Dissociation method influences immune cell composition in porcine tonsil cell suspensions.

Introduction: Porcine tonsils are follicle-rich secondary lymphoid organs positioned at the oropharyngeal interface. In pigs, a major livestock species, they serve as a primary barrier against inhaled and ingested pathogens and play a central role in mucosal immunity. Despite their importance as a point of entry and as a reservoir for numerous pathogens, the cellular landscape driving tonsillar immune responses in pigs remains insufficiently defined. Methods: As a first step toward improving our understanding of tonsil cellularity and enabling advanced Results: Mechanical dissociation increased total cell yield by 27%, while enzymatic digestion improved cell viability by 21%. During short-term storage immune cell frequencies decreased over time, with mechanical dissociation detecting higher cell numbers and enzymatic digestion providing higher viability. Mechanical isolation improved detection of CD172α+ myeloid cells and Blimp-1+IRF-4+ plasma B cells, whereas enzymatic digestion increased relative frequencies of CD79α+ B cells and ICOS+Bcl6+ T follicular helper cells (Tfh). Surprisingly, enzymatically isolated cells showed impaired cytokine production compared to mechanically isolated cells and presented reduced viability in 3D hanging drop cultures. Discussion: These findings indicate that mechanically isolated tonsillar cells may be preferentially suited for spheroid cultures. However, to establish a robust 3D culture system modelling GC reaction, further optimization is needed. Our study highlights the importance of tailoring tissue dissociation strategy to specific experimental endpoints.