Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy

Held, M ORCID: 0000-0003-0118-5898, Santeramo, Ilaria, Wilm, bettina ORCID: 0000-0002-9245-993X, Murray, patricia and Levy, raphael ORCID: 0000-0001-5728-0531 (2018) Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy. PLoS One, 13 (7). e0199918-.

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Abstract

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.

Item Type:	Article
Uncontrolled Keywords:	organoids, kidneys, light microscopy, gels, fluorescence microscopy, basement membrane, focal planes, fluorescence imaging
Depositing User:	Symplectic Admin
Date Deposited:	30 Jul 2018 09:29
Last Modified:	19 Jan 2023 01:29
DOI:	10.1371/journal.pone.0199918
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3024360