Abstracts from the 26th European Society for Animal Cell Technology Meeting - Cell culture technologies: bridging academia and industry to provide solutions for patients

BURGI, MARÍA DE LOS MILAGROS; APARICIO, GABRIELA; KRATJE RICARDO; SCORTICATI, CAMILA; OGGERO, MARCOS

BMC Proceedings

BioMed Central

Lugar: Londres; Año: 2020 vol. 14 p. 18 - 20

Background Neurodegenerative diseases are incurable conditions that progressive affect nerve cells. According to the World Health Organization (WHO), neurodegenerative diseases affect millions of people around the world, mainly due to the increase in life expectancy and the concomitant increase in the world's elderly population. There is not treatment for these pathologies. In this sense, human erythropoietin (hEPO) has a leading role because its antiapoptotic, antiinflammatory, and antioxidant effects have been observed in neural tissues [2, 3]. In order to evaluate the in vitro neuroprotection and neuroplasticity of rhEPO and putative derivatives, two cell culture-based procedures were optimized. Materials and methodsThe in vitro neuroprotective activity of different concentrations of CHO-derived rhEPO was determined as the capacity of the cytokine to reverse the staurosporine (STP)-induced apoptosis in cultures of: murine neuroblastoma cell line (N2a) and hippocampal neurons at 5 and 11 days in vitro (DIV). The neuroprotection was measured as the percentage of viable cells (N2a cells) or apoptotic nuclei (HPNC) after STP-induced apoptosis. Neuroplasticity was determined by measuring neuritogenesis, filopodia and synapses formation. N2a cells were incubated for 3 h with 10, 50 and 300 ng/ml of rhEPO and then, the longest neurite, the average neurite length and the number of neurites per cell were quantified using the NeuronJ plugin for ImageJ (NIH). Hippocampal neurons treated with rhEPO were used to evaluate filopodia (5 DIV) and synapses (15 DIV) formation. Briefly, the number of filopodia per 20 µm of neurite length was quantified in 40?50 neurites per group. Synapse formation was measured by colocalization of puncta, along 25 µm of dendrite length, between pre- and post-synaptic markers in 20-30 neurons per condition, using three dendritic regions per neuron. The selected neurons were at least two cell diameters away from their nearest neighbor [4]. Colocalization of puncta were determined using the plugin Puncta Analyzer of ImageJ. For statistics, significant differences were determined using one-way ANOVA followed by the Dunett´s test or two tails test, as appropriate.Results rhEPO was capable to significantly reverse the STP-induced apoptosis in a dose-response effect in both N2a cells and neurons (between p