(394) Development of a Programmable Automated Cell Culture System to Mimic Cystic Fibrosis-Related Diabetes Conditions In Vitro

Background: The most common comorbidity that affects cystic fibrosis (CF) patients is cystic fibrosis-related diabetes (CFRD). CFRD patients experience approximately a six-fold higher rate of lung function decline compared to their non-diabetic CF counterparts. Despite its severe clinical impact, the mechanism that drives accelerated CFRD lung function decline is still unknown. Thus, it is essential to identify the pathway(s) underlying CFRD pathophysiology and its connection to lung disease to improve patient care. The first challenge to be addressed is the fact that current in vitro cell culture models do not resemble CFRD-like conditions. Cells in vivo experience fluctuations of glucose levels after every meal. In contrast, cells cultured in vitro receive media with a very high initial glucose concentration which gets progressively depleted until the next media change (after multiple days). To address this need, we manufactured a programmable and automated cell culture system (PACCS) capable of changing cell culture media multiple times a day to simulate changes in blood glucose after meals.

Methods: As a proof-of-concept experiment to validate the PACCS system, CF human bronchial epithelia expressing WT CFTR (WT-CFBE) were plated in Corning 3460 Transwells at a density of 100,000 cells per well and cultured at liquid-liquid interface for a day. Transwells were transferred to the PACCS system the next day and cultured at air-liquid interface for 3 days with daily media changes performed by PACCS. Cells cultured manually were used as a control. PACCS relies on pinch valves for fast opening and closing of solution lines. Media changes are scheduled using a commercially available controller. Further, we designed and 3D printed a cell culture plate capable of holding multiple Transwells in place while allowing multiple media changes. The plate was 3D printed with a biocompatible polylactic acid (PLA) material and used for media exchange experiments. For simplicity, we cultured cells with 5.5 mM (100 mg/dL) glucose media to simulate normal glycemic control. Cells cultured by hand received the same media at the same time. As an end-point measurement, all samples were evaluated by Ussing chamber recordings. Electrophysiology properties such as transepithelial electrical resistance (TEER) and current from CFTR were recorded and compared between culture conditions.

Results: Results show comparable TEER and CFTR current between the two conditions. Overall, WT-CFBE cells cultured with PACCS behaved similarly to cells cultured by hand. Moving forward, we are using the same set-up to program multiple media changes per day with different glucose concentration inputs to simulate meals. The meal-like glucose fluctuations will better mimic in vivo conditions.

Conclusions: In conclusion, preliminary data show that PACCS can be successfully used for cell culture to simulate a more physiologically relevant CFRD-like cell culture approach. Improving our model systems in vitro would allow us to better understand the effects of chronic and acute hyperglycemia in airway cells and will bring us one step closer to understanding the connection between CFRD and accelerated CF lung disease.

Acknowledgements: This work was supported by the Marcus Professorship in CF, and the CF Foundation (MCCART21G0).

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