Data from: Serum-free and xenobiotic-free preservation of cultured human limbal epithelial cells

Aim/Purpose of the Study: 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. Materials and Methods: 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). Results: 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). Conclusion: 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.研究目的：开发一种无血清且无异种外源性生物制剂的一周存储方案，以维持体外培养的人角膜缘上皮细胞片（human limbal epithelial sheets）的细胞活力、形态与表型。 材料与方法：将完整人羊膜（amniotic membrane）作为载体，培养人角膜缘组织外植体（human limbal explants），培养时长为两周。将所得上皮细胞片置于密封容器中，于23℃下分别在含特定动物血清源性组分（Quantum 286）的无血清培养基，或无异种外源性生物制剂的培养基（Minimal Essential Medium）中存储4天与7天。分别对存储组与未存储组的培养物进行检测分析：检测指标包括细胞活力、羊膜与上皮细胞片厚度，以及一系列免疫组化标志物——涵盖未成熟细胞标志物（ΔNp63α、p63、Bmi-1、C/EBPδ、ABCG2及K19）、分化细胞标志物（K3与Cx43）、增殖标志物（PCNA）及凋亡标志物（Caspase-3）。 结果：培养物的初始细胞活力为98±1%，且存储后仍维持在较高水平。未存储的角膜缘上皮细胞片平均中央厚度为23±3 μm，存储后未观察到明显的细胞丢失。未存储的上皮细胞片主要呈现未成熟表型：13例培养物中有9例的ΔNp63α阳性率超过3%。存储处理后，采用Quantum 286培养基存储7天的组别中，ABCG2与C/EBPδ的表达量显著下调（P=0.04）；采用该培养基存储4天的组别中，Bmi-1的表达量显著下调（P=0.02）；其余标志物的表达均无显著变化。分化标志物的表达与上皮组织及羊膜厚度均无显著关联，仅ABCG2与角膜缘上皮厚度呈显著负相关（R=-0.69，P=0.01），ΔNp63α与羊膜厚度呈显著负相关（R=-0.59，P=0.03）。 结论：以羊膜为载体、由组织外植体培养得到的角膜缘上皮细胞片，可在23℃下于无血清及无异种外源性生物制剂的培养基中进行存储，且在存储4天与7天后，其细胞活力、超微结构及ΔNp63α阳性率均可得到维持。