Towards a defined, serum- and feeder-free culture of stratified human oral mucosal epithelium for ocular surface reconstruction.

PURPOSE: Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. METHODS: In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration. RESULTS: In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. CONCLUSION: In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.

Immunohistochemical staining of estrogen and progesterone receptors: aspects for evaluating positivity and defining the cutpoints.

BACKGROUND: Estrogen (ER) and progesterone (PgR) receptors are predictive and prognostic factors in breast cancer. The most suitable immunohistochemical cutpoints for dividing the tumors in hormone receptor-negatives and -positives may, however, need more consideration. We examined the association between breast cancer survival and cutpoints assessed by four different models. We looked for evidence for which patient subgroups could be handled best through applying different cutpoints. MATERIALS AND METHODS: Three hundred and twenty-four samples of invasive breast cancer were immunohistochemically stained for ER and PgR, bcl-2 and erbB2. Fractions of ER- and PgR-positive cells and also ER and PgR staining scores were assessed. The fractions of stained cells and staining scores, respectively, were determined on the whole section area, and the area of most intense staining. Candidate cutpoints, dividing the patients into good and poor prognosis groups, were tested among all patients group, N+ and N- groups, premenopausal and postmenopausal patient groups. The correlation between immunohistochemistry results of ER, PgR, bcl-2 and erbB2 as well as SMI (standardized mitotic index), patient age, tumor size and axillary lymph node status were tested. RESULTS: The ER score was correlated with age, SMI and bcl-2 positivity. The PgR score was correlated with erbB2 and bcl-2. Lobular carcinomas had higher staining scores of ER and PgR than ductal carcinomas. CONCLUSION: In this material, ER was correlated with factors reflecting the differentiation of the tumor. On the basis of the ER and PgR immunohistochemistry cutpoint analysis, we found that the optimal cutpoints in different patient groups may not be the same.