Evaluation of distinct freezing methods and cryoprotectants for human amniotic fluid stem cells cryopreservation.
Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me(2)SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me(2)SO and glycerol presented workable viability ratios.
Laboratory of Genetics and Molecular Hematology (LIM-31), University of São Paulo Medical School, 05403-000 São Paulo, SP, Brazil.
Felipe de Lara
Fde L
Janz
Adriana de Aguiar
Ade A
Debes
Rita de Cássia
Rde C
Cavaglieri
Sérgio Aloísio
SA
Duarte
Carolina Martinez
CM
Romão
Antonio Fernandes
AF
Morón
Marcelo
M
Zugaib
Sérgio Paulo
SP
Bydlowski
D000653:Amniotic Fluid / Q000166:cytology*; D000704:Analysis of Variance; D015703:Antigens, CD / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005260:Female; D005434:Flow Cytometry; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015703:Antigens, CD; D003451:Cryoprotective Agents; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3