Supplementary Material for: Establishment of cell cultures from the cavefish Astyanax mexicanus: a resource for in vitro studies of supernumerary B chromosome biology

Introduction
Supernumerary B chromosomes have been extensively investigated using diverse in vivo approaches, revealing insights into their origin, nature, evolutionary dynamics, maintenance mechanisms, and effects on their carriers. Despite its broad applicability across various biological research fields, in vitro cell culture remains underexplored as a tool for studying B chromosome biology, with studies limited to using cell cultures only as a source of chromosome preparations.
Methods
In the present study, cell cultures of the fish Astyanax mexicanus were established, with (AMEcfB) and without (AMEcf) the B chromosome, using caudal fin tissue collected through non-lethal procedures. These cultures were compared in terms of cell proliferation and in response to environmental stress (pH and temperature) using the MTT and RT-qPCR assay.
Results
The AMEcf and AMEcfB cell lines exhibited karyotypic stability and high proliferative potential, demonstrating their suitability for diverse scientific applications. The B chromosome, found exclusively in a subpopulation of AMEcfB cells, influenced cell physiology by affecting cell division dynamics. Experiments under extreme pH and temperature conditions revealed differences in cell viability and gene expression between the two lines, highlighting the role of the B chromosome in modulating environmental responses.
Conclusion
These findings align with previous reports on the effects of B chromosomes in living organisms. Thus, the A. mexicanus cell cultures established here represent a promising resource for investigations into B chromosome biology, offering significant ethical, economic, and methodological advantages.