Human tonsillar tissue block cultures differ from autologous tonsillar cell suspension cultures in lymphocyte subset activation and cytokine gene expression.

Lymphoid tissues cultured either as tissue blocks or as cell suspensions are used to study the behaviour of immune cells within their habitat. The preservation of tissue structures in tissue blocks, which is considered to be a major advantage, has been poorly defined. We characterised the morphological evolution of tissue cultures from human palatine tonsils and compared their lymphocyte subsets and the constitutive cytokine gene expression to those in autologous tonsillar single-cell suspension cultures over time, and after adding cyclosporin A (CsA) to mimic the situation in individuals treated with immunosuppressive drugs.

Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were developed for the quantification of expression of the genes for human interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, IL-12, IL-15, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta and for the endogenous reference hydroxymethylbilane synthase (HMBS). The assays detected as little as five plasmid copies and were 100% specific. The creation and integration of a calibration sample into the assays permitted their calibration across experiments.