Megaly, Michael

Abstract Hepatic spheroids are widely used in bioartificial liver (BAL) systems, hepatotoxicology, and pharmacology due to improved functional stability compared with monolayer cultures. However, many existing methods rely on specialized equipment or static platforms that limit scalability and dynamic culture applications. Primary rat hepatocytes were cultured as three‐dimensional spheroids using a rocker‐based suspension system. Spheroid formation was evaluated across a range of rocking speeds (7–18 cycles per minute [cpm]) and inoculation densities. Spheroid morphology was quantified over time using two‐dimensional imaging. Functional performance was assessed by urea and albumin production on days 1, 4, 7, and 10. Spheroid formation efficiency was evaluated using volumetric modeling. Mean spheroid diameter decreased from 199 ± 41 μm at 7 cpm to 80.2 ± 17.6 μm at 18 cpm. The spheroid area was significantly influenced by both culture day ( p < 0.0001) and rocker speed, with significant differences observed across speeds at all time points ( p < 0.001, days 1–7). Mean spheroid area at 10 cpm was 13,750 ± 9051 μm 2 (day 1), 26,730 ± 8658 μm 2 (day 4), and decreased to 22,526 ± 8705 μm 2 (day 7). Urea production differed across speeds (ANOVA, p = 0.043), with peak performance at 12 cpm (median 3.47 μg/μg DNA) compared to 7 cpm (median 0.11 μg/μg DNA). Albumin production was 1086.8 ng/μg DNA on day 1 and 1534.4 ng/μg DNA on day 10. Urea production was 0.62 μg/μg DNA on day 1 and 1.25 μg/μg DNA on day 10. Median hepatocyte incorporation was 86% (range 73%–116%) by day 4. This rocker‐based method provides a simple, low‐cost, and scalable approach for generating hepatic spheroids with tunable size distributions under dynamic conditions. The system supports progressive functional maturation and is compatible with established scale‐up strategies, supporting its potential for translational applications.

Background. Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising noninvasive biomarker for detecting allograft rejection in solid organ transplantation. However, its diagnostic utility in pancreas transplantation, including in multiorgan recipients, remains underexplored. Methods. We conducted a retrospective analysis of 42 pancreas transplant recipients (38 simultaneous pancreas-kidney, 4 pancreas transplant alone) who underwent for-cause organ biopsy and dd-cfDNA testing between April 2020 and December 2024. dd-cfDNA was measured using the Prospera assay, with a threshold of ≥1% considered high risk and <1% low-risk. Biopsy results were correlated with dd-cfDNA to assess test performance. Results. A total of 50 dd-cfDNA tests and 45 organ biopsies were analyzed. Among simultaneous pancreas-kidney recipients, dd-cfDNA demonstrated a sensitivity of 86.7%, specificity of 52.2%, positive predictive value (PPV) of 54.2%, and negative predictive value (NPV) of 85.7% for rejection. In pancreas transplant alone recipients, all high-risk dd-cfDNA results corresponded to biopsy-proven rejection, yielding 100% sensitivity, specificity, PPV, and NPV. When combining both cohorts, dd-cfDNA testing achieved an overall sensitivity of 88.2%, specificity of 57.7%, PPV of 57.7%, and NPV of 88.2%. Additionally, dd-cfDNA kinetics suggested its potential utility as a surveillance tool posttreatment, potentially reducing the need for closure biopsies. Conclusions. dd-cfDNA is a valuable noninvasive adjunct for monitoring of rejection in pancreas transplant recipients. Further prospective studies are needed to validate these findings and define the optimal role of dd-cfDNA in posttransplant care.