Microglia replenished OHSC: A culture system to study in vivo like adult microglia

Abstract

Recent data suggest that ramified microglia fulfil various tasks in the brain. However, to investigate this unique cell type cultured primary microglia are only a poor model. We here describe a method to deplete and repopulate organotypic hippocampal slice cultures (OHSC) with ramified microglia isolated from adult mouse brain creating microglia-replenished OHSC (Mrep-OHSC). Replenished microglia integrate into the tissue and ramify to a degree indistinguishable from their counterparts in the mouse brain. Moreover, wild-type slices replenished with microglia from TNFα-deficient animals provide similar results as OHSC prepared from microglia-specific TNFα-knockout mice (CX3CR1(cre) /TNFα(fl/fl) ). Furthermore, this study demonstrates that replenished microglia in OHSC maintain original functions and properties acquired in vivo. Microglia from ERCC1(Δ/ko) mice, a mouse model of accelerated aging, maintain enhanced Mac2 expression and their activated phenotype after replenishment to wild-type OHSC tissue. Thus, the present study demonstrates that Mrep-OHSC are a unique tool to construct chimeric brain slices allowing studying the function of different phenotypes of in vivo like microglia in a tissue culture setting. GLIA 2016 GLIA 2016;64:1285-1297.

Characteristics of primary rat microglia isolated from mixed cultures using two different methods

“…In 2003, another method was introduced that involved mild trypsinization [19]. Although some new methods have been demonstrated to achieve high-yield isolation of microglial cells from postnatal and adult brains [44, 45], shaking remains the most commonly used approach to date (see Fig. 1).…”

Activation of EP2 receptor suppresses poly(I: C) and LPS‐mediated inflammation in primary microglia and organotypic hippocampal slice cultures: Contributing role for MAPKs

“…OHSCs were prepared from 1–3 days old C57BL/6 wild‐type mice as described previously (Masuch et al, ). In brief, mice were decapitated, brains were collected and hippocampi were isolated.…”

Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures

“…Similarly, microglia in cultured brain slices rapidly lose their characteristic ramified morphology and mature marker expression (Haynes et al, 2006). Microglia-depleted organotypic hippocampal slices can be repopulated to generate highly ramified microglia in a culture paradigm (Masuch et al, 2016), but the presence of other cells limits some applications. Along a similar vein, microglial morphology and some other properties more closely resemble mature, quiescent microglia if the cells are cultured on an astrocyte feeder layer or in the presence of astrocyte-conditioned medium (ACM) (Sievers et al, 1994; Tanaka and Maeda, 1996).…”