Characterization of a rat primary retinal cell culture model

MICHELIS, G.; GERMAN, O. L.; VILLASMIL, R.; SOTO, T. B.; ROTSTEIN, N.; POLITI, L. E.; BECERRA, P.

Nashville

Simposio; XIXth International Symposium on Retinal Degeneration Meeting; 2021

Purpose: The purpose of this study is to characterize the photoreceptors and amacrine neuronal cells inthe primary rat retinal cell culture model to serve as a biological system for the evaluation of relevantneurotrophic factors for the retina.Methods: Dishes were sequentially coated with poly-ornithine and the conditioned media from RN22schwannoma cultures before seeding the cells. Dissected neonatal rat retinas (postnatal day 1, PN1)were mechanically and enzymatically dissociated before being plated. Dissociated cells were cultured ina chemically defined medium for 5 days prior to being assayed. Retinal cell types were identified bymorphological and immunochemical methods. Antibody to HPC-1 was used to specifically recognizeamacrine neurons and antibodies to CRX and Rhodopsin were used to identify photoreceptors; and thelabeled cells were quantified by microscopy and flow cytometry. PEDF-R was detected byimmunocytochemical methods. Recombinant human PEDF protein was diluted in Hanks? Balanced SaltSolution and added to the cultures at 2 days in vitro. Cell death was assayed by flow cytometry todetermine cells labeled with Annexin V-Mitotracker and terminal deoxynucleotide transferase dUTP nickend labeling (TUNEL) at 5 days in vitro (i.e., three days of PEDF treatment). To determine transcriptionalchanges, RT² Profiler PCR Arrays for cell death were performed with cultures treated with and withoutPEDF for 6 hours.Results: Crx- and HPC-1-positive cells represented about 80% and 15% of cells present in the primary ratretinal cell cultures, respectively, with the remaining cells showing pyknotic nuclei. The Crx-positive cellswere round in shape, with a cell body of 5-10 µm in diameter and with a single short axon. HPC-1-positive cells had a cell body of about 10-30 µm and had multiple neurites, making connections witheach other. A fraction of the photoreceptor precursor cells (5-10%) contained rhodopsin. Opsindistributed in the cells in a polarized fashion towards an outer segment-like precursor structure in aminority of the rhodopsin-positive population. Both cell types produced PEDF-R, a receptor for theneurotrophic factor PEDF, with a high degree of colocalization with the plasma membrane markerNa+/K+ pump. Additions of PEDF to the cultures regulated the expression levels of genes involved in celldeath relative to those without the factor, such as repression of genes for pro-apoptotic factors andinduction of genes for anti-apoptotic factors. PEDF decreased the number of cells that died by apoptosis,when compared to cell cultures treated with only vehicle as control.Conclusion: Our primary rat retinal cell cultures are comprised mainly of photoreceptors with a lowpercentage of amacrine neurons that resemble most features of their in vivo counterparts. Thephotoreceptors in culture respond to the neurotrophic stimuli of PEDF, by decreasing the apoptosis rateand regulating the expression of genes involved in cell survival. The model provides a platform forscreening trophic factor candidates, and other agents that can affect neural retinal cell survival anddevelopment.