Evaluation of therapeutic sensitivity of retinal ganglion cells to targeted peptide bioregulator in culture

Abstract

To study how therapeutically sensitive retinal cell culture is to the peptide bioregulator isolated from cattle retina (Retinalamin) in models of glaucomatous optic neuropathy (GON). The cells were isolated from the retinae separated from newborn mice. Cell sheets were disaggregated and transformed into a suspension. Removal of the off-target cell population was done by adding antibodies to deplete the cells with CD48 marker, and magnetic microbeads that attach to them. Selection of ganglion cells and obtainment of its enriched fraction was done by immunomagnetic separation. To assess the toxicity of Retinalamin, a cytotoxic test was performed on the culture of skin fibroblasts with sequential dilution of the drug into concentrations of 5.0-0.009 mg/mL. The cells were seeded at 5000 per plate well and exposed to the drug for 24 hours. To study the excitotoxic damage, the first group of plate wells with retinal cells had solution of sodium glutamate added in concentration of 20 mM, Retinalamin was added into the second group of wells in concentration of 1.25 mg/mL; both substances were added into the third group of wells. The control group consisted of intact plate wells. The cells were exposed to substances for 24 hours. Cell vitality was then evaluated using colorimetry. Optic density was measured using an automatic photometer with detection wavelength of λ=490 nm. The cell culture achieved by immunomagnetic separation is mixed and consists of ganglion and glial cells. Retinalamin does not display significant cytotoxicity in any of the studied concentrations. The excitotoxic damage caused significant decrease of the amount of viable cells in the culture (9% of the control wells). The concomitant addition of glutamate and 1.25 mg/mL Retinalamin resulted in a 51.6% increase in the amount of viable cells. The intergroup differences were statistically significant by Student's t-test. Retinalamin is not cytotoxic. The concomitant addition of glutamate and Retinalamin reliably decreases the toxic action of glutamate on isolated retinal cells.