Geisen Pete, McColm Janet R, King Bradley M, Hartnett M Elizabeth
Department of Ophthalmology, University of North Carolina Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Curr Eye Res. 2006 Sep;31(9):739-48. doi: 10.1080/02713680600837408.
To investigate and compare the characteristics of four different types of retinal pigment epithelium (RPE) cells cultured for 2 to 5 weeks to provide guidance when choosing RPE cells for experimentation.
Human cell lines ARPE-19 (ARPE) and D407, primary RPE cells from C57Bl/6 mouse (mRPE), and primary human fetal RPE (hfRPE) cells were grown in respective media previously reported to be optimal for each cell type. Two methods to obtain hfRPE were used: one isolated outside and transported to our laboratory, and one isolated primarily within our laboratory from donor human fetal eyes. Barrier function was determined by transepithelial electrical resistance (TER) and permeability and structure by localization of Na+,K+-ATPase alpha-1, ZO-1, and actin. VEGF expression, determined by real-time polymerase chain reaction (PCR) for mRNA and ELISA for protein, was determined after exposure to 24 h of 1% oxygen. Madin-Darby canine kidney (MDCK) cells were compared as a non-RPE epithelial cell line.
ARPE at passage 15, but not passage 32, maintained steady low TER measurements (up to 30 ohms x cm(2)) despite forming a monolayer with apical Na+,K+-ATPase alpha-1 labeling after 35 days. mRPE developed and maintained a TER of 30 ohms x cm(2) for 2 weeks but did not localize ATPase. hfRPE showed two phenotypes. hfRPE isolated remotely and sent to us appeared more mesenchymal and undifferentiated (hfRPE-U) and had a slow but steady increase in measured TER to approximately 25 ohms x cm(2), whereas hfRPE isolated from donor eyes in our laboratory showed well-differentiated monolayers (hfRPE-D) with TER measurements > 500 ohms x cm(2) within 1 month of culture. TER measurements reflected permeability determined by the measurement of paracellular movement of sodium fluorescein. All human RPE cell types showed expression of VEGF mRNA and protein, and expression was upregulated by hypoxia in hfRPE and D407, but not in ARPE, which had constitutively high expression. ARPE expressed high levels of VEGF protein in media and cell lysates (777.2; 54.4 pg/mg protein, respectively), whereas hfRPE and D407 produced significantly less (media: 5.7 [p = 0.001], 323.6 pg/mg protein [p = 0.01]; lysate: 0 [p < 0.001], 3.5 pg/mg protein [p < 0.001], respectively).
Primary RPE cells and those from cell lines had different responses to medium-term culture or hypoxic stress. Primary isolation of hfRPE cells with careful control of culture conditions to assure adequate differentiation is recommended when using this cell as an example of a highly polarized epithelium. For disease, use of RPE cells that do not require long-term culture are more efficient and may be more relevant to study certain pathologies.
研究并比较培养2至5周的四种不同类型视网膜色素上皮（RPE）细胞的特性，为实验选择RPE细胞提供指导。
人细胞系ARPE - 19（ARPE）和D407、C57Bl/6小鼠的原代RPE细胞（mRPE）以及原代人胎儿RPE（hfRPE）细胞在先前报道的对每种细胞类型最佳的相应培养基中培养。获取hfRPE的方法有两种：一种是在外部分离并转运至我们实验室，另一种是主要在我们实验室从供体人胎儿眼睛中分离。通过跨上皮电阻（TER）测定屏障功能，通过Na⁺,K⁺ - ATP酶α - 1、紧密连接蛋白1（ZO - 1）和肌动蛋白的定位测定通透性和结构。在暴露于1%氧气24小时后，通过实时聚合酶链反应（PCR）检测mRNA以及酶联免疫吸附测定（ELISA）检测蛋白来测定血管内皮生长因子（VEGF）表达。将麦迪逊 - 达比犬肾（MDCK）细胞作为非RPE上皮细胞系进行比较。
第15代而非第32代的ARPE，尽管在35天后形成了具有顶端Na⁺,K⁺ - ATP酶α - 1标记的单层细胞，但TER测量值保持稳定较低水平（高达30欧姆×厘米²）。mRPE在2周内形成并维持TER为30欧姆×厘米²，但未定位到ATP酶。hfRPE表现出两种表型。远程分离并送至我们实验室的hfRPE显得更具间充质特性且未分化（hfRPE - U），其测量的TER缓慢但稳定增加至约25欧姆×厘米²，而在我们实验室从供体眼睛分离的hfRPE在培养1个月内显示出分化良好的单层细胞（hfRPE - D），TER测量值>500欧姆×厘米²。TER测量反映了通过测量荧光素钠的细胞旁移动所确定的通透性。所有人类RPE细胞类型均显示VEGF mRNA和蛋白表达，并且在hfRPE和D407中，缺氧上调了表达，但在组成性高表达的ARPE中未上调。ARPE在培养基和细胞裂解物中均表达高水平的VEGF蛋白（分别为777.2；54.4 pg/mg蛋白），而hfRPE和D407产生的明显较少（培养基：5.7 [p = 0.001]，323.6 pg/mg蛋白 [p = 0.0１]；裂解物：0 [p < 0.001]，3.5 pg/mg蛋白 [p < 0.001]，分别）。
原代RPE细胞和细胞系来源的RPE细胞对中期培养或缺氧应激有不同反应。当将hfRPE细胞用作高度极化上皮细胞的示例时，建议在仔细控制培养条件以确保充分分化的情况下进行原代分离。对于疾病研究，使用不需要长期培养的RPE细胞更有效，并且可能与研究某些病理情况更相关。
