改良组织块培养法体外培养人角膜上皮干细胞

In vitro culture of human corneal epithelial stem cells using modified explant culture

背景:角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的:探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法:人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于37℃、体积分数为5%CO 2细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论:①改良组织块培养组出膜时间明显短于常规组织块培养组(P<0.05),出膜率明显高于常规组织块培养组(P<0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。

BACKGROUND:Corneal epithelial stem cells,also known as limbal stem cells,are distributed in the basal layer of limbal epithelium.It is extremely difficult to deal with limbal stem cell deficiency or dysfunction that is caused by severe thermal burn,chemical burn,and chronic inflammation of ocular surface.At present,in vitro culture of corneal epithelial stem cells using tissue engineering technology followed by clinical transplantation is a new and effective therapeutic direction.OBJECTIVE:To explore the feasibility of serum-free culture of human corneal epithelial stem cells in vitro using modified explant culture method.METHODS:The remaining donor corneal tissues after keratoplasty(less than 8 mm in diameter)were obtained from Henan Eye Bank,and the outer and middle limbus were dissected under surgical microscope.Two culture methods were used to culture human corneal epithelial stem cells.In the conventional explant culture group,the limbal tissues were adhered to the dish with the epithelium being upward,then Keratinocyte-serum free medium(K-SFM)was added into dishes,followed by incubation at 37°C in a 5%CO2 incubator.In the modified explant culture group,limbal tissues were dissected to immerse in the K-SFM culture medium and incubated at 37°C in the 5%CO2 incubator for 12 hours.The limbal tissues were then adhered to the dish with the epithelium being downward.The day whenever the cells from the limbal tissues adhered to the dish was marked as the 1^st day of culture,and changes in cell morphology and growth were recorded by phase contrast microscopy every day.Immunofluorescent staining was used to detect the expression of K3 and p63 in primary cells on the 5th,10th and 14^th day of the modified explant culture.RESULTS AND CONCLUSION:The mean early stage of growth in the modified explant culture group was shorter than that of the conventional explant culture group(P<0.05),and the mean growth rate of the modified explant culture group was higher than that of the conventional explant culture group(P<0.05).In the modified explant culture group,cells had a good growth state,and many cells with small size gathered together on the 10th day of culture.On the 14^th day,cell clones were formed,and the cells in the clone showed uniform morphology.On the 5^th day,K3 highly expressed,while p63 lowly expressed in primary cells.On the 10th day,both of K3 and p63 had an increased expression.On the 14th day,there was no significant increase in the K3 expression,but the expression of p63 increased significantly.In the in vitro serum-free culture condition,the modified explant culture could significantly promote the growth of corneal epithelial stem cells,and expand corneal epithelial stem cells in vitro,which could provide sufficient seed cells for enriching corneal epithelial stem cells and constructing human limbal multilayered epithelial sheets.