Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue

Mason, SL, Stewart, RMK, Sheridan, CM ORCID: 0000-0003-0100-9587, Keshtkar, F, Rooney, P, Austin, E, Schloetzer-Schrehardt, U, Kruse, FE and Kaye, SB ORCID: 0000-0003-0390-0592 (2016) Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue. Investigative Ophthalmology and Visual Science, 57 (8). pp. 3708-3713.

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Abstract

Purpose: We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. Methods: Cell suspensions were formed from fresh and organ culture–stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. Results: Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). Conclusions: Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.

Item Type:	Article
Uncontrolled Keywords:	limbal epithelium, corneal organ culture, transplantation
Depositing User:	Symplectic Admin
Date Deposited:	24 Oct 2016 10:08
Last Modified:	19 Jan 2023 07:27
DOI:	10.1167/iovs.16-19354
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3003954