Comparative Study of Xenobiotic-free Media for the Cultivation of Human Corneal Epithelial Stem Cells

Abstract

Purpose : The culture of human corneal epithelial stem cells or limbal stem cells (LSCs) using animal components poses the risk of cross-species contamination in clinical applications. We quantitatively compared different xenobiotic-free culture media for the ex vivo expansion of human LSCs.

Methods : LSCs were cultured from 2x2 mm limbal tissue explants (n=6) on thermolysin-denuded amniotic membrane (AM) using xenobiotic-free culture media: CnT-Prime with 5% of human serum (CnT-PR), embryonic stem cell medium and the standard SHEM (control) at 1:1 ratio (ESCM), and modified SHEM (mSHEM) in which cholera toxin and dimethyl sulfoxide (DMSO) were removed. LSC phenotype was analyzed as follows: morphology, proliferation rate, cell size, outgrowth size, percentage of p63α high-expressing (p63α^bright) cells and expression of cytokeratin (K) 14, K12, pancytokeratin (PanK) and the stromal cell marker vimentin (Vim). Data were analyzed with the pairwise t-test.

Results : mSHEM supported compact cell outgrowths that contained small and cuboidal limbal epithelial-like cells; CnT-PR and ESCM provided a slightly more heterogeneous morphology. CnT-PR and mSHEM reduced the cell proliferation rate by 76.6% (p=0.03) and 39.4% (p=0.002), respectively compared to the standard SHEM control. However, mSHEM was superior at maintaining the small limbal epithelial population (≤12 µm, 7.0% ± 3.4% vs. 3.5% ± 0.1% in control, p=0.04), and producing the highest amount of transplantable outgrowths (>13 mm in the smallest diameter, 68.8% vs. 94.1% in control, p=0.05). Outgrowths from all three conditions contained a similar percentage of PanK⁺ and K14⁺ cells, (>95% in all cases, p>0.05), and K12⁺ cells (ranging from 0.9% to 2.7% vs. 2.5% ± 1.2% in control, p>0.05). Cultures grown in CnT-PR contained a percentage of p63α^bright cells (17.8% ± 5.7%) similar to control (22.8% ± 2.4%, p>0.05); however, ESCM and mSHEM produced a smaller amount of p63α^bright cells (6.7% ± 3.6%, p=0.04, and 12.5% ± 2.2%, p=0.04, respectively). Only the cultures in ESCM contained an amount of Vim⁺ stromal cells (1.6% ± 0.8%) significantly larger than that of the control (0.6% ± 0.3%, p=0.04).

Conclusions : mSHEM showed an efficient maintenance of the LSC phenotype. Other supplements to replace cholera toxin and DMSO are needed to increase the expansion efficiency of mSHEM.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.