Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 µm depth) and well bioreactors (35 µm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays. Biotechnol. Bioeng. 2009;102: 1222–1230. © 2008 Wiley Periodicals, Inc.