Introduction/Aims Penetrating traumatic brain injury (pTBI) is a surgical emergency, with poor clinical outcomes. Management is supportive including surgical wound debridement, anti-epileptic administration and infection prophylaxis; no clinical neuroregenerative therapies exist. Surgical biomaterial scaffolds have therapeutic potential, but developmental testing relies heavily on highly invasive live animal injury models which are low throughput, expensive, technically challenging, and present significant ethical challenges. We recently developed a high throughput, multi-glial and facile in vitro pTBI model within which neural cell interactions with a surgical grade scaffold, DuraGen PlusTM could be investigated. A key limitation was the lack of a neuronal component, meaning axonal transection and regeneration could not be assessed. Here, we report an advanced version of the model, which (i) encompasses all the major neural cell types including neurons; (ii) within which penetrating lesions can be induced and biomaterials implanted. Methods Cerebral cortices from postnatal mice were extracted and cultured in vitro with a novel supporting medium. Cultures were transected at 8 days post-culture simulating penetrating injury in vitro, with immunocytochemistry used to analyse neural-cell responses/interactions. Results We demonstrate the utility of the model in reproducibly simulating pTBI in vitro with key pathological features such as glial scarring, microglial invasion and axonal outgrowth, p&lt;0.05, n=5). The benefit of the model to study nanomaterial-neural cell interactions is demonstrated. Conclusion Our new model offers significant benefits for high throughput, facile developmental testing of novel nano/biomaterials in preclinical studies.