title: Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables.
abstract: Microglia are key players in the central nervous system in health and disease. Much pioneering research on microglia function has been carried out in vivo with the use of genetic animal models. However, to fully understand the role of microglia in neurological and psychiatric disorders, it is crucial to study primary human microglia from brain donors. We have developed a rapid procedure for the isolation of pure human microglia from autopsy tissue using density gradient centrifugation followed by CD11b-specific cell selection. The protocol can be completed in 4 h, with an average yield of 450,000 and 145,000 viable cells per gram of white and grey matter tissue respectively. This method allows for the immediate phenotyping of microglia in relation to brain donor clinical variables, and shows the microglia population to be distinguishable from autologous choroid plexus macrophages. This protocol has been applied to samples from over 100 brain donors from the Netherlands Brain Bank, providing a robust dataset to analyze the effects of age, post-mortem delay, brain acidity, and neurological diagnosis on microglia yield and phenotype. Our data show that cerebrospinal fluid pH is positively correlated to microglial cell yield, but donor age and post-mortem delay do not negatively affect viable microglia yield. Analysis of CD45 and CD11b expression showed that changes in microglia phenotype can be attributed to a neurological diagnosis, and are not influenced by variation in ante- and post-mortem parameters. Cryogenic storage of primary microglia was shown to be possible, albeit with variable levels of recovery and effects on phenotype and RNA quality. Microglial gene expression substantially changed due to culture, including the loss of the microglia-specific markers, showing the importance of immediate microglia phenotyping. We conclude that primary microglia can be isolated effectively and rapidly from human post-mortem brain tissue, allowing for the study of the microglial population in light of the neuropathological status of the donor.
authors.affiliation: Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands. m.mizee@nin.knaw.nl.|Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands. m.mizee@nin.knaw.nl.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Mizee
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Suzanne S M
authors.initials: SS
authors.lastname: Miedema
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Marlijn
authors.initials: M
authors.lastname: van der Poel
authors.affiliation: Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.lastname: Adelia
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Karianne G
authors.initials: KG
authors.lastname: Schuurman
authors.affiliation: Department of Astrocyte Biology and Neurodegeneration, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Miriam E
authors.initials: ME
authors.lastname: van Strien
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Melief
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Smolders
authors.affiliation: Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Debbie A
authors.initials: DA
authors.lastname: Hendrickx
authors.affiliation: Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Kirstin M
authors.initials: KM
authors.lastname: Heutinck
authors.affiliation: Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.|Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Jörg
authors.initials: J
authors.lastname: Hamann
authors.affiliation: Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.|Department of Neuroimmunology, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Inge
authors.initials: I
authors.lastname: Huitinga
mesh_terms: D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D001921:Brain / Q000378:metabolism / Q000473:pathology; D039481:CD11b Antigen / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002555:Cerebrospinal Fluid / Q000737:chemistry; D015925:Cryopreservation; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008297:Male; D017628:Microglia / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014015:Tissue Banks; D014019:Tissue Donors
chemical_list: D039481:CD11b Antigen; C509295:ITGAM protein, human; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: Biobanking; Post-mortem human brain; Primary human microglia; Primary microglial cell culture; Rapid cell isolation protocol