Optimal cell concentration for cryopreservation of banked human adipose tissue-derived stem cells.

Human adipose tissue-derived stem cells (hADSCs) are an attractive source for regenerative medicine. However, cryopreservation protocols-particularly with respect to optimal cell concentration-remain inadequately defined. hADSCs were isolated from adipose tissue of 12 donors (mean age: 31.8 ± 8.9 years; BMI: 22.9 ± 4.2). Second-passage cells were cryopreserved for two weeks at concentrations of 0.5 × 10⁶/mL, 1 × 10⁶/mL, 2 × 10⁶/mL, 5 × 10⁶/mL, and 10 × 10⁶/mL. Post-thaw viability, apoptosis, immunophenotype, proliferation, and tri-lineage differentiation were evaluated using standard assays. Cell viability increased significantly with higher cryopreservation concentrations, reaching 94.2 ± 2.0% at 10 × 10⁶/mL (p < 0.05 vs. 0.5 × 10⁶/mL). Early apoptosis decreased with increasing concentration, reaching its lowest level at 5 × 10⁶/mL (2.9 ± 0.5%; p < 0.05 vs. 10 × 10⁶/mL), but showed a slight increase at 10 × 10⁶/mL. Proliferation and tri-lineage differentiation into adipocytes, osteoblasts, and chondrocytes were maintained across all groups, as confirmed by histological staining and molecular analyses. Cryopreservation at 5 × 10⁶/mL offers the most favorable balance between high viability and minimal apoptosis while preserving proliferative and differentiation potential. This concentration likely represents an optimal condition for hADSC biobanking and clinical use. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00877-6.