39] Preparation of inside-out thyroid follicles for studies on transcellular transport (transcytosis)

Abstract

This chapter describes the preparation of inside-out thyroid follicles for studies on transcellular transport also referred to as “transcytosis.” Studies on the mechanism of transepithelial passage of thyroglobulin can be facilitated by using an in vitro system where access to the apical plasma membrane is possible. Such a system is provided by preparations of inside-out follicles from pig thyroid gland. The chapter describes the conditions that lead to the formation of inside-out follicles and allows studying the transepithelial vesicular traffic from the apical to the basolateral cell surfaces. Collagenase digestion of thyroid tissue and application of shearing forces by pipetting result in the dissociation of tissue into intact segments of follicles opened during pipetting. Fibroblasts and parafollicular and endothelial cells are separated from follicle segments by low-speed differential centrifugation. Purified follicle segments form closed follicular spheres with a reversed polarity within 24 hr of suspension culture. The polarity of all follicles in suspension is reversed so that the apical plasma membrane is oriented toward the culture medium, and the basal cell surface is directed toward the central cavity.