Development and characterization of scaffolds for 3D cell culture

In recent years the study of three-dimensional (3D) cell culture has undergone a progressive development. The 3D models have shown characteristics more similar to the overall conditions in vivo with respect to 2D cultures (Hutmacher DW., 2001). The 3D approach decreases the gap between cell culture system and the cellular physiology, helping to understand better various cell functions such as proliferation, adhesion, viability, morphology, microenvironment, and response to drugs. Compared to 2D cultivation, the cells in 3D are completely different in terms of morphology, signaling, and microenvironmental metabolism (Astashkina A et al., 2014). The scaffold provides the support necessary for the cells to attach, proliferate, and maintain their differentiated function. As a drawback, the structural characteristics of 3D scaffolds make their characterization quite complex. In my project a new hydrogel scaffold (HS) has been produced and several methods have been modified and/or developed for characterizing its cell compatibility, porosity, mechanical and optical properties and molecular diffusion. The first condition that has been verified was that the absence of toxicity for the cell types used and the capacity of the scaffolds to promote cell adhesion and growth for a period suitable for biological tests. Different cell lines have been evaluated, like human endothelial vascular cell line (HECV), human keratinocyte cell line (NCTC), human fibroblast cell line and human liver carcinoma cell line. HECV and NCTC have been cultured in 3D for more than 30 days. The porosity of the lyophilized scaffold has been calculated by liquid displacement method, using acetonitrile as the displacement liquid, and resulted to be higher than 95%. The mechanical properties of the HS have been analysed and Young’s Modulus (YM) and yield strength (YS) were evaluated in dried state and in liquid immersion. The YM of HS ranges from 15 to 120 kPa, and the YS ranges from 3 to 21 kPa. The transparency of HS has been easily evaluated by UV-VIS transmittance study and by observing letters printed on a paper sheet placed under the scaffold completely immersed in PBS, measuring the sharpness of the typographical sign, with respect to a reference read through a PBS solution. The molecular diffusion through the scaffold has been evaluated using Franz diffusion cells at 37 °C, with metformin hydrochloride as diffusing molecule. The diffusion coefficient D was calculated, its value being approx. 1.2×10-9 m²/s.