Enhanced Cell Proliferation and Maturation Using Carboxylated Bacterial Nanocellulose Scaffolds for 3D Cell Culture

Abstract

Developing scaffolds for three-dimensional (3D) cell culture and tissue regeneration with biopolymers requires the creation of an optimal nanobiointerface. This interface must possess suitable surface chemistry, biomechanical properties, and fibrillar morphology across nano- to microscale levels to support cell attachment and growth, enabling a biomimetic arrangement. In this study, we developed a hydrogel scaffold made from bacterial nanocellulose (BNC) functionalized with carboxylic acid groups (BNC–COOH) through a reactive deep eutectic solvent (DES), offering a sustainable approach. The surface properties and fibrillar structure of BNC–COOH facilitated the formation of hydrogels with significantly enhanced water uptake (1.4-fold) and adhesion force (2.3-fold) compared to BNC. These hydrogels also demonstrated tissue-like rheological properties in both water with G′ exceeding G″, suggesting predominantly elastic (solid-like) characteristics and viscosities in the range of 8– 15 Pa·s. The BNC–COOH hydrogel scaffold demonstrated excellent biocompatibility, supporting significant cell growth and anchorage for the 3D growth of mammalian cells and enhancing preadipocyte growth by up to 7.3 times. Furthermore, the BNC–COOH hydrogel facilitates the maturation of 3T3-L1 preadipocytes into mature adipocytes, inducing typical morphology changes, such as decreased filopodia extensions, rounded cell shape, and lipid droplet accumulation without any additional chemical induction stimulus. Therefore, we demonstrated that a reactive DES composed of oxalic acid and choline chloride represents a mild reaction medium and a suitable approach for designing biocompatible 3D hydrogel scaffolds with improved physicochemical properties and biological activities for 3D cell culture.