Mesenchymal Stem Cell Seeding on 3D Scaffolds.

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.