Dissociation method influences immune cell composition in porcine tonsil cell suspensions

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.

As a first step toward improving our understanding of tonsil cellularity and enabling advanced in vitro models, we evaluated how different tissue dissociation methods affect the relative frequencies of both, abundant and rare innate and adaptive immune cell subsets from swine tonsils. We investigated the influence of mechanical vs. enzymatic dissociation methods on the isolation of tonsillar leukocytes, particularly those involved in the GC reaction: T follicular helper cells (Tfh), germinal center (GC) B cells and plasma cells, as well as myeloid subsets.

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.

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.