Process automation for the differentiation of induced pluripotent stem cells into induced chondrocytes

Abstract

Osteoarthritis (OA) presents a growing health burden while current treatments remain largely symptomatic. Advanced Therapy Medicinal Products (ATMPs) based on chondrocytes have emerged as promising candidates to restore articular cartilage and address the underlying pathology. However, chondrocyte-based therapies remain limited by proliferative capacity, donor variability, and phenotypic instability. Induced pluripotent stem cells (iPSCs) represent a renewable cell source capable of directed differentiation into chondrocytes, offering the potential for standardized and scalable production. This thesis establishes an automated workflow on the StemCellDiscovery (SCD) platform for iPSC cultivation and chondrogenic differentiation into induced chondrocytes (iCHOs), translating a manual protocol into modular routines (coating, remove coating, seeding, media change, washing, and harvesting). Quality control included media-fill sterility testing, immunofluorescence of pluripotency markers, and Alcian Blue staining. Technical modules for coating, seeding, and media change operated reliably. Washing and harvesting were more sensitive but improved after refining TrypLE concentration and handling parameters. During cultivation, automated conditions maintained cell growth and pluripotency comparable to manual culture. Differentiation under both conditions yielded similar outcomes. The automated workflow produced higher cell numbers and slightly improved viability at the replating stage, while both approaches showed progression toward chondrogenic-like phenotypes. During late-stage differentiation, the culture phase was discontinued due to declining cell numbers and a contamination event. Alcian Blue staining showed only faint signals, preventing conclusive assessment of chondrogenic differentiation. Overall, automation reproducibly executed key operations and preserved essential cell properties while identifying challenges in harvesting and long-term differentiation. Future priorities include refining detachment parameters, expanding validation runs, integrating molecular analyses, and enhancing contamination control toward Good Manufacturing Practice (GMP)-compliant, scalable iCHO production for OA therapy.