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Abstract: A combination of hydrogels and microfluidics allows the construction of biomimetic three-dimensional (3D) tissue models in vitro, which are also known as organ-on-a-chip models. The hydrogel patterning with a well-controlled spatial distribution is typically achieved by embedding sophisticated microstructures to act as a boundary. However, these physical barriers inevitably expose cells/tissues to a less physiologically relevant microenvironment than in vivo conditions. Herein, we present a novel dissolvable temporary barrier (DTB) strategy that allows robust and flexible hydrogel patterning with great freedom of design and desirable flow stimuli for cellular hydrogels. The key aspect of this approach is the patterning of a water-soluble rigid barrier as a guiding path for the hydrogel using stencil printing technology, followed by a barrier-free medium perfusion after the dissolution of the DTB. Single and multiple tissue compartments with different geometries can be established using either straight or curved DTB structures. The effectiveness of this strategy is further validated by generating a 3D vascular network through vasculogenesis and angiogenesis using a vascularized microtumor model. As a new proof-of-concept in vasculature-on-a-chip, DTB enables seamless contact between the hydrogel and the culture medium in closed microdevices, which is an improved protocol for the fabrication of multiorgan chips. Therefore, we expect it to serve as a promising paradigm for organ-on-a-chip devices for the development of tumor vascularization and drug evaluation in the future preclinical studies.