Overcoming obstacles in three-dimensional cell culture model establishment: Approaches for growing A549 non-small cell lung cancer spheroids using a clinostat system

Abstract

Introduction: Non-small cell lung cancer (NSCLC) accounts for 80-85 % of lung cancer cases globally. And the A549 cell line is widely used in pharmacological and toxicity screening. Due to its popularity as a NSCLC model, it was inevitable that three-dimensional (3D) cultures of A549 cells would be established. 3D models increase physiological relevance, and their advanced structure allows researchers to obtain more translatable and reliable results. However, establishing this cell line as a 3D model may come with challenges, like clumping. Methods: In this study, A549 spheroids were established using a clinostat-based rotating bioreactor system and were characterised in terms of morphology, planimetry, and viability. Results: The main challenge faced included continuous aggregation of the spheroids, which constrained growth and development. This challenge was successfully overcome by supplementation with ascorbic acid, foetal bovine serum coating, and minimising handling, and a NSCLC mini-tumour model was established and semi-characterised. The spheroids survived for 25 days and had a significant increase in growth. Conclusion: The A549 spheroid model cultured in a clinostat-based microgravity system was shown to be stable, viable, and suitable to be used in pharmacological and toxicological investigations.