Protocol to generate species-specific astrocyte-conditioned medium for human organoid neuron maturation

Brain organoids have emerged as promising models for neuroscience research; however, their utility is often limited by inadequate maturation. Here, we present a protocol for enhancing the maturation of human 2D and 3D neural cultures using astrocyte-conditioned medium (ACM). We outline the steps for isolating primary mouse astrocytes, culturing both mouse and human astrocytes, and preparing ACM. Additionally, we provide detailed procedures for quality control and downstream applications of ACM derived from different species. For complete details on the use and execution of this protocol, please refer to Zheng et al.¹.