Characterisation and application of primary cultured retinal mononuclear phagocytes

Abstract

Purpose : Resident microglia and recruited macrophages, collectively known as mononuclear phagocytes (MNPs), are known to be involved in retinal development, maintaining homeostasis and contributing to retinal degenerations. Therefore, culturing primary retinal microglia in vitro is an important tool for understanding their pathophysiology and function. We present a new method using fluorescence-activated cell sorting for isolating and culturing MNPs from both adult rodent and human retinas allowing for further investigation of their role in the retina.

Methods : Rodent and human retinas were subjected to mechanical dissociation and enzymatic digestion (2.5mg/ml papain). MNPs were stained with a CD11b-PE labelled antibody and isolated via fluorescence-activated cell sorting. MNPs were sorted directly into a 48-well plate containing DMEM-F12, 0.25ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 2.5ng/ml macrophage-CSF (M-CSF). Primary MNPs were profiled for microglial markers CD11b, CD45, CX3CR1, CD64, F4/80, and exclusion markers CCR1, CD86, MHCII, at 0 days, 2 weeks and 4 weeks in culture using flow cytometry. Human MNPs were profiled using IBA-1 and CD11b immunocytochemistry at 4 weeks. The expression of pro-inflammatory cytokines (Il-1β, Il-6 and Ccl2) was assessed using RT-qPCR following inflammatory stimulation with 1μg/ml lipopolysaccharide (LPS) for 24h. Phagocytosis was measured periodically over a 96h incubation with 2μm fluorescent latex beads using an IncuCyte.

Results : Rodent MNPs at 0 days were profiled as CD11b⁺, CD45⁺, CX3CR1⁺, CD64^lo, F4/80^lo, CCR1^-, CD86^-, MHCII^-. Over a 4 week period, cell numbers increased by at least 400% (P<0.05), with a reversion to a ‘microglia-like’ profile, CD11b⁺, CD45⁺, CX3CR1⁺, CD64⁺, F4/80⁺, CCR1^-, CD86^lo, MHCII^-. Following LPS stimulation, MNPs demonstrated increased expression of Il1β, Il6 and Ccl2 (P<0.05), as well as increased phagocytosis. Human MNPs at 4 weeks in culture stained positive for microglial markers IBA-1 and CD11b.

Conclusions : We have demonstrated that using our novel culturing and extraction technique, cultured MNPs over a 4-week period increased in numbers and reverted to a profile synonymous to that previously reported for microglia. The protocol presented will prove valuable for developing a greater understanding of MNPs and their role in retinal degenerations, and provides an in vitro model for testing novel therapeutics.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.