394 Development of a programmable automated cell culture system to mimic cystic fibrosis-related diabetes conditions in vitro

chamber analysis and investigated changes in messenger ribonucleic acid (mRNA) and protein expression.Results: Results from our Ussing chamber experiments show that hyperglycemia mildly decreased transepithelial electrical resistance (TEER) of WT and ΔF508 CFTR cells, although only ΔF508 CFTR cells had a significant decrease in TEER when also challenged with lipopolysaccharide.Differential expression analysis of the subset of tight-junction proteins included in our study showed, for example, that the mRNA transcript level of claudin-4 was downregulated in WT and ΔF508 CFTR cells cultured under hyperglycemic conditions.Analysis of claudin-4 expression using immunostaining also showed downregulation of this tight-junction protein in cells cultured under hyperglycemic conditions.Overall, our preliminary data show that mammalian airway cells experience gene expression changes in response to hyperglycemia.The importance of cell culture conditions when conducting in vitro research was particularly evident with the ΔF508 CFTR cells, which were less capable of forming tight junctions, even when using normal glucose media.This was found to be consistent even with new vials of cells.Troubleshooting efforts suggested that thawing these cells in Dulbecco's modified eagle medium (DMEM) rather than minimum essential media (MEM) might be a solution to this problem.After the initial thaw, we cultured cells in DMEM for 2 weeks before switching them back to the recommended MEM, which led to consistently greater TEER.We are now comparing gene expression of cells thawed in MEM with that of cells thawed in DMEM to determine mechanisms by which culture conditions may control tight junction function.Conclusions: This work aims to identify novel pathways that play a role in CFRD pathophysiology and inform therapeutic development to mitigate CF lung disease progression exacerbated by CFRD, emphasizing the importance of in vitro culture conditions.