While the importance of autocrine-paracrine signaling in vivo is clear, the ability to study the effects of secreted endogenous factors in vitro is hampered by canonical culture platforms. In multi-well plates, the large air-liquid interface gives rise to convective flows that continually mix the fluid disrupting the local diffusion-based accumulation. Simple microchannels provide a more controlled microenvironment that can be used to study secreted factor effects. Here, we utilize microchannel

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... culture to examine basic culture parameters and their interactions using normal mammary gland epithelial cells (NMuMG). The following parameters were studied: (1) cell density (80 vs. 240 cells mm 22 ), (2) exogenous growth factors (epidermal growth factor [EGF] vs. fetal bovine serum), (3) medium change frequency (1 h, 4 h, 12 h), and (4) culture platform (microchannels vs. 96-well plates). The cells exhibited increased growth rates in microchannels as compared to 96-well plates. Cell proliferation increased as the frequency of media change decreased. For the microchannel geometries used, important threshold concentrations were reached in a few hours. In aggregate, the results indicate that the function of the four factors and their interactions on NMuMG growth are spatially and temporally related by molecular diffusion in the controlled microchannel space. The convective-free microchannel environment may prove useful for studying soluble factor signaling in vitro, and to test models and predictions of autocrine-paracrine signaling.