Biopsy harvesting site and distance from the explant affect conjunctival epithelial phenotype ex vivo

Abstract

The purpose of the study was to investigate if the number of goblet cells expanded ex vivo from a conjunctival explant is affected by the biopsy harvesting site on the conjunctiva and the distance from the explant. Conjunctival explants from six regions: superior and inferior bulbus, fornix, and tarsus of male Sprague–Dawley rats were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for eight days. Histochemical and immunofluorescent staining of goblet (CK-7/UEA-1/1MUC5AC), stratified squamous, non-goblet (CK-4), proliferating (PCNA) and progenitor (ABCG2) cells were analyzed by epifluorescence and laser confocal microscopy. Outgrowth was measured with NIH ImageJ. For statistical analysis the Mann–Whitney test and Spearman’s rank–order correlation test were used. Cultures from superior and inferior fornix contained the most goblet cells as indicated by the presence of CK-7+, UEA-1+ and MUC5AC+ cells. Superior and inferior forniceal cultures displayed 60.8% ± 9.2% and 64.7% ± 6.7% CK-7+ cells, respectively, compared to the superior tarsal (26.6% ± 8.4%; P < 0.05), superior bulbar (31.0% ± 4.0%; P < 0.05), inferior bulbar (38.5% ± 9.3%; P < 0.05) and inferior tarsal cultures (27.7% ± 8.3%; P < 0.05). While 28.4% ± 6.3% of CK-7+ goblet cells co-labeled with PCNA, only 7.4% ± 1.6% of UEA-1 + goblet cells did (P < 0.01). CK-7+ goblet cells were located at a lower concentration close to the explant (39.8% ± 3.1%) compared to near the leading edge (58.2% ± 4.5%; P < 0.05). Both markers for goblet cell secretory product (UEA-1 and MUC5AC), however, displayed the opposite pattern with a higher percentage of positive cells close to the explant than near the leading edge (P < 0.05). The percentage of CK-4+ cells was higher near the explant compared to near the leading edge (P < 0.01). The percentage of CK-7+ goblet cells in the cultures did not correlate with the outgrowth size (rs = −0.086; P = 0.435). The percentage of UEA-1 + goblet cells correlated negatively with outgrowth size (rs = −0.347; P < 0.01), whereas the percentage of CK-4+ cells correlated positively with the outgrowth size (rs = 0.473; P < 0.05). We conclude that forniceal explants yield the highest number of goblet cells ex vivo and thereby seem to be optimal for goblet cell transplantation. We also suggest that CK-7+/UEA-1− cells represent highly proliferative immature goblet cells. These cells could be important during conjunctival migration as they are mostly located close to the leading edge and their density does not decrease with increasing outgrowth size.

Molecular layer deposition builds biocompatible substrates for epithelial cells

“…In the He et al's study, we characterized the cells using the goblet cell‐specific cytokeratin (CK) 7 compared to the stratified squamous cell‐specific CK4 . In a previous study, we found that CK7 was the best way to identify goblet cells in actively proliferating and migrating cultures as goblet cells lose their secretory product including MUC5AC as they migrate and proliferate. Based on our previous work, we are confident that the cells growing on the MLD substrates are conjunctival epithelial cells that are most likely goblet cells.…”

“…In cases of conjunctival epithelial stem cell deficiency, for example, following removal of extensive conjunctival tumors or chemical burns, cultured conjunctival transplants containing goblet cells can be used to restore the conjunctiva. As MLD substrates support the growth of goblet cells, this technique may potentially be used for production of goblet cell transplants …”

“…The method for culturing conjunctival goblet cells in this study has been reported previously by the research group. [42][43][44] In this study, the phenotype of cells cultured from rat conjunctiva was investigated by RT-PCR and immunofluorescence microscopy. For RT-PCR analysis, cells were pooled from six explant cultures from one animal.…”

“…Goblet cells are one of the two main types of epithelial cells present in the conjunctival epithelium, which covers the posterior surface of the eyelids and the anterior surface of the globe (except the cornea). A reduction in amount of goblet cells is associated with tear film instability and eventually in severe ocular surface disease when these cells are lost . In some cases where the conjunctiva is lost or severely damaged, transplantation of cultured conjunctiva containing goblet cells can restore ocular surface homeostasis …”

“…A reduction in amount of goblet cells is associated with tear film instability and eventually in severe ocular surface disease when these cells are lost. 37,38 In some cases where the conjunctiva is lost or severely damaged, transplantation of cultured conjunctiva containing goblet cells can restore ocular surface homeostasis. 39,40 In this study, to assess potential bioactivity of MLD substrates, we investigated phenotype, attachment, proliferation, and viability of rat conjunctival goblet cells cultured on substrates made by MLD compared to that of cells grown on uncoated coverslips, the positive control.…”