SeedEZ 3D cell culture methods and protocols – cell seeding. User guidelines and protocols for cell seeding into the SeedEZ three-dimensional (3D) cell culture system. Learn how to make consistent 3D cell culture and co-culture models with the SeedEZ substrate using spot-a-culture, dip-and-culture and stack-and-culture methods. Learn how to use SeedEZ for contact co-culture and non-contact co-culture. Next, learn how to make 3D sandwich cultures and 3D tissue reconstructions modeling multi-layered tissues such as cerebral cortex in a SeedEZ stack. Detailed protocols for generation of brain 3D co-cultures using primary cortical neurons and mixed population of glia seeded with and without Matrigel extracellular matrix into the SeedEZ are provided.