In this paper, we present a culture of A549 and MRC-5 spheroids in a microfluidic system. The aim of our work was to develop a good lung cancer model for the evaluation of drug cytotoxicity. Our research was focused on determining the progress of cell aggregation depending on such factors as the depth of culture microwells in the microdevices, a different flow rate of the introduced cell suspensions, and the addition of collagen to cell suspensions. We showed that these factors had a significant influence on spheroid formation. It was found that both MRC-5 and A549 cells exhibited higher aggregation in 500 μm microwells. We also noticed that collagen needs to be added to A549 cells to form the spheroids. Optimizing the mentioned parameters allowed us to form 3D lung tissue models in the microfluidic system during the 10-day culture. This study indicates how important an appropriate selection of the specified parameters is (e.g., geometry of the microwells in the microsystem) to obtain the spheroids characterized by high viability in the microfluidic system.