Enhanced Cell Penetration and Pluripotency Maintenance of hiPSCs in 3D Natural Chitosan Scaffolds

Human-induced pluripotent stem cells (hiPSCs) cultured in 3D matrices hold great promise in disease modeling, drug discovery, and tissue regeneration. Uniform cell distribution in a 3D structure is critical to the growth and function of hiPSCs, yet cell seeding in 3D matrices often remains superficial, leading to limited cell proliferation and compromised pluripotency. Here, an approach to improve cell penetration depth of hiPSCs in 3D scaffolds modified with hiPSCs conditioned medium (CM) is reported. It is shown that extracellular matrix components are successfully deposited onto the scaffold wall surface after CM treatment and promoted homogeneous cell adhesion during initial seeding. Compared to plain, unmodified scaffolds, the CM treated scaffold improves spatial cell distribution uniformity and upregulates pluripotency markers. Notably, the expression of 29 genes associated with 11 signaling pathways participated in the pluripotency maintenance of hiPSCs exhibits >2-fold change in hiPSCs grown in the CM treated scaffolds than 2D counterparts, demonstrating that CM treated scaffolds can support a more primitive and undifferentiated phenotype of hiPSCs. This study introduces a simple and effective method to enhance cell penetration and maintain cell pluripotency in 3D matrices.