To generate in vitro lens tissues with normal structural and functional features, we modified the decades-old rat lens explant culture system.
29 During lens morphogenesis, cells of the lens vesicle face each other; anterior cells differentiate into the lens epithelium, and the posterior cells, exposed to vitreous humor, differentiate into fibers.
30 To mimic aspects of this cellular arrangement, we set up paired epithelial explants that were asymmetrically exposed to vitreous. Our results show that when cultured for more than 30 days, the EPs produced an approximately biconvex tissue capable of focusing light and magnifying images. Functional EPs contained epithelial-like and fiberlike cells that had structural, ultrastructural, and molecular features similar to those seen in the normal lens. For instance, by approximately 40 days of culture, EPs generally contained monolayers of cuboidal, epithelial-like cells that were primarily located in the region of the tissue that had not been in direct contact with the vitreous fluid (the part of the EP that faced the plastic surface of the culture dish). These cells had large, lobulated nuclei similar to those seen in lens epithelial cells in vivo.
25 26 27 Furthermore, these cells expressed proteins normally present in lens epithelial cells. They included α-crystallin, the cytoskeletal protein vimentin, and the endoplasmic reticulum protein PDI.
17 19 20 21 22 23 24 In some cases, however, the epithelial cells extended toward the vitreous-exposed surface and, on rare occasions, were multilayered and encircled the tissue. Why the epithelial cells varied in location in this way is unknown. One possibility may be related to the different batches of vitreous used during this study. Although every effort was made to standardize vitreous collection, some variations between vitreous batches were unavoidable. For example, the ages of the animals from which the vitreous was collected varied from batch to batch and could therefore have introduced variations in growth and/or differentiation factor concentrations between experiments. Note also that the vitreous was routinely diluted 1:1 with culture medium, so that concentrations of such factors would already have been reduced by 50% at the outset of these experiments.