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Three dimensional (3D) cultivation provides means of evaluating the efficiency of microcarrier-based suspension systems for cell expansion, demonstrating its importance for the development of cell processing in industry. In this study, functional properties of novel microcarriers derived from Klebsiella oxytoca were evaluated and, based on those results, 3D cultivation was performed using a novel neffs system. The novel microcarriers were prepared by a biofilm-based operation, allowing a more stable operation with lower energy consumption. The novel microcarriers had diameters between 500 and 1000 μm, a rough surface, and a relatively high amount of carbohydrate (average 62.5%). All microcarriers demonstrated excellent cell adsorption capacity, with around 91.8% of cells adsorbing within 2 h. Additionally, bacterial cells were found to survive on the microcarriers, resulting in prolonged cell activity. 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