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Vascular smooth muscle cells (VSMCs) in the normal aorta are described as having a contractile phenotype because they can contract and do not proliferate. VSMCs in pathological conditions such as atherosclerosis and restenosis can proliferate and migrate, but lose their ability to contract, which is referred to as a synthetic phenotype. VSMCs show plasticity by changing their phenotype according to the surrounding environment. When VSMCs are cultured on a plastic plate, which is a normal two-dimensional culture system, they display the synthetic phenotype because they proliferate and migrate without contraction. Recently, we successful‐ ly cultured VSMCs that display features similar to the contractile phenotype, using type I collagen three-dimensional matrices, “honeycombs,” in the presence of abundant fetal bovine serum albumin. VSMCs cultured in honeycombs stop proliferating and can contract. The honeycomb culture system can maintain VSMCs in the contractile phenotype for a long period of time. In this chapter, we show the method of this new culture system and the characteristics of VSMCs in honeycombs. It is expected that the use of this culture system will generate new information on the characteristics of VSMCs.

Three-Dimensional “Honeycomb” Culture System that Helps to Maintain the Contractile Phenotype of Vascular Smooth Muscle Cells