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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\u00a0\u00b5g\/mL for 12\u00a0h), 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.<\/jats:p>","DOI":"10.1007\/s11064-026-04777-9","type":"journal-article","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T07:57:08Z","timestamp":1779350228000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Primary Spinal Cord Mixed Culture Method for In Vitro Analysis of Glial Heterogeneity and Inflammatory Responses"],"prefix":"10.1007","volume":"51","author":[{"given":"Cinthia Cristina","family":"de Oliveira Santos Costa","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catarina","family":"de Jesus Nunes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Clara Neves Reis","family":"Pedreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia Lima","family":"Costa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravena Pereira","family":"do Nascimento","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,21]]},"reference":[{"key":"4777_CR1","doi-asserted-by":"publisher","first-page":"108742","DOI":"10.1016\/j.jneumeth.2020.108742","volume":"340","author":"NM Agalave","year":"2020","unstructured":"Agalave NM, Lane BT, Mody PH, Szabo-Pardi TA, Burton MD (2020) Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord. 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