Format

Send to

Choose Destination
See comment in PubMed Commons below
Invest Ophthalmol Vis Sci. 2014 Jan 20;55(1):375-86. doi: 10.1167/iovs.13-12517.

Optimal isolation and xeno-free culture conditions for limbal stem cell function.

Author information

1
The Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Abstract

PURPOSE:

To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions.

METHODS:

Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype.

RESULTS:

Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2.

CONCLUSIONS:

The LSC function can be maintained in vitro under appropriate Xeno-free conditions.

KEYWORDS:

cell culture; limbal; oxygen; stem; xeno-free

PMID:
24030457
PMCID:
PMC3900275
DOI:
10.1167/iovs.13-12517
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems Icon for PubMed Central
    Loading ...
    Support Center