Open AccessXeno‐Free Extraction, Culture, and Cryopreservation of Human Adipose‐Derived Mesenchymal Stem Cells
Author(s) -
Escobar Carlos Hugo,
Chaparro Orlando
Publication year - 2016
Publication title -
stem cells translational medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.781
H-Index - 71
eISSN - 2157-6580
pISSN - 2157-6564
DOI - 10.5966/sctm.2015-0094
Subject(s) - fetal bovine serum , mesenchymal stem cell , cryopreservation , adipose tissue , stem cell , andrology , microbiology and biotechnology , immunophenotyping , platelet lysate , adipogenesis , biology , cell therapy , immunology , chemistry , cell , flow cytometry , biochemistry , medicine , embryo
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. Significance This study was performed to standardize a complete ordered protocol to produce xeno‐free human adipose‐derived mesenchymal stem cells (hASCs) as a safe therapeutic alternative. Cells were extracted by adipose tissue explants and then cultured and cryopreserved using human platelet lysate (hPL). Different scientific journals have published data regarding the use of hPL as a safe fetal bovine serum substitute for hASC culture, using heparin to avoid clot formation. This article reports the use of hPL for extracting, culturing, and cryopreserving hASCs without anticoagulant.