Figure 1. Comparison of lung airway morphogenesis in 3D culture.

(A) Phase contrast image of monocultured NHBE cells at day 8. NHBE cells formed spherical colonies, but no branching morphogenesis occurred. (B) Phase contrast image of NHBE cells co-cultured with HUVECs. No branching morphogenesis occurred, but the growth rate of NHBE cells was slightly higher than controls. (C) Phase contrast image of NHBE cells co-cultured with MSCs. No branching morphogenesis occurred. The size of the NHBE cells is double that of the control case at day 8. (D) Phase contrast image of NHBE cell clots in Matrigel at day 1, day 4, and day 8. The clot consisted of 2 × 10⁵ cells at day 1. The branches grew out from the clot at day 4, and the growth rate was much faster than the cultures that were homogeneously distributed. At day 8, the maximum length of branches reached 700 μm and number of branches from the clot was increased. (E) Higher magnification image of branches at day 8. Secondary branching was observed. (F) Quantitation of cell growth rate. Co-culture of NHBE cells increased the growth rate, but the cells grew much faster in NHBE cell clots. The size of branches from cell clots at day 3 were 3 times higher than the cell sizes cultured homogeneously (p = 0.027). Error bars indicate standard deviation (n = 20). Scale bars 100 μm (A–E).