We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-microFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, the multi-channel 3D-microFCCS was designed for culturing different 3D cellular aggregates simultaneously to mimic multiple organs in the body. Four human cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-beta1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-beta1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-microFCCS could be potentially used to supplement or even replace animal models in drug screening.