The implementation of in vitro results to in vivo applications has limitations due to conventional two-dimensional (2D) in vitro conditions lacking the ability to create a physiologically representative model. This study investigated a three-dimensional (3D) cell culture technique to model lung tumors in vitro. A 3D lung cancer model was created by applying collagen (a semi-non-adhesive material) to a transwell, which allowed for nutrient transfer through the collagen. Two lung cancer cells lines (H358, a bronchioalveolar carcinoma and A549, a lung adenocarcinoma) were seeded on top of the collagen. The non-adhesive collagen allowed the cells to preferentially attach to one another rather than to the surface, thus creating multicellular spheroids (MCS). To better mimic the environment for lung cancer specifically, an air-interface culture (AIC) as opposed to the commonly used liquid-covered culture (LCC) was created. For AIC conditions, the cell media on the apical side of the Transwell was removed and the basolateral side of the well was filled with cell media to allow for effective nutrient transport to the cells while also exposing the cells to air. A comparison of 2D and 3D cell behavior and viability was completed using paclitaxel and aerosol particles containing paclitaxel as a representative model for drug delivery. As evidenced with brightfield and fluorescent microscopy imaging, the AIC model proved to yield viable MCS at sizes similar to MCS formed in LCC conditions (100 to 200μm in diameter). With the optimized 3D model, LCC cells were exposed to paclitaxel in media. In another drug delivery method, paclitaxel-loaded dry powder aerosol particles were delivered to AIC cells through direct application with an insufflator. This alternative delivery method using direct delivery of dry powder particles was be used as the drug application method on AIC since paclitaxel cannot be delivered through media as in LCC conditions. The nanoparticles containing paclitaxel are comprised of a PEGylated phospholipid excipient mixture which encapsulates the drug. Using viability analysis, it was shown that the applications of paclitaxel in LCC conditions show variance in efficacy when comparing 2D and 3D culture conditions (where the IC50 values for paclitaxel were higher for 3D compared to 2D). Transepithelial electrical resistance (TEER) across Calu-3 (another lung adenocarcinoma cell line) monolayers was evaluated before and after particle delivery to illustrate that the particle application does not affect the permeability of the cells, which indicated that this form of drug therapy will not affect the permeability of lung tissue. Overall, a much more representative in vitro model has been developed that is expected to be an improved predictor of efficacy of alternative drug delivery methods such as direct pulmonary delivery for lung cancer, which could lead to more efficient drug therapies for lung cancer patients.

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