Introduction: End-stage liver disease is life-threatening and primarily treated by transplantation, which carries surgical risks and requires lifelong immunosuppression to prevent immune rejection. Inflammation and ischemic injury from transplantation can cause early graft damage despite immunosuppressants. To address donor shortages and mitigate immunosuppression side effects, bioengineered liver substitutes, such as hiPSC-derived liver organoids, show great promise. This study investigates the use of slow-release alpha-1 antitrypsin (A1AT)-loaded nanoparticles incorporated into liver organoids to reduce early post-transplant inflammation and support tissue repair in vitro, to enhance immediate graft function and survival.

Method: Human embryoid bodies (hEBs) were produced using our custom agarose micro-molding platform, and then directed through a hepatic lineage specification protocol to yield liver-like organoids. Nanoparticles encapsulating alpha-1 antitrypsin (A1AT) for controlled release (SR-A1AT-NP) were fabricated via a dextran-based formulation and subsequently applied to the organoid cultures. These engineered organoids were then subjected to an in vitro inflammatory challenge using a cytokine mix (TNF-α, IL-1β, and IL-6, collectively referred to as Cytomix), and their viability and cellular health were assessed via MTT and G6PD assays, providing metrics on live and apoptotic cell populations. For in vivo validation, SR-A1AT-NP-containing liver organoids were transplanted into a rat model of acute liver failure to evaluate their capacity to mitigate inflammatory damage post-transplant. Engraftment efficiency was determined by quantifying human albumin in the host serum, and the therapeutic benefit was gauged by comparing survival and liver function outcomes between treated and control groups.

Results: Liver organoids incorporating SR-A1AT-NP exhibited significantly enhanced viability upon Cytomix exposure for 24 hours in vitro, relative to their nanoparticle-free counterparts (Fig. 1). In addition, total protein content was maintained at levels comparable to untreated controls (Fig. 2). Following in vivo transplantation, SR-A1AT-NP organoids led to marked improvements in graft function, graft and recipient survival with sustained albumin production (Table. 1).

Conclusion: Liver organoids loaded with SR-A1AT-NPs exhibited a protective response against cytokine-induced damage in vitro, as evidenced by sustained overall cell viability and preserved liver function. This was confirmed through the consistent expression of key hepatic markers, including albumin and HNF-4α, in comparison to Cytomix-treated controls, along with a reduction in STAT-6 levels observed in both untreated and treated organoids.