A Novel Method Using Quantum Dots for Testing the Barrier Function of Cultured Epithelial Cell Sheets
A Novel Method Using Quantum Dots for Testing the Barrier Function of Cultured Epithelial Cell Sheets
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DOI：
10.1167/iovs.14-15579
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发表时间：
2015-04-01
影响因子：
4.4
通讯作者：
Nishida, Kohji
中科院分区：
文献类型：
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作者：
Duncan, Thomas J.;Baba, Koichi;Nishida, Kohji
目的。角膜上皮提供了一个屏障，可以保护眼睛的深层结构免受任何颗粒或病原体的侵害。培养的上皮细胞片用于移植手术以进行角膜修复或再生。本研究的目的是开发一种使用荧光量子点纳米粒子来验证培养的上皮细胞片的质量和屏障功能的新方法。从口腔粘膜或角膜缘细胞培养的人类功能上皮细胞片在正常含钙培养基或不含钙和钙螯合剂的培养基中孵育。介质中还包含两种不同尺寸的量子点的悬浮液。孵育后，使用共焦显微镜进行量子点渗透分析。 结果。与在含钙培养基中孵育的细胞片相反，细胞外钙的去除导致紧密连接的破坏，损害细胞片的屏障功能。这导致跨上皮电阻降低，量子点更深、更普遍地渗透到细胞旁空间和细胞片内部。结论。该方法提供了易于解释的关于细胞片紧密连接功能的定性和定量数据，以及关于其表面和内部形态以及任何局部损伤或异常区域的纳米级和微米级结构信息。这项新技术可用作移植用培养上皮细胞片验证系统的一部分。
PURPOSE. The corneal epithelium provides a barrier to protect the deeper structures of the eye from any particles or pathogens. Cultured epithelial cell sheets are used in transplantation surgery for corneal repair or regeneration. The purpose of this study was to develop a novel method using fluorescent quantum dot nanoparticles for validating the quality and barrier function of cultured epithelial cell sheets.METHODS. Human function epithelial cell sheets, cultured from oral mucosal or corneal limbal cells, were incubated in either normal calcium-containing medium or medium containing no calcium with a calcium chelator. Also contained in the media were suspensions of two different sizes of quantum dots. Following incubation, analysis of quantum dot penetration was carried out using confocal microscopy.RESULTS. In contrast to the cell sheets incubated in calcium-containing medium, removal of extracellular calcium resulted in the disruption of tight junctions, compromising the cell sheet's barrier function. This caused a reduction in transepithelial electrical resistance and deeper, more ubiquitous penetration of the quantum dots into the paracellular space and interior of the cell sheet.CONCLUSIONS. This method provides easy to interpret qualitative and quantitative data on the functionality of a cell sheet's tight junctions, as well as nanoscale and microscale structural information on its surface and interior morphology, and any localized areas of damage or abnormality. This novel technique could be used as part of the validation system for cultured epithelial cell sheets for use in transplantation.