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J. Eidet, I. G. Fostad, M. Shatos, T. P. Utheim, O. A. Utheim, S. Raeder, D. A. Dartt; Effect of Biopsy Location and Size on Phenotype and Proliferative Ability of Cultured Rat Conjunctival Tissue. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1912.
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To evaluate the effect of location and size of biopsy on phenotype and proliferative ability of cultured rat conjunctival tissue.
Pieces of conjunctiva were used from six areas: superior and inferior bulbus, fornix, and tarsus of male Sprague-Dawley rats (n=17). Explants were grown in RPMI 1640 with 10 % serum on coverslips for 8 days or assayed for colony forming efficiency (CFE). Analysis included histochemical and immunofluorescence microscopy. Outgrowth was analyzed using ImageJ software. The following antibodies were used: CK7 (goblet cell body), UEA-1 (goblet cell secretory product), PCNA (proliferating cells), and ABCG2 (undifferentiated cells). For statistical analysis the Mann-Whitney test and simple linear regression were used.
Forniceal tissue yielded the largest outgrowth with respect to explant size (21.9-23.9 fold increase, p < 0.01). Division of large explants into multiple smaller pieces correlated with increased total sum of outgrowth area (r = 0.38, p < 0.001). CFE was higher for cells from superior fornix compared to cells in the bulbar areas, with 3.6 % and 1.1-1.6 % colonies formed, respectively (p < 0.05). Forniceal cultures had more goblet cells compared with cultures from other areas with 60.8-64.7 % being CK7+ (p < 0.05). 28.4 % ± 6.3 % of CK7+ goblet cells co-labeled with PCNA, while only 7.4 % ± 1.6 % of UEA-1+ goblet cells did (p < 0.01). CK7+ goblet cells, but not UEA-1+ goblet cells, occasionally co-labeled with ABCG2.
Forniceal conjunctiva divided into small pieces appears to be the most successful for transplantations with substantial numbers of goblet cells. CK7+/UEA-1- cells appear to have the most proliferative potential for goblet cell transplantation.
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