Figure 1.
Apical-basal polarities of epithelial cells in 2-D or 3-D culture.
(A) Polarized epithelial cells in a 2-D sheet. Cells are on extracellular matrix (ECM, orange) coated artificially or deposited by the cells themselves. Plasma membranes facing the ECM or adjacent cells are called basolateral membranes (red). The remaining membrane areas are called apical membranes (green). Apical junctions (blue) are formed at the border between basolateral and apical membranes. (B) Polarized epithelial cells forming a spheroid in the ECM gel. Basolateral membranes are formed on the outside surface of the spheroid facing the ECM. Apical membranes are formed inside the spheroid. (C) Polarized epithelial cells forming a spheroid in suspension culture. Concentration of the ECM deposited by the cells themselves appears higher within the spheroid. Apical membranes are formed on the outside surface of the spheroid facing the culture medium. Basolateral membranes are formed on inside the spheroid.
Figure 2.
Apical-basal polarities of epithelial cells in 2-D sheets.
Confluent MDCK II, EpH4, and R2/7 α-cate cells cultured on coverslips without ECM-coating were stained for ZO-1, Scrib, and PKC zeta, shown in blue, red and green, respectively in the merged images (the most right). In black-and-white images, samples were focused at the level of the apical junctions. In the merged images, series of confocal optical sections were reconstructed to show horizontal images (center) and vertical images (right and bottom). Directions of the arrows indicate from basal to apical. In all cases, PKC zeta and Scrib accumulated at apical membranes and basolateral membranes, respectively. ZO-1 was localized exclusively to the border between the two membranes, indicating the apical junction regions (yellow arrowheads). The combination of antibodies to these proteins clearly reveals similar polarization of epithelial cells of different species and origins. Bar, 10 µm.
Figure 3.
Apical-basal polarity and lumen formation of epithelial spheroids in Matrigel.
Spheroids of epithelial cells after culturing for 2 days (EpH4 cells) or 3 days (MDCK II and R2/7 α-Cate cells) in Matrigel. They were stained for DNA (yellow), ZO-1 (blue), Scrib (red), and PKC zeta (green). Series of confocal optical sections were obtained and projected on a single plane. In MDCK II cells, single lumens lined with PKC zeta (yellow arrowhead) and enclosed by a ZO-1 network (yellow arrow) were observed. EpH4 cells typically had no lumen and ZO-1 was distributed on the surface of the spheroid (yellow arrow). R2/7 α-Cate cells had several small lumens inside the spheroid (yellow arrows and arrowheads). Bar, 10 µm.
Figure 4.
Apical-basal polarity and lumen formation of epithelial spheroids in collagen gels.
Spheroids of MDCK II, EpH4, and R2/7 α-cate cells after 3-day culture in type I collagen gels. Cells were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells, single lumens lined with PKC zeta (yellow arrowhead) and enclosed by ZO-1 network (yellow arrow) were observed. EpH4 cells had no lumen, and the ZO-1 network (yellow arrow) was distributed on the surface of the spheroid. PKC zeta also accumulated at the surface of the spheroid (yellow arrowhead). R2/7 α-Cate cells had several small lumens inside the spheroid (yellow arrows and arrowheads). Bar, 10 µm.
Figure 5.
Apical-basal polarity formation of epithelial spheroids in hanging drops.
Spheroids of MDCK II, EpH4 and R2/7 α-Cate cells after 1.5–2 day culture in hanging drops. In this case, the ECM deposited by those cells is considered to be concentrated within the spheroids. Spheroids were stained for DNA (yellow), ZO-1 (blue), Scrib (red) and PKC zeta (green). Series of confocal optical sections were obtained and projected onto a single plane. In MDCK II cells PKC zeta accumulated on the surface of the spheroid (yellow arrowhead). A ZO-1 network was also found at the surface (yellow arrow). The epithelial cell polarity is opposite to that of cells in the ECM gels. EpH4 and R2/7 α-Cate cells showed similar protein distributions, and also showed similar polarities. Bar, 10 µm.
Figure 6.
Quantitation of apical-basal polarity and lumen formation in epithelial spheroids.
Polarity and lumen formation of spheroids cultured under different conditions shown in Figs. 3–5 was quantified by microscopic observation. “TJ outside” means a spheroid with apical membranes connected with tight junctions facing the outside (see Fig. 1C). “Single lumen” means a spheroid with basolateral membrane facing the outside, apical membrane inside, and only one lumen inside (see Fig. 1B). “Multiple lumens” means a spheroid with basolateral membrane facing the outside, apical membrane inside, and several lumens inside. (A) Cells were cultured in Matrigel for 3 days with the exception of Eph4 cells which were cultured for 2 days. While MDCK II and R2/7 α-Cate spheroids show the same polarity (apical inside), MDCK II spheroids typically have a single lumen whereas R2/7 α-Cate spheroids have multiple lumens. Most of the EpH4 spheroids showed opposite polarity. n = 442, 409 and 570 for MDCK II, EpH4 and R2/7 α-Cate spheroids, respectively, from 3–4 independent experiments. (B) Cells were cultured in collagen gel for 3 days. The results were similar to those cultured in Matrigel. n = 339, 280 and 425 for MDCK II, EpH4 and R2/7 α-Cate spheroids, respectively, from independent 3–4 experiments. (C) Cells were cultured in hanging drops for 1.5–2 days. Spheroids of all cell types showed the same polarity (apical outside). n = 387, 153 and 378 for MDCK II, EpH4 and R2/7 α-Cate spheroids, respectively, from independent 3 experiments.
Figure 7.
Reversal of apical-basal polarity of MDCK II spheroids.
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.
Figure 8.
Spheroid formation in Lipidure-coated plates.
Cells were cultured in non-adherent Lipidure-coated 96-well U-bottom plates for time-lapse recording over 24 hrs. MDCK II cells attached together quickly to form several spheroids, each consisting of a small number of cells. The spheroids themselves did not fuse together, remaining separate from each other (white arrowheads). EpH4 cells did not attach together quickly, and tended to form a single spheroid. R2/7 α-Cate cells attached together quickly and tended to form a single spheroid. Bar, 100 µm.