Abstract
Conventional two-dimensional (2D) cell culture relies on a flat adhesive substrate, which not only restricts cellular three-dimensional growth space but also lacks the complex interactions and multiple signaling pathways between cells and the extracellular matrix (ECM), resulting in significant differences in the survival state of cells cultured in vitro compared to that in their natural three-dimensional (3D) microenvironment in vivo, presenting inherent constraints for investigating complex biological questions in vitro. In this study, we introduce the DNA-inspired biomimetic nanomaterial, Janus base nanotubes (JBNTs), designed to simulate the long collagen fibers in natural tissues to promote 3D spheroid formation within a conventional 2D system. Under physiological conditions, the lysine groups in JBNTs confer positive charges to JBNTs, allowing negatively charged cells and proteins to adhere to the material surface via electrostatic interactions, thereby providing rapid anchorage sites for cells and facilitating spontaneous cell aggregation into 3D structures. We believe that this engineered microenvironment triggers cellular self-regulation, upregulating the expression of cell membrane receptor proteins and key extracellular matrix components. Cells engage with these microenvironmental elements to facilitate the formation of stable 3D cellular spheroids. Additionally, the JBNTs-induced spheroids exhibit a cell viability exceeding 90% and can be readily dissociated into single cells using trypsin for subculture, demonstrating excellent cytocompatibility and high cell reusability, which are advantageous for constructing 3D in vitro models. This study presents an innovative approach for constructing 3D microenvironments, offering new pathways for guiding cellular behavior and advancing 3D cell culture techniques.