Idrees Ayesha, Chiono Valeria, Ciardelli Gianluca, Shah Siegfried, Viebahn Richard, Zhang Xiang, Salber Jochen
1 Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Turin, Italy.
2 Universitätsklinikum Knappschaftskrankenhaus Bochum, Ruhr-Universität Bochum, Bochum, Germany.
Int J Artif Organs. 2018 Nov;41(11):779-788. doi: 10.1177/0391398818775519. Epub 2018 May 29.
Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially "murine in vitro dermal construct" based on L929 cells was generated, leading to the development of "human in vitro dermal construct" consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue, RealTime-Glo MT, and CellTiter-Glo (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the "shaking time" to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.
生物材料的细胞相容性测试迫切需要三维细胞培养系统。这项工作旨在开发三维体外真皮皮肤模型，并对其进行优化以用于细胞相容性评估。最初生成了基于L929细胞的“小鼠体外真皮构建体”，进而开发出了由正常人真皮成纤维细胞与大鼠尾腱I型胶原组成的“人体外真皮构建体”。为了评估细胞的活力，对不同的检测方法（CellTiter-Blue、RealTime-Glo MT和CellTiter-Glo，普洛麦格公司）进行了评估，以针对相应的细胞类型和三维系统优化最适合的检测方法。进行了Z轴堆叠成像（活/死和鬼笔环肽/ DAPI - 普洛麦科公司），以可视化基质内的正常人真皮成纤维细胞，显示出丝状伪足样形态以及正常人真皮成纤维细胞在基质中的均匀分布。在分析的这些检测方法中，发现CellTiter-Glo是最佳的细胞活力检测方法。CellTiter-Blue试剂影响正常人真皮成纤维细胞的细胞形态（与L929细胞不同），表明对细胞生物学活性有干扰，导致活力数据不太可靠。另一方面，RealTime-Glo仅在细胞密度非常低时提供线性信号，这使得该检测方法不适用于此系统。通过优化“振荡时间”使CellTiter-Glo适用于三维真皮构建体，以增强试剂渗透和最大程度地释放三磷酸腺苷，结果表明振荡60分钟时的活力值比振荡5分钟时高2.4倍。此外，活力结果表明细胞在基质内是有活力的。该模型将通过增加更多皮肤层进一步改进以制成全层模型。
