithelial+cell+model.">Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model.</a> Biol. Reprod. 82, 617-617 (2010).</li><li>Straub, T. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+tissue+culture+model+for+the+study+of+attach+and+efface+lesion+formation+by+enteropathogenic+and+enterohaemorrhagic+Escherichia+coli.">A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.</a> Cell. Microbiol. 7, 1771-1771 (2005).</li><li>Sainz, B., TenCate, V., Uprichard, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+Huh7+cell+culture+system+for+the+study+of+Hepatitis+C+virus+infection.">Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection.</a> Virol. J. 6, 103-103 (2009).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>Lelkes, P. I., Ramos, E., Nikolaychik, V. V., Wankowski, D. M., Unsworth, B. R., Goodwin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTSF-2:+a+new,+versatile+cell+culture+medium+for+diverse+normal+and+transformed+mammalian+cells.">GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.</a> In Vitro Cell. Dev. Biol. Anim. 33, 344-344 (1997).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcarrier+Cell+Culture+-+Principles+and+Methods.">Microcarrier Cell Culture - Principles and Methods.</a> Handbooks. , (2005).</li></ol>

우리는 공개 아무것도 없어.

여기에 제시 문화 기술은 고도로 침입 EVT 같은 세포와 수사관을 제공합니다. 그것은 지금은 차별화의 손실 세 차원 (꼭대기의, 기저, 그리고 측면 세포 표면) 10,13의 화학 및 분자 단서에 세포 반응의 억제에 의한 monolayers에서 발생되는 인정되었습니다. 이 기술은 EVT 세포를 침해에 대한 utero에 명시된 바와 같이 특징을 반영합니다. 절차는 기존의 monolayer 조직 문화 시간 속도론을 모?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Cytodex microcarrier 비즈 </td><td> 시그마 - 올드 리치 </td><td> C3275 </td><td></td></tr><tr><td> 회전 세포 배양 시스템 (RCCS) </td><td> Synthecon </td><td> RCCS - D </td><td> 회전자베이스, 전원 공급 장치, 4 일회용 RCCS 단위를 포함 </td></tr><tr><td> RCCS 일회용 단위 </td><td> Synthecon </td><td> 연락처 Synthecon </td><td></td></tr><tr><td> 3ml Luer - 잠금 팁 주사기 </td><td> BD </td><td> 309,585 </td><td></td></tr><tr><td> 10ml 넓은 팁 serological 피펫 </td><td> BD </td><td> 357,504 </td><td></td></tr><tr><td> 가상 알파 </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> Leibovitz의 L - 15 배지, 분말 </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, 내독소 무료 </td><td> 어부 </td><td> MT - 25-055 - CM </td><td></td></tr><tr><td> 나트륨 중탄산염 </td><td> 시그마 - 올드 리치 </td><td> S - 7795 </td><td></td></tr><tr><td> 펩톤 </td><td> 피셔 과학 </td><td> BP1420 - 100 </td><td></td></tr><tr><td> Fructose와 </td><td> 시그마 - 올드 리치 </td><td> F3510 - 100 </td><td></td></tr><tr><td> 갈락토 오스 </td><td> 시그마 - 올드 리치 </td><td> G5388 - 100 </td><td></td></tr><tr><td> 포도당 </td><td> 시그마 - 올드 리치 </td><td> G7528 - 250 </td><td></td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L - 글루타민 </td><td> Invitrogen </td><td> 25,030 </td><td></td></tr><tr><td> 인슐린 - 트랜스페린 - 나트륨 Selenite (ITS) </td><td> 시그마 - 올드 리치 </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10,437 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Invitrogen </td><td> 15,140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

인간 trophoblast 연구 분야는 placentation 동안 설립 복잡한 환경을 이해 에이즈. 이러한 연구의 성격으로 인해, 생체내 실험에서 인간이 불가능합니다. 차 문화, explant 문화와 trophoblast 세포 라인 1 조합은 질 벽에 2의 침략과 임신의 성공 구축에 필요한 extravillous trophoblast 세포 (EVTs)에 의해 자궁 나선형 동맥 3,4의 리모델링에 대한 우리의 이해를 지원합니다. 이러한 모델에서 gleaned 지식의 풍부한에도 불구하고, 그것은 자신의 생체내의 대응에 5,6에 비해 EVT 같은 세포 라인을 사용하여 시험 관내 세포 배양 모델에서 세포 속성을 변경 표시 허용됩니다. 회전 세포 배양 시스템 (RCCS)에서 교양 세포가보다 밀접하게 U에서 차별 모방되는 EVT 같은 세포 라인의 형태학의 phenotypic 및 기능 속성을 표시침략 (예 : 매트릭스 metalloproteinases (MMPs))와 trophoblast 차별 7, 8, 9을 중재 유전자의 증가 표현과 tero의 EVTs. 셍 조르즈 병원 Placental 세포 라인 4 (SGHPL - 4) (친절하게 박사 가이 휘틀리 박사 주디스 카트 라이트에 의해 기증) RCCS의 테스트에 사용되었다 EVT 같은 세포 라인입니다. RCCS 문화 선박의 디자인은 장기와 조직은 3 차원 (3 - D) 환경에서 작동하는 원리에 따라 달라집니다. 3 차원에 재배 생리학 관련 전단 조건을 포함한 선박의 동적 문화 조건, 세포에 의한 자연 세포 동질성을 기반으로 집계를 형성하고 organotypic 조직과 같은 어셈블리 10,11,12으로 구분. 유체 궤도의 유지 보수 생체내에서 발견 조건과 비슷한 낮은 전단, 낮은 난기류 환경을 제공합니다. 배양해 세포의 침전이 회전을 조정하여 반박입니다세포의 지속적인 자유 낙하를 보장하기 위해 RCCS 속도. 가스 교환은 생물 반응기의 뒷면에있는 투과 소수성 막을 통해 발생합니다. 생체내 자신의 부모의 조직과 마찬가지로, RCCS - 성장 세포들은 다공성 microcarrier 비즈의 표면에 양식 때문에 삼차원 (그들, 혀끝의 기초, 그리고 측면 표면에서 IE)에서 화학 및 분자 그라디언트에 응답할 수 있습니다. 플라스틱처럼 스며들지 않는 표면에 2 차원 monolayers으로 성장하면 세포들은 기저 표면에서이 중요한 커뮤니케이 션의 박탈됩니다. 따라서, 환경에 의해 부과된 공간적 제약이 심히 따라서 3 차원 환경 13 중요한 역할을 암시, 주변 microenvironment에서 세포의 감각과 디코딩 신호 방법에 영향을 미칩니다. 우리는 다양한 인간의 상피 조직 7,14,15,16의 생물 학적 의미 3 - D 모델을 엔지니어링하는 RCCS를 사용했습니다. 사실, 많은 이전 리포트는 민주 공화국을 가지고RCCS의 교양 세포가 다른 모델 10,17-21 가능하지 않은 생리학 관련 phenotypes을지지 수 onstrated. 요약에서 RCCS의 문화는 실험 조작의 다양한 의무 아르 차별화된 세포의 큰 번호를 제공하는 쉽고 재현성, 높은 처리량 플랫폼을 나타냅니다. 다음과 같은 프로토콜, 예를 들어 EVTs를 사용하여, 우리는 명확하게 RCCS 세 - 치수 문화 자기편 세포에 필요한 단계를 설명합니다.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

전통적인 2 차원 세포 배양 기술은 종종 차별화 마커, 크린 시토킨 및 성장 요인과 관련하여 변경된 특성의 결과. extravillous trophoblast 세포 라인과 함께 그림과 같이 회전 세포 배양 시스템의 세 가지 차원 세포 배양 (RCCS)는 이러한 요소 중 많은 표현 reestablishes.

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

rotating-cell-culture-systems-for-human-cell-culture-human

Research

JoVE Journal

Bioengineering

16.4K 조회수.  Tulane University Medical School. 전통적인 2 차원 세포 배양 기술은 종종 차별화 마커, 크린 시토킨 및 성장 요인과 관련하여 변경된 특성의 결과. extravillous trophoblast 세포 라인과 함께 그림과 같이 회전 세포 배양 시스템의 세 가지 차원 세포 배양 (RCCS)는 이러한 요소 중 많은 표현 reestablishes.

비디오: Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

기계 다이나믹 셀 문화 Microfabricated 플랫폼

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

연구

생체공학

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

활성 세포 배양을위한 형상 기억 폴리머

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

다른 Lineages로 인간 배아 줄기 세포 (hESC)의 전파 및 차별 화를위한 3D 플랫폼으로서 알긴산의 Microcapsule

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

종양 세포 침략에 중간 유체 흐름의 효과를 측정하는 세 가지 차원 세포 배양 모델

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

대량 세포 계측법 : CyTOF 질량 Cytometer에 매일 조정하고 실행 세포 샘플에 대한 프로토콜

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

수성 2 단계 시스템을 사용하여 셀 공동 문화 패턴

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

독성 흡입 화학 시험에 대한 시험 관내 세포 배양 모델에서

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

인간의 유선 상피의 호르몬 응답 3D 문화 모델

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

듀얼에 대 한 생체 모방 지하실 멤브레인으로 ultrathin Porated 탄성 Hydrogels 세포 배양

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...