Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

Abstract

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.

Identification of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

“…Previously, a negative depletion method using antifibroblast magnetic beads was used to purify cultured HCECs from stromal fibroblasts, since no cell-surface antibody specific for HCECs were described. 20 In our study, we adopted a positive selection strategy to isolate HCECs from a mixed population of HCECs and stromal fibroblasts by FACS. Results in our study showed that cells separated by FACS were viable and retained pump-associated marker Na þ /K þ -ATPase, tight junction protein ZO-1, as well as GPC4 and CD200.…”

“…18 Alternatively, stromal fibroblasts could be depleted from contaminated cultures through a negative cell-selection strategy by magnetic affinity cell separation (MACS) using antifibroblast magnetic microbeads. 20 A positive cell-selection approach, using magnetic microbeads or a more sensitive fluorescence-activated cell sorting (FACS), is plausible, but this is limited by the specificity of cell-surface markers for HCECs. For example, the two highly used markers reported for cultured HCECs are pump-associated protein Na þ /K þ -ATPase (NaK) and tight-junction protein ZO-1.…”

Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach

“…Therefore, hexagonal CECs will have a profile closer to 1.0, whereas an increasingly elongated fibroblast-like CEC will have a circularity value closer to zero (42). Digital micrographs of hCECs cultured using M4 alone or using the dual media approach were taken at the third passage at confluence.…”

Corneal endothelium: developmental strategies for regeneration

“…To overcome this problem, magnetic cell separation improves HCECs yield, allowing for a high separation efficacy. 75 The non-enzymatic method is based on the use of ethylenediamine tetraacetic acid (EDTA) to release cell-cell junctions at the same time as it promotes cell division upon exposure to mitogens. 30,70,72,76 In this process, EDTA can also cause cell damage and decrease cell yield.…”