Introduction: Mosquito cell lines such as SuaE1 (An. gambiae), MSQ43 (An. stephensi), and 4a-2 (An. coluzzii) differ from mammalian cell models in that they achieve immortality through spontaneous immortalization rather than deliberate genetic transformation. The establishment of cell lines benefit from a regimented consistent approach which should allow for consistent repeatable results over time. While this makes them valuable and accessible tools, it may also predispose them to variability that complicates reproducibility. In this project we will be investigating the key corecomponents seen across different cell culture media for mosquito cell lines, and determining which components increase or may decrease the variability and potential dedifferentiation of these cells.

Methods: To investigate this, we continuously cultured these three mosquito cell lines and documented their phenotypes across passages. We imaged each cell line with brightfield microscopy in triplicate over several months. We further evaluated culture media and the lab environment for effects that may have altered cell differentiation in our cultured cell species.

Results: We observed significant, fully penetrant morphological changes in two of our three cell lines (SuaE1: high severity; 4a-2: moderate severity), including stratification, size and shape changes, differences in flask adherence, and cyst-like division. These phenotypes were absent in one line (MSQ-43).

Conclusion/Discussion: These findings suggest that spontaneous immortalization contributes to phenotypic instability between mosquito cell lines, raising important concerns for reproducibility in downstream applications such as transcriptomics, proteomics, and pathogen interaction studies. We also found that different media cell conditions can lead to insufficient supplementation of requirements for cell growth for initial establishment after thawing versus subsequent long-term maintenance growth.