Tobolt, Diane

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.

ABSTRACT Pancreatic islet transplantation can restore endogenous insulin production and offers a potential cure for diabetes, but its clinical impact has been limited by the inability to preserve large numbers of functional islets for timely use. Cryopreservation could provide an on-demand supply, yet conventional methods cause ice formation, cell injury, and loss of insulin secretion. We developed CryoMesh, a vitrification platform that combines a low-toxicity cryoprotectant with a thermally conductive, biocompatible mesh to enable ultra-rapid cooling and rewarming for ice-free cryopreservation. This approach supports long-term, clinical-scale preservation of both human pancreatic and stem cell–derived islets while maintaining viability, architecture, mitochondrial integrity, and glucose-responsive insulin secretion. Human islets preserved for up to one year restored normoglycemia in diabetic mice, with complete recovery of function and no increase in immunogenicity or loss of potency. The combination of high viability, recovery, and functional preservation at clinical scale has not been achieved previously. By decoupling islet isolation and manufacture from transplantation, CryoMesh enables extended quality, potency, and safety testing, cost-effective batch production, and global banking and distribution. These capabilities remove a major barrier to curative cell therapy for diabetes and establish a generalizable strategy for preserving complex multicellular therapeutics. One Sentence Summary A clinical scale vitrification platform enables long-term banking of human pancreatic and stem cell–derived islets for transplantation.