RESUMEN
The aim of this study was to construct a full-thickness artificial cornea substitute in vitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400 µm thickness, 11 mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K + ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea.
Asunto(s)
Órganos Bioartificiales , Córnea/citología , Córnea/crecimiento & desarrollo , Endotelio Corneal/citología , Epitelio Corneal/citología , Células Madre Embrionarias Humanas/citología , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Técnicas de Cocultivo/métodos , Trasplante de Córnea/métodos , Endotelio Corneal/crecimiento & desarrollo , Epitelio Corneal/crecimiento & desarrollo , Células Madre Embrionarias Humanas/fisiología , Humanos , Conejos , Células Madre , Ingeniería de Tejidos/instrumentación , Andamios del TejidoRESUMEN
Utilizando a técnica de RIECKMANN e col., os autores realizaram 20 microtestes de sensibilidade, procurando verificar a capacidade da violeta de genciana em impedir, na cultura "in vitro", o desenvolvimento habitual do Plasmodium falciparum. Os resultados mostraram que houve inibiçäo da evoluçäo do protozoário nas concentraçöes de 1/1000, 1/1500, 1/2000, 1/2500, 1/3000 e 1/4000, significando que nas condiçöes da experiência o corante atuou sobre as formas sanguíneas assexuadas do protozoário. Estas verificaçöes sugerem que a violeta de genciana poderia ser usada na profilaxia da malária transfusional