Glycerol-Based Cryopreservation of CELT-Fat: Identification of the Optimal Concentration in a GMP-Compatible Protocol

Background: Autologous fat grafting is widely used in reconstructive, aesthetic and regenerative surgery and often requires repeated applications. Cryopreservation of lipoaspirate enables autologous fat banking and off-the-shelf availability; however, its clinical implementation is limited by freezing-induced tissue injury, regulatory requirements and uncertainties regarding the optimal preservation protocol. Glycerol is a biocompatible cryoprotective agent with promising preliminary data. Nevertheless, the optimal concentration for lipoaspirate cryopreservation remains unknown. The aim of this study was to determine the optimal glycerol concentration for preservation of adipose tissue processed according to the Cell-Enriched Lipotransfer (CELT) protocol in clinically relevant volumes under GMP-compatible conditions. Methods: Lipoaspirates from 10 patients were processed by centrifugation according to the CELT protocol and allocated into experimental groups: fresh unfrozen control, frozen samples without cryoprotectant, frozen samples with PBS, and frozen samples supplemented with glycerol in concentrations ranging from 10% to 60%. Samples were cryopreserved using a controlled freezing rate at a temperature of −80 °C for 24 h. Large-volume cryopreservation was additionally performed with the best concentration of glycerol. Post-thaw tissue quality was assessed by resazurin assay of whole tissue, stromal vascular fraction (SVF) cell live/dead counting, and resazurin assay after short-term cell culture. Results: Glycerol supplementation improved post-thaw tissue viability compared with cryopreservation without cryoprotectant or with PBS alone. An optimal concentration range between 10% and 30% glycerol was identified, with highest preservation of metabolic activity and surviving cell yield observed at 20%. Higher glycerol concentrations resulted in a marked decline in tissue quality. Cryopreservation in large volume was feasible and did not impair post-thaw viability compared with small-volume samples. Conclusions: Glycerol-based cryopreservation allows effective and GMP-compatible preservation of human lipoaspirate. An optimal glycerol concentration range was identified, enabling large-volume fat banking without compromising tissue quality. This protocol provides a clinically applicable strategy for autologous fat storage and may facilitate repeated reconstructive and regenerative treatments.