Dynamic suspension culture enhances scalable maturation of hiPSC-derived cartilage organoids for regenerative medicine

Abstract

BackgroundCartilage tissue engineering requires scalable culture strategies to produce high-quality organoids. Human induced pluripotent stem cells (hiPSCs) provide a renewable source of chondrogenic cells. However, conventional static 3D culture limits tissue maturation, reproducibility, and scalability. Dynamic culture systems may help overcome these limitations, although their application for hiPSC-derived cartilage maturation remains poorly explored. MethodsIn this study, we established and validated a dynamic suspension bioreactor culture platform (CERO, OLS) for scalable maturation of hiPSC-derived chondroprogenitor cells (hiCPCs) into cartilage organoids populated by biomimetic human induced chondrocytes (hiCHOs). Key culture parameters, including aggregate preparation strategy, agitation speed, and maturation duration, were systematically evaluated. Cartilage maturation under dynamic and conventional static culture conditions was assessed by histology and immunohistochemistry, biochemical assays, organoid size measurements, and gene expression (RT-qPCR). In addition, the functional integration of optimized organoids was evaluated in a human osteochondral explant model. ResultsPre-formed manually picked hiCPC aggregates showed improved cartilage formation compared with single-cell seeding or pelleted aggregates in the bioreactor. Dynamic suspension culture promoted increased construct growth, enhanced ECM deposition, and a more favourable cartilage-associated molecular phenotype compared with static culture. HiCHO organoids matured under dynamic suspension conditions displayed increased sulphated glycosaminoglycan and proteoglycan deposition together with higher expression of cartilage-associated genes ACAN, COMP, MGP, and COL2A1. Although prolonged static maturation alone supported continued cartilage development, introducing dynamic suspension culture during later maturation stages further reinforced favourable molecular and matrix-associated features. Importantly, hiCHO organoids generated under optimized dynamic culture conditions successfully filled human cartilage defects and established matrix continuity with surrounding native tissue in a human osteochondral ex vivo explant model. ConclusionsThis study shows that dynamic suspension culture is an effective and scalable strategy for maturation of hiPSC-derived cartilage organoids. Consequently, this approach supports reproducible neo-cartilage production and allows functional testing in human tissue models. These findings support the use of dynamic culture systems for cartilage repair and in vitro/ex vivo cartilage research.