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A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents

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Abstract

Ependymal cells form an epithelium lining the ventricular cavities of the vertebrate brain. Numerous methods to obtain primary culture ependymal cells have been developed. Most of them use foetal or neonatal rat brain and the few that utilize adult brain hardly achieve purity. Here, we describe a simple and novel method to obtain a pure non-adherent ependymal cell culture from explants of the striatal and septal walls of the lateral ventricles. The combination of a low incubation temperature followed by a gentle enzymatic digestion allows the detachment of most of the ependymal cells from the ventricular wall in a period of 6 h. Along with ependymal cells, a low percentage (less than 6 %) of non-ependymal cells also detaches. However, they do not survive under two restrictive culture conditions: (1) a simple medium (alpha-MEM with glucose) without any supplement; and (2) a low density of 1 cell/µl. This purification method strategy does not require cell labelling with antibodies and cell sorting, which makes it a simpler and cheaper procedure than other methods previously described. After a period of 48 h, only ependymal cells survive such conditions, revealing the remarkable survival capacity of ependymal cells. Ependymal cells can be maintained in culture for up to 7–10 days, with the best survival rates obtained in Neurobasal supplemented with B27 among the tested media. After 7 days in culture, ependymal cells lose most of the cilia and therefore the mobility, while acquiring radial glial cell markers (GFAP, BLBP, GLAST). This interesting fact might indicate a reprogramming of the cell identity.

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Acknowledgments

The authors are grateful to José Esteban Casares Mira for valuable technical assistance and David Navas Fernández for his help with confocal microscopy. Leica confocal microscope (SP5 II) was funded by FEDER funds of the European Union. This work was supported by grants from Ministerio de Ciencia, Tecnología, Innovación y Sanidad (Spain) (BFU 2006-11754; SAF2010-19087; PNSD2010-143), and Junta de Andalucía (Spain) (P07-CVI-03079; SAS-S 0742; SAS08-0029; SAS 2010-111224).

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  1. Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Campus de Teatinos, Universidad de Málaga, 29071, Málaga, Spain

    J. M. Grondona, P. Granados-Durán, P. Fernández-Llebrez & M. D. López-Ávalos

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  1. J. M. Grondona
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  2. P. Granados-Durán
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  3. P. Fernández-Llebrez
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  4. M. D. López-Ávalos
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Correspondence to J. M. Grondona or M. D. López-Ávalos.

Electronic supplementary material

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Online resource 1 and 2 Videos taken from the ependymal surface of an explant 2 hours after the enzymatic treatment. Ependymal cell clumps of different sizes (3-5 cells up to about 20-30 cells) partially detached from the surface can be observed. Few cells are already free in the nearby media. Clumps and individual cells display a prominent circular movement as a consequence of the active ciliary beat (MPG 11702 kb)

Supplementary material 2 (MPG 11830 kb)

Online resource 3 Video of isolated ependymal cells 6 h after the enzymatic treatment. Most of the cells are in motion, which indicates that they are ciliated ependymal cells (MPG 2078 kb)

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Grondona, J.M., Granados-Durán, P., Fernández-Llebrez, P. et al. A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents. Histochem Cell Biol 139, 205–220 (2013). https://doi.org/10.1007/s00418-012-1008-2

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