PROBLEM TO BE SOLVED: To provide production methods of three-dimensional cell cultures having a structure close to a capillary of a living body.SOLUTION: A production method of a three-dimensional cell culture having a microvessel-like structure comprises the following step 1 to step 3. Step 1: Seeding shook fibroblasts in a culture vessel to form a first fibroblast layer. Step 2: Adding collagen IV to the first fibroblast layer, and then seeding and culturing vascular endothelial cells to form a blood vessel-like structural layer on the first fibroblast layer. Step 3: Seeding the shook fibroblasts together with VEGF and FGF on the blood vessel-like structural layer and forming a second fibroblast layer with a blood vessel-like structure extending upward from the blood vessel-like structural layer.SELECTED DRAWING: Figure 1【課題】生体の毛細血管に近い構造を有する三次元細胞培養物の製造方法を提供する。【解決手段】下記の工程１～工程３を含む、微小血管様構造を有する三次元細胞培養物の製造方法工程１：振とう処理された線維芽細胞を培養容器に播種して第１線維芽細胞層を形成する工程、工程２：第１線維芽細胞層上にコラーゲンIVを添加し、次いで血管内皮細胞を播種して培養し、第１線維芽細胞層の上に血管様構造層を形成する工程工程３：振とう処理された線維芽細胞をVEGF及びFGFとともに血管様構造層上に播種し、血管様構造層から上方に血管様構造が伸びた第２線維芽細胞層を形成する工程。【選択図】図１
