In this preliminary study, the long-term effects of calcium chloride crosslinking concentration on viability of 16HBE14o- human bronchial epithelial cells embedded in alginate-extracellular matrix (ECM) or alginate–methylcellulose–ECM hydrogels have been investigated. There is currently a limited understanding regarding the effects of crosslinking solution concentration on lung epithelial cells embedded in hydrogel. Furthermore, the effects of calcium chloride concentration in crosslinking solutions on other cell types have not been reported regarding whether the addition of viscosity and stiffness tuning agents such as methylcellulose will alter the responses of cells to changes in calcium chloride concentration in crosslinking solutions. While there were no significant effects of calcium chloride concentration on cell viability in alginate–ECM hydrogels, there is a decrease in cell viability in alginate–methylcellulose–ECM hydrogels crosslinked with 300 mM calcium chloride crosslinking solution. These findings have implications in the maintenance of 16HBE14o- 3D cultures with respect to the gelation of alginate with high concentrations of ionic crosslinking solution.

We introduce Group Isolation Gauge Effect Metrics (GIGEM), a high-throughput software suite for Drosophila sleep analysis designed to manage large population datasets from multi-batch experiments. In this study, we characterized social isolation-induced sleep phenotypes across 38 Sleep Inbred Panel (SIP) lines after both acute and chronic social isolation. Beyond this specific study, GIGEM is broadly suited for diverse Drosophila sleep research involving large-scale and multi-batch datasets. GIGEM enabled an extensive analysis of sleep parameters and allowed systematic identification of sleep changes in response to social isolation. Following acute social isolation, SIP line animals showed heterogeneous responses, including sleep loss, sleep gain, and no change in sleep. In contrast, chronic social isolation led to significant sleep loss in nearly all lines, though to varying degrees. While most SIP lines were sensitive to chronic social isolation, a select few were resistant to it. These results were obtained across 38 SIP lines and wild-type controls (totaling 5670 animals). Collectively, our results demonstrate that chronic social isolation-induced sleep loss is a robust yet variable trait and establish GIGEM as a powerful, generalizable tool for Drosophila sleep phenotyping in large-scale, multi-batch studies.