Long-term mouse spinal cord organotypic slice culture as a platform for validating cell transplantation in spinal cord injury

Spinal cord injury (SCI) is an extremely invalidating condition with a severe physical and psychological impact. Resolutive cures are still lacking, due to its complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCI. Standard SC organotypic cultures are maintained for up to 2 or 3 weeks in vitro . Here, we describe an optimized protocol for long-term maintenance for up to three months (90 days). The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a GFP reporter were transplanted into mouse SC-slices. 30 days after the transplant, cells still show GFP expression, and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an e x vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments. SUMMARY In this paper, we provide a reproducible method to generate and maintain long–term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies. ### Competing Interest Statement The authors have declared no competing interest.