Purchase this article with an account.
H. Levis, T. Alekseeva, H. Jawad, R. A. Brown, F. Tully, J. T. Daniels; Plastic Compressed Collagen as a Novel Carrier for Cultured Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1934.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To develop an alternative substrate to amniotic membrane for cultured limbal epithelial cell ocular surface reconstruction.
A mixed population of human limbal epithelial cells (LECs) and a population of limbal fibroblasts were isolated from donor corneal rims and expanded in culture. Neutralised rat-tail type I collagen, 10x MEM and medium containing fibroblasts was cast into circular gels for 30 min at 37°C and subjected to plastic compression (5min: Brown et al., 2005). Human LECs were seeded onto the surface of the compressed gels and maintained submerged in LEC culture medium for 13d before being subjected to airlifting for a further 5d. Gels were then fixed and processed for immunochemical analysis and electron microscopy (EM) for comparison with normal human cornea.
LECs cultured on plastic compressed collagen closely resemble the human corneal epithelium. Immunocytochemical analysis of wholemount cellular collagen gels revealed that fibroblasts within the gels maintained a typical fibroblastic morphology with extended processes. Immunohistochemical analysis of transverse sections indicated that β-tubulin was predominantly expressed in the basal epithelial layer whereas cytokeratin 3 was seen throughout. The basement membrane proteins collagen IV and laminin were present in a thin layer between the cells and collagen substrate. Scanning EM showed polygonal superficial epithelial cells with numerous microvilli and microplicae. Transmission EM showed that epithelial cells with a keratin rich cytoplasm had adopted a multilayered conformation and revealed the presence of desmosomes between highly interdigitated adjacent epithelial cells.
Limbal epithelial cells seeded onto plastic compressed collagen substrates adopt many of the typical characteristics of native human corneal epithelium. This rapidly reproducible corneal epithelial construct has great potential as both an in vitro model and cell carrier for ocular surface reconstruction.
This PDF is available to Subscribers Only