Development of a method for the isolation and culture of astrocytes from the canine cerebral cortex

Background: Astrocytes are considered key players in neuroimmunopathological processes, and they play a certain role in neuroinflammation. Rodent primary astrocyte cultures are commonly used in the study of human neuroinflammation. However, gene sequence homologies are closer between humans and dogs than between humans and rodents.

New method: We established protocols to isolate astrocytes from the canine forebrain. Cerebral hemispheres of 3-4-week-old dogs were used. The isolation procedure included the use of the Neural Tissue Dissociation Kit P, demyelination by the magnetic bead method, and separation and preparation by differential adhesion.

Results: We found a 96% astrocyte purification rate after isolation by differential adhesion. Purified canine astrocytes increased the secretion of interleukin-1β, interleukin-6, and tumor necrosis factor-alpha, and increased the expression of glial fibrillary acidic protein after lipopolysaccharide stimulation. We sequenced the transcriptome of the purified canine astrocytes and analyzed the differentially expressed genes among the rodent, human, and canine astrocytes. Transcriptome profiling and gene ontology analysis of the genes co-expressed in humans and canines indicate that human and canine astrocytes may be different from their rodent counterparts in terms of mediated interactions with metals.

Compared with the existing methods: The cells prepared by our method allow for the rapid separation of astrocytes with a relatively small resource scheme. The method also retains the cell phenotype and has an in vitro culture lifetime of approximately 2-3 months.

Conclusion: We established a method for preparing canine astrocytes with high purity, which can be used to study the biological function of astrocytes in vitro.