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 hPLsupplemented 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. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:358-365 SIGNIFICANCEThis 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.

Human platelet lysate to substitute fetal bovine serum in hMSC expansion for translational applications: a systematic review

“…In the vast majority of reports the ability of hMSCs to be differentiated along pre-determined lineages was not affected by being cultured using hPL: adipogenic, osteogenic and chondrogenic differentiation were demonstrated with no relevant difference in effectiveness or potential to the same hMSCs cultured with FBS. In three cases MSCs, expanded in hPL, were more effectively differentiated along osteogenic, chondrogenic and adipogenic routes [ 37 , 49 , 58 ]; others reported a difference only for osteogenic [ 17 , 40 , 42 , 47 , 54 ] or adipogenic differentiation respectively [ 19 , 31 ]. Only one publication reported that FBS supported osteogenic and adipogenic differentiation to a higher degree [ 57 ].…”

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues

“…Cryopreservation is currently the best way for long‐term cell storage; MSCs, isolated by explant method, can be successfully revitalized after cryopreservation while maintaining their characteristics . In addition, after cryopreservation of UC tissue, it is possible to isolate MSCs by explant method from post‐thaw tissue pieces …”

“…For removing animal serum from MSC culture, it is suggested to use human serum obtained from the donor, volunteers or UC blood . Human platelet lysate is also recently recommended to be used instead of animal serum …”

Concise Review: Mesenchymal Stem Cell Therapy for Pediatric Disease: Perspectives on Success and Potential Improvements

“…Several groups have evaluated these different PRF or PRP preparations, studying growth factor concentrations , , the effect of leukocytes , and the variability between donors . Finally, recent studies comparing platelet lysate, defined xenobiotic‐free supplements, and FBS have been published .…”