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

Abstract

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.

A Chitosan Scaffold Supports the Enhanced and Prolonged Differentiation of HiPSCs into Nucleus Pulposus-like Cells

“…Additionally, we assessed the surface charge of the chitosan scaffold across a pH range of 6.5–8.5 (Figure d). Prior studies have indicated that chitosan scaffolds with near-neutral charge enhance the adhesion and proliferation of human neural stem cells, outperforming negatively charged collagen scaffolds and chitosan–hyaluronic hybrid scaffolds. , Additionally, it has been reported that pure chitosan materials enhance the proliferation and pluripotency of hiPSCs. , Here, the zeta potential of our chitosan scaffolds showed minimal variation, maintaining near-neutrality despite changes in pH, which suggests a favorable setting for iPSCs attachment.…”

“…The resulting solution was cast into commercial tissue culture microplates, refrigerated at 4 °C for 1 h, at −5 °C for 20 min, at −20 °C for 1 h, and then anneal at −2 °C for 2 h, followed by lyophilization using a SP VirTis Genesis Pilot lyophilizer (SP Scientific, USA). 30 Prior to cell seeding, the chitosan scaffolds were sectioned into 400 μm slices and disinfected with 70 and 90% ethanol, followed by rinsing with PBS 3 times for 10 min each. After the disinfection process, the scaffolds were transferred into a 24-well plate, ready for cell seeding.…”

“…Chitosan is a naturally derived polysaccharide with intrinsic advantages for tissue engineering, such as its biocompatibility and structural congruence with native extracellular matrix (ECM) components including glycosaminoglycans and collagen . The chitosan-based 3D culture platform has proven highly effective in supporting the growth, renewal, and expansion of both human induced pluripotent stem cells (hiPSCs) and human neural stem cells. , Additionally, their ease of processing and cost-effectiveness make chitosan scaffolds uniquely well-suited as a promising platform for stem cell differentiation. We engineer chitosan scaffolds with optimal stiffness to replicate the natural environment of a healthy nucleus pulposus, ensuring sufficient mechanical strength for potential biomaterial implantation.…”