The Development of Three-Dimensional Lung Multicellular Spheroids in Air and Liquid Interface Culture for the Evaluation of Anti-Cancer Therapeutics

The translation 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. The utilization of three-dimensional (3D) cell culture in Air Interface Culture (AIC) conditions creates an extremely beneficial model of lung cancer tumors via the development of multicellular spheroids (MCS). This study investigated a new 3D cell culture technique to model lung tumors in vitro.  The first step in the creation of this model was to optimize 3D culture by applying collagen (a semi-non-adhesive material) to a Transwell in a 12-well plate. Lung cancer cells were seeded (H358, bronchioalveolar carcinoma, type II alveolar cells and A549, lung adenocarcinoma, type II alveolar cells) on the collagen. The semi-non-adhesive collagen allowed the cells to preferentially attach to one another rather than to the surface, thus creating MCS. To better mimic the conditions of lung cancer specifically, the next step was to create AIC as opposed to the commonly used Liquid Covered Conditions (LCC). Once the cells attached to the collagen, the cell media on the apical side of the Transwell was removed. 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. Through the use of brightfield microscopy imaging and Nikon Software area measurement of the MCS that formed in both AIC and LCC conditions, the AIC model proved to yield healthy MCS at sizes similar to MCS formed in LCC conditions (100-200μm).

Once the 3D model was optimized, a comparison of 2D and 3D cell behavior and viability was completed using the common anti-cancer therapeutic paclitaxel and aerosol particles containing paclitaxel as a representative model for drug delivery. LCC spheroids were exposed to paclitaxel in media, and dry powder aerosolized lipospheres containing paclitaxel were applied to AIC spheroids using an insufflator in order to create a model for direct pulmonary drug delivery with a dry powder inhaler. The lipospheres containing paclitaxel were comprised of a PEGylated phospholipid excipient mixture which encapsulated the drug. Viability analysis demonstrated different drug efficacy when comparing the 2D and 3D systems.  Specifically, 2D spheroids were more sensitive to paclitaxel treatment as indicated by lower IC50 values for both cell lines. Transepithelial electrical resistance (TEER) was measured with an electrode across a monolayer of another lung cancer cell line Calu-3 (lung adenocarcinoma) to illustrate that the culture conditions as well as particle application did not affect the permeability of the cell monolayer, which indicated that this form of drug therapy would not affect the permeability of lung tissue. Overall a much more representative in vitro model has proved a largely beneficial 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.