Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue
Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue
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DOI：
10.1167/iovs.05-1622
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发表时间：
2006-08-01
影响因子：
4.4
通讯作者：
Miller, Sheldon S.
中科院分区：
文献类型：
--
作者：
Maminishkis, Arvydas;Chen, Shan;Miller, Sheldon S.
目的。提供一种可重复的方法来培养 hfRPE 细胞汇合单层，这些细胞表现出与天然组织相似的形态、生理学、极性和蛋白质表达模式。方法。人胎儿眼睛在到达时被解剖，RPE细胞片从脉络膜机械分离并在完全由市售成分组成的专门设计的培养基中培养。使用先前描述的技术进行生理学实验。使用标准技术进行免疫组织化学、电子显微镜和通过ELISA测量细胞因子。 结果。 RPE 细胞培养物的汇合单层表现出上皮形态和严重色素沉着，电子显微镜显示广泛的顶膜微绒毛。通过各种紧密连接蛋白的免疫荧光标记来鉴定连接复合物。平均跨上皮电位 (TEP) 为 2.6 +/- 0.8 mV，顶端阳性，平均跨上皮电阻 (RT) 为 501 +/- 138 Omega (.) cm(2)（平均值 +/- SD；n = 35）。向根尖浴中添加 100 μM 三磷酸腺苷 (ATP) 可使净液体吸收从每小时 13.6 +/- 2.6 增加到 18.8 +/- 4.6 μL (.) cm(-2)（平均值 +/- SD；n = 4）。在其他实验中，VEGF 主要分泌到基底浴中 (n = 10)，而 PEDF 主要分泌到顶浴中 (n = 10)。 结论。开发了一种新的细胞培养程序，可产生具有天然组织形态和生理特征的汇合原代 hfRPE 培养物。这些易于复制的原代培养物的上皮极性和功能与先前研究的天然人类胎儿和牛 RPE 脉络膜外植体非常相似。
PURPOSE. Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue.METHODS. Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA.RESULTS. Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance ( R T) was 501 +/- 138 Omega (.) cm(2) (mean +/- SD; n = 35). Addition of 100 mu M adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 mu L (.) cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10).CONCLUSIONS. A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.