A novel method to prepare highly enriched primary cultures of chicken retinal horizontal cells

Abstract

The retina is a heterogeneous tissue composed of different neuronal cells (photoreceptor (PRC), horizontal (HC), amacrine, bipolar and retinal ganglion RGC cells) and glial cells. Our aim in this work was to purify HCs from the chicken embryonic retina to obtain primary cultures highly enriched in these cells for further characterization. To this end, disaggregated retinas of chicken embryos at day 15 were subjected to a discontinuous bovine serum albumin (BSA) gradient of concentrations raging from 1 to 4%. After centrifugation, cells collected from the different phases were cultured for 4 days and characterized by immunochemistry and cell morphology. Phases were examined with specific antibodies against HC markers such as PROX-1, Islet-1 and calretinin, together with markers for other retinal cell populations. The results show that only in the fraction corresponding to 2.5% BSA did most of the cells display PROX-1 and Islet-1 positive immunoreactivities with a typical HC morphology. Moreover, Western blot assays indicate that the 2.5 % BSA phase exhibits the strongest PROX-1 immunolabeling, denoting the typical molecular weight (MW) of ~ 83KDa. Based on an accurate morphological analysis, a number of cells in this fraction resembled H1- and H3-type HCs (axon-bearing "brush-shaped" and axon-less "candelabrum-shaped" HCs respectively). In conclusion, the BSA gradient has so far proved to be a simple yet very useful method to selectively separate specific retinal cell types for the further study and characterization of their molecular, biochemical and electrophysiological properties.