Abstract：
Evaluation of mesenchymal stem cell seeding efficiency in three-dimensional (3D) scaffolds is a critical step for constructing a potent and useful tissue engineering product for regenerative medicine. To determine the quantity of cells seeded on a scaffold, their condition and viability, and/or to confirm cell adhesion to the scaffold surface, a number of cellular assays are used. The assays are most often based on a direct or indirect colorimetric-, fluorimetric-, bioluminescent-, or isotope-based measurement of changes reflecting the activity of cellular processes. This chapter presents a selection of assays measuring the efficiency of cell seeding on scaffolds, that is, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the ATP (adenosine triphosphate), DAPI (4\',6-diamidino-2-phenylindole) assay, the Alamar Blue (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, resazurin) assay and the Pico Green dsDNA (N\'-[3-(dimethylamino)propyl]-N,N-dimethyl-N\'-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]propane-1,3-diamine) assay. These assays monitor the number of viable cells, sometimes in conjunction with specifying cell membrane integrity, determine enzymatic activity associated with cell metabolism, measure cell proliferation rate, and assess the total protein or DNA content in the cell-scaffold construct. The choice of the appropriate methods and the details for testing 3D cultures are of utmost importance to properly evaluate tissue engineering products. Still, developing standards for assessment of cell-scaffold constructs remains a challenge in tissue engineering.
摘要：
在三维（3D）支架中评估间充质干细胞的接种效率是构建用于再生医学的有效且有用的组织工程产品的关键步骤。为了确定接种在支架上的细胞数量，他们的条件和生存能力，和/或确认细胞粘附到支架表面，使用了许多细胞测定。这些测定通常基于直接或间接比色法-，氟酰亚胺-，生物发光体-，或基于同位素的测量反映细胞过程活动的变化。本章介绍了测量细胞接种在支架上的效率的选择，也就是说,MTS（3-（4,5-二甲基噻唑-2-基）-5-（3-羧基甲氧基苯基）-2-（4-磺基苯基）-2H-四唑）测定，MTT(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物)测定，ATP（三磷酸腺苷），DAPI(4',6-二氨基-2-苯基吲哚)测定，Alamar蓝（7-羟基-10-氧化苯恶嗪-10-ium-3-酮，刃天青）测定和PicoGreendsDNA（N'-[3-（二甲基氨基）丙基]-N，N-二甲基-N'-[4-[(E)-(3-甲基-1,3-苯并噻唑-2-亚基)甲基]-1-苯基喹啉-1-铵-2-基]丙烷-1,3-二胺)测定。这些试验监测活细胞的数量，有时结合指定细胞膜完整性，确定与细胞代谢相关的酶活性，测量细胞增殖率，并评估细胞支架构建体中的总蛋白或DNA含量。选择合适的方法和测试3D培养物的细节对于正确评估组织工程产品至关重要。尽管如此，开发评估细胞支架构建体的标准仍然是组织工程中的一个挑战。
