Practical Method for Excised Adipose Tissue Cryopreservation Using Commercial Freezers: Optimized Thawing and Evaluation of Isolated SVF Cells

BACKGROUND: Adipose tissue is vital for plastic and reconstructive surgery due to its accessibility and regenerative potential via abundant stromal vascular fraction (SVF) and adipose-derived stem cells (ADSCs). However, unpredictable graft resorption remains a major limitation, for which cryopreservation offers a potential solution. Yet, cryopreserving excised adipose tissue in commercial freezers with mechanical isolation of ADSCs remains unexplored. METHODS: Human adipose tissue from 22 donors was preserved as tissue blocks or lipoaspirates at -20 °C or -80 °C for up to 4 months. Across 284 technically replicated experiments, oil release, mitochondrial activity (XTT assay), SVF yield and viability (trypan blue and flow cytometry), and ADSC multilineage differentiation were assessed. Fresh samples served as controls. RESULTS: Rapid thawing at 37 °C (thawing method 3) minimized oil leakage (0.02 ml) and better preserved SVF yield with viability (3.03 and 0.64 × 10 4 cells/ml) after 2 months of tissue block preservation at -80 °C compared with lipoaspirates, per gram of fat. After 4 months, -80 °C-stored samples retained more SVF cells (88%) with 68% viability and showed stronger ADSC marker expression (CD73⁺/CD90⁺/CD105⁺: 70-80%) than -20 °C samples. ADSCs from -80 °C stored blocks differentiated into adipogenic, chondrogenic, and osteogenic lineages, whereas -20 °C stored samples showed poor adhesion and no differentiation. CONCLUSION: Cryopreserving adipose tissue blocks at -80 °C, followed by rapid thawing at 37 °C and mechanical isolation, provides a practical, xeno-free, and scalable approach to long-term storage. This strategy effectively preserves viable ADSCs, supporting their potential clinical application in regenerative therapies.