Abstract
Purpose :
To evaluate the viability of using Descemet’s membrane (DM) as a culture substrate for ex vivo expansion of limbal stem cells (LSC).
Methods :
Fresh cadaveric donor corneas were obtained for the study. Endothelium was mechanically debrided from the posterior surface of DM. DM was then peeled and isolated from the donor corneas. The residual corneoscleral rim was then cut into small limbal explants fragments. The limbal explants were then randomly cultured on either anterior DM (fetal banded layer) or tissue culture plastic (TCP) using fetal bovine serum-based culture media. Cultures were maintained out to two weeks. Outgrowth of LSC from the cultured limbal explants was then compared between the two culture substrates (TCP vs peeled DM). DM was then fluid dissected off the posterior stroma from a fresh set of corneas. The overlying corneal stroma was then excised, leaving DM attached peripherally to the corneoscleral rim, but exposed anteriorly to an intact limbal ring. LSC were then cultured from the intact limbal ring directly onto DM and monitored for outgrowth. Cultured cells from all three culture conditions were then characterized for presence of limbal stem cells using immunohistochemistry to evaluate the relative expression of putative stem cells markers, ΔNp63 and ABCG2.
Results :
In total 5 donor corneas were used for limbal explant cultures and 3 donor corneas were used to culture LSC from intact limbus onto DM. One set of limbal explant cultures from a single donor was excluded due to contamination. Of the remaining limbal explant cultures, outgrowth was noted from 84% (21/25) of explant fragments on TCP compared to 91% (30/33) of explant fragments on peeled DM. All corneas (100%, 3/3) cultured directly onto DM from intact limbal rings, showed rapid outgrowth onto DM. On phase contrast microscopy, there was no difference in cellular morphology based on culture condition. Overall, for cells cultured on TCP, 52% expressed ΔNp63, 74% expressed ABCG2, and 45% showed co-localization of ΔNp63 and ABCG2 expression. In comparison, for cells cultured on peeled DM, 86% expressed ΔNp63, 70% expressed ABCG2, and 64% showed co-localization. And for intact DM, 98% expressed ΔNp63, 90% expressed ABCG2, and 86% showed co-localization.
Conclusions :
Anterior DM (fetal banded layer) is a viable culture substrate for ex vivo expansion of limbal stem cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.