047 – RNA-transcriptome guided optimization of in vitro culture conditions for adult primary microglia
Raissa Timmerman (1) – Ella Zuiderwijk-Sick (1) – Nynke Oosterhof (2) – Anke Õt Jong (1) – Jennifer Veth (1) – Saskia Burm (1) – Tjakko van Ham (2) – Jeffrey Bajramovic (1)
Alternatives Unit, Biomedical Primate Research Centre, Lange Kleiweg 161, 2288 GJ Rijswijk, The Netherlands (1) – Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands (2)
Microglia, the resident tissue macrophages of the central nervous system (CNS), are key players during brain homeostasis as well as during neurodevelopmental and neurodegenerative disorders. Detailed knowledge of their cell biology is therefore of pivotal importance, and primary cell cultures provide an excellent means to obtain such knowledge.
However, recent studies have demonstrated that the transcriptome of classical primary microglia culture models only partially recapitulates that of mature homeostatic microglia. Aiming to optimize cell culture conditions, we have compared the transcriptomes of primary adult rhesus microglia that were cultured in vitro for 7 days under different conditions with the transcriptome of ex vivo mature homeostatic microglia.
In line with earlier published data, major differences in the gene expression profiles of in vitro and ex vivo microglia were observed, regardless of cell culture conditions. Gene ontology analysis showed that in particular genes associated with proliferation were upregulated in vitro, possibly caused by exposure to serum. Interestingly, we further observed that the different transcriptomes clustered better to donor origin than to culture condition. Consistent with literature, transcriptomes of microglia that were exposed in vitro to TGF-beta were slightly enriched for microglia signature genes Sal1 and Tmem119. Relative expression values were however still significantly lower than those of ex vivo microglia.
Based on these results we have tested a variety of different cell culture approaches that have now resulted in cultures of primary adult microglia that both morphologically as well as molecularly more closely resemble ex vivo microglia. Upcoming analyses of their RNA transcriptomes will guide our further developments.