ABSTRACT
Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250-300 µm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/ß6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.
