Figure 6
Human buccal cells. Fibroblasts underwent automated digestion and were plated onto fibronectin coated tissue culture plasticware for 3 days before media was changed to DMEM + HPL for further passages. Fibroblasts underwent MMC treatment for 0, 2, 4 and 6 h. 2 h stops cell proliferation without promoting apoptosis (a) n = 1. The epithelial cells underwent automated digestion and were plated onto a growth arrested human fibroblast feeder layer in HPL + Y-27632 media. At passage 3 they were seeded into a non-contact co-culture with human growth arrested fibroblast feeder layer. They underwent submerged culture for 3 days and were lifted to ALI for 14 days to allow differentiation. Phase image shows cells at p0 (b) H &E (c), E-cadherin staining (d) and cytokeratin 4 (green) found in intermediate and differentiated cells and Basal cell mark integrin β4 (red) (e) when differentiated at ALI. Population doublings (f), population doubling time (g), viability (h) and cell number (i), up to passage 3 were recorded, Scale bar shows 200 µm and 100 µm, n = 2.
