Human eyelid adipose-derived stem cells (hEAs) and amniotic mesenchymal stem cells (hAMs) are very valuable sources for the cell therapeutics. Both types of cells have a great proliferating ability in vitro and a multipotency to differentiate into adipocytes, osteoblasts and chondrocytes. In the present study, we evaluated their stem cell characteristics after long-time cryopreservation for 6, 12 and 24 months. When frozen-thawed cells were cultivated in vitro, their cumulative cell number and doubling time were similar to freshly prepared cells. Also they expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM, and immune-related genes of HLA-ABC and 2M. Following differentiation culture in appropriate culture media for 2-3 weeks, both types of cells exhibited well differentiation into adipocyte, osteoblast, and chondrocyte, as revealed by adipogenic, osteogenic or chondrogenic-specific staining and related genes, respectively. In conclusion, even after long-term storage hEAs and hAMs could maintain their stem cell characteristics, suggesting that they might be suitable for clinical application based on stem cell therapy.

The human eyelid adipose-derived stem cells (HEACs) are known as a candidate source for stem cell-based therapy. HEACs possess the ability to proliferate in vitro and multipotency to differentiate into adipogenic, osteogenic and chondrogenic cells. To be used later than the time of collection, a long-term storage is needed. In this study, we investigated stem cell characteristics after cryopreservation of HEACs for 6 months and 1 year in liquid nitrogen. Frozen-thawed stem cells have shown that cumulative cell and doubling numbers were similar to those of fresh HEACs. After thawing, HEACs expressed stem cell-related genes of SCF, NANOG, OCT4, and TERT, ectoderm-related genes of NCAM and FGF5, mesoderm/endoderm-related genes of CK18 and VIM. They also consistently expressed transcripts of the immune-related genes of HLA-ABC and β2M. To induce mesodermal differentiation, cell were cultivated in adipogenic, osteogenic or chondrogenic medium for 2～3 weeks. After each differentiation culture, HEACs expressed adipocyte-, osteocyte- and chondrocytespecific genes. They were also stained with Oil red O, von Kossa, or alcian blue, revealing adipogenic, osteogenic, or chondrogenic character, respectively. The results suggest that long-term storage up to 1 year do not affect their biological properties, HEACs may be suitable for clinical application on cell-based therapies.