Isolation and Immortalisation of Tonsil Keratinocytes for Three-Dimensional Tissue Engineered Models

Objectives: The ability to isolate and culture primary cells is important for the in vitro investigation of specific tissues and diseases as these are more representative than cancer-derived cell lines. This study aimed to isolate primary keratinocytes from tonsils removed during routine tonsillectomies (ethics ref 09/H1308/66) using enzymatic digestion and use these to create functional tissue-engineered tonsillar tissue.
Methods: The efficiency of cell isolation using two different enzymes (trypsin and dispase) was compared. The growth of primary tonsil keratinocytes was measured and compared to cells cultured in the presence of the Rho kinase inhibitor Y27632. Keratinocytes were used to develop a tissue-engineered model of tonsil epithelium using primary tonsil fibroblasts and de-epithelialized dermis and these models were then incubated with Streptococcus pyogenes to model tonsillitis.
Results: Enzymatic digestion of tonsillar tissue with trypsin resulted in the isolation of significantly more keratinocytes compared to dispase isolation. Keratinocytes cultured without the Rho kinase inhibitor Y27632 survived in culture for less than 10 population doublings whereas cells cultured in the presence of this inhibitor grew for over 30 population doublings without changing their phenotype. Tonsil keratinocytes and fibroblasts cultured in three dimensions produced a multi-layered differentiated epithelium that histologically resembled the surface epithelium of normal tonsils and responded to S. pyogenes by increasing expression of pro-inflammatory cytokines.
Conclusions: Tonsil keratinocytes can be successfully isolated and cultured in vitro. Y27632 was able to markedly prolong the life span of keratinocytes without any deleterious consequences to the cell phenotype making these cells useful for a number of applications that require longer term culture. A functional tissue engineered model of tonsil epithelium was generated which will provide a useful tool for studying cells in a more physiologically relevant way.