Name: observing and quantifying fibroblast-mediated fibrin gel compaction
Uploaded: 2014-01-16T13:45:00
Description: Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

우리는 조지 Giudice와 스티븐 엘리아슨이 변형 추적 알고리즘에 도움을 준 인간의 진피 섬유아세포와 라메쉬 라구병증을 기부해 주셔서 감사합니다. 이 작업에 대한 지원은 국가 필요 펠로우십 분야의 미국 교육부 대학원 지원 (GAANN P200A120071)에 의해제공되었다.

1. 스텐실 준비
파라필름에 스텐실을 준비하여 원하는형상(그림 1A 및1B)에 따라 각 각 각 절제물의 위치를 레이아웃합니다. 공간은 각각 약 1-2mm 떨어져 익스플. 이 거리는 각성 사이의 섬유 정렬을 생성하기위한 이상적인 간격에 해당합니다. 샘플이 테이프로 준비될 커버글래스 영역 아래에 스텐실을 부착합니다.
2. 살균...

<ol>
	<li>Grinnell, F., Petroll WM, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+and+mechanics+in+three-dimensional+collagen+matrices.">Cell motility and mechanics in three-dimensional collagen matrices.</a> Annu. Rev. Cell. Dev. Biol. 26, 335-361 (2001).</li><li>Sander, E. A., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomimetic collagen tissues: collagenous tissue engineering and other applications. In: Collagen: Structure and Mechanics. , (2008).</li><li>Pedersen JA, ., Swartz MA, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+in+the+third+dimension.">Mechanobiology in the third dimension.</a> Ann. Biomed. Eng. 33 (11), 1469-1490 (2005).</li><li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro.">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 76 (3), 1274 (1979).</li><li>Guidry, C., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+hydrated+collagen+gel+reorganization+by+human+skin+fibroblasts.">Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts.</a> J. Cell. Sci. 79 (1), 67-81 (1985).</li><li>Fluck, J., Querfeld, C., Cremer, A., Niland, S., Krieg, T., Sollberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+primary+fibroblasts+undergo+apoptosis+in+three-dimensional+contractile+collagen+gels.">Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels.</a> J. Invest. Dermatol. 110 (2), 153-157 (1998).</li><li>Rosenfeldt, H., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+quiescence+and+the+disruption+of+ERK+signaling+in+mechanically+unloaded+collagen+matrices.">Fibroblast quiescence and the disruption of ERK signaling in mechanically unloaded collagen matrices.</a> J. Biol. Chem. 275 (5), 3088-3092 (2000).</li><li>Nakagawa, S., Pawelek, P., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+fibroblasts+in+contracted+collagen+gels:+Effects+on+cell+growth+and+biosynthetic+activity.">Long-term culture of fibroblasts in contracted collagen gels: Effects on cell growth and biosynthetic activity.</a> J. Invest. Dermatol. 93 (6), 792-798 (1989).</li><li>Harris, A. K., Stopak, D., Wild, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+traction+as+a+mechanism+for+collagen+morphogenesis.">Fibroblast traction as a mechanism for collagen morphogenesis.</a> Nature. 290, 249-251 (1981).</li><li>Stopak, D., Harris AK, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+morphogenesis+by+fibroblast+traction:+I.+tissue+culture+observations.">Connective tissue morphogenesis by fibroblast traction: I. tissue culture observations.</a> Dev. Biol. 90 (2), 383-398 (1982).</li><li>Sawhney, R. K., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+local+movements+of+collagen+fibers+by+fibroblasts+drive+the+rapid+global+self-organization+of+collagen+gels.">Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels.</a> J. Cell. Biol. 157 (6), 1083-1092 (2002).</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 (11), 5374 (2008).</li><li>Sander, E., Barocas, V., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39 (2), 714-729 (2011).</li><li>Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anisotropic+biphasic+theory+of+tissue-equivalent+mechanics:+the+interplay+among+cell+traction,+fibrillar+network+deformation,+fibril+alignment,+and+cell+contact+guidance.">An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.</a> J. Biomech. Eng. 119, 137-146 (1997).</li><li>Neidert, M. R., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+valves+with+commissural+alignment.">Tissue-engineered valves with commissural alignment.</a> Tissue Eng. 12 (4), 891-903 (2006).</li><li>Robinson PS, ., Robinson PS, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+biaxial+behavior+of+fibrin-based+tissue-engineered+heart+valve+leaflets.">Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.</a> Tissue Eng. Part A. 15 (10), 2763-2772 (2009).</li><li>Grassl, E., Oegema, T., Tranquillo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+as+an+alternative+biopolymer+to+type+I+collagen+for+the+fabrication+of+a+media+equivalent.">Fibrin as an alternative biopolymer to type I collagen for the fabrication of a media equivalent.</a> J. Biomed. Mater. Res. 60 (4), 607-612 (2002).</li><li>Paten, J. A., Zareian, R., Saeidi, N., Melotti, S. A., Ruberti, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+an+optically+accessible,+low-volume,+mechanobioreactor+for+long-term+study+of+living+constructs.">Design and performance of an optically accessible, low-volume, mechanobioreactor for long-term study of living constructs.</a> Tissue Eng. Part C Methods. 17 (7), 775-788 (2001).</li><li>Ye KY, ., Sulli van KE, ., Black LD, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+cardiomyocytes+in+a+fibrin+hydrogel+for+cardiac+tissue+engineering.">Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering.</a> J. Vis. Exp. (55), (2011).</li><li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16 (10), 3261-3270 (2010).</li><li>Raghupathy, R., Witzenburg, C., Lake, S. P., Sander, E. A., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+regional+mechanical+anisotropy+in+soft+tissue+analogs.">Identification of regional mechanical anisotropy in soft tissue analogs.</a> J. Biomech. Eng. 133, (2011).</li><li>Robinson, P. S., Johnson, S. L., Evans, M. C., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue-engineered+valves+from+cell-remodeled+fibrin+with+commissural+alignment+of+cell-produced+collagen.">Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.</a> Tissue Eng. Part A. 14 (1), 83-95 (2008).</li><li>Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblast+in+wound+healing+and+fibrocontractive+diseases.">The myofibroblast in wound healing and fibrocontractive diseases.</a> J. Pathol. 200 (4), 500-503 (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 (3), 241-254 (2005).</li><li>Grinnell, F., Rocha L, B., Iucu, C., Rhee, S., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nested+collagen+matrices:+A+new+model+to+study+migration+of+human+fibroblast+populations+in+three+dimensions.">Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions.</a> Exp. Cell. Res. 312 (1), 86-94 (2006).</li><li>Miron-Mendoza, M., Seemann, J., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+regulation+of+cell+motile+activity+through+matrix+stiffness+and+porosity+in+three+dimensional+collagen+matrices.">The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices.</a> Biomaterials. 31 (25), 6425-6435 (2010).</li><li>Wakatsuki, T., Kolodney MS, ., Zahalak GI, ., Elson EL, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+studied+by+a+reconstituted+model+tissue.">Cell mechanics studied by a reconstituted model tissue.</a> Biophys. J. 79 (5), 2353-2368 (2000).</li><li>De Jesus, A., Aghvami, M., Sander, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-mediated+fiber+realignment+in+fibrin+gels.">Fibroblast-mediated fiber realignment in fibrin gels.</a> , (2013).</li><li>Aghvami, M., Barocas, V. H., Sander, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+mechanical+simulations+of+cell+compacted+collagen+gels.">Multiscale mechanical simulations of cell compacted collagen gels.</a> J. Biomech. Eng. 135, (2013).</li></ol>

이 프로토콜은 세포 매개 ECM 리모델링과 관련된 역학을 관찰하고 정량화하기 위한 목적으로 개발되었다. 이러한 과정은 다수의 생물학적 현상의 기초와 엔지니어링 조직에 대한 중요한 의미를 갖는다2,22,흉터1,23을감소시키고, 병리학 조직을 리모델링12,24. 시간 경과 DIC 현미경 검사법의 사용은 세포 견인력의 결과로 발생하는 피브린 섬유의 변위와 정렬을 해결하고 정량?...

세포 매트릭스 상호 작용을 연구하기위한 중요한 도구는 세포 채워진 콜라겐 젤1,2이다. 겔은 조직의 생체 내 특성에 더 가깝고 전통적인 2D 배양3에서제공하는 것보다 세포 행동을 이해하는 데 더 적합한 3D 환경을 제공한다. 섬유아세포가 콜라겐 젤 내에서 균질하게 분포된 초기 연구에 따르면 세포는 콜라겐 섬유를 빠르게 통합하고 겔4,5을압축하는 것으로 나타났다. 자유 부동 젤의 수축 섬유 아세포는 겔이 완전히 다짐1,6,7에도달 한 직후 정지 상태로 전환한다. 경계에 제약이 있는 겔의 섬유아세포는 활성, 합성 상태8에 남아 있으며 겔 형상 및 외부 제약 에 의존하는 방식으로 섬유 정렬을 생성합니다5,9. 세포 활동의 차이는 세포가 젤의 콜라겐 섬유에 대한 통합을 통해 견인력을 발휘함에 따라 발생하는 내부 장력(또는 그 부족)의 결과로 나타난다.
이 기술의 변이체는 콜라겐 젤 내에서 떨어져 섬유아세포 이질(즉, 세포의 덩어리)을 배치하고 세포 매트릭스 상호 작용을 관찰하고 각기 (때로는 인대 와 같은 스트랩이라고도 함)10-12사이의 섬유 정렬의 점진적 개발을 포함한다. 각기 시스템의 주요 장점은 세포를 간단한 기하학적 패턴으로 배열할 수 있게 해주므로 세포 중심의 섬유 재조정의 메커니즘을 쉽게 시각화하고 조사할 수 있다는 것입니다. 이러한 정렬 패턴은 주로 세포 견인력, 세포 공간 분포, 겔 형상 및 젤의 기계적 제약 사이의 상호 작용에 의존하며 글로벌 조직, 기계적 기능 및 국소 기계환경에서중심적인 역할을 하기 때문에 이해하는 것이 중요하다.
조직 공학 분야에서, 기계적으로 기능적, 엔지니어링 조직을 생산하기위한 하나의 전략은 엔지니어링 조직이 네이티브 조직의 것을 모방 섬유 정렬을 소유할 수 있도록 세포 다짐에서 발생하는 섬유 정렬 패턴을 제어하는 것을 포함한다14,15. 이러한 정렬은 엔지니어링 된 조직이 네이티브 조직의 복잡한 기계적 행동을 복제하는 데 필요하다고 믿습니다. 이 전략의 변형은 콜라겐 젤을 피브린젤(16)으로대체하는 것이다. 피브린 젤은 다짐 시 콜라겐 젤과 유사한 정렬 패턴을 개발합니다. 시간이 지남에 따라 피브린은 분해되고 초기 피브린 섬유 정렬 패턴을 따르는 세포 합성 ECM로 대체됩니다. 그 결과 엔지니어링 된 구조는 콜라겐 젤 유래 구문(17)에비해 기계적 특성을 크게 향상시켰다.
피브린 젤의 정렬 프로세스 및 후속 리모델링 이벤트는 복잡하고 제대로 이해되지 않은 방식으로 진행됩니다. 이러한 상호 작용과 세포 거동 및 ECM 리모델링에 미치는 영향을 더 잘 특성화하기 위해, 우리는 각성 방법에 기초한 절차를 개발했습니다. 이 방법에서, 섬유아세포 각질은 다른 기하학적 패턴의 피브린 젤에 위치한다. 젤은 환경 제어, 현미경 장착 바이오 반응기(18)에서유지되며, 압축 및 섬유 재정렬 과정은 시간 경과 차동 간섭 콘트라스트 (DIC) 현미경 검사법으로 모니터링됩니다. 변위 필드는 사용자 지정 알고리즘으로 정량화됩니다. 이러한 실험에서 얻은 데이터는 조직 엔지니어링 전략 최적화, 상처 치유 개선 및 병리학 조직 리모델링 을 포함하여 여러 과정에 광범위한 영향을 미칩니다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

콜라겐과 피브린 젤에 내장된 세포는 겔 섬유에 견인력을 부착하고 발휘합니다. 이러한 힘은 젤 미세 구조의 로컬 및 글로벌 재구성 및 재조정으로 이어질 수 있습니다. 이 과정은 세포의 위치, 젤의 형상 및 젤의 기계적 제약 사이의 상호 작용에 부분적으로 의존하는 복잡한 방식으로 진행됩니다. 이러한 변수가 글로벌 섬유 정렬 패턴을 생성하는 방법을 더 잘 이해하기 위해, 우리는 기하학적으로 간격이 있는 각질(섬유아세포의 클러스터) 사이의 압축 공정을 관찰하기 위해 환경 제어 생물 반응기와 결합된 시간 경과 차동 간섭 콘트라스트(DIC) 현미경 을 사용합니다. 그런 다음 이미지를 사용자 지정 이미지 처리 알고리즘으로 분석하여 변형맵을 가져옵니다. 이 기술에서 얻은 정보는 상처 치유, 질병 개발 및 조직 공학 응용 분야의 프로세스를 이해하는 데 중요한 영향을 미치는 다양한 세포 매트릭스 상호 작용의 기계생물학을 조사하는 데 사용할 수 있습니다.

조직 리모델링은 세포와 주변 매트릭스 사이의 상호 물리적 상호 작용에 의해 부분적으로 구동되는 복잡한 과정입니다. 세포는 주변 섬유를 재구성하고 섬유 네트워크에서 장력을 생성합니다. 섬유와 기계적 환경의 정렬은 차례로 세포 행동을 제어하므로 세포와 매트릭스 가 전 세계적으로 재구성되어 리모델링 된 조직을 생성합니다. 본 실험에서, 항종의 세포는, 처음에 형태학에서 둥근, 겔로 ...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

시간 경과 현미경 검사법과 이미지 처리 기술은 48시간 동안 환경적으로 통제된 생물 반응기에서 섬유아세포 매개 젤 다짐 및 피브린 섬유 재정렬을 관찰하고 분석하는 데 사용되었습니다.

섬유아세포 매개 피브린 겔 다짐 관찰 및 정량화

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

세포 수축을 Quantifying에 대한 불만 사항 매트릭스의 준비

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

연구

생체공학

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 ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

엔지니어링 섬유소 기반 조직의 면역 조직 및 제약 조건의 적용에서 생성하고 세포와 콜라겐 (재) 조직을 유도하는 스트레인

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

백혈구 - 혈소판 풍부한 섬유소, 소설 생체 재료의 특성

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

젤 보조 재수를 사용하여 거대한 크기의 Polymersomes 형성

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

생물학적 설정에서 생성 및 관측 화학 발광위한 새로운 기술

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

젤-seq: DNA와 RNA 히드로 매트릭스를 사용 하 여 동시 시퀀싱 라이브러리 준비를 위한 방법

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...