The authors present a hydrogel/poly-dimethylsiloxane (PDMS) hybrid bioreactor. The bioreactor enables a low shear stress 3D culture by integrating a hydrogel as a barrier into a PDMS casing. The use of PDMS allows the reversible adhesion of the device to a commercially available microelectrode array. A two-step molding process facilitates this relatively simple, cost effective, and leakage-free add-on microculture system. Agarose (2%) is used as hydrogel barrier material and mass transport is evaluated by fluorescein isothiocyanate-albumin fluorescence under static conditions which yields a diffusion coefficient of average value of 2.2 × 10-7 cm 2 s-1 across the barrier. To validate our bioreactor for diffusion limited 3D cell culture, rat cortical cells were successfully cultured in Matrigel for 6 days. © 2013 American Vacuum Society.