Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23 °C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23 °C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell–cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03 ± 0.38 microvilli/μm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69 ± 0.54 microvilli/μm; P = 0.98 and 0.89 ± 1.0 microvilli/μm; P = 0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07 ± 1.0 microvilli/μm; P = 0.47 and 0.07 ± 0.07 microvilli/μm; P = 0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4 ± 0.3 cell layers, as opposed to 4.0 ± 0.9 cell layers; P = 0.89 after 4 days of HEPES-MEM storage and 2.8 ± 0.6 cell layers; P = 0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7 ± 0.2 cell layers; P = 0.46 and 3.4 ± 0.4 cell layers; P = 0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4% ± 3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1% ± 4.5%; P = 0.99 and 85.1% ± 13.7%; P = 0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7% ± 15.2%; P = 0.97 and 79.8% ± 15.7%; P = 0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23 °C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

► We investigated serum-free storage of cultured conjunctival epithelium at 23 °C.
► The serum-free storage media included HEPES-MEM and Optisol-GS.
► Viability and phenotype were maintained after 7 days of storage in both media.
► The number of epithelial microvilli tended to decrease during storage.
► The number of cell layers appeared to decline in the stored epithelium.