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Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury.An ex vivo spinal cord culture method to study cell responses to injury.Adult spinal cord slices cultured ex vivo mimic spinal cord injury in vivo.The ependymal cell marker profile is maintained ex vivo.Ependymal cells are activated, proliferate, differentiate, and migrate ex vivo.Ependymal cells/progeny exhibit spontaneous Ca2+ activity and respond to ATP.