Biopharmaceutical production would benefit from rapid methods to optimize production of therapeutic proteins by screening host cell line/vector combination, culture media, and operational parameters such as timing of induction. Miniaturized bioreactors are an emerging research area aiming at improving the development speed. In this work, a 3 mm thick mini-bioreactor including two 12 mm wide culture chambers connected by a 5 mm wide channel is described. Active mixing is achieved by pressure shuttling between the two chambers. Gas-liquid phase exchange for oxygen and carbon dioxide is realized by molecular diffusion through 50 mu m thick polymethylpentene membranes. With this unique design, a velocity difference between the middle area and the side areas at the interfaces of the culture chambers and the connecting channel is created, which enhances the mixing efficiency. The observed mixing time is on the order of 100 s. The combination of high permeability toward oxygen of polymethylpentene membranes and fluid movement during active pressure shuttling enables higher volumetric oxygen transfer coefficients, 5.7 +/- 0.4-14.8 +/- 0.6 h(-1), to be obtained in the mini-bioreactors than the values found in traditional 50 mL spinner flasks, 2.0-2.5 h-1. Meanwhile, the calculated volume averaged shear stress, in the range of 10(-2)-10(-1) N/m(2), is within the typical tolerable range of animal cells. To demonstrate the applicability of this mini-bioreactor to culture suspended animal cells, the insect cell, Spodoptera frugiperda, is cultured in mini-bioreactors operated under a K(L)a value of 14.8 +/- 0.6 h(-1) and compared to the same cells cultured in 50 mL spinner flasks operated under a KLa value of 2.2 h(-1). Sf-21 cells cultured in the mini-bioreactors present comparable length of lag phases and growth rates to their counterparts cultured in 50 ml, spinner flasks, but achieve a higher maximum cell density of 5.3 +/- 0.9 x 10(6) cell/mL than the value of 3.4 +/- 0.4 x 10(6) cell/mL obtained by cells cultured in 50 ml, spinner flasks. Sf-21 cells infected with SEAP-baculovirus produce a maximum SEAP concentration of 11.3 +/- 0.7 U/ml, when cultured in the mini-bioreactor. In contrast, infected Sf-21 cells cultured in 50 mL spinner flasks produce a maximum SEAP concentration of 7.4 +/- 0.9 U/mL and onset of production is delayed from 18 h in minibioreactor to 40 h in spinner flasks.