Abstract
Purpose: :
The ex vivo expansion of human limbal epithelium for transplantation in patients with limbal stem cell deficiency conventionally requires the presence of foetal calf serum (FCS) in the growth medium. Human serum (HS) has been shown to contain growth factors which promote wound healing. Moreover, when used topically on the ocular surface, autologous serum drops have been shown to promote corneal epithelial healing. Therefore, in order to progress towards the eventual elimination of animal products in the culture of human limbal epithelium, we investigated the use of HS as a replacement for FCS.
Methods: :
Three adult human limbal rings, donated for research, were obtained from the UK Transplant Service. Limbal epithelial cells, retrieved from each limbal ring by trypsinisation, were co–cultured with 3T3–J2 mouse embryonic fibroblasts, either in the presence of growth medium containing FCS or HS. Sub–confluent primary passage cultures were assessed by flow cytometry for the putative limbal stem cell marker p63, and the terminally differentiated corneal epithelial cell marker cytokeratin (CK) 3/12. The limbal epithelial cultures were also serially passaged for a maximum of three passages. After each passage, cell counts were performed and colony forming efficiency (CFE) assays were set up. After 12 days in culture, the CFE assays were fixed, stained with 1% Rhodamine B, and counted.
Results: :
Flow cytometry revealed no statistically significant difference in the expression of p63 or CK3/12 between the FCS and HS containing cultures (p>0.5). The cell counts were higher (p=0.1) in the FCS containing cultures (650,000) as compared to the HS containing cultures (500,000). The CFEs were significantly higher (p=0.01) in the FCS containing cultures (16.5%) as compared to the HS containing cultures (12.5%).
Conclusions: :
The use of animal cells and products for the ex vivo expansion of human limbal epithelium is sub–optimal for transplantation purposes. However, this study shows that although HS can be used to establish limbal epithelial cultures, it is not as effective as FCS. The identification of growth factors specific to FCS, which further enhance limbal epithelial cultures, will require further investigation.
Keywords: cornea: epithelium • cornea: basic science • wound healing