이 작품은 NIH R01 GM47607, CA163296 및 CET에 NSF 1,334,493에 의해 지원됩니다. 우리는 원고의 중요한 읽기 터너 실험실의 구성원을 감사드립니다. 이 보고서에 표시된 모든 데이터는 Wormer 등. 2014 (11)의 허가에 의해 재현되었다.

Durotaxis 챔버 1. 준비 <ol><li> 하나의 6- 웰 조직 배양 플레이트를 준비하기 위해, 50ml의 원뿔형 튜브 용기를 설정. 50ml의 튜브에서 PDMS 염기 용액의 약 10g을 칭량 (솔루션은 매우 점성). </li><li> 90 : 1 기판 (1 ~ kPa로의 적합성), 필요에 가교 결합제 용액의 정확한 양을 결정 (90)에 의해 튜브에 PDMS 염기 용액의 중량을 측정 나눈다. 같은 튜브에 PDMS의 가교제 용액의 계산 된 금액을 추가합니다. 예 : 10g / 90 = 0.11 G. PDMS 염기 용액에 PDMS 가교제 용액 0.11 g을 추가한다. 참고 : 기본 및 가교제 솔루션은 모두 PDMS 키트에 제공됩니다. </li><li> 격렬 작은 주걱을 사용하여 실온에서 5 분 동안 PDMS 기재 / 가교 결합제의 혼합물을 혼합한다. 이 단계에서 혼합물을 공기 방울 다수를 포함 할 것이다. </li><li> BUB을 제거하기 위해 실온에서 50 XG에 5 분 동안 벤치 탑 원심 분리기에서 PDMS 기판을 원심 분리기bles. 이 경우 여전히 다시 5 분, 원심 분리 후 거품. </li><li> 90 피펫 1ml의 : 조직 배양의 각 웰에 1 PDMS 기판은 6 웰 플레이트를 처리 하였다. PDMS에 존재하는 임의의 잔류 기포는 21 G 바늘을 사용하여 팝핑함으로써,이 단계에서 제거 될 수있다. PDMS가 아니라 30 분 동안 전파하도록 허용합니다. </li><li> 종기 12mm 유리 # 5 분간 증류수 1 커버 슬립. 두 번 반복하고 증류수 된 커버를 저장합니다. </li><li> 부드럽게 한 후 PDMS에 커버 슬립을 드롭 PDMS 솔루션에 coverslip에의 한 쪽을 터치하여 조직 배양 플레이트의 각 웰에 하나, 건조 coverslip에 배치합니다. 커버 슬립이 침전으로, PDMS는 커버 슬립의 가장자리를 통해 잠식하기 시작하지만, 완전히 포함되지 않습니다. 이것은 (, B를 그림 1A 참조) 경화 후 PDMS와 유리 사이의 인터페이스를 생성합니다. </li><li> PDMS를 치료 (경화) 16 시간 동안 오븐에서 70 ° C에서 접시를 품어. 작은지면 배치세포 배양 후드에서 UV 및 E를 10 분 동안 살균. 참고 : 사용의 몇 일 이내에 판을 만드는 것이 가장 좋습니다. 그러나 판은 버퍼없이 파라 필름에 싸여과 품질에 띄는 감소없이 최대 2 주 동안 4 ℃에서 저장 될 수있다. </li></ol> 2. 셀 도금 주 : siRNA를 매개 녹다운의 효과를 연구하는 경우, 제조자의 지시 나 선택의 세포 유형에 대한 최적의 프로토콜을 이용하여 최저을 수행한다. <ol><li> 각 코트는 37 ℃에서 1 시간 동안 칼슘 및 마그네슘이없는 / ml의 PBS에 피브로넥틴을 10 μg의 1 ml의 챔버 durotaxis. 대안 적으로, 피브로넥틴은 4 ℃에서 16 시간 동안 / ml의 PBS에 피브로넥틴을 10 μg의 PDMS와 인큐베이션하여 분석 전에 일을 적용 할 수있다. 전체 표면을 PBS 용액에 피브로넥틴에 빠져들되어 있는지 확인합니다. </li><li> 열 변성 1 % BSA를 준비합니다. 0.5 g의 무게 BSA 및 PBS 50 ㎖에 용해.필터는 12 분 동안 80 ℃에서 0.22 ㎛의 필터와 열교환을 통해 살균 용액. 참고 :이 솔루션 4 ℃에서 도금 및 저장 셀하기 전에 하루를 준비합니다. </li><li> 피브로넥틴 솔루션을 기음과 PBS로 3 회 반복한다. 각 웰에 PBS에 열 변성 BSA 1 ㎖를 추가하고 실온에서 30 분 동안 품어. </li><li> durotaxis 챔버가 열 변성 BSA로 차단하는 동안를 Trypsinize 및 선택의 세포를 계산합니다. </li><li> 플레이트 원하는 특정 세포 유형에 필요한 매체를 사용 durotaxis 챔버의 각 웰에 2 ㎖를 1 × 105 세포. 세포 부착 및 5 % CO 2, 37 ℃에서 가습 된 배양기에서 4 시간 동안 기판에 도포 할 수있다. 주 : U2OS 세포는 일상적 2 mM L- 글루타민, 1 mM 피루브산 나트륨, 10 IU / ml의 페니실린, 10 μg의 / ml의 스트렙토 마이신이 보충 된 10 % FBS DMEM으로 유지된다. 주 :이 예에서 사용되는 세포 수에 최적화U2OS 세포. 다른 유형의 세포에 대한 최적화가 요구 될 수있다. 이 밀도는 세포를 다른 세포와 중요한 상호 작용없이 마이그레이션 할 수있는 충분한 공간을 제공합니다. </li></ol> 3. 라이브 셀 이미징 <ol><li> 10X 목표와 위상 콘트라스트를 사용하여, 거꾸로 현미경에 라이브 세포 이미징을 수행합니다. 현미경은 장기 촬상 동안 37 ℃ 온도의 제어 및 5 % CO 2 있도록 밀폐 환경, 습한 챔버가 장착되어야한다. </li><li> 세포를 약 3.5 시간 동안 분산 한 후, 현미경 실 플레이트를 조립한다. 샘플 (S)이 30 분 동안 챔버에서 평형화 허용한다. </li><li> 가능한 경우 현미경에 방문 자동화, 멀티 포인트를 설정합니다. (90) 사이의 인터페이스에 초점 : 1 PDMS와 유리 coverslip에 모든 durotaxis 챔버 당 40 점의 평균과의 인터페이스 주위에 이미지에 포인트를 선택합니다. 이미지 셀 최대 16 시간 동안 매 10 분. 참고 : 재관심의 기온은 커버 슬립의 가장자리와 PDMS와 커버 슬립 사이의 실제 인터페이스에 대응하는 내부 라인에 해당하는 외부 줄, 두 줄로 표시됩니다. 그림 1B를 참조하십시오. </li></ol> 4. 데이터 분석 <ol><li> 표 1과 같은 스프레드 시트를 생성한다. </li><li> 생성 된 각각의 동영상에서 유리 표면과 그 반대로 PDMS에서 교차 이벤트의 수를 카운트. 엑셀 스프레드 시트의 해당 열에 교차 이벤트의 수를 기록한다. 주 : 교차 이벤트가 어느 한 방향으로 PDMS와 유리 사이의 경계 위에 전달 세포핵으로 정의된다. </li><li> 다중 교차를 정량화하기 위해, 셀은 인터페이스 교차 회수를 카운트. 그 숫자는 셀이 영화의 단부에 위치 된 기판 상에 대응하는 엑셀 열에 기록되어야한다. t의 인터페이스를 가로 지르는 모든 셀에 대한 분석을 반복그는 영화. 촬영하는 동안 시야에서 마이그레이션 세포를 제외합니다. 주 : 세포가 피브로넥틴 - 코팅 된 PDMS와 유리 커버 슬립의 표면에 균일하게 부착 할 수있는 것을, 실험의 초기에 셀 카운팅함으로써 확인하는 것이 중요하다. </li><li> 유리 표면 (즉, durotaxis을 시행)에 PDMS에서 마이그레이션 세포의 비율을 계산합니다. 소프트에서 하드 및 교차 이벤트의 총 수에 의해 하드 및 분할에 끝났 여러 교차 이벤트에 이벤트를 건너의 수를 추가합니다. </li><li> 교차점의 총 수에 의해 다중 교차의 수를 분할하여 복수의 횡단의 비율을 계산한다. </li></ol>

<ol>
	<li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Paszek, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Levental, K. R., 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=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79 (1), 144-152 (2000).</li><li>Plotnikov, S. V., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+cell+migration+by+tugging.">Guiding cell migration by tugging.</a> Current Opinion in Cell Biology. 25 (5), 619-626 (2013).</li><li>Choquet, D., Felsenfeld, D. P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+rigidity+causes+strengthening+of+integrin-cytoskeleton+linkages.">Extracellular matrix rigidity causes strengthening of integrin-cytoskeleton linkages.</a> Cell. 88 (1), 39-48 (1997).</li><li>Balaban, N. Q., 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=Force+and+focal+adhesion+assembly:+a+close+relationship+studied+using+elastic+micropatterned+substrates.">Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.</a> Nature Cell Biology. 3 (5), 466-472 (2001).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Wang, Y. L., Pelham, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+flexible,+porous+polyacrylamide+substrate+for+mechanical+studies+of+cultured+cells.">Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.</a> Methods in Enzymology. 298, 489-496 (1998).</li><li>Prager-Khoutorsky, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+polarization+is+a+matrix-rigidity-dependent+process+controlled+by+focal+adhesion+mechanosensing.">Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing.</a> Nature Cell Biology. 13 (12), 1457-1465 (2011).</li><li>Wormer, D. B., Davis, K. A., Henderson, J. H., Turner, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+focal+adhesion-localized+CdGAP+regulates+matrix+rigidity+sensing+and+durotaxis.">The focal adhesion-localized CdGAP regulates matrix rigidity sensing and durotaxis.</a> PloS ONE. 9 (3), e91815 (2014).</li><li>Trichet, L., 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=Evidence+of+a+large-scale+mechanosensing+mechanism+for+cellular+adaptation+to+substrate+stiffness.">Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness.</a> Proceedings of the National Academy of Sciences. 109 (18), 6933-6938 (2012).</li><li>Eyckmans, J., Boudou, T., Yu, X., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hitchhiker&#8217;s+guide+to+mechanobiology.">A hitchhiker&#8217;s guide to mechanobiology.</a> Developmental Cell. 21 (1), 35-47 (2011).</li><li>Martinac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensitive+ion+channels:+molecules+of+mechanotransduction.">Mechanosensitive ion channels: molecules of mechanotransduction.</a> Journal of Cell Science. 117 (12), 2449-2460 (2004).</li><li>Jafar-Nejad, H., 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=Sec15,+a+component+of+the+exocyst,+promotes+notch+signaling+during+the+asymmetric+division+of+Drosophila+sensory+organ+precursors.">Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors.</a> Developmental Cell. 9 (3), 351-363 (2005).</li></ol>

The authors have no conflicts to disclose.

여기에서 우리는 세포를 마이그레이션하는 durotaxis을 연구하는 간단한 분석을 설명합니다. 이 분석의 주요 장점은 PDMS를 사용 durotaxis 챔버 제조의 용이성이다. 기판의 강성을 용이하게 분석에서 다양한 강성의 연구를 허용하도록하기 위해 가교제 PDMS 염기 용액의 비율을 변경함으로써 조작 될 수있다. 그러나, 시스템의 하나의 잠재적 인 한계는 더 정교한 시스템 (4)에 의해 제공되는 강도...

세포 외 기질 (ECM)의 콜라겐, 피브로넥틴 및 라미닌을 포함하고, 가교 구조 단백질의 복잡한 배열로 구성된다. 이 아니라 ECM 셀룰러 조직 중요한 구조적지지를 제공한다는 확립되어 있지만, 세포가 활발히 세포 생존, 분화 및 세포 이동을 포함한 다양한 세포 과정을 조절하는 그들의 ECM 환경에서 물리적 인 변화에 대응 것을 나타내는 증거가 증가하고있다. 딱딱한 (~ 25 kPa로) 기판 조골 세포 분화를 촉진 한 예를 들어, ECM의 강성 차이는 연질 기판 (~ 1 kPa의) 신경성 계통 촉진과 함께, 서로 다른 계통으로 중간 엽 줄기 세포를 구동 할 수있다. 마찬가지로 기질 매트릭스 강성 증가는 주변 조직에 2,3- 유방 상피 세포 및 종양 침윤을 촉진하는 것으로 나타났다. 이 mechanosign의 특히 흥미로운 양태세포가 더 견고한 기판 4,5- 향해 우선적으로 마이그레이션하는 durotaxis로 알려진 프로세스에서 aling 활동 결과. 세포는 지속적 ECM 결합 인테그린 수용체를 통해 세포 외 환경의 물리적 특성을 감지. 이것은, 차례로, 국소 유착 또는 초점 콘택트 -6,7-라고도 접착제 구조의 형성을 구동 할 그들의 세포질 도메인 수많은 구조 및 시그널링 단백질의 축적을 촉진한다. 인테그린이 더 고유의 효소 활성이 없기 때문에, 신호는 변화하는 환경에 8 셀의 응답을 조정하기 위해 이러한 액세서리 단백질을 통해 ECM에서 릴레이됩니다. 따라서, mechanosignaling durotaxis 및 조절에 관여하는 주요 단백질의 동정 및 특성 규명은 수사의 중요한 영역이다. 다양한 모델 시스템 durotaxis 관내 연구 개발되어 왔지만, 대부분은 할이용 콜라겐 코팅 된 폴리 아크릴 아미드 기판 (4). 그러나, 폴리 아크릴 아미드의 기판의 제조는 기술적으로 어려울 수 있으며, 상기 분석 방법에 사용되는 콜라겐은 기판 (9)와 화학적으로 가교되어야한다. 폴리 디메틸 실록산 (PDMS) 기판은 폴리 아크릴 기판 (10)에 필적하는 기계적 성질을 나타내는 것으로 밝혀졌다. 그러나, PDMS 기판을 간단히 가교제 염기의 혼합 비율에 의해 제조되며, 이러한 기판은 이에 PDMS 세포 행동에 강성의 영향을 연구하기 쉬운 도구를 만드는 화학적 가교의 필요성없이 ECM 단백질로 코팅 될 수있다. 여기서, 우리는 부드러운 PDMS 기판이 경질 유리 커버 슬립과 통합되는 간단한 durotaxis 실을 준비하는 방법에 대해 설명합니다. 분석은, 아래에 설명 된대로, durotaxis을 연구하는 빠르고 간단한 방법을 제공합니다. 이 연구를 위해 우리는 siRNA를 매개로 결합 된 인간의 U2OS의 골육종 세포를 사용cdGAP의 최저은 durotaxis (11)이 초점 접착 단백질의 역할을 연구합니다. 중요한 것은,이 프로토콜은 개별적인 요구에 쉽게 적응 될 수있다. 다른 유형의 세포는 U2OS 세포를 대체 할 수 있고, 어떤 단백질은 뜨거나 durotaxis 동안 셀의 동작에 미치는 영향을 결정하기 과발현 될 수있다. 더욱이,이 프로토콜은 역학 거동 사용 FRAP 또는 접근법 FRET을 분석 형광 태그로 단백질을 포함하도록 구성 될 수있다.

<table><tbody><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>3097358-1004</td><td>Sylgard 184 Silicone Elastomer Kit</td></tr><tr><td>#1 Cover glass 12 mm</td><td>Fisher Scientific</td><td>12-545-82</td><td></td></tr><tr><td>6-well plate</td><td>Celltreat</td><td>229106</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>15-017-CM</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Fisher Scientific</td><td>BP356-100</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>610077</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>21600-044</td><td></td></tr><tr><td>Falcon tubes</td><td>Celltreat</td><td>229456</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>U2OS cells</td><td>ATCC</td><td>HTB-96</td><td></td></tr></tbody></table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

세포 외 매트릭스의 조성과 기계적 성질이 조직 유형 사이에서 매우 가변적이다. 이 크게 결합 조직 기질 다이버 영향 셀 동작은 세포 증식, 분화, 접착 시그널링 및 이동 방향을 포함하여 정상 및 병리학 적 과정을 조절한다. 이와 관련하여, 특정 세포 유형의 타고난 능력 durotaxis라고도 단단, 이하 호환 매트릭스 기판을 향해 이동한다. 이 현상은 배아 발생, 상처 복구 및 암세포 침윤 동안 중요한 역할을한다. 여기, 우리는 폴리 디메틸 실록산 (PDMS) 기판을 사용하여 시험 관내에서, durotaxis을 연구하는 간단한 분석을 설명합니다. 챔버 durotaxis 설명의 준비는 상대적으로 부드러운 PDMS 젤 단단한 유리 커버 슬립 사이에 강성 인터페이스를 만듭니다. 제공된 예에서, 우리는 cdc42의 역할을 입증하는 이들 durotaxis 챔버를 사용하고 / RAC1는 GTPase 활성 보호 자전거cdGAP에서, mechanosensing 인간 U2OS의 골육종 세포에서 durotaxis 규제. 이 분석은 mechanosignaling과 durotaxis에서 각각의 역할을 탐구하는 다른 세포 유형 및 / 또는 관심의 다른 단백질의 최저에 쉽게 적응할 수있다.

durotaxis 챔버의 개략도는도 1a에 도시되어있다. 소프트 PDMS 기판 (90 : 솔루션 가교제 PDMS 기재 1 혼합물)을 6 웰 접시에서 확산되고 유리 커버 슬립시켜 인터페이스를 생성하고 부분적으로 커버 슬립의 상부 표면을 덮는 PDMS, 위에 배치되는 다른 준수의 두 기판 사이. 소프트 PDMS 기판의 강성은 유리의 강도가 약 1 GPa의 1-2 반면, 뇌 조직의 일반적인 적합성에 필적 ~ 1 kPa로한다. PDMS...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

평가 셀 Mechanosensing 및 Durotaxis에 대한 단순화 된 시스템<em&gt; 체외</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

a-simplified-system-for-evaluating-cell-mechanosensing-durotaxis

Research

JoVE Journal

Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.2K 조회수.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

비디오: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

마이크로 실리콘 캔틸레버의 이용은 세포 수축 기능을 평가하기 위해<em&gt; 체외</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

연구

생체공학

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

초파리 배아에 구슬 없는 광학 핀셋으로 셀 역학 조사

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

발생학

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

광학 핀셋을 사용하여 감금된 세포 전 역학의 직접 힘 측정

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

의 Mechanosensation에 대한 행동 분석 클론의 MARCM은 기반<em&gt; 초파리</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

신경과학

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS / 택시와 셀룰러 Chemotaxis 측정

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

생물학

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Mechano-Node-Pore 감지: 다중 파라미터 단일 셀 점탄성 측정을 위한 신속한 무표지 플랫폼

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

고처리량 미세유체 압축 시스템을 통한 다세포 유기체의 기계자극

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

자석 구슬을 사용하여 기계 인장에 대한 응답으로 세포 표면 접착 리모델링 분석

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum 응답 심각한 기계적인 자극의 평가

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

원뿔형 플레이트 점도를 통한 인간 혈소판 및 세포 표면 수용 체에 대 한 균일 한 전단 분석

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

평가 셀 Mechanosensing 및 Durotaxis에 대한 단순화 된 시스템

요약

더 많은 비디오 탐색

이 비디오의 챕터

마이크로 실리콘 캔틸레버의 이용은 세포 수축 기능을 평가하기 위해