title: Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants.
abstract: A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.
authors: Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu, Republic of Korea.
authors: Hyun-Jung
authors: HJ
authors: Cho
authors: Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu, Republic of Korea.
authors: Seung Hee
authors: SH
authors: Lee
authors: Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu, Republic of Korea; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
authors: James J
authors: JJ
authors: Yoo
authors: Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu, Republic of Korea. Electronic address: yhshon@hmail.knu.ac.kr.
authors: Yun-Hee
authors: YH
authors: Shon
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D000653:Amniotic Fluid / Q000166:cytology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D061945:Caspase Inhibitors / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000494:pharmacology*; D053686:Fetal Stem Cells / Q000187:drug effects*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D003451:Cryoprotective Agents; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
keywords: Amniotic fluid-derived stem cells; Apoptosis; Caspase inhibitor; Catalase; Cryopreservation; Dimethylsulfoxide; Trehalose