Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables

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. Electronic supplementary material The online version of this article (doi:10.1186/s40478-017-0418-8) contains supplementary material, which is available to authorized users.


Supplemental figure 2.
Viable microglia yield shows no overall significant difference between both isolation methods, in both WM and GM isolations (unpaired Mann-Whitney test).

Detailed protocol for the isolation of primary human microglia
Tissue -Collected at autopsy and stored in 50 ml tube containing 25 ml hibernate-A medium at 4°C.
Tissue pieces over 5 grams should be cut into smaller sections to maintain enough surface to volume ratio. -Put tissue in 10 cm petri dish with hibernate, remove large vessels/membranes. After this step pieces should be pipettable with 10 ml pipette.

Media
o WM: chop tissue in little chunks using 2 scalpels.
o GM: grind the tissue over a 100 mesh tissue sieve with a plunger from a 50ml disposable syringe. Flush out any attached tissue from the sieve with Hibernate.
the final volume is 30ml.
-Add 2 ml FBS (at least much ml as trypsin added), resuspend/mix well and put suspension in 50 ml tube. If working with more than 4 grams of tissue, split sample in as many tubes as necessary (max ~4 gr per Percoll tube ). Spin sample 10'@1800 rpm 4°C (do not fast cool the centrifuge, so cooling starts when sample is being centrifuged).
-Pour off supernatant and resuspend pellet in ~15 ml cold dDMEM. Filter cell suspension through a clean 100 mesh filter/cup in a petri dish. Add dDMEM if needed (for rinsing petri dish). Collect cells (and "dish-rinse") in a 50 ml tube. Final volume should be 20 ml, also when working with smaller tissue quantities.
-Slowly drip cold Percoll directly on the cell suspension. Use half the volume in tube (eg 20 ml cell suspension: add 10 ml Percoll). Do not mix! Spin tube(s) 30'@4000 rpm 4°C, acc 9, brake 4. After this step carefully remove the tubes from the centrifuge. You should see 3 layers: myelin on top, erythrocytes almost in the bottom, Percoll layer on the bottom. Glial cells are between myelin and erythrocytes.
-Using a glass pipette, transfer cells to a new 50 ml tube (either remove the myelin layer with a pipette or carefully push aside), try not to incorporate any erythrocytes/myelin. Wash cells by filling up tube with cDMEM, spin 10'@1500 rpm 4°C.
-Pour off supernatant and resuspend cells in beads buffer when continuing with bead isolation. Count the number of viable cells (cell chamber) using Trypan Blue.
-During incubation set up magnet and MS columns -Wash MS column with 500 µl BB, when reservoir is empty put cells on column. Wash column 3 times by adding 500 µl BB whenever column reservoir is empty. Collect flowthrough and wash steps in one tube (this contains all CD11b-negative cells).
-Elute column: take column off magnet, add at least 1 ml BB and push cells out using plunger immediately after addition of BB.
-For fractions of interest: add buffer/medium to your cells, spin, resuspend and count cell number in a cell chamber using trypan blue. Always reserve cells from cd11b elution (10.000 -20.000 cells) for staining (CD45, CD11b, CD15) and analysis on FACS. -