We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluiclic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37degreesC. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of similar to2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. (C) 2004 Wiley Periodicals, Inc.