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Rayne Lim, Arkasubhra Ghosh, Rajiv R Mohan, Shyam S Chaurasia; Primary Culture and Characterization of Porcine Retinal Microglial Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1498. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal microglial cells are the first responders to retinal insult, and play a key role in innate immune responses. Microglial responses in an in vitro setting will allow us to delineate cellular responses, and provide valuable insight into its role in the pathogenesis of diabetic retinopathy. This study was designed to establish an in vitro primary culture of pig retina microglial cells (pMicroglia).
Whole eye globes from five-month old domestic pigs were collected from the local abattoir. Globes were hemisected and retina was isolated under aseptic conditions. Retinas were dissociated by aspiration, digested with collagenase type A, strained through a 100µm membrane and seeded on Primaria coated plates (BD Biosciences) in DMEM/F-12 containing 20% FBS, 1% penicillin/streptomycin and 25ng/ml M-CSF. Porcine retinal microglial cells between passage 1-3 were used for experiments. Microglial functionality was tested using several microglial markers and FITC-conjugated photoreceptor outer segment (POS) phagocytosis. pMicroglia were activated with LPS (10ng/ml) or hyperglycemia (25mM glucose) for 24hours. Cells were harvested for analysis using real-time PCR (qPCR), western blot (WB), and immunocytochemistry (ICC).
pMicroglia exhibited amoeboid morphology and phagocytic activity. ICC of untreated pMicroglia showed positive staining for microglial markers – Iba-1 and CD68, negative for glial cell markers – GFAP and GS. Treatment with LPS or hyperglycemia showed upregulation of microglial activation markers – CD11b, MFG-E8, TSPO and CD68. Proinflammatory cytokines TNFα and IL-6 were also elevated.
We succeeded in culturing primary porcine retinal microglial cells, which exhibit microglial markers, maintain phagocytic activity and release proinflammatory cytokines under hyperglycemic conditions.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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