The application of in vitro isolation, cryopreservation and patch-clamp microelectrode recording methods to adult rat thymic nurse cells

Abstract

Characterisation of the ionic mechanisms possessed by immune cells has begun to reveal a range of transmembrane ion channel properties which may have immunological significance. Since thymic epithelial cells appear to influence selection of the T cell repertoire, understanding their membrane physiology may be of importance. A method is therefore outlined for the targeting of patch-clamp electrophysiological measurements to acutely isolated, fresh and cryopreserved adult rat thymic nurse cells. These cells represent a discrete and predominantly cortical population of epithelium. Cells were separated by enzymatic and mechanical dispersal of thymus, enriched by sedimentation and identified on the basis of their characteristic lympho-epithelial, multi-cellular morphologies. Phase-bright cells retaining this anatomical form survived freezing and the voltage-gated conductances in such cells are described. The merits of this approach include the preparation and storage of mature differentiated phenotypes of immune cells for in vitro studies. Their preservation permits temporal separation of experiments, correlative experimentation on identical samples, optimises cell preparation in terms of cost and labour, and reduces animal and tissue requirements.