Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy.
Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy.
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DOI：
10.1371/journal.pone.0199918
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Lévy R
中科院分区：
文献类型：
--
作者：
Held M;Santeramo I;Wilm B;Murray P;Lévy R
筛选细胞的分化潜力需要组织培养模型和成像方法的组合，这些方法允许长期跟踪细胞的位置和功能。已经建立了胚胎肾再聚集体外测定，其允许监测再聚集和嵌合肾类器官中的器官型细胞行为。然而，标准荧光显微镜的高光子负载阻碍了细胞整合的评估，这对在一段时间内实时成像三维系统提出了挑战。因此，我们采用了光片显微镜，一种大大减少光漂白和光毒性效应的技术。我们还开发了一种培养再聚集体的新方法，该方法涉及浸没培养，产生更能反映体内发育的类器官。为了便于从不同角度成像，我们将类器官嵌入可自由旋转的水凝胶圆柱体中。进行终点固定和染色以提供额外的生物分子信息。我们成功地在15小时内对重新聚集的肾类器官内的标记细胞进行了成像，并跟踪了它们的命运，同时监测了器官型形态结构的发育。我们的研究结果表明，从转基因小鼠获得的表达Wt1的胚胎肾细胞可以整合到重新聚集的嵌合肾类器官中，并有助于肾单位的发育。此外，使用新的培养方法在重新聚集的组织中形成的新生近端小管显示出分泌功能，如通过它们将有机阴离子模拟物分泌到管腔中的能力所证明的。
Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.
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影响因子：
7.7
作者：
Lindström NO;Lawrence ML;Burn SF;Johansson JA;Bakker ER;Ridgway RA;Chang CH;Karolak MJ;Oxburgh L;Headon DJ;Sansom OJ;Smits R;Davies JA;Hohenstein P
通讯作者：
Hohenstein P
GUDMAP数据库 - 用于泌尿生殖器研究的在线资源。
DOI：
10.1242/dev.063594
发表时间：
2011-07
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Harding SD;Armit C;Armstrong J;Brennan J;Cheng Y;Haggarty B;Houghton D;Lloyd-MacGilp S;Pi X;Roochun Y;Sharghi M;Tindal C;McMahon AP;Gottesman B;Little MH;Georgas K;Aronow BJ;Potter SS;Brunskill EW;Southard-Smith EM;Mendelsohn C;Baldock RA;Davies JA;Davidson D
通讯作者：
Davidson D
影响因子：
48
作者：
Chung, Kwanghun;Deisseroth, Karl
通讯作者：
Deisseroth, Karl
影响因子：
2.3
作者：
Kreidberg, Jordan A.
通讯作者：
Kreidberg, Jordan A.
影响因子：
4.6
作者：
Kaufmann, Anna;Mickoleit, Michaela;Huisken, Jan
通讯作者：
Huisken, Jan