A Primary Spinal Cord Mixed Culture Method for In Vitro Analysis of Glial Heterogeneity and Inflammatory Responses

Traditional in vitro models used in spinal cord research often rely on simplified cell systems, such as PC-12 cells, which do not reproduce the mixed cellular environment or glia-neuron interactions of spinal cord tissue. Although primary spinal cord cultures have been described, many protocols focus on purified populations and require technically demanding or costly procedures. In this study, we established a low-cost mixed primary spinal cord culture from neonatal Wistar rats and characterized its cellular composition and inflammatory responsiveness in vitro. At 7 days in vitro, immunofluorescence analysis showed the presence of neurons (42.5%), astrocytes (17.2%), and microglia (4.4%), together with an unresolved non-labeled cell population (35.9%). GFAP-positive astrocytes displayed marked morphological heterogeneity, including process-bearing, flattened, and ramified forms. To examine the inflammatory responsiveness of the preparation, cultures were exposed to lipopolysaccharide (LPS, 5 µg/mL for 12 h), which increased nitrite levels and was associated with a higher abundance of amoeboid-like cells and increased Iba1-positive and CD68-positive cell labeling. The culture could also be maintained for prolonged periods in vitro, although its biological interpretation changed over time as non-neuronal cells became more prominent. These findings support the use of this preparation as an accessible mixed primary spinal cord platform for studying glial-associated inflammatory responses in vitro and for future screening of anti-inflammatory compounds.