Three-dimensional (3D) cellular spheroids have attracted much attention as a transplantation procedure because the increased cell-to-cell interaction in spheroids enhances cell survival and its functions after the transplantation into the body. Furthermore, the potency of spheroidal cells may be further improved by introducing transgenes to augment cellular functions as well as enhance the paracrine effects by secreting key proteins involved in the essential cellular signaling cascades. In this study, we organized a new platform for genetically-modified cell transplantation by combining a microfabricated culture system for 3D spheroid formation with a newly developed non-viral transfection system, polyplex nanomicelle. After transfection of Gaussia luciferase using the nanomicelle, the prolonged luciferase expression was obtained for more than a month with continuous albumin secretion from the hepatocyte spheroids to the level comparable with control spheroids receiving no transfection. In contrast, by the transfection using FuGENE HD, a commercially available lipid-based reagent, the luciferase expression was obtained, yet the albumin secretion was significantly decreased with disintegration of the spheroid architecture. To assess the feasibility of the hepatocyte spheroids for in vivo transplantation, the spheroids were recovered by the use of micropatterned culture plate functionalized with thermosensitive polymer and dispersed into Matrigel(TM) Matrix. The luciferase expression as well as albumin secretion was maintained for more than a month from the spheroids in the Matrix. Thus, the combination of spheroid cell culture on micropatterned plates with gene introduction using polyplex nanomicelle is a promising platform for genetically-modified cell transplantation to achieve sustained transgene expression with maintaining innate cell functions.