%0 Journal Article
%T Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation
%A Gary S. L. Peh
%A Man-Xin Lee
%A Fei-Yi Wu
%A Kah-Peng Toh
%A Deepashree Balehosur
%A Jodhbir S. Mehta
%J International Journal of Biomaterials
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/601302
%X The culture of human corneal endothelial cells (CECs) is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs) that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS) technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated ¡°labeled¡± and ¡°flow-through¡± cell fractions were collected separately, cultured, and morphologically assessed. Cells from the ¡°flow-through¡± fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the ¡°labeled¡± fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs. 1. Introduction The inner monolayer of the human cornea¡ªthe corneal endothelium (CE)¡ªfunctions both as a barrier and a pump and plays a critical role in the regulation of corneal hydration. The CE layer prevents excessive fluids from entering the glycosaminoglycan-rich stromal layer while actively pumping fluid out to prevent corneal edema [1¨C3]. This maintains corneal thickness and keeps the corneal transparent [3, 4]. Cells of the CE do not have the capacity to undergo functional regeneration in vivo [5¨C7]. Hence, when corneal endothelial cell loss occurs, the existing cells spread out to maintain functional integrity of the CE. However, a critical threshold must be maintained to preserve corneal clarity. If endothelial dysfunction develops, there will be an inability to efficiently pump fluid out of the stroma, resulting in stromal and epithelial edema, loss of corneal clarity, and visual acuity [4], which will eventually lead to corneal blindness¡ªa situation where the retina is normal but the cornea becomes edematous. This is the second leading cause of visual blindness worldwide [8]. Restoration of vision in these situations is only possible by replacing the dysfunctional CE layer with healthy donor CE through corneal transplantation. There is a global shortage of transplant-grade donor corneal tissues, and this greatly restricts the number of corneal transplantation performed yearly [9, 10]. Hence, significant efforts have
%U http://www.hindawi.com/journals/ijbm/2012/601302/