In vitro, pancreatic β-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell-cell and cell-matrix interactions that are essential for β-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of β-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic β-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.