Name: mammalian cell division in 3d matrices via quantitative confocal reflection microscopy
Uploaded: 2017-11-29T14:00:00
Description: Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Ce travail a été soutenu par les NIH accorde R01CA174388 et U54CA143868. Les auteurs aimerait remercier le prix PURA de l’Université Johns Hopkins, pour le soutien de Chen Wei-tong. Ce matériel est basé sur le travail soutenu par la National Science Foundation recherche bourse d’études supérieures au titre de la subvention no 1232825.

Le protocole fourni respecte les directives de la Homewood institutionnels Review Board (HIRB).
1. stable Expression de H2B-mCherry comme un marqueur de cellule mitose
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Génération de Particules Lentivirales rein embryonnaires humaines 293 t (HEK 293 t) cellules
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<li>Les cellules HEK 293 t sur un plat de culture cellulaire de 10 cm à la densité de 5 x 106 cellules/plat au milieu de culture cellulaire (de Dulbecco Medium (DMEM) contenant hyperglycé...

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	<li>Ly, D. H., Lockhart, D. J., Lerner, R. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+misregulation+and+human+aging.">Mitotic misregulation and human aging.</a> Science. 287 (5462), 2486-2492 (2000).</li><li>Gascoigne, K. E., Taylor, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+anti-mitotic+drugs+kill+cancer+cells?.">How do anti-mitotic drugs kill cancer cells?.</a> J Cell Sci. 122 (Pt 15), 2579-2585 (2009).</li><li>Brinkley, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+the+centrosome+numbers+game:+from+chaos+to+stability+in+cancer+cell+division.">Managing the centrosome numbers game: from chaos to stability in cancer cell division.</a> Trends Cell Biol. 11 (1), 18-21 (2001).</li><li>Phillip, J. M., Aifuwa, I., Walston, J., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanobiology+of+Aging.">The Mechanobiology of Aging.</a> Annu Rev Biomed Eng. 17, 113-141 (2015).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Martin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ablation+or+inhibition+of+stromal+matrix+metalloproteinase-9+on+lung+metastasis+in+a+breast+cancer+model+is+dependent+on+genetic+background.">Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background.</a> Cancer Res. 68 (15), 6251-6259 (2008).</li><li>Knox, J. J., Hotte, S. J., Kollmannsberger, C., Winquist, E., Fisher, B., Eisenhauer, 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=Phase+II+study+of+Triapine+in+patients+with+metastatic+renal+cell+carcinoma:+a+trial+of+the+National+Cancer+Institute+of+Canada+Clinical+Trials+Group+(NCIC+IND.161).">Phase II study of Triapine in patients with metastatic renal cell carcinoma: a trial of the National Cancer Institute of Canada Clinical Trials Group (NCIC IND.161).</a> Invest New Drugs. 25 (5), 471-477 (2007).</li><li>Komlodi-Pasztor, E., Sackett, D. L., Fojo, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+targeting+mitosis:+tales+of+how+great+drugs+against+a+promising+target+were+brought+down+by+a+flawed+rationale.">Inhibitors targeting mitosis: tales of how great drugs against a promising target were brought down by a flawed rationale.</a> Clin Cancer Res. 18 (1), 51-63 (2012).</li><li>Tong, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+and+pharmacologic+study+of+SNS-032,+a+potent+and+selective+Cdk2,+7,+and+9+inhibitor,+in+patients+with+advanced+chronic+lymphocytic+leukemia+and+multiple+myeloma.">Phase I and pharmacologic study of SNS-032, a potent and selective Cdk2, 7, and 9 inhibitor, in patients with advanced chronic lymphocytic leukemia and multiple myeloma.</a> J Clin Oncol. 28 (18), 3015-3022 (2010).</li><li>Matulonis, U. A., 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=Phase+II+study+of+MLN8237+(alisertib),+an+investigational+Aurora+A+kinase+inhibitor,+in+patients+with+platinum-resistant+or+-refractory+epithelial+ovarian,+fallopian+tube,+or+primary+peritoneal+carcinoma.">Phase II study of MLN8237 (alisertib), an investigational Aurora A kinase inhibitor, in patients with platinum-resistant or -refractory epithelial ovarian, fallopian tube, or primary peritoneal carcinoma.</a> Gynecol Oncol. 127 (1), 63-69 (2012).</li><li>Boss, D. 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=Clinical+evaluation+of+AZD1152,+an+i.v.+inhibitor+of+Aurora+B+kinase,+in+patients+with+solid+malignant+tumors.">Clinical evaluation of AZD1152, an i.v. inhibitor of Aurora B kinase, in patients with solid malignant tumors.</a> Ann Oncol. 22 (2), 431-437 (2011).</li><li>Griffith, L. G., Swartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+complex+3D+tissue+physiology+in+vitro.">Capturing complex 3D tissue physiology in vitro.</a> Nat Rev Mol Cell Biol. 7 (3), 211-224 (2006).</li><li>Cukierman, E., Pankov, R., Stevens, D. 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=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Lu, P., Weaver, V. M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+a+dynamic+niche+in+cancer+progression.">The extracellular matrix: a dynamic niche in cancer progression.</a> J Cell Biol. 196 (4), 395-406 (2012).</li><li>Giri, A., 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=The+Arp2/3+complex+mediates+multigeneration+dendritic+protrusions+for+efficient+3-dimensional+cancer+cell+migration.">The Arp2/3 complex mediates multigeneration dendritic protrusions for efficient 3-dimensional cancer cell migration.</a> FASEB J. 27 (10), 4089-4099 (2013).</li><li>Fraley, S. I., Feng, Y., Giri, A., Longmore, G. D., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+and+temporal+controls+of+three-dimensional+cell+migration+by+zyxin+and+binding+partners.">Dimensional and temporal controls of three-dimensional cell migration by zyxin and binding partners.</a> Nat Commun. 3, 719 (2012).</li><li>Fraley, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distinctive+role+for+focal+adhesion+proteins+in+three-dimensional+cell+motility.">A distinctive role for focal adhesion proteins in three-dimensional cell motility.</a> Nat Cell Biol. 12 (6), 598-604 (2010).</li><li>Lesman, A., Notbohm, J., Tirrell, D. A., Ravichandran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+forces+regulate+cell+division+in+three-dimensional+environments.">Contractile forces regulate cell division in three-dimensional environments.</a> J Cell Biol. 205 (2), 155-162 (2014).</li><li>He, 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=Local+3D+matrix+confinement+determines+division+axis+through+cell+shape.">Local 3D matrix confinement determines division axis through cell shape.</a> Oncotarget. 7 (6), 6994-7011 (2016).</li><li>Held, 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=CellCognition:+time-resolved+phenotype+annotation+in+high-throughput+live+cell+imaging.">CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.</a> Nat Methods. 7 (9), 747-754 (2010).</li><li>Fallica, B., Maffei, J. S., Makin, G., Zaman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+cellular+behavior+and+response+to+PI3K+pathway+inhibition+by+culture+in+3D+collagen+gels.">Alteration of cellular behavior and response to PI3K pathway inhibition by culture in 3D collagen gels.</a> PLoS One. 7 (10), e48024 (2012).</li><li>Meli, L., Jordan, E. T., Clark, D. S., Linhardt, R. J., Dordick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+a+three-dimensional,+microarray+environment+on+human+Cell+culture+in+drug+screening+systems.">Influence of a three-dimensional, microarray environment on human Cell culture in drug screening systems.</a> Biomaterials. 33 (35), 9087-9096 (2012).</li><li>Artym, V. V., Matsumoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+three-dimensional+collagen+matrix.">Imaging cells in three-dimensional collagen matrix.</a> Curr Protoc Cell Biol. 10, 1-20 (2010).</li><li>Gunzer, M., Kampgen, E., Brocker, E. B., Zanker, K. S., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+dendritic+cells+in+3D-collagen+lattices.+Visualisation+of+dynamic+interactions+with+the+substratum+and+the+distribution+of+surface+structures+via+a+novel+confocal+reflection+imaging+technique.">Migration of dendritic cells in 3D-collagen lattices. Visualisation of dynamic interactions with the substratum and the distribution of surface structures via a novel confocal reflection imaging technique.</a> Adv Exp Med Biol. 417, 97-103 (1997).</li><li>Geraldo, S., Simon, A., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+cytoskeletal+organization+of+invasive+cancer+cells+in+3D.">Revealing the cytoskeletal organization of invasive cancer cells in 3D.</a> J Vis Exp. (80), e50763 (2013).</li><li>Lleres, D., James, J., Swift, S., Norman, D. G., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+chromatin+compaction+in+living+cells+using+FLIM-FRET.">Quantitative analysis of chromatin compaction in living cells using FLIM-FRET.</a> J Cell Biol. 187 (4), 481-496 (2009).</li><li>Poincloux, 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=Contractility+of+the+cell+rear+drives+invasion+of+breast+tumor+cells+in+3D+Matrigel.">Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel.</a> Proc Natl Acad Sci U S A. 108 (5), 1943-1948 (2011).</li><li>Carey, S. P., Kraning-Rush, C. M., Williams, R. M., Reinhart-King, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+control+of+invasive+tumor+cell+behavior+by+extracellular+matrix+microarchitecture.">Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture.</a> Biomaterials. 33 (16), 4157-4165 (2012).</li><li>Langan, T. J., Chou, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+mammalian+cell+cultures+by+serum+deprivation.">Synchronization of mammalian cell cultures by serum deprivation.</a> Methods Mol Biol. 761, 75-83 (2011).</li><li>Jackman, J., O'Connor, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+synchronizing+cells+at+specific+stages+of+the+cell+cycle.">Methods for synchronizing cells at specific stages of the cell cycle.</a> Curr Protoc Cell Biol. 8, (2001).</li><li>Zwanenburg, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+shake-off+to+synchronize+cultured+CHO+cells.">Standardized shake-off to synchronize cultured CHO cells.</a> Mutat Res. 120 (2-3), 151-159 (1983).</li><li>Harper, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+cell+populations+in+G1/S+and+G2/M+phases+of+the+cell+cycle.">Synchronization of cell populations in G1/S and G2/M phases of the cell cycle.</a> Methods Mol Biol. 296, 157-166 (2005).</li><li>Kihara, T., Ito, J., Miyake, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+biomolecular+diffusion+in+extracellular+matrix+condensed+by+fibroblasts+using+fluorescence+correlation+spectroscopy.">Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.</a> PLoS One. 8 (11), e82382 (2013).</li><li>Ramanujan, 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=Diffusion+and+convection+in+collagen+gels:+implications+for+transport+in+the+tumor+interstitium.">Diffusion and convection in collagen gels: implications for transport in the tumor interstitium.</a> Biophys J. 83 (3), 1650-1660 (2002).</li><li>Harjanto, D., Maffei, J. S., Zaman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+effect+of+cancer+invasiveness+and+collagen+concentration+on+3D+matrix+remodeling.">Quantitative analysis of the effect of cancer invasiveness and collagen concentration on 3D matrix remodeling.</a> PLoS One. 6 (9), e24891 (2011).</li><li>Wolf, K., 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-based+cell+migration+models+in+vitro+and+in+vivo.">Collagen-based cell migration models in vitro and in vivo.</a> Semin Cell Dev Biol. 20 (8), 931-941 (2009).</li><li>Petroll, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+interference+contrast+and+confocal+reflectance+imaging+of+collagen+organization+in+three-dimensional+matrices.">Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices.</a> Scanning. 28 (6), 305-310 (2006).</li><li>Fraley, S. I., 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=Three-dimensional+matrix+fiber+alignment+modulates+cell+migration+and+MT1-MMP+utility+by+spatially+and+temporally+directing+protrusions.">Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions.</a> Sci Rep. 5, 14580 (2015).</li><li>Ikeda, Y., Collins, M. K., Radcliffe, P. A., Mitrophanous, K. A., Takeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transduction+efficiency+in+cells+of+different+species+by+HIV+and+EIAV+vectors.">Gene transduction efficiency in cells of different species by HIV and EIAV vectors.</a> Gene Ther. 9 (14), 932-938 (2002).</li><li>Ma, H. T., Poon, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+HeLa+cells.">Synchronization of HeLa cells.</a> Methods Mol Biol. 761, 151-161 (2011).</li></ol>

L’étude précédente de la division cellulaire en 3D n’était pas efficace en raison de limitations expérimentales et défis techniques18,19. Les étapes essentielles pour une étude efficace de la division cellulaire chez les mammifères dans les matrices de collagène 3D sont : (1) l’incorporation de marqueurs mitotiques marqués par fluorescence aux cellules ; (2) la synchronisation de la division cellulaire ; et (3) le suivi des événements de la ...

Mitose de la cellule est un événement critique dans la vie cellulaire, dont la réglementation joue un rôle crucial dans le développement de tissus et organes. Une mitose anormale est impliquée dans des variations génétiques naturelles, les processus de vieillissement humain et la progression du cancer1,2,3,4,5. L’augmentation du taux de prolifération des cellules tumorales par rapport aux cellules normales est une des caractéristiques du cancer, malgré le fait que les comportements cellulaires sont assez hétérogènes entre les différents types de tumeurs et même chez les patients. En dépit des résultats précliniques prometteurs, certaines drogues antimitotiques nouvellement développé n’ont pas démontré d’être efficaces dans les essais cliniques6,7,8,9,10 ,11. La pertinence des modèles expérimentaux et précliniques doit être considérée. Plusieurs types de cellules normales de mammifères et cancer divisent dans des matrices en trois dimensions (3D), tels que les fibroblastes et cellules de Fibrosarcome en collagène riche j’ai 3D des tissus conjonctifs et cellules cancéreuses métastatiques dans la 3D stromales la matrice extracellulaire (MEC). Cependant, la grande majorité de la division cellulaire chez les mammifères des expériences et des essais ont été réalisée sur les cellules cultivées sur des substrats à deux dimensions (2D). Une matrice 3D ingénierie pourrait mieux récapituler la microstructure et propriétés mécaniques des signaux biochimiques de l’ECM 3D de deux tissus normaux et pathologiques12,13,14, 15,16,17.
L’étude des mammifère comment la division cellulaire est régulée en 3D des environnements demeure largement inexploré malgré la pertinence physiologique tant la signification thérapeutique18,19. Les raisons possibles incluent les difficultés techniques et expérimentales défis associés à étudier la division cellulaire dans des matrices 3D. La mitose cellulaire constitue une petite fraction temporelle dans le cycle cellulaire complet20. Travaux antérieurs ont montré que le taux de prolifération de plusieurs cellules de mammifères, tels que l’adénocarcinome du sein MCF-7, ostéosarcome humain U2OS et foie humain HepG2, est beaucoup plus faible dans des matrices 3D par rapport à leurs homologues sur des substrats 2D21, 22. En outre, les cellules incorporés dans des matrices 3D se déplacent dans et hors de discussion au cours de l’imagerie de cellules vivantes. Tous ces facteurs contribuent à la très faible efficacité de capture d’événements de la division cellulaire en 3D à l’aide de techniques d’imagerie de la culture.
Interactions entre les cellules et ECM jouent un rôle crucial dans la régulation des divisions cellulaires. Nous décrivons ici une approche pour étudier efficacement la division cellulaire chez les mammifères dans des matrices 3D de collagène. La méthode comprend l’incorporation des marqueurs mitotiques pour les cellules, la synchronisation de la division cellulaire, ainsi que le suivi des événements de la division dans des matrices 3D à l’aide de la technique d’imagerie de cellules vivantes, de la microscopie de réflexion confocal résolution temporelle, et analyse d’imagerie quantitative. Protéine de fluorescence-étiquetée histone H2B est tout d’abord introduit dans les cellules comme marqueurs pour différencier mitose et interphase de cellules. Puis les cellules sont synchronisées à l’aide de la combinaison du traitement de blocage et la nocodazole thymidine, suivie d’une technique mécanique de shake-off. Cellules synchrones sont alors directement encapsulées dans des matrices 3D de collagène. Événements de la division cellulaire des cellules multiples sont surveillées efficacement à l’aide de l’imagerie de cellules vivantes Time-lapse faible grossissement. La déformation des fibres de collagène, qui est un indicateur de l’interaction cellule-matrice, est contrôlée au moyen de la microscopie confocale réflexion à fort grossissement.
Nous avons déjà utilisé cette technique pour surveiller et de quantifier l’interaction cellule-matrice avant, pendant et après la mitose de deux lignées de cellules de cancer métastatique, carcinome canalaire invasif humain MDA-MB-231 et les cellules humaines Fibrosarcome HT1080 en collagène 3D 19de matrices. Les méthodes présentées ici fournissent une approche efficace et générale pour étudier les deux mammifères division cellulaire dans une interaction environnement et cellule-matrice 3D. La lignée de cellules MDA-MB-231 est utilisée à titre d’exemple dans le présent document. Ce protocole prévoit de nouveaux aperçus de la base moléculaire du développement des tissus normaux et les maladies et pourrait également permettre la conception de nouvelles approches diagnostiques et thérapeutiques.

<table>
	<tbody>
		<tr>
			<td>Human embryonic kidney 293T</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>Physical Sciences Oncology Center, NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>ThermoFisher Scientific</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life technologies</td>
			<td>11668-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding H2B-mCherry in a lentiviral vector</td>
			<td>Addgene</td>
			<td>plasmid 21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>GibcoBRL</td>
			<td>11810-025</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>113375-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Bake</td>
			<td>3722-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV syringe filter unit, 0.45-&#956;m, PVDF, 33 mm</td>
			<td>Millipore</td>
			<td>SLHVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000E epifluorescence microscope</td>
			<td>Nikon</td>
			<td>TE2000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 1K CCD camera</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 confocal microscope</td>
			<td>Nikon</td>
			<td>A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

L’étude de la division cellulaire chez les mammifères comment est réglementée dans une 3D environnement demeure largement inexploré malgré sa pertinence physiologique et sa signification thérapeutique. Les raisons possibles de l’absence d’exploration sont les limitations expérimentales et les défis techniques qui rendent l’étude de la division cellulaire en culture 3D inefficace. Nous décrivons ici une méthode axée sur l’imagerie pour étudier efficacement la division cellulaire chez les mammifères et les interactions cellule-matrice dans des matrices 3D de collagène. Cellules marquées avec la H2B fluorescents sont synchronisées à l’aide de la combinaison du traitement de blocage et la nocodazole thymidine, suivie d’une technique mécanique de shake-off. Cellules synchrones sont ensuite incorporées dans une matrice de collagène 3D. La division cellulaire est contrôlée au moyen de la microscopie de cellules vivantes. La déformation des fibres de collagène pendant et après la division cellulaire, qui est un indicateur de l’interaction cellule-matrice, peut être surveillée et quantifiées à l’aide de la microscopie quantitative réflexion confocale. La méthode fournit une approche efficace et générale pour étudier la division cellulaire chez les mammifères et les interactions cellule-matrice dans un environnement 3D physiologiquement pertinent. Cette approche non seulement fournit de nouveaux aperçus de la base moléculaire du développement des tissus normaux et les maladies, mais permet également à la conception de nouvelles approches diagnostiques et thérapeutiques.

L’objectif de cet article est de présenter une méthode axée sur l’imagerie pour étudier les processus de la division cellulaire chez les mammifères dans des matrices 3D et de quantifier les interactions entre la cellule et la matrice extracellulaire 3D pendant et après la division cellulaire. Afin de faciliter l’imagerie de la mitose de la cellule, nous avons incorporé H2B-mCherry dans les cellules MDA-MB-231 à l’aide de transduction des gènes. H2B conjugué avec protéin...

Watch this Scientific Journal Video about Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy at JoVE.com

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

Ce protocole a efficacement étudie la division cellulaire chez les mammifères dans les matrices de collagène 3D en intégrant la synchronisation de la division cellulaire, la surveillance des manifestations de la division dans des matrices 3D à l’aide de la technique d’imagerie de cellules vivantes, microscopie de réflexion confocal résolution temporelle et analyse d’imagerie quantitative.

Division cellulaire chez les mammifères dans des Matrices 3D par microscopie Quantitative réflexion Confocal

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 T-cells in a 100 milliliter cell culture dish in 10 milliliters of normal cell culture medium. Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day just before starting transfections, remove a vial of the transfection reagent from four degrees Celsius and leave it out to reach room temperature.Then to start, add one milliliter of reduced serum medium to a sterile microfuge tube. To this, add specified amounts of lentiviral vector plasmids used for transfection and mix by pipetting. Incubate the tube at room temperature for five minutes.After five minutes, add 48 microliters of the transfection reagent into this tube and mix gently by pipetting. Subsequently, incubate this mixture at room temperature for at least 15 minutes. Once the incubation is complete, remove the plate with HEK-293 T-cells from the incubator.Add the prepared DNA transfection reagent mixture drop wise onto the cells and gently swirl the plate to ensure even distribution in the plate. Place the plate back into the incubator. Approximately six hours after transfection, remove the plate and aspirate the medium.Add 10 milliliters of fresh culture medium and transfer the plate back into the incubator. At multiple time points, 24, 48, and 72 hours post-transfection, harvest the culture medium containing the lentiviral particles in separate tubes. Next, filter the harvested culture medium through a sterile 0.45 micron filter to remove cellular debris.Replace with fresh culture medium following each harvest. The filtrate containing the lentiviral particles can be used for transduction right away or can be stored at minus 80 degrees Celsius. Plate human breast carcinoma cells MDA-MB-231 in a 35 millimeter culture dish in normal culture medium.Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day, aspirate the culture medium from the plate and add one milliliter of the lentiviral particles together with one milliliter of fresh culture medium. Incubate the cells in the incubator for about 16 hours.After the incubation with viral particles is complete, remove the plate to aspirate the medium. Then add two milliliters of fresh medium and transfer back to the incubator for 24 to 72 hours. Check the expression of H2B-mCherry in these transduced cells using an epifluorescence microscope.To start the synchronization experiments, first plate MDA-MB-231 cells expressing H2B-mCherry at 50 to 60%confluency in a 24-well plate. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, replace the culture medium with 0.5 milliliters of medium containing two millimolar Thymidine.Leave the plate in the incubator for 24 hours. Once the incubation is complete, wash the cells three times with PBS to release them from the Thymidine treatment. Then leave the cells in 0.5 milliliters of normal culture medium for five hours in the incubator.After this, to block these cells in mitosis, add Nocodazole to the culture medium. Leave the cells in the incubator for 12 hours. After the Nocodazole treatment is complete, take out the plate and using an orbital shaker, shake the cells for 45 seconds to one minute to release mitotic cells.Subsequently, transfer the medium containing the extracted mitotic cells into a new centrifuge tube. Then add 0.5 milliliters of fresh medium to each well. After, repeat the shaking and extraction of cells two more times.Pool the total collected medium and then centrifuge it to pellet the mitotic cells. After the spin, resuspend the cell pellet in 0.25 milliliters of fresh cell culture medium. Using a hemocytometer, count the number of cells.At the start of this step, prepare 50 milliliters of 10x DMEM solution and filter sterilize it. Next, determine the volumes of all the components that will be used to make the 3D collagen matrix as specified in the text protocol. Pre-chill all these components on ice to slow down the gelation of collagen.Now, to a pre-chilled 1.5 milliliter centrifuge tube, add the required numbers of cells in medium then 10x DMEM, FBS, deionized water, collagen, and finally sodium hydroxide. Mix carefully using a one milliliter pipette. Once the solution is well mixed, add 500 microliters of the mixture into each well of the 24-well plate.Place the 24-well plate in a 37 degree Celsius incubator. After 30 minutes, take out the plate and add 500 microliters of pre-warmed culture medium on top of the gel. Prior to imaging, turn on the live cell units of the phase contrast and confocal microscopes.To capture dividing cells, place the 24-well plate containing the cells embedded in collagen matrices into the live cell unit of the fluorescence microscope. Using the 10X objective, find the bottom of the plate and then move it up until the microscope focuses at about 500 micrometers from the bottom of the plate. Move the stage around to find cells in different stages of mitosis and select multiple positions for imaging.Set up the timelapse experiment with two-minute intervals. Next, to image the collagen network, configure the confocal microscope to capture only reflected light from the 488 nanometer laser. Then place the plate containing cells in collagen matrices into the live cell unit of this microscope.Find the bottom of the plate and then move the objective lens up until it focuses at about 100 micrometers from the bottom of the plate. Move the stage around to find cells and select multiple positions for imaging. Set up the timelapse experiment with five-minute intervals.Fluorescent images of a dividing H2B-mCherry expressing MDA-MB-231 cell embedded in collagen matrix are shown here. Different phases of mitosis are readily observed as expected. The distribution of collagen fibers in white visualized simultaneously using confocal reflection microscopy changes very little during the course of mitosis.Cell synchronized G2-M phase and embedded in a collagen matrix complete cell division in the first two hours while control asynchronous cells divide randomly. Quantification of the distribution of collagen fibers during interphase and mitotic phases suggest that matrix deformation is minimal during cell division and higher in interphase cells. Once mastered, this technique can be completed in seven days starting from generating the stable cell line to analyzing the video cell division in a 3D matrix.After watching this video, you hopefully have a good understanding of how to synchronize and monitor mammalian cell division in 3D matrices using live cell imaging and how to determine matrix deformation using confocal reflection microscopy and quantitative imaging analysis techniques.

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