A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach.

Cell injection therapy is emerging as an alternative to treat corneal endothelial dysfunction (CED) and to avoid corneal scarring due to bullous keratopathy. However, establishing a standardized culture procedure that provides appropriate cell yield while retaining functional features remains a challenge. Here, we describe a detailed framework obtained from in vitro culture of human corneal endothelial cells (HCECs) and comparative in vivo experimental models for CED treatment with a new cell tracking approach. Two digestion methods were compared regarding HCEC morphology and adhesion. The effect of Y-27632 (ROCKi) supplementation on final cell yield was also assessed. Cell adhesion efficacy with two cell delivery systems (superparamagnetic embedding and cell suspension) was evaluated in an ex vivo human cornea model and in an in vivo rabbit CED model. The injection of supplemented culture medium or balanced salt solution (BSS) was used for the positive and negative controls, respectively. HCEC isolation with collagenase resulted in better morphology and adhesion of cultured HCEC when compared to EDTA. Y-27632 supplementation resulted in a 2.6-fold increase in final cell yield compared to the control. Ex vivo and in vivo adhesion with both cell delivery systems was confirmed by cell tracker fluorescence detection. Corneal edema and opacity improved in both animal groups treated with cultured HCEC. The corneas in the control groups remained opaque. Both HCEC delivery systems seemed comparable as treatments for CED and for the prevention of corneal scarring.

Amniotic membrane with living related corneal limbal/conjunctival allograft for ocular surface reconstruction in Stevens-Johnson syndrome.

OBJECTIVE: To report the use of preserved amniotic membrane and living related corneal limbal/conjunctival transplantation in total limbal stem cell and conjunctival deficiency secondary to severe Stevens-Johnson syndrome. DESIGN: Prospective, noncomparative, interventional case series. PATIENTS AND METHODS: Ten eyes of 10 patients with total limbal stem cell and conjunctival deficiency secondary to Stevens-Johnson syndrome underwent excision of cicatricial tissue followed by amniotic membrane and living related corneal limbal/conjunctival transplantation. MAIN OUTCOME MEASURES: Reconstruction of corneal epithelium (clear appearance without epithelial defect, normal fluorescein permeability, and the absence of conjunctiva-derived goblet cells on impression cytologic testing), decrease in corneal vascularization, and improvement in visual acuity. RESULTS: During a mean follow-up of 16.7 months, satisfactory ocular surface reconstruction was obtained in 2 eyes (20%), with reduced inflammation and vascularization and a mean epithelialization time of 3 weeks. Surgical failure was observed in 4 cases (40%) and complications (infection) in 4 cases (40%). Visual acuity improved in 4 eyes (40%), remained stable in 5 eyes (50%), and decreased in 1 eye (10%). CONCLUSIONS: Amniotic membrane and living related corneal limbal/conjunctival transplantation were successful in 20% of severe cases of total limbal stem cell and conjunctival deficiency secondary to Stevens-Johnson syndrome. A high proportion of postoperative complications, in particular, infection, seemed to jeopardize a favorable outcome.

Imunofluorescência direta no diagnóstico de casos suspeitos de penfigóide cicatricial ocular / Direct immunofluorescence investigation in patients with clinical suspicion of ocular cicatricial pemphigoid

Objetivo: O objetivo deste estudo é caracterizar por meio do exame oftalmológico os pacientes com suspeita clínica de penfigóide cicatricial ocular e apresentar os resultados obtidos com a técnica de imunofluorescência direta. Métodos: Estudo prospectivo realizado no setor de Córnea e Doenças Externas da Universidade Federal de São Paulo. Foram examinados 18 pacientes com suspeita clínica de penfigóide cicatricial ocular e realizadas biópsias de conjuntiva para o processamento da imunofluorescência direta em 13 pacientes (26 olhos), que não estavam na vigência de imunossupressão sistêmica ou terapêutica antiglaucomatosa tópica. Resultados: Segundo o estadiamento de Foster, a proporção de olhos classificados de I a IV foram: 3,8 por cento, 3,8 por cento, 15,4 por cento respectivamente. Apenas 3 (23 por cento) pacientes apresentaram imunofluorescência direta positiva. Conclusão: A maioria dos pacientes encontravam-se em estágio avançado da doença, o que denota retardo no diagnóstico. A imunofluorescência direta apresentou baixa sensibilidade em detectar depósitos de imunocomplexos na membrana basal da conjuntiva, quando comparada aos dados da literatura.