Introduction: Effective cryopreservation of porcine islet cells is crucial for advancing research and enabling their therapeutic use through long-term storage and global distribution. However, traditional methods often compromise cell viability and function. This study evaluates a novel cryopreservation medium optimized to preserve islet cells in single-cell form. Post-thaw, we assessed their viability, reaggregation into pseudoislets, and functional performance.
Methods:

• Pseudoislet Generation: Native islets were dissociated into single cells using Accumax and seeded into microwell plates. After 5-7 days in culture, pseudoislets (PIs) were harvested for analysis.
• Cryopreservation: Dissociated cells were washed in D-PBS and resuspended in cryopreservation media, then stored in liquid nitrogen for 6–24 weeks before thawing.
• Dynamic Glucose-Stimulated Insulin Secretion (GSIS): Islets were pre-incubated in 3 mM glucose for 2 hours, then sequentially exposed to 3 mM (basal), 16.7 mM (stimulatory), 60 mM KCl, and basal glucose again. Total insulin content was extracted using RIPA buffer. Insulin levels were quantified using the Revvity HTRF insulin kit and Synergy Neo2 plate reader.
• Flow Cytometry: Islets were dissociated, stained with a viability dye, fixed, permeabilized, and blocked. Intracellular markers for insulin, glucagon, and somatostatin were used to assess endocrine cell composition. Data were collected using an LSR Fortessa.

Results: Cryopreserved cells in the test medium demonstrated a significantly higher post-thaw viability (87%) compared to traditional ''FBS + 5% DMSO'' (44%) and other commercial media: NutriFreez® D5 (46%), Synth-a-Freeze™ (39%), and CryoStor® CS5 (41%).
Pseudoislets derived from cryopreserved and fresh cells were morphologically similar, maintaining dense, rounded structures indicative of intact architecture. Diameter measurements were consistent across both groups.
FDA/PI staining revealed similar viability after 5–7 days in culture: 92% in fresh and 87% in cryopreserved-derived pseudoislets.
Flow cytometry revealed similar proportions of somatostatin⁺, insulin⁺ and glucagon⁺ cells.
Dynamic insulin secretion profiles were also comparable between pseudoislets generated from fresh and cryopreserved cells.
Conclusion: We developed a cryopreservation medium that significantly improves post-thaw cell viability compared to conventional and commercial alternatives. Thawed islet cells effectively reaggregated into structurally intact pseudoislets that maintained viability and dynamic insulin secretion comparable to fresh ones. Importantly, cryopreservation had no significant impact on the proportions of key endocrine cell types. These findings support the utility of the developed medium for reliable islet preservation and downstream applications.