Reversal of apical-basal polarity of MDCK II spheroids.

<p>MDCK II spheroids cultured in hanging drops for 1.5–2 days were collected and further cultured in Matrigel. Spheroids were stained for ZO-1 (blue), Scrib (red), and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. On day 0 just before starting culture in Matrigel, spheroids showed polarity, with the apical membrane facing the outside. Spheroids cultured for 2 days in Matrigel had several lumens inside associated with PKC zeta (yellow arrowheads) and ZO-1 accumulation (yellow arrows), indicating that the reversal of epithelial apical-basal polarity is taking place. Scrib began to be redistributed to the outside surface of the spheroids (orange arrowheads). Spheroids cultured for 4 days in Matrigel showed fusion of multiple lumens to form a single lumen (yellow arrow and yellow arrowhead). Spheroids cultured for 7 days in Matrigel showed an expanded single lumen associated with PKC zeta (yellow arrowhead) and ZO-1 network (yellow arrow). The epithelial apical-basal polarity has been completely reversed. Bar, 20 µm.