RNAseq profiling of primary microglia and astrocyte cultures in near-term ovine fetus: A glial in vivo-in vitro multi-hit paradigm in large mammalian brain

•After decades of relative neglect, microglia and astrocytes (also known as glia) are emerging as key players in understanding the brain function and pathophysiology, well beyond their originally conceived “neuronal support” role. Glia shape neuronal development and function in health and disease.•In fetal brain, so far methods were limited to study glial function.•Here we present a new protocol and paradigm to derive pure primary microglia and astrocytes cultures from a mature fetal sheep brain, a powerful and widely used animal model of human brain development bearing high resemblance to human brain.•This technique allows versatile studies of glia function including assessing the genomic properties using next-generation sequencing approaches.•With such model we aim to provide a useful novel platform to tackle the challenge of understanding the diversity in glial phenotype and function in a large developing brain. The chronically instrumented fetal sheep is a widely used animal model to study fetal brain development in health and disease, but no methods exist yet to interrogate dedicated brain cell populations to identify their molecular and genomic phenotype. For example, the molecular mechanisms whereby microglia or astrocytes contribute to inflammation in the brain remain incompletely understood. Here we present a protocol to derive primary pure microglial or astrocyte cultures from near-term fetal sheep brain, after the animals have been chronically instrumented and studied in vivo. Next, we present the implementation of whole transcriptome sequencing (RNAseq) pipeline to deeper elucidate the phenotype of such primary sheep brain glial cultures. We validate the new primary cultures method for cell purity and test the function of the glial cells on protein (IL-1β) and transcriptome (RNAseq) levels in response to a lipopolysaccharide (LPS) challenge in vitro. This method represents the first implementation of pure microglial or astrocytes cultures in fetal sheep brain. The presented approach opens new possibilities for testing not only supernatant protein levels in response to an in vitro challenge, but also to evaluate changes in the transcriptome of glial cells derived from a large mammalian brain bearing high resemblance to the human brain. Moreover, the presented approach lends itself to modeling the complex multi-hit paradigms of antenatal and perinatal cerebral insults in vivo and in vitro.