title: Xeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.
abstract: Molecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are commonly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no sequential and orderly protocol for producing human adipose-derived stem cells (hASCs) under xeno-free conditions. After standardizing a human platelet lysate (hPL) production protocol, four human adipose tissue samples were processed through explants with fetal bovine serum (FBS)-supplemented or hPL-supplemented media for extracting the adipose-derived stem cells. The cells were cultivated in cell culture medium + hPL (5%) or FBS (10%). The cellular replication rate, immunophenotype, and differentiation potential were evaluated at fourth passage. Cellular viability was evaluated before and after cryopreservation of the cells, with an hPL-based solution compared with an FBS-based solution. The explants cultured in hPL-supplemented media showed earlier and faster hASC proliferation than did those supplemented with FBS. Likewise, cells grown in hPL-supplemented media showed a greater proliferation rate, without losing the immunophenotype. Osteogenic differentiation of xeno-free hASC was higher than the hASC produced in standard conditions. However, adipogenic differentiation was reduced in xeno-free hASC. Finally, the cells cryopreserved in an hPL-based solution showed a higher cellular viability than the cells cryopreserved in an FBS-based. In conclusion, we have developed a complete xeno-free protocol for extracting, culturing, and cryopreserving hASCs that can be safely implemented in clinical studies.
authors.affiliation: Basic Science Department, Medicine School, Fundación Universitaria de Ciencias de la Salud, Bogotá, Colombia Physiology Department, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia.
authors.forename: Carlos Hugo
authors.initials: CH
authors.lastname: Escobar
authors.affiliation: Physiology Department, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia ochaparrog@unal.edu.co.
authors.forename: Orlando
authors.initials: O
authors.lastname: Chaparro
mesh_terms: D017667:Adipocytes / Q000166:cytology*; D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects; D000818:Animals; D001792:Blood Platelets / Q000166:cytology; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D015925:Cryopreservation; D003470:Culture Media / Q000494:pharmacology*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis / Q000187:drug effects
chemical_list: D003470:Culture Media
keywords: Adipose; Adult stem cells; Cryopreservation; Explant culture; Human platelet lysate; Xeno-free production
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