Microfluidic Chamber Array for Continuously-Perfused Cell Culture and Assay

Continuous perfusion during cell culture is desirable for greater control of the microenvironment, particularly when controlling for the effects of autocrine or paracrine signaling. Microfluidic devices are well-suited for continuous perfusion because they offer dramatic savings of reagents due to their small volumes. A variety of microfluidic culture devices has been developed for continuous perfusion, yielding impressive demonstrations of high-throughput data collection. However, these systems typically link culture chambers with shared flow paths, leading to cross-contamination. In addition, the integration of other functions, such as independent addressing (or isolation) of chambers, becomes challenging because of the need for more sophisticated flow networks.

We realize a rectangular chamber array in a PDMS device with three attributes: (i) continuous perfusion; (ii) independently-addressable chambers and subarrays; and (iii) flow paths that forbid cross-chamber contamination. The combination of these attributes is made possible by a bridge-and-underpass architecture, where flow streams travel vertically to pass over (or under) channels and on-chip valves. The array achieves considerable versatility due to subarray, row, column, or single chamber addressing for the following: incubation with adsorbed molecules; perfusion of differing media; seeding or extraction of cells; and assay staining. In addition, culture surfaces are shielded from direct perfusion to minimize shear-induced cell behavior. We have thus far used the device to classify different phenotypes of alveolar epithelial type II (ATII) cells, particularly the extent of epithelial-to-mesenchymal transition (EMT), which is a highly suspected pathway in tissue regeneration, fibrosis, and cancer metastasis. Cells are cultured on combinations of matrix proteins (fibronectin or laminin by row) and soluble signals (TGF-β1 +/- by column) in a 2x4 array with two subarray repeats per chip. We perform cultures up to 4 days, as well as vital and end-point fluorescent assays by subarrays to quantify viability, transdifferentiation (EMT), and F-actin organization. Assay and morphological data are used to tease-out effects of cues driving each phenotype, confirming this as an effective and versatile combinatorial screening platform for trials requiring high microenvironmental control. -11-2009-->