Abstract

We herein designed a 64-chamber laminar-flow microfluidic chip with independently addressable culture units capable of establishing spatially heterogeneous chemical environments to mimic tissue microenvironments. Stable chemical gradients were successfully generated within the chip through controlled laminar flow, allowing precise spatial modulation of cellular exposure. Using TNF-α as a model stimulus, we observed a clear time delay in NF-κB activation between cells directly exposed to the cytokine and those located on the medium-only side, confirming the establishment of spatially distinct chemical conditions. Notably, even cells not directly exposed to TNF-α eventually responded, indicating that molecular diffusion along the static solid–liquid interface serves as an effective delivery route for bioactive molecules. To further demonstrate the platform’s utility, we constructed a skin-mimetic co-culture model of HaCaT keratinocytes and human skin fibroblasts (HSFs) to assess the diffusion and cytotoxic effects of 5-fluorouracil (5-FU). The results revealed that fibroblasts provided protective effects against 5-FU-induced cytotoxicity, likely via paracrine signaling or direct cell–cell interactions. These findings highlight the platform’s capacity for probing not only spatial drug-delivery dynamics but also intercellular interactions under physiologically relevant conditions. This system offers a powerful and versatile tool for studying spatiotemporal signaling, drug screening, and topical therapeutic development.

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