When the fibroblasts contracted, and no changes were
observed in IECs, digestion was terminated by the addition of
complete DMEM. Fibroblasts were detached with D-Hank’s
buffer, followed by adding medium. Cell clones were obtained
after three passages by screening only a single IEC in each well of
96-well plates where IECs had been diluted to ensure that each
well contained only a single cell. Cell populations were expanded
after the cloning and transferred to mass culture in cell flasks. The
IECs were kept frozen after 8–9 passages in liquid nitrogen.