Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System.

Abstract

Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs). Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction. Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period. Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.