Digital light processing-printed macroencapsulated human liver organoids preserve hepatic stellate cell quiescence for transplantation in immunocompetent mice

Abstract

Digital light processing (DLP) bioprinting enables the fabrication of stem cell-derived liver organoids into biomimetic architectures. However, preserving quiescent hepatic stellate cells (HSCs) within human liver organoids during DLP printing remains challenging, as the mechanical requirements for print fidelity often conflict with the compliant microenvironment necessary for maintaining HSC quiescence. To address this limitation, we delineated the printable window of induced pluripotent stem cell (iPSC)-derived HSCs that balances printability with resistance to activation. Notably, day 10 HSCs retained a quiescent phenotype, contributing to the assembly of multicellular organoids without fibrotic activation. We subsequently evaluated the stress-relaxation properties of poly(ethylene glycol) diacrylate (PEGDA)–gelatin methacryloyl (GelMA) and F127DA–GelMA hydrogels, demonstrating that fast-relaxing inner hydrogels preserved the compact cellular morphology of liver organoids, suppressed activation-associated gene expression, and protected HSCs from fibrotic conversion. This highlights that stress relaxation, rather than stiffness alone, is critical for cellular adaptation. Then, we developed a DLP-bioprinted macroencapsulation platform integrating stage-selected iPSC-derived HSCs, stress relaxation-tuned hydrogels, and a protective outer shell. The DLP-printed outer shell conferred structural integrity to the macroencapsulated construct, enabling successful implantation in immunocompetent mice with high graft viability and minimal alpha-smooth muscle actin (α-SMA) expression in iPSC-derived liver organoids containing HSCs. In summary, this study establishes a coordinated strategy harmonizing HSC selection, matrix mechanics, and bioprinting design to balance biological and mechanical demands, yielding structurally stable, physiologically relevant liver organoids while preventing fibrotic activation during biofabrication.

Keywords

Human liver organoids / Hepatic stellate cells / Digital light processing bioprinting / Stress relaxation / Macroencapsulation

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