Characterization of Human Subcutaneous Adipose Tissue and Validation of the Banking Procedure for Autologous Transplantation

Abstract

Adipose tissue (AT) is composed of a heterogeneous population which comprises both progenitor and differentiated cells. This heterogeneity allows a variety of roles for the AT, including regenerative functions. In fact, autologous AT is commonly used to repair soft tissue defects, and its cryopreservation could be a useful strategy to reduce the patient discomfort caused by multiple harvesting procedures. Our work aimed to characterize the cryopreserved AT and to validate its storage for up to three years for clinical applications. AT components (stromal vascular fraction-SVF and mature adipocytes) were isolated in fresh and cryopreserved samples using enzymatic digestion, and cell viability was assessed by immunofluorescence (IF) staining. Live, apoptotic and necrotic cells were quantified using cytometry by evaluating phosphatidylserine binding to fluorescent-labeled Annexin V. A multiparametric cytometry was also used to measure adipogenic (CD34+CD90+CD31−CD45−) and endothelial (CD34+CD31+CD45−) precursors and endothelial mature cells (CD34−CD31+CD45−). The maintenance of adipogenic abilities was evaluated using in vitro differentiation of SVF cultures and fluorescent lipid staining. We demonstrated that AT that is cryopreserved for up to three years maintains its differentiation potential and cellular composition. Given our results, a clinical study was started, and two patients had successful transplants without any complications using autologous cryopreserved AT.

Good Manufacturing Practice-Compliant Cryopreserved and Thawed Native Adipose Tissue Ready for Fat Grafting

“…The flow cytometry of SVF cells showed similar percentages of ASCs in fresh and thawed samples, whereas leukocytes were reduced and EPCs increased in the thawed samples. A recent study obtained similar results in terms of cell count, viability, and SVF sub-population percentages, using a CM with DMSO and human albumin [ 23 ]. In summary, we developed a standardized GMP protocol that could lead to the optimization of AT cryopreservation to guarantee a safer and more controlled quality of thawed native lipoaspirate, in order to use it further in reconstructive and cell-based treatments.…”