Despite wide use and approval of poly lactic-coglycolic acid (PLGA) for surgical applications, there have been very few studies on tissue constructions that mimic physiological multilayer structures by combining PLGA scaffolds with tissue engineering. In our study, we developed a bioreactor system to maintain, and to train two types of three-layered vascular-like structures. Then we examined how the perfusion conditions and different tissue engineering approaches affected the formation of the layered structure and degradation of the PLGA scaffolds. In the proposed Distributed Method, the cells were seeded layer by layer on a single scaffold, using spheroids bigger than scaffold fiber gaps and we achieved the higher cell density compared with the Stratified Method where we stacked three PLGA sheets seeded with individual vascular cell types. At the histological level, scaffold degradation was more prominent in the bioreactor compared to the same time interval in vivo. In addition, the faster flow accelerated the decomposition of PLGA fibers. Moreover, bioreactor perfusion culture at lower flow rates could balance cell adhesion and survival, improve the cell density and promote self-organization of multilayer structure with desirable rate of PLGA scaffolds degradation.