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Protocol for purification and culture of astrocytes: useful not only in 2 days postnatal but also in adult rat brain

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Abstract

Astrocytes play the key roles in the physiology and pathology of the CNS. Thereupon, in this manuscript, we aim to demonstrate that the protocol for purification and culture of astrocytes is useful not only in 2 days postnatal but also in adult rat brain. Also, the mentioned protocol is a simple and efficient primary cell culture technique. The whole-brain was isolated from the skull and the meninges were removed carefully. Afterward, the cerebral hemispheres were mechanically and enzymatically digested. Then, the cell suspension was seeded in T25 culture flask and was incubated at 37 °C in the CO2 incubator. The first shaking was performed after 7–8 days and on day 14, second shaking was done. After 2–3 passage, the culture was analyzed. By passaging, the majority of extracted cells were astrocytes presenting with a polygonal to fusiform and flat morphology that expressed GFAP, GLAST, and S100β. The expression of neural, neuronal and oligodendrocyte markers was not detected in extracted cells. The patch-clamp recording comfirmed the purity of isolated astrocytes as well. The isolated cells from adult rat brain were astrocytes that expressed specific astrocyte markers after 3 and 10 passages. This method is suggested to obtain a population of astrocytes that may provide a beneficial tool for different neurophysiological and pathophysiological studies.

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Acknowledgements

The present study was funded by ROYAN Institute for Stem Cell Biology and Technology, ACECR. I am especially indebted to Prof. Hossein Baharvand, Head of the ROYAN Institute for Stem Cell Biology and Technology, who have been supportive of my research goals.

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Authors and Affiliations

  1. Department of Brain and Cognitive Sciences, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 1665659911, Iran

    Masoumeh Zarei-Kheirabadi & Sara Mirsadeghi

  2. Department of Orthopaedics, Rothman Orthopaedic Institute, Thomas Jefferson University, Philadelphia, PA, USA

    Alexander R. Vaccaro

  3. Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran

    Vafa Rahimi-Movaghar

  4. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, 1665659911, Iran

    Sahar Kiani

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  1. Masoumeh Zarei-Kheirabadi
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  2. Sara Mirsadeghi
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  3. Alexander R. Vaccaro
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  4. Vafa Rahimi-Movaghar
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  5. Sahar Kiani
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Correspondence to Sahar Kiani.

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11033_2020_5272_MOESM1_ESM.tif

Supplementary material 1 Figure S1. The characterization of extracted cells. The immunostaining of neural marker (NESTIN), oligodendrocyte marker O4, neuronal marker TUJ1, fibroblast marker Thy-1, and microglial markers (CD45and CD68) in extracted cells. The expression of these markers was not detected in extracted cells after 3 passages. (TIF 288 kb)

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Zarei-Kheirabadi, M., Mirsadeghi, S., Vaccaro, A.R. et al. Protocol for purification and culture of astrocytes: useful not only in 2 days postnatal but also in adult rat brain. Mol Biol Rep 47, 1783–1794 (2020). https://doi.org/10.1007/s11033-020-05272-2

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  • DOI: https://doi.org/10.1007/s11033-020-05272-2

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