Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Norway.
Exp Eye Res. 2012 Apr;97(1):1-9. doi: 10.1016/j.exer.2012.01.013. Epub 2012 Feb 7.
In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.
在边缘干细胞缺乏症 (LSCD) 患者中，体外扩增的人角膜缘上皮细胞 (HLEC) 的移植可以恢复角膜表面的结构和功能完整性。然而，HLEC 的培养和移植方案有很大的不同，在大多数方案中，生长添加剂，如霍乱毒素、外源性生长因子、激素和胎牛血清，都被使用。在本文中，我们首次比较了在包含胎牛血清的复杂培养基 (COM) 中培养的人角膜缘上皮细胞 (HLEC) 和以人血清为单一生长补充剂的培养基 (HSM)，并报告了我们首次在自体 HSM 中扩增并用于 LSCD 患者移植程序的 HLEC 检查结果。通过全基因组微阵列、RT-PCR、Western blot 检测和细胞活力、形态、免疫组织化学标记物表达和集落形成效率检测扩增的 HLEC。在 HSM 中培养 HLEC 产生了多层上皮，在基底层中检测到与 LESC 相关的细胞标志物。在 HSM 和 COM 中培养的细胞之间几乎没有转录差异，细胞活力也相当。与 LESC 相关的 p63 基因在 HSM 中的表达量比 COM 高 3.5 倍，Western blot 也证实了 HSM 培养物中 p63α 带更强。角蛋白 CK12 是角膜特异性的，在两种培养条件下都有发现，而在 HSM 中 CK3 阳性细胞明显更多。LSCD 患者移植手术后留在 HAM 上的上皮片上的细胞表达了中央上皮特征，而在生长抑制的成纤维细胞上以低密度培养的分离细胞产生了包含 21±12% p63α 阳性细胞的克隆（n=3）。总之，不含动物来源或动物细胞培养物来源的生长添加剂的培养基，用人血清作为单一生长补充剂，可作为在 HAM 上体外扩增 HLEC 的常用复杂培养基的等效替代品。
