Posters

Ex vivo expansion of limbal stem cells using Descemet’s membrane as a culture substrate

Poster Details

First Author: J.Hou USA

Co Author(s):    P. Bedard   C. Yuan   -. -              

Abstract Details

Purpose:

To evaluate the viability of using Descemet’s membrane (DM) as a culture substrate for ex vivo expansion of limbal stem cells (LSC).

Setting:

Investigational laboratory study. Department of Ophthalmology & Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA. Lions Gift of Sight Eye Bank, St. Paul, MN, USA.

Methods:

Limbal explants fragments were obtained from the corneoscleral rim of donor corneas after peeling DM. The explants fragments were then cultured on either the peeled DM or tissue culture plastic (TCP). Outgrowth of LSC from the cultured fragments was then compared between the two substrates (TCP vs DM). DM was then bubbled off another set of corneas. Corneal stroma was excised, leaving DM attached and exposed to the intact limbal ring. LSC were then cultured from the intact limbus onto DM. Immunohistochemistry was used to compare expression of stem cell markers, ΔNp63 and ABCG2, between LSC cultured on each substrate.

Results:

In total, 4 donor corneas were used for limbal explant cultures and 3 donor corneas were used to culture from intact limbus. Among the explant cultures, outgrowth was noted from 84% of limbal explant fragments on TCP compared to 91% on DM. All 3 cultures using intact limbus, showed rapid outgrowth onto DM. Overall, for cells cultured on TCP, 52% expressed ΔNp63, 74% expressed ABCG2, and 45% showed co-localization. In comparison, for cells cultured on peeled DM, 86% expressed ΔNp63, 70% expressed ABCG2, and 64% showed co-localization. For intact DM, 98% expressed ΔNp63, 90% expressed ABCG2, and 86% showed co-localization.

Conclusions:

DM is a viable culture substrate for ex vivo expansion of limbal stem cells. DM supported more efficient outgrowth in limbal explant cultures compared to TCP. DM preserved the stem cell phenotype of cultured LSC better than TCP.

Financial Disclosure:

None