Over the years, a number of nano-engineered cargo systems have been explored for their capability to encapsulate, deliver on-target, and release bioactive compounds (1). Whilst the widely used microcapsule drug encapsulation has successfully combined several functionalities (2), uncontrollable capsule spreading within the target tissue remains a serious issue. Recent advances in the fabrication of patterned nanostructured films appear to overcome the scattering issue, by providing arrays of micro-wells (microchambers) with a predetermined size and spatial patterns. This in turn enables release of loaded bioactive compounds, controlled in space and time. There have been, however, only a few attempts to functionally test such delivery systems in live cells (3-5). We, therefore, carried out a physiological study of the fabricated polylactic acid (PLA)-based microchamber arrays in human cells of neuronal phenotype, Neuro2A cell line, in order to examine the efficiency of cargo delivery for various bioactive compounds. First, we documented the biocompatibility of the fabricated microchamber arrays by setting human neuroblastoma cells to grow on the surface of the microchamber arrays. There were no signs of excitotoxicity in differentiating Neuro2A cells on either empty PLA-based arrays (no payload) or those loaded with a fluorescent indicator or principal excitatory neurotransmitter glutamate, for up to ten days in vitro. Scanning electron microscopy confirmed the effective cargo packing inside individual microchambers when loading i) soluble substances of low molecular weight (glutamic acid or its analogue, L-glutamic acid hydrochloride) or ii) peptides (nerve growth factor, NGF). Next, we demonstrated the site-specific cargo release on-demand by the laser-triggered opening of individual microchamber(s). This resulted in physiological cellular responses in local cells. For the laser-triggered glutamate release from PLA-based microchambers, we monitored intracellular Ca2+ dynamics in differentiated Neuro2A cells using two-photon excitation imaging and detected a robust intracellular Ca2+ rise in local cells in response to opening microchambers loaded with glutamate (n=102 cells tested, p<0.001 compared to group of the cells on empty microchamber arrays, n=33 cells); the Ca2+ rise was repeatable, reproducible, and within physiological range. For the laser-triggered opening of microchambers loaded with NGF, we observed directed neurite outgrowth of the local cells toward the opening sites (n=5 microchamber array samples). In summary, our results indicate that microchamber arrays can provide a versatile nanostructured drug delivery system, which is biologically safe and efficient in packaging bioactive compounds for geometry-constrained action in the target tissue.