Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin

Background & Aim Three-dimensional (3D) suspension culture system is commonly used for mass production of human induced pluripotent stem cells (hiPSCs). In this system, hiPSCs form spheroid aggregate structure and expand with increasing the size. However, hiPSC aggregates form collagen type I shell-like structure surrounding the aggregates in the late phase of culture, and the outside-in compressive stress inhibits cell growth. In this study, we designed a scalable culture strategy to prolongate hiPSCs aggregate growth in a 3D suspension culture system. To improve the proliferation potential of hiPSC aggregates, we prevent the collagen structure formation by using botulinum hemagglutinin (HA) which can bind and disrupt E-cadherin mediated cell-cell adhesion. The difference of collagen structure and mechanical force generated from cells were observed and we discussed the fundamental mechanisms. Methods, Results & Conclusion hiPSCs suspension culture was performed using a single-use 5 mL stirred bioreactor. After 72 h from seeding, HA was treated in the medium and the final concentration were adjusted to 0, 0.5,1.0, and 5.0 nM. Time profile data of cell growth and the expression of related proteins inside the aggregates was observed. The aggregate size was continuously increased throughout the culture with higher HA concentration conditions. The final cell density was increased with the increasing HA concentration and that saturated from 1.0 nM (Figure). The peripheral expression of phosphorylated myosin light chain, an active domain of myosin motor proteins, was decreased after HA treatment. The collagen shell-like structure formation was also prevented, and proliferation maker Ki-67 positive cell population was increased by HA treatment.These results suggest that the relaxation of cell-cell adhesion by HA can prolongate hiPSC aggregates growth and this simple culture system has a potential to improve the scalable production of hiPSCs for 3D suspension cultures.