The mechanical stress state of an organ is a critical, but still poorly understood, driver of organogenesis and regeneration. Here we report a chip-based regulated environment for micro-organs (REM-Chip) that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under a multitude of genetic and chemical perturbations. This method has enabled us to identify essential conditions for generating organ-scale intercellular calcium (Ca 2+ ) waves (ICWs) in Drosophila wing imaginal discs that are also observed in vivo. Spontaneous ICWs require the presence of components in fly extract-based growth serum (FEX). Using the REM-Chip, we demonstrate that the release and not the initial application of mechanical compression is sufficient but not necessary to initiate ICWs. Further, the extent of the Ca 2+ response is heterogeneous between discs and correlates with the degree of spontaneous ICWs activity in the pre-stress state. This system and method enable detailed examinations of the interplay between mechanical stress state, biochemical regulatory networks, and physiology in complex, hierarchically organized organ cultures. SIGNIFICANCE STATEMENTWe present a first-of-class microfluidic chip that can perturb a developing organ both chemically and mechanically. Here we advance the field of organogenesis by presenting precise mechanical perturbation methods for whole-organ explants. This enables researchers to systematically interrogate critical relationships between mechanical stress state and biochemical signaling. Our methods advance available modes of studying Drosophila wing disc development, a powerful model for examining pathways critical for human development. We elucidate the causative links between mechanical perturbations and ICWs. Spontaneous ICWs require fly extract (FEX), a growth serum derived from flies, to be included in the culture media. Further, we find that the extent of ICW response to mechanical perturbation is determined by the spontaneous ICW activity prior to stimulation.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/081869 doi: bioRxiv preprint first posted online 3 Organs develop in a diverse landscape of signals. However, the connections between exogenous forces, gene expression, and signal transduction in organ culture are still poorly understood. The calcium ion (Ca 2+ ) has been demonstrated as a universal second messenger that regulates and coordinates a diverse range of intracellular processes such as proliferation and morphogenesis (1). Dysregulation of Ca 2+ signaling via genetic and epigenetic modifications has been implicated in human diseases including cardiomyopathies (2), cancer metastasis (3), and neurodegenerative disorders (4). Furthermore, Ca 2+ signaling has been shown to act as a central signal integrator in stem cell proliferation and homeostasis (5). The ubiquity of ...