Characterization of cultivated nasal mucosal epithelial cell sheets for ocular surface reconstruction: an in vitro...

To investigate the in vivo and in vitro epithelial phenotypes of cultivated nasal mucosal epithelial cell (CUNMEC) sheets generated by a microspheroidal suspension culture technique.

Human nasal mucosal tissues were obtained during endoscopic dacryocystorhinostomy (DCR). The tissues were digested with 2 mg/mL collagenase A and shaken at 37 °C and 1,200 rpm for 16 h. Afterward, the cell aggregates were seeded onto a de-epithelialized human amniotic membrane (AM) and cultivated for 2 weeks. The CUNMEC sheets were then transplanted into 6 New Zealand albino rabbit (M : F = 3 : 3) corneas after partial or total keratectomy. The animals were subjected to immunosuppression for two weeks and then sacrificed. The samples were subjected to light and electron microscopy (EM) and immunoconfocal microscopy examinations and a BrdU label retention assay.

Following collagenase digestion, the tissue fragments formed cell aggregates of various sizes. The individual microspheres attached strongly to the AM and subsequently spread out and coalesced to form a confluent cell sheet. Staining of the cell sheets revealed positive expression of keratins 3, 4, and 13, and MUC5AC, as well as positive Alcian blue and PAS staining, indicating the presence of goblet cells (GCs). The signals for p63 and BrdU detection were concentrated within the microspheres. The presence of CUNMECs in rabbit eyes was confirmed by anti-human cytoplasmic antibody staining. Keratins 3, 4, and 13 were positively expressed in the suprabasal region, whereas p63 expression was observed only in the basal layer. Transmission EM revealed stratified epithelium with the formation of desmosomes and hemidesmosomes. Scanning EM revealed a cobble stone-like epithelial surface without cilia formation. α-Tubulin, a major component of cilia, was expressed only in the apical region of the nasal mucosa and showed diffuse cytoplasmic staining in the CUNMEC sheets. ZO-1, a component of tight junctions, was expressed in the apical region of the nasal mucosa and was expressed at intercellular borders in CUNMECs.

Following transplantation, the CUNMEC sheet presented a stratified epithelial layer containing GCs but without cilia and expressed progenitor cell markers, which are morphologically similar to those of ocular surface epithelia and may have the potential to function as a surrogate epithelium for ocular surface reconstruction, especially in dry eye conditions.