Cell culture is a well-established standard technique and a fundamental tool in biology and medicine. Establishment of a novel culture method by meeting various challenges can sometimes open up new fields of cell biology and medicine. An artificial microenvironment for cultured cells is made up of complicated factors, including cytokines, scaffold material type, cell-cell interactions, and physical stress. To replicate the tissue architecture, cell-cell interactions, and specific physical microenvironment, we previously demonstrated the effectiveness of a three-dimensional culture system, and further established two simple culture systems: air-liquid interface (ALI) and fluid flow stress (FFS). A three-dimensional collagen gel culture system can replicate cell-cell interactions in vitro. As skin is constantly exposed to air, the ALI system closely mimicked the skin microenvironment and maintained the homeostasis of the epidermis and dermis. The ALI culture system also revealed the possibility of skin regeneration through ectopic mesenchymal cell involvement. Fluid streaming and shear stress were recently demonstrated to constitute the critical microenvironment for various cell types. The FFS system demonstrated that fluid streaming induced epithelial-mesenchymal transition of mesothelial cells, leading to peritoneal fibrosis. Our novel culture systems will hopefully open up new fields of regenerative medicine and pathological research.