A serum- and feeder-free technique of culturing human corneal epithelial stem cells on amniotic membrane

Abstract

Purpose: To describe a simple technique of cultivating human corneal epithelial stem cells using an Epilife culture medium under serum- and feeder-free conditions.

Methods: Cadaveric donor limbal corneal epithelial cells were cultured on denuded amniotic membranes using an explant technique that was free of serum and feeder cells in the Epilife medium containing a growth supplement of defined composition. These cells were assessed by phase contrast microscope. The expressions of the proposed corneal epithelial stem cell markers (p63, ATP-binding cassette member 2 (ABCG2), and cytokeratin 15 and 19) and differentiation markers (cytokeratin 3, 12, connexin 43, and p75) were analyzed using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical staining.

Results: Successful cultures were obtained, resulting in a monolayer to double layer cell sheets with a cobblestone-like morphology. RT-PCR and immunocytochemistry disclosed an expression of both putative limbal stem cell (LSC) markers and differentiation-associated markers in the cultured cells. Most of the cultured corneal epithelial cells that were immunopositive for putative LSC markers were smaller, more uniform, and closer to the limbal explant than cells positively stained with differentiation-associated markers.

Conclusions: A serum- and feeder-free culture system using Epilife medium may grow human corneal epithelial equivalents, minimizing the risk of contamination during culture. The technique may also be useful for the clinical application of limbal stem cell culture.

Figure 1

Relationship between the donor ages, time from death to culture and time to initial growth in cultivated corneal epithelial stem cells. A: Correlation graph between the donor ages versus time to initial growth. Cultured cells from younger donors significantly grew faster than cells from older donors (p=0.0069). B: Correlation graph between the time from death to culture versus time to initial growth. The shorter the time from death to culture, the faster initial cell growth was observed, although not significantly (p=0.0749).

Figure 2

Demonstration of limbal cultures as observed under phase contrast microscope. The corneal epithelial cells were proliferating from the periphery of the explant (red arrow) onto the denuded human amniotic membrane in the absence of feeder cells and serum. The cells adjacent to the explant appeared to be smaller and more uniform and had large nuclei (white arrow) compared to the cells that expanded further away from the explant (black arrow). Magnification: 200X.

Figure 3

RT-PCR for putative LSC markers and differentiation associated markers. K3, K12, K15, K19, connexin 43, p63, p75, and ABCG2 were all expressed by the cultured corneal epithelial cells. GAPDH, a housekeeping gene, was used as an internal control.

Figure 4

Immunocytochemical staining of human corneal epithelial culture from limbal explants. A: Cultured corneal epithelial cells expressed cytokeratin 3 (K3) which was a marker of differentiated corneal epithelium. Positive K3 staining was confined to the cytoplasm. B: There was no staining in negative control. Magnification: 400X.

Figure 5

Immunofluorescent staining of human corneal epithelial culture from limbal explants. A: Staining of K15 in cytoplasm of cultured corneal epithelial cells was observed. B: Cells also showed immunoreactivity for K19 in cytoplasm. C: Expression of ABCG2 in the cell membrane and cytoplasm was seen. D: Some cells revealed positive staining for p63 in nucleus. E: No staining was observed with negative control. Magnification: 400X.

Figure 6

Expression of ABCG2 on cultured corneal epithelial cells. ABCG2, the putative LSC marker, was expressed on the smaller and more uniform cells.