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B. Balasubramaniam, D. A. Carter, E. J. Mayer, E. Nicholson, A. D. Randall, A. D. Dick; Functional and Phenotypic Analysis of Primary Adult Human Retinal Microglia. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6251.
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© ARVO (1962-2015); The Authors (2016-present)
Microglia (MG) play a central homeostatic role in the retina. They contribute to inflammatory and degenerative states and counteract infection. To better understand how these processes contribute to human disease we have been developing methods to isolate, culture and characterise adult microglia from post mortem human retina. Here we present phenotypic and functional studies of such primary human retinal MG.
Primary MG were isolated using CD11b-dependent magnetic sorting of retinal cell suspensions generated from human retinal explants pre-conditioned in fractalkine-supplemented culture media (CM) with M-CSF. Morphological and phenotypic analyses were supplemented with investigations of cytokine responses and real-time [Ca2+]i imaging. Purified MG cultured in CM fed M-CSF for 3 days, were exposed to CM supplemented with activating or inhibitory cytokines or cognate signals for 4 hours and 24 hours, loaded with Fura-2 and ratiometrically imaged for [Ca2+]i responses to ATP, synthetic ligands for the protease-activated receptor 1-3 (PAR1-3) and LPS-IFNγ.
FACS analysis demonstrated these isolated primary human MG expressed CD45, CD11c, CD11b, TLR-4 and NOS2. A CD200R agonist (DX109) and conditioning with IL-4/TGFβ maintained the morphology of these cells in a ramified state. Primary retinal MG exhibited robust [Ca2+]i responses to 50uM ATP and selective agonists for PAR1-3. Such responses were typically greater in ramified cells. This enabled assessment of functional responses to inflammatory or anti-inflammatory signals. CBA-analysis indicated that these primary human adult MG release IL-1β, IL-6 and IL-8.
We have successfully purified and initialised characterisation of a population of adult human retinal MG. We believe cells produced in this fashion have the potential to aid our understanding of microglial signalling and function in man. We are also using similar methods to compare our primary adult cells with transformed MG cell lines originally derived from human embryonic CNS.
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