저자는이 작품을 자금에 대한 허가 번호 UO1CA143069, R01CA142833, R01CA114462, RO1CA179556, T32-AG000213-24 및 T32-GM008692-18을 인정하고 싶습니다. 우리는 또한 CT-화재 분석의 개발 및 지원 궤적의 제레미 Bredfelt 및 Yuming 리우를 인정합니다.

1. 중화, 희석 및 3D 조사 및 세포 수축 분석 실험을위한 콜라겐 솔루션의 중합 <ol><li> 무균 조직 배양 후드에서 얼음에, 콜라겐 (1 : 1) 중화 2 배 PBS에서 살균, 얼음처럼 차가운 100 mM의 HEPES, 15 ML 원뿔 튜브의 pH 7.4,와. 용액이 균질하지 않고 혼합 소용돌이가 더 이상 볼 수 있습니다 때까지 플라스틱 피펫 충분히 섞는다. 혼합 과정에서 공기 방울을 소개 않도록주의하십시오. 얼음에 잠시 저장합니다. </li><li> 세포 미디어와 적절한 농도 (등 RPMI, DMEM, 등)에 중화 된 콜라겐을 희석. <ol><li> N은 = (D은 V *), V는 / (S / 2), N이 필요 중화 콜라겐 양이고는 D 원하는 콜라겐 농도는 인 등식을 사용하여 목적하는 콜라겐의 농도를 확인하는 데 필요한 중화 콜라겐의 양을 계산하는데 바람직한 콜라겐 농도의 최종 부피 및 S는 t이며그는 재고 콜라겐 농도를 시작. </li><li> 세포 및 세포 배지 용액을 첨가하여 부피로 중화 콜라겐의 양을 준비한다. 철저하게 혼합하고 주조 할 준비가 될 때까지 얼음에 저장합니다. <ol><li> 예 : (6 웰 플레이트 또는 50mm 유리 바닥 접시 겔 캐스팅을위한 일반적인 양), 2 ㎎ / ㎖의 1 ㎖의 겔 부피 젤 먼저 원하는 최종 부피 원하는 농도 (2 ㎎ / ㎖)을 곱 (1 mL)을 첨가 하였다. 이 번호 (2 mg)을 가지고 병에 상장 주식 농도 (9.49 ㎎ / ㎖)의 절반을 나눕니다. 이 경우, 중화 된 콜라겐 0.42 mL의 미디어 / 세포 혼합액 0.58 ㎖로 희석한다. </li></ol></li></ol></li><li> 피펫이 아닌 조직 배양 중 하나에 얼음처럼 차가운 콜라겐 / 셀 / 미디어 혼합물을 6 웰 플레이트 또는 50mm의 유리 바닥 접시를 처리 하였다. 균등하게 솔루션을 확산 피펫 팁을 사용합니다. 주 : collag 외부 세포의 부착 또는 성장을 최소화하기 위해 처리 된 플레이트가 아닌 조직 배양을 사용하는 것이 중요젤 갖추고 있습니다. </li><li> 15 분 - 약 10 상온에서 중합 할 수 있습니다. 겔 중합에 불투명 설정해야합니다. </li><li> 중합을 완료 60 분 -이 불투명 설정 한 후, 추가로 45 37 ˚C에 플레이트 접시를 이동합니다. </li><li> 45 후 - 잘 또는 접시의 경계 주위 P200 피펫 팁을 실행하여 잘의 측면에서 젤 미디어의 3 ㎖ 및 해제 - 60 분, 2를 추가합니다. 소용돌이 요리 부드럽게 젤을 분리합니다. 콜라겐 젤은 미디어에 떠되어야한다. </li></ol> 2. 웨스턴 블로 팅, 세포 형태 형성 및 젤 Gontractility 분석은 <ol><li> 십일 분석 - 7에 대한 세포와​​ 시드, 1.6 - 강성을 ECM 관련하여 단백질 수준, 형태 또는 휴대 수축력을 평가하기 위해, 1.1에 따라, 콜라겐 젤을 부어 시작합니다. 성장률 및 포화 상태에 따라 경험적으로 각 세포주 시드 레이트를 결정한다. 100,000 세포 / 겔 - 10 일 시험의 경우, 파종 밀도에서 20000의 범위 일 수있다. </li><li> 측정세포 수축은 룰러 또는 카메라 매 24 시간을 사용하거나, 적당한 시간 간격으로 겔 직경을 측정한다. 주 : 또한 현미경 수집 대응하는 이미지는 꽈리 형 구조, 세뇨관 상피 세포 돌기 및 lameliapodia 같이 겔 시드 세포주의 형태 적 특징의 특성을 검사 할 수있다. </li><li> , 단백질 수준을 평가 워즈니악 등. (17)와 갤러거 등. (18)의 설명에 따라 관심의 단백질의 웨스턴 블롯 분석을 위해 RIPA에서 젤은 버퍼 용균 및 방법에 관한 것이다. <ol><li> 중요 : 세포 라인 사이의 수축의 비교에서, 루이에 의해 설명 된대로 젤에서 추출 할 수있는 전체 DNA에 수축력을 정상화, 등 (19), 또는 서양에 의해 변하지 않는, 가사 단백질 (히스톤, GAPDH, 등). 블롯 분석, 워즈니악 등. (17)와 갤러거 등의 설명에 따라. (18). 겔 내에서 계산 세포는 혈구를 사용하는 경우, 세포를 집중 계약 젤 등 총 젤 지역에 휴대폰 번호를 정상화해야합니다. </li></ol></li><li> 매체 1 ㎖를 제거하고 신선한 미디어 1 ㎖로 교체하여 4 일 - 피드 겔 매 3. 측정이 수축에 스파이크가 발생할 신선한 미디어 / 혈청의 첨가로 촬영 한 후 젤을 공급해야합니다. </li></ol> 콜라겐 섬유 정렬을위한 PDMS 마이크로 채널 3 세대 참고 : 정렬 된 콜라겐 매트릭스를 생성하려면, PDMS 마이크로 채널 (그림 2A)을위한 금형은 소프트 리소그래피 (15)를 통해 만들어진 SU-8 실리콘 마스터가 필요합니다. <ol><li> PDMS 채널을 만들기 위해, 공예 막대기로 일회용 컵에 철저하게 PDMS를 섞는다. 6 인치 실리콘 마스터를 들어, 경화제 2 g의 엘라스토머베이스 20g을 혼합한다. </li><li> 탈 가스를 진공 압력으로 진공 챔버의 일회용 컵을 배치하여 PDMS 혼합물550mm 수은. 드 가스 1 - 1.5 시간. </li><li> PDMS의를 드 가스 배출하지만, 쏟아져의 실리콘 마스터를 준비합니다. 실리콘 마스터이어서, 핫 플레이트 상에 투명 필름의 깨끗한 시트를 배치하여 준비한다. 마이크로 몰드가 직면하고 있는지 확인합니다. 주 : PDMS 주조 및 경화 동안 실리콘 마스터 투명 필름 두 장 사이에 개재된다. </li><li> 한 후 - 1.5 시간, 진공 챔버에서 탈기 PDMS를 제거하고 천천히 실리콘 마스터 위에 붓는다. 중요 : 공기 방울을 피하십시오. 마스터의 중심에 부어, 그리고 중력이 균등하게 확산 할 수 있도록 계속합니다. 주 : PDMS 드롭 마스터 가장자리 끝까지 연장 할 필요는 없다. </li><li> PDMS 마스터에 부은 후 실리콘 마스터와 PDMS 위에 투명 필름의 제 2 시트를 적용한다. 조심 기포 않도록 아래 PDMS / 실리콘 마스터 위에 제 투명성 시트 롤. 서두르지 마. PDMS는 지금 균등 O를 확산해야버전 마스터. </li><li> 부드럽게 1/8 &quot;아크릴 시트 뒤에 투명성의 상단에 고무 시트를 배치합니다. </li><li> 아크릴 시트의 상단에 세 10 파운드 무게를 추가합니다. 초기에, 가중치는 &quot;부유&quot;한다. 그들이 더 진행하기 전에 정착 및 안정화 할 수 있습니다. </li><li> 4 시간 동안 70 ℃ 및 치료 PDMS에 열판의 온도를 설정합니다. 방해하기 전에 1 추가 시간의 최소 냉각시킵니다. </li><li> 조심스럽게 웨이퍼에서 투명 필름 위에 시트를 벗겨, 그리고 집게로 채널을 제거합니다. </li><li> 준비가 될 때까지 먼지가없는 접시에 스토어 사용할 수 있습니다. </li></ol> 사용 4. 준비하고 PDMS 마이크로 채널 <ol><li> 거꾸로 새로운, 깨끗한 투명 필름에 배치 채널. 날카로운 집게로 원 운동을 사용하여 청소 모든 포트 (그림 2b). PDMS의 조각을 제거합니다. </li><li> 압정으로 천 테이프 포장의 일부를 이용하여 채널 면도. 벤치의 표면 (최대 접착면)에 테이프를 적용하고 채널 O를 설정n 개의 최고. 좋은 접촉을 보장하기 위해 집게의 둥근 끝 채널 아래로 누릅니다. 분리 보이는 이물질이 제거 될 때까지 양쪽 반복한다. </li><li> 전사 세정은, 70 % EtOH로 50 ml의 원추형으로 PDMS 채널 수험 공부. 30 초 동안 최대 속도로 소용돌이. EtOH를 폐기하고 신선한 70 % EtOH로 교체합니다. 준비가 될 때까지 70 %의 EtOH에 보관 사용하기. </li><li> 조직 문화 후드 및 무균 기술을 사용하여, 깨끗하고 무균 커버 유리 또는 유리 바닥 요리에 PDMS 채널을 전송할 수 있습니다. EtOH로 증발 할 때까지 채널면이 위로와 UV 치료를 넣습니다. </li><li> 채널은 커브 글라스를 향해 아래로 향하게되도록 일단 건조의 PDMS를 뒤집습니다. 좋은 도장을 확인하기 위해 유리에 PDMS 채널을 누르십시오. 커버하는 멸균 PDMS의 패치를 추가 / 중앙 포트습니다 (포트 B를 그림 2B). 진행하기 전에에 철저하게 건조 할 수 있습니다. </li><li> 프리 코트 멸균 10 μg의 / ㎖의 콜라겐으로 채널 내부. 코팅하는 chann의 상부에 100 μL 방울을 배치엘, 그리고 진공을 통해 그립니다. 37 ℃에서 1 시간 후, 냉장고 콜라겐 코팅 용액으로 채워져 전송 채널. 30 분 - 약 15 침착 해. </li><li> 흡입기 또는 피펫으로 콜라겐 코팅 용액을 제거하고, 콜라겐의 제조를 시작한다. </li></ol> 마이크로 채널 사용 5. 콜라겐 준비 <ol><li> 배 PBS, pH를 7.4에서 얼음처럼 차가운 100 mM의 HEPES와 : 얼음에, 콜라겐 (1 일)을 중화. 균질 할 때까지 철저하게 믹스 (자세한 내용은 섹션 1.1 참조). </li><li> 세포 미디어와 적절한 농도 중화 콜라겐을 희석 (자세한 내용은 1.2 절 참조). 얼음에 15 분 동안 품어. </li><li> 동시에, 냉각 얼음에 채널을 탑재. 참고 : 목표 아래 4 C 또는에서 채널 프로세스의 모든 구성 요소를 가지고있다. 콜라겐 핵 생성 온도 및 시간은 중합 공정에 중요한 파라미터이고, 필요한 경우, 추가의 최적화를위한 시작점이 될 수있다. </li><li> COUN이때 적절한 접종 밀도에서 hemacytometer와 함께 재현 탁 t 세포. 3,000,000 세포 / ml - 계산의 편의를 위해, (1)에 재현 탁. (자세한 내용은 또한 2.1 절 참조). </li><li> 세포가 계산되었고, 15 분 경과하면, 쏟아지는 콜라겐을 진행합니다. </li></ol> 6. 정렬 및 임의의 콜라겐 마이크로 채널 붓는 <ol><li> 채널을 통해 콜라겐을 그리기 전에, 조정​​ 및 인라인 진공 레귤레이터 진공 압력을 설정합니다. 진공 압력은 유도 및 정렬의 정도를 결정 콜라겐 유량을 제어 할 수있는 힘을 제공한다. <ol><li> 랜덤 또는 정렬되지 않은 매트릭스를 들어 10 mmHg의 이하의 진공 압력으로 다양한 채널 (3mm x 세로 200 ㎛의 높이)를 사용한다. </li><li> 정렬 행렬를 들어, 60mm 수은 이상의 진공 압력 좁은 채널 (1mm x 세로 200 μm의 높이)를 사용합니다. </li></ol></li><li> 얼음에서 장착 채널을 제거하고 층류 시간의 청소, 소독 표면에 배치짐 우드. </li><li> (그림 2B) 포트 A에 중화 콜라겐 150 μl를 - 빠르게 작업하고 (120)를 넣습니다. </li><li> 포트 C (도 2b)를 통해 진공 라인에 연결된 25 ㎖ 피펫을 배치하여 채널을 통해 콜라겐을 그린다. 하나의 균일 한 모션을 통해 콜라겐을 그립니다. 중요 : 랜덤 또는 정렬되지 않은 행렬를 들어, 채널을 통해 서서히 콜라겐을 그립니다 (약 0.5-1 초당 mm), 그것은 끝에 도달하면 중지합니다. 정렬 행렬를 들어, 더 빠르게에서 콜라겐을 그릴하지만 공기 방울을 피하기 위해주의하십시오. </li><li> 조심스럽게 P200의 pipetman 또는 흡입기와 포트 지역에서 초과 콜라겐을 제거합니다. </li><li> A와 C. 모든 포트가 지금 적용해야 두 포트를 통해 멸균 PDMS 패치를 놓습니다. </li><li> 이 후 - 3 분, 센터 PDMS 패치 (포트 B)를 제거하고 2 추가 - 중앙 포트 - (10,000 세포 5) 세포의 3 μl를. 15 분 - 부분적으로 또 다른 10 실온에서 (불투명 설정) 중합 할 수 있습니다. </li><li> 10 후- 15 분, 추가로 15 37 ˚C로 전송 - 중합을 완료하는 데 30 분. PDMS 커버를 제거하고 필요에 따라 완전히 채널 및 문화 세포를 충당하기 위해 미디어를 추가 할 수 있습니다. 세포는 이전 미디어 mL를 제거하고, 새로운 미디어 ㎖로 교체하여 공급 될 수있다. </li></ol>

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	<li>Conklin, M. W., 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=Aligned+collagen+is+a+prognostic+signature+for+survival+in+human+breast+carcinoma.">Aligned collagen is a prognostic signature for survival in human breast carcinoma.</a> Am J Pathol. 178, 1221-1232 (2011).</li><li>Provenzano, P. P., 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=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4, 38 (2006).</li><li>Riching, K. 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=3D+collagen+alignment+limits+protrusions+to+enhance+breast+cancer+cell+persistence.">3D collagen alignment limits protrusions to enhance breast cancer cell persistence.</a> Biophys J. 107, 2546-2558 (2014).</li><li>Even-Ram, S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+in+3D+matrix.">Cell migration in 3D matrix.</a> Curr Opin Cell Biol. 17, 524-532 (2005).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cell+migration:+a+multiscale+tuning+model.">Plasticity of cell migration: a multiscale tuning model.</a> J Cell Biol. 188, 11-19 (2010).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197, 439-455 (2012).</li><li>Cukierman, E., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+with+three-dimensional+matrices.">Cell interactions with three-dimensional matrices.</a> Curr Opin Cell Biol. 14, 633-639 (2002).</li><li>Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J., 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=ROCK-generated+contractility+regulates+breast+epithelial+cell+differentiation+in+response+to+the+physical+properties+of+a+three-dimensional+collagen+matrix.">ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix.</a> J Cell Biol. 163, 583-595 (2003).</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, 241-254 (2005).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., 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=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Tlsty, T. D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stroma+and+regulation+of+cancer+development.">Tumor stroma and regulation of cancer development.</a> Annu Rev Pathol. 1, 119-150 (2006).</li><li>Provenzano, P. P., 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=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., 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=Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+Rho/ROCK-dependent+matrix+reorganization.">Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization.</a> Biophys J. 95, 5374-5384 (2008).</li><li>Guo, C., Kaufman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+and+magnetic+field+induced+collagen+alignment.">Flow and magnetic field induced collagen alignment.</a> Biomaterials. 28, 1105-1114 (2007).</li><li>Sung, K. E., 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=Control+of+3-dimensional+collagen+matrix+polymerization+for+reproducible+human+mammary+fibroblast+cell+culture+in+microfluidic+devices.">Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices.</a> Biomaterials. 30, 4833-4841 (2009).</li><li>Lee, P., Lin, R., Moon, J., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+alignment+of+collagen+fibers+for+in+vitro+cell+culture.">Microfluidic alignment of collagen fibers for in vitro cell culture.</a> Biomed Microdevices. 8, 35-41 (2006).</li><li>Wozniak, M. A., 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=Use+of+three-dimensional+collagen+gels+to+study+mechanotransduction+in+T47D+breast+epithelial+cells.">Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells.</a> Biol Proced Online. 7, 144-161 (2005).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G., Ausubel, F. 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=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Current protocols in molecular biology. , 18 (2008).</li><li>Liu, X., Harada, S., Ausubel, F. 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=DNA+isolation+from+mammalian+samples.">DNA isolation from mammalian samples.</a> Current protocols in molecular biology. , 14 (2013).</li><li>Roeder, B. A., Kokini, K., Sturgis, J. E., Robinson, J. P., Voytik-Harbin, 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=Tensile+mechanical+properties+of+three-dimensional+type+I+collagen+extracellular+matrices+with+varied+microstructure.">Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure.</a> J Biomech Eng. 124, 214-222 (2002).</li><li>Bredfeldt, J. S., 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=Computational+segmentation+of+collagen+fibers+from+second-harmonic+generation+images+of+breast+cancer.">Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer.</a> J Biomed Opt. 19, 16007 (2014).</li><li>Bischel, L. L., Beebe, D. J., Sung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+model+of+ductal+carcinoma+in+situ+with+3D,+organotypic+structure.">Microfluidic model of ductal carcinoma in situ with 3D, organotypic structure.</a> BMC Cancer. 15, 12 (2015).</li></ol>

저자는 공개 아무것도 없어

3D 콜라겐 젤은 세포가 해석하는 방법을 이해하고 지역의 미세 환경에 대응하는 우리의 도구 상자에 추가되었다. 이 원고는 3D 콜라겐 매트릭스 내에서 세포를 포함하기위한 아주 기본적인 프로토콜을 제공하고, 재현성 랜덤 또는 정렬 된 콜라겐 섬유와 매트릭스를 생성 할 수 있습니다. 두 프로토콜은 다른 콜라겐 이성체, 가교제, 기타 매트릭스 단백질 잠재적 중합시에 첨가 할 수 있으므로 적응...

Many cellular processes have been extensively studied in 2 dimensions, thereby forming a collective knowledge of basic cell signaling mechanisms. These studies, however, neglect important cellular cues received from the structural and mechanical properties of the local cellular microenvironment and extracellular matrix (ECM). To better understand how cells interact within a physiological context, it is important to study them in 3-dimensional (3D) assays. The ECM for these 3D assays can either be cell-derived or reconstituted from purified proteins. Regardless of the source of the ECM, 3D matrix assays have proven to be invaluable for understanding how cells navigate and interact within the physiological world. For example, cells grown in 3D matrices display distinct modes of locomotion that depend on the mechanical nature of their surrounding ECM which are not observed in 2D experiments4-6. Moreover, cells cultured in 3D also have fewer and less pronounced stress fibers and focal adhesions than their counterparts grown on hard surfaces such as glass or plastic7. 
The importance of contextual 3D assays is not limited to cell migration, however. Some other cell signaling events can only be investigated through the use of 3D assays. During tissue and cell differentiation, the stiffness of the extracellular environment and ECM provides signals that can influence morphogenic events. For example, mammary epithelial tubulogenesis only occurs in low stiffness 3D matrices, but not in stiff matrices nor on 2D substrata8,9. When cultured within stiff 3D matrices, these same epithelial cells take on an aberrant phenotype with increased proliferation and cell membrane protrusions driven through altered FAK and ERK signaling10. Many other signaling pathways and cellular processes are known to be similarly affected by the stiffness of the local cellular environment, and these signaling cascades highlight the importance of investigating signaling events and cellular phenotype in the context of appropriate local mechanical properties of a 3D ECM. 
Collagen I is a particularly relevant protein to use for in vitro studies as it is the most abundant component of the ECM and is responsible for many of the mechanical properties of the cellular microenvironment. While it was originally thought of as merely a structural protein, its role is now known to be much more complex. Collagen fiber composition, architecture, orientation, density, and stiffness all provide a concentrated milieu of signaling information5. During the progression of certain diseases, such as chronic inflammation and tumorigenesis, the collagen network is dramatically remodeled2,11. More specifically in breast cancers, increased collagen deposition and tissue stiffness accompany and likely contribute to tumor progression. In these early tumors, the stiffened collagen network appears strained and more aligned, such that most of the fibers encapsulate the growing tumor2. As the tumor progresses, the collagen continues to reorganize, and regions of the fibrillar network become orientated perpendicular to the tumor boundary2,12. Perpendicular alignment serves as a prognostic biomarker where these patients have a poorer disease free progression and overall survival1. One explanation for this correlation is that the poor outcomes are a consequence of increased dissemination of tumor cells via persistent cell migration in aligned collagen networks3. 
To understand how cells specifically respond to alignment and organization that is observed in tumor progression, it is necessary to generate both random and aligned 3D collagen matrices for experimentation. There are three basic methodologies to induce alignment within fibrillar networks. The first technique utilizes a strain-inducing device where the collagen between two points is contracted or stretched to generate alignment. Fibers parallel to the axis of force are pulled taut while fibers perpendicular to the axis are compressed and buckled. While strain-induced techniques typically offer superb alignment, this approach requires bulky equipment that is not easily adaptable to many platforms3,13. Alternatively, cell-induced strain can be created by placing localized plugs of cells that subsequently contract and align the collagen13. This method has the problem of being variable, as many parameters may be subject to change. The second method utilizes magnetic beads and a magnetic field during polymerization to induce collagen alignment13,14. Good results can be obtained from this method with unsophisticated equipment, but it does require the use of antibodies or some other method to magnetize the polymer. Therefore, it can be somewhat expensive to use, and the stiffness of the collagen gel is potentially modified by the increased connections in the network. Moreover, the magnetic beads used in this process are often autofluorescent, which is problematic for imaging experiments. Lastly, alignment can be generated by PDMS microfluidic channels3,15,16. In this method, collagen alignment is achieved by flowing polymerizing collagen through small microfluidic channels. These microfluidic channels can be made in a multitude of designs, and are easily adaptable to many platforms. Moreover, they are very economical as very small quantities of collagen and other reagents are used due to their diminutive sizes.
Provided here is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. In addition, a more advanced technique, wherein PDMS microfluidic channels are used to control the organization and alignment of the collagen matrix is also provided. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a 3-dimensional, physiological microenvironment.

<table><tbody><tr><td>High Concentration Rat Tail Collagen</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>SylGard184 elastomer kit</td><td>Corning</td><td>NC9285739</td><td>Elastomer for PDMS channels</td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310</td><td>For HEPES neutralization buffer</td></tr><tr><td>KCl&amp;nbsp;</td><td>Fisher</td><td>BP366</td><td>For HEPES neutralization buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>BP362</td><td>For HEPES neutralization buffer</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S374</td><td>For HEPES neutralization buffer</td></tr><tr><td>NaCl</td><td>Fisher</td><td>BP358</td><td>For HEPES neutralization buffer</td></tr><tr><td>Levy Improved Neubauer Hemacytometer</td><td>Fisher</td><td>15170-208</td><td>cell counting</td></tr><tr><td>6-well non-tissue culture plate&amp;nbsp;</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>50 mm glass bottom dish</td><td>MatTek</td><td>P50g-1.5-30-f</td><td></td></tr><tr><td>Bel-Art Plastic Vacuum Desiccator</td><td>Bel-Art</td><td>F4200-2021</td><td>Degassing chamber for PDMS</td></tr><tr><td>transparency film&amp;nbsp;</td><td>3M</td><td>pp2950</td><td>Plastic film for pouring pdms channels</td></tr><tr><td>ThermoScientific CimaRec</td><td>ThermoScientific&amp;nbsp;</td><td>HP141925</td><td>Hot plate for curing PDMS microchannels</td></tr><tr><td>Vacuum regulator</td><td>Precision Medical</td><td>PM3100</td><td>Vacuum regulator for collagen microchannels</td></tr><tr><td>8&quot; x 8&quot; rubber sheet</td><td>Amazon - Rubber-Cal</td><td>Silicone - 60A</td><td>rubber sheet for pouring PDMS microchannel</td></tr><tr><td>8&quot; x 8&quot; x 0.125&quot; acrylic sheet</td><td>Amazon</td><td>Plexiglass sheets</td><td>for pouring PDMS microchannels</td></tr><tr><td>10 lb weights</td><td>Amazon</td><td>CAP Barbell</td><td>for pouring PDMS microchannels</td></tr><tr><td>15 ml Conical tubes</td><td>Fisher</td><td>352097</td><td></td></tr><tr><td>50 ml Conical tubes</td><td>Fisher</td><td>352098</td><td></td></tr><tr><td>Plastic pipets</td><td>Dot Scientific</td><td>229202B, 229206B, and 667225B</td><td>2&amp;nbsp;ml, 5&amp;nbsp;ml, and 25&amp;nbsp;ml</td></tr><tr><td>70% EtOH</td><td>Fisher</td><td>NC9663244</td><td></td></tr></tbody></table>

preparation of 3d collagen gels and microchannels for the study of 3d interactions in vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

3D 분석법 콜라겐 겔 강성 동일한 범위 내에서 실시 할 수 있지만, 겔 강도를 변화하는 세포들은 세포 미세 환경에서의 기계적 변화에 응답하는 방법을 결정하는데 사용될 수있다. 뻣 뻣 한 콜라겐 하이드로 겔이 포함 된 세포가 로컬 주변의 콜라겐을 수축에없는 젤로 정의된다. 상이한 세포 유형의 고유 수축 독특하고, 따라서 그것은 호환 단단한로 감지 될 콜라겐 농도를...

Watch this Scientific Journal Video about Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo at JoVE.com

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

콜라겐은 ECM의 핵심 구성 요소이며, 이전의 분화와 증식에 이르기까지 여러 세포 과정에 대한 중요한 단서를 제공한다. 3D 콜라겐 히드로 겔 내에 세포를 포함하는 프로토콜 및 PDMS 마이크로 채널을 사용하여 무작위 또는 정렬 된 콜라겐 매트릭스를 생성하는 더 진보 된 기술이 여기에 제공된다.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

preparation-3d-collagen-gels-microchannels-for-study-3d-interactions

Research

JoVE Journal

Bioengineering

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

16.9K 조회수.  University of Wisconsin-Madison.  콜라겐은 ECM의 핵심 구성 요소이며, 이전의 분화와 증식에 이르기까지 여러 세포 과정에 대한 중요한 단서를 제공한다. 3D 콜라겐 히드로 겔 내에 세포를 포함하는 프로토콜 및 PDMS 마이크로 채널을 사용하여 무작위 또는 정렬 된 콜라겐 매트릭스를 생성하는 더 진보 된 기술이 여기에 제공된다. 

비디오: Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

콜라겐의 모듈을 사용하여 미세 조직의 제조가 포함 세포를 젤

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

연구

생체공학

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

3D로 침윤성 암 세포의 세포 골격 조직을 공개

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

의학

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

동심 젤 시스템은 3 차원 세포의 이동에 매트릭스 미세 환경의 생물 물리학 역할을 연구하는

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

평면 그라데이션 확산 시스템은 3D 콜라겐 매트릭스에 화성을 조사하기 위해

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

미세화 및 3D 미세 혈관의 조립

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Microengineering 3D 콜라겐 하이드로겔(장거리 섬유 정렬 포함)

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

신경계 발달 중 축산 성장과 행동을 분석하는 3-D 콜라겐 기반 하이드로겔 생성

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비

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