Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes

Laura Zelenka; Dennis Pägelow; Christina Krüger; Jana Seele; Friederike Ebner; Sebastian Rausch; Manfred Rohde; Seija Lehnardt; Kira van Vorst; Marcus Fulde

doi:10.1016/j.jneumeth.2021.109420

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes

J Neurosci Methods. 2022 Jan 15:366:109420. doi: 10.1016/j.jneumeth.2021.109420. Epub 2021 Nov 20.

Authors

Affiliations

¹ Institute of Microbiology and Epizootics, Centre of Infection Medicine, Freie Universität Berlin, Robert-von-Ostertag-Straße 7-13, 14163 Berlin, Germany.
² Institute of Cell Biology and Neurobiology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
³ University Medical Center Göttingen, Institute of Neuropathology, Göttingen, Germany.
⁴ Freie Universität Berlin, Institute of Immunology, Berlin, Germany.
⁵ Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
⁶ Institute of Cell Biology and Neurobiology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany; Department of Neurology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
⁷ Institute of Microbiology and Epizootics, Centre of Infection Medicine, Freie Universität Berlin, Robert-von-Ostertag-Straße 7-13, 14163 Berlin, Germany. Electronic address: Marcus.Fulde@fu-berlin.de.

PMID: 34808220
DOI: 10.1016/j.jneumeth.2021.109420

Abstract

Background: The crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable.

New method: We modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension.

Results: We generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation.

Comparison with existing method(s): Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99-100%).

Conclusions: We have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

Keywords: Astrocytes; Glial cells; Magnetic-activated cell sorting (MACS®); Microglia; Primary cell culture; Purity; Viability.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Astrocytes* / metabolism
Cell Separation / methods
Cells, Cultured
Mice
Microglia*
Neuroglia