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Purpose of the Study: Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors., Patients and Methods: MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control., Results: It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype., Conclusions: Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)