From Monolayer to Spheroid: Assessing Influenza a Virus Infection in 2D and 3D Cell Culture of A549 and Hek293 Publisher Pubmed



Shokouhi Targhi H ; Gholizadeh F ; Sokhandan H ; Mollapoursisakht MM ; Mehrbod P
Source: Virology Journal Published:2025

Abstract

Background and aim: Three-dimensional (3D) culture models better mimic cell-to-cell interactions compared to traditional two-dimensional (2D) cultures, providing more physiologically relevant alternative for virus infection studies. This study aimed to explore the effectiveness of 3D culture models for studying viral propagation using A549 and HEK293 cell lines in spheroid form with two different matrices: alginate (Alg) and a combination of alginate with methylcellulose (Alg + MC). Methods: The 3D cultures of A549 and HEK293 cells were generated in 2 matrices. The cultures were characterized by proliferation assay and size assessment. The matrices were further analyzed by scanning electron microscopy (SEM) and immunofluorescence microscopy. Influenza A virus/PR/8/34 (H1N1) was propagated in MDCK cell and virus infectious dose was determined. A549 and HEK293 cells were grown in 2D form and virus was adapted to these 2 cell lines in serial passages. The best yields were inoculated to 2D and 3D forms. The supernatants and cells were collected in 48 h and subjected to qPCR to determine and compare the virus propagation in 2D and 3D formats. Results: Spheroids derived from A549 and HEK293 cell lines were successfully developed in 2 different matrices and characterization confirmed assembly of the cells together with considerable growth rate and viability. In case of HEK293, in dissolved patterns, external supernatant of Alg + MC and in undissolved ones, external supernatant in Alg and external and internal supernatants in Alg + MC showed the lowest decrement in viral load. Regarding A549, among dissolved ones, internal supernatants in Alg and Alg + MC and in undissolved samples, external and internal supernatants in Alg and internal supernatants in Alg + MC showed the least reduction. In both cell samples reduction was observed in all matrices, which was significant in A549 cell (P<0.05). Conclusion: We conclude that Alg + MC matrix, with its increased porosity and lower cohesion compared to Alg alone, was easier to dissolve but more difficult to re-solidify. One possible explanation for the observed higher viral replication in this matrix is that it may have facilitated improved viral access to the cells. Future modifications that increase virus–cell interaction time in this system could further enhance infection efficiency. © 2025 Elsevier B.V., All rights reserved.