EMAIL THIS PAGE TO A FRIEND

PloS one

Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells.


PMID 25734654

Abstract

To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.

Related Materials

Product #

Image

Description

Molecular Formula

Add to Cart

C106
CGS-12066 maleate salt, solid
C17H17F3N4 · C4H4O4
94563
Dimethyl sulfoxide, analytical standard
C2H6OS
01934
Dimethyl sulfoxide, for inorganic trace analysis, ≥99.99995% (metals basis)
C2H6OS
1211006
Dimethyl sulfoxide, United States Pharmacopeia (USP) Reference Standard
C2H6OS
D2050000
Dimethyl sulfoxide, European Pharmacopoeia (EP) Reference Standard
C2H6OS
46043
Ethidium homodimer, suitable for fluorescence, ~90% (HPCE)
C46H50Cl4N8
54461
HEPES, ≥99.0% (T)
C8H18N2O4S
PHR1428
HEPES, Pharmaceutical Secondary Standard; Certified Reference Material
C8H18N2O4S
1316004
Hydrocortisone, United States Pharmacopeia (USP) Reference Standard
C21H30O5
PHR1014
Hydrocortisone, Pharmaceutical Secondary Standard; Certified Reference Material
C21H30O5
31719
Hydrocortisone, VETRANAL, analytical standard
C21H30O5
H1300000
Hydrocortisone, European Pharmacopoeia (EP) Reference Standard
C21H30O5
Y0001092
Hydrocortisone for peak identification, European Pharmacopoeia (EP) Reference Standard
C21H30O5
76523
L-Glutamine, certified reference material, TraceCERT®
C5H10N2O3
PHR1125
L-Glutamine, Pharmaceutical Secondary Standard; Certified Reference Material
C5H10N2O3
209643
Selenium, pellets, <5 mm, ≥99.99% trace metals basis
Se
204307
Selenium, pellets, <5 mm particle size, ≥99.999% trace metals basis
Se
229865
Selenium, powder, −100 mesh, 99.99% trace metals basis
Se
209651
Selenium, powder, −100 mesh, ≥99.5% trace metals basis
Se
HT-SE101
Selenium, pellets, < 5mm, ≥99.999%
Se
GF66395212
Selenium, foil, 25x25mm, thickness 3mm, 99.95%
Se