Cancer-stromal cell interaction is a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, are limited in three aspects: 1) Limited control of cell microenvironment; 2) Inability of studying cell behavior in a single-cell manner, and; 3) Difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is centered on improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells, while maintaining nutrient renewal. In this work, two-phase flow was used as the simple isolation method, separating the microenvironments of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows the area reduction in vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between cell culture layer and media exchange layer. The selectivity of the semi-permeable membrane can retain the signaling proteins within the chamber, while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk with cells in other chambers. Utilizing these innovations, we demonstrated co-culture of UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to recapitulate tumor proliferation enhancement in the vascular endothelial niche.