Shafaie Sara, Hutter Victoria, Brown Marc B, Cook Michael T, Chau David Y S
Research Centre in Topical Drug Delivery and Toxicology, Department of Pharmacy, Pharmacology and Postgraduate Medicine, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, United Kingdom.
MedPharm Ltd, Unit 3 Chancellor Court, Surrey, Guildford, United Kingdom.
PLoS One. 2017 Oct 30;12(10):e0186799. doi: 10.1371/journal.pone.0186799. eCollection 2017.
In vitro cell based models have been invaluable tools for studying cell behaviour and for investigating drug disposition, toxicity and potential adverse effects of administered drugs. Within this drug discovery pipeline, the ability to assess and prioritise candidate compounds as soon as possible offers a distinct advantage. However, the ability to apply this approach to a cell culture study is limited by the need to provide an accurate, in vitro-like, microenvironment in conjunction with a low cost and high-throughput screening (HTS) methodology. Although the geometry and/or alignment of cells has been reported to have a profound influence on cell growth and differentiation, only a handful of studies have directly compared the growth of a single cell line on different shaped multiwell plates the most commonly used substrate for HTS, in vitro, studies. Herein, the impact of various surface geometries (flat, round and v-shaped 96 well plates), as well as fixed volume growth media and fixed growth surface area have been investigated on the characteristics of three commonly used human cell lines in biopharmaceutical research and development, namely ARPE-19 (retinal epithelial), A549 (alveolar epithelial) and Malme-3M (dermal fibroblastic) cells. The effect of the surface curvature on cells was characterised using a combination of a metabolic activity assay (CellTiter AQ/MTS), LDH release profiles (CytoTox ONE) and absolute cell counts (Guava ViaCount), respectively. In addition, cell differentiation and expression of specific marker proteins were determined using flow cytometry. These in vitro results confirmed that surface topography had a significant effect (p < 0.05) on cell activity and morphology. However, although specific marker proteins were expressed on day 1 and 5 of the experiment, no significant differences were seen between the different plate geometries (p < 0.05) at the later time point. Accordingly, these results highlight the impact of substrate geometry on the culture of a cell line and the influence it has on the cells' correct growth and differentiation characteristics. As such, these results provide important implications in many aspects of cell biology the development of a HTS, in vitro, cell based systems to further investigate different aspects of toxicity testing and drug delivery.
基于细胞的体外模型一直是研究细胞行为以及研究给药药物的处置、毒性和潜在不良反应的宝贵工具。在这个药物发现流程中，尽快评估候选化合物并对其进行优先级排序的能力具有明显优势。然而，将这种方法应用于细胞培养研究的能力受到以下因素的限制：需要提供一个准确的、类似体外的微环境，同时还要结合低成本和高通量筛选（HTS）方法。尽管据报道细胞的几何形状和/或排列对细胞生长和分化有深远影响，但只有少数研究直接比较了单一细胞系在不同形状的多孔板（HTS体外研究中最常用的底物）上的生长情况。在此，研究了各种表面几何形状（平底、圆形和V形96孔板）以及固定体积的生长培养基和固定的生长表面积对生物制药研发中三种常用人类细胞系（即ARPE-19（视网膜上皮细胞）、A549（肺泡上皮细胞）和Malme-3M（皮肤成纤维细胞））特性的影响。分别使用代谢活性测定（CellTiter AQ/MTS）、LDH释放曲线（CytoTox ONE）和绝对细胞计数（Guava ViaCount）相结合的方法来表征表面曲率对细胞的影响。此外，使用流式细胞术测定细胞分化和特定标记蛋白的表达。这些体外结果证实，表面形貌对细胞活性和形态有显著影响（p < 0.05）。然而，尽管在实验的第1天和第5天表达了特定的标记蛋白，但在后期时间点不同板几何形状之间未观察到显著差异（p < 0.05）。因此，这些结果突出了底物几何形状对细胞系培养的影响以及它对细胞正确生长和分化特性的影响。因此，这些结果在细胞生物学的许多方面都具有重要意义，即在开发基于HTS体外细胞系统以进一步研究毒性测试和药物递送的不同方面。
