gms | German Medical Science

Objective: The role of microglia in brain cancer is increasingly recognized (Wei et al., 2013) and there is urgent need to isolate highly pure microglia with high yield from human brain tumor samples to further analyse their contribution to tumor development and perturbation. Here, we demonstrate that this can be achieved by a method previously described for embryonic rodents and adult rats.

Methods: Brain tumor samples (n=3) were obtained from patients during surgery, which exhibited the typical morphological appearance of gliogenic tumors as diagnosed by contrast-enhanced MRI. The tissues were mechanically dissected removing vascular remnants and blood clots. After filtration through 100-µm pore nylon mesh Cell strainers and removal of debris by centrifugation cells were allowed to adhere to Aclar® plastic films placed at the bottom of the wells of a six-well-plates. After an incubation period of 10-12 days during which medium was exchanged every second day an anti-F4-80 antibody and an Iba-1 antibody were employed to detect microglial markers. In addition, an anti-GFAP-antibody was used as a control for astrocytic and glioma cells and DAPI-staining was used for staining of nuclei.

Results: 10 to 12 days after isolation about 90 % of cells exhibited the typical needle-shape morphology of microglia in Aclar® covered and uncovered wells. Although a small amount of GFAP-positive debris was detectable, no viable cells positive for GFAP were detected. Around 80-90% of the cells stained positive for both F4/80 and Iba-1. Most importantly, we obtained ~5 times more cells from equal amounts of starting material using Aclar® covered wells in contrast to plain wells. In addition, we did not detect GFAP-positive cells.

Conclusion: Here we demonstrate that Iba-1 and F4/80-positive microglia can be isolated in high yield and purity from human glioma using Aclar® film. The method presented is less time consuming and avoids mechanical stress which is an obstacle observed by using other frequently applied methods. As Aclar® film is microscopy-compatible, cells can directly be monitored, treated during the 10 to 12-day incubation period and can also be removed by mild trypsinisation for further experiments.

Figure 1 [Fig. 1], Figure 2 [Fig. 2]