Large-scale mammalian cell culture is essential in cell therapy, vaccine production, and the manufacturing of therapeutic protein drugs. Due to the adherent growth characteristic of most mammalian cell types, the combination of cell carrier and bioreactor is a common choice in large-scale mammalian cell culture. Current cell carriers developed by polymer crosslinking, lithography, or emulsion drops are unable to obtain a structure with uniformed porous structure and porous interior design, which results in an inhomogeneous culture condition for cells and therefore cannot ensure an optimal dynamic culture condition for cell proliferation, matrix production, and cell differentiation. In addition, the fluidic shear stress (a standard mechanical stimulation in bioreactor culture) and inner-carrier velocity (to ensure nutrient transport and waste exchange), which influence cell viability and growth, are not well-controlled/analyzed due to an irregular porous structure with these traditionally synthesized cell carriers. To solve these problems, we designed four types of hollow porous spheres (HPS, 1.0 cm diameter) with different porous structures. To investigate the impacts of porous structure on surface shear stress and inner velocity, computational fluid dynamics (CFD) simulations were conducted to analyze the liquid flow behavior in HPSs, based on which an optimal structure with minimal surface shear stress and best inner velocity was obtained and fabricated using fused deposition modeling three-dimensional (3D) printing technology. Inspired by the industrial large-scale culture system, a novel 3D dynamic culture system was then established using HPSs to seed the cells, which were then placed in a mini bioreactor on a tube roller. CFD analysis showed that under 0.1 m/s water flow, the shear stress at most surface areas from four HPSs was lower than 20 dynes/cm2, which suggests that the HPSs should provide protection against physical stress to the cells living on the scaffold surface. A dynamic cell seeding was developed and refined using the 3D culture system, which increased the 32% seeding efficiency of MC3T3 cells compared to the traditional static cell seeding method. The cell proliferation analysis demonstrated that HPSs could speed up cell growth in dynamic cell culture. The HPS with a honeycomb-like structure showed the highest inner pore velocity (CFD analysis) and achieved the fastest cell proliferation and the highest cell viability. Overall, our study, for the first time, developed a 3D printed HPS cell culture device with a uniformed porous structure, which can effectively facilitate cell adhesion and proliferation in the dynamic cultural environment, thereby could be considered an ideal carrier candidate.
Keywords: 3D cell culture; 3D printing; bioreactor; cell carrier; cellular viability; computational fluid dynamics simulation; dynamic cell culture; porous hollow spheres.