Development of substrates for the ex vivo expansion of conjunctival epithelium

Abstract

The conjunctiva is a mucous membrane lining the ocular surface and is crucial to ocular surface homeostasis. Ocular surface diseases lead to a poor tear film, irreversible conjunctival scarring and continual corneal desiccation that may result in painful loss of vision. Conjunctival integrity and a tear film with appropriate consituents are crucial to the survival of corneal and limbal stem cell transplants. I hypothesised that two novel substrates could be developed for the ex vivo expansion of conjunctival epithelium to address a range of transplantation requirements: 1) a degradable biological substrate from the decellularisation of human conjunctiva and 2) a synthetic non-degradable substrate from expanded polytetrafluoroethylene (ePTFE). This study demonstrated that conjunctival epithelial cells (HCjE-Gi cell line) were supported at a greater cell density on ePTFE subjected to ammonia gas plasma treatment. Flow cytometry determined the phenotype of conjunctival epithelium developed on treated ePTFE was similar to that developed on an established cell culture product; Thincert, a chemically modified polyethylene terephthalate (PET) membrane. Primary conjunctival epithelium was also expanded ex vivo on ammonia plasma treated ePTFE, however, the cell density declined after 14 days in culture. No significant differences were found in terms of intracellular marker expression between primary conjunctival epithelium developed on ammonia plasma treated ePTFE and the positive control (PET membrane). Human conjunctiva was successfully decellularised (99% DNA removal). There was no demonstrable cytotoxicity, evidence of collagen denaturation, change in tensile strength or change in the qualitative detection of extracellular matrix proteins collagen IV, laminin and fibronectin. The development of stratified conjunctival epithelium of an appropriate phenotype was also demonstrated following explant culture on a freshly decellularised conjunctival tissue substrate. This is the first study to develop decellularised conjunctiva and plasma modified ePTFE as substrates for the ex vivo expansion of conjunctival epithelium. Novel conjunctival constructs developed from both substrates may be further developed to address a range of transplantation requirements.