Systems Design Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Alcon/CIBAVision, Duluth, Georgia, United States of America.
PLoS One. 2014 May 16;9(5):e96448. doi: 10.1371/journal.pone.0096448. eCollection 2014.
To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing.
Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression.
The reconstructed curved corneal epithelium was composed of 3-5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression.
The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.
为了进一步改进角膜的体外模型，本研究专注于创建一个三维、分层、弯曲的上皮组织；并随后对其进行特征描述和评估，以确定其作为生物相容性测试模型的适用性。
将永生化的人角膜上皮细胞培养至七天达到融合状态，然后在空气-液体界面上分化和分层培养七天，再进行测试。该模型测试了不同浓度的含有苯扎氯铵（BAK）的商业眼科溶液，BAK 是一种已知的细胞毒性剂，还有两种相关的眼用表面活性剂。还使用整个浸泡在磷酸盐缓冲盐水（BA PBS）中的 balafilcon A 镜片来评估生物相容性并验证模型的有效性。通过细胞活力测定和流式细胞术研究细胞死亡和整合素表达的变化来检测细胞活力。
重建的弯曲角膜上皮组织由 3-5 层细胞组成。BAK 的浓度增加表现出剂量依赖性的细胞活力降低和整合素表达增加和细胞死亡增加。在存在表面活性剂的情况下，细胞活力没有明显变化。正如预期的那样，BA PBS 组合似乎具有非常好的生物相容性，细胞活力或整合素表达没有任何不良变化。
分层、弯曲的上皮模型被证明对细胞毒性的明显变化敏感，适合继续评估隐形眼镜的生物相容性测试。我们的结果表明，流式细胞术可以为生物材料或细胞毒性化合物对上清液和贴壁细胞群体的细胞反应提供定量测量。作为角膜上皮的专门设计的体外模型，这种眼表面生物相容性的定量模型可能有助于我们更好地理解细胞-材料相互作用，并减少动物试验的使用。
