Tokuda, Kazunori Tokushima University
Ikemoto, Tetsuya Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Saito, Yu Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Miyazaki, Katsuki Tokushima University
Yamashita, Shoko Tokushima University KAKEN Search Researchers
Yamada, Shinichiro Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Imura, Satoru Tokushima University KAKEN Search Researchers
Morine, Yuji Tokushima University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
adipose-derived stem cell
The aim of our study is to determine whether insulin-producing cells (IPCs) differentiated from adipose-tissue-derived stem cells (ADSCs) can be cryopreserved. Human ADSCs were differentiated into IPCs using our two-step protocol encompassing a three-dimensional culture and xenoantigen-free method. Thereafter, IPCs were frozen using three different methods. First, IPCs were immediately frozen at −80°C (−80°C group). Second, IPCs were initially placed into a Bicell freezing container before freezing at −80°C (BICELL group). Third, a vitrification method for oocytes and embryos was used (CRYOTOP group). Cell counting kit-8 (CCK-8) assay showed that cell viability was decreased in all groups after cryopreservation (P < 0.01). Corroboratively, the amount of adenosine triphosphate was markedly decreased after cryopreservation in all groups (P < 0.01). Immunofluorescence staining showed a reduced positive staining area for insulin in all cryopreservation groups. Furthermore, 4′,6-diamidino-2-phenylindole and merged immunofluorescence images showed that cryopreserved cells appeared to be randomly reduced in the −80°C group and CRYOTOP group, while only the central region was visibly reduced in the BICELL group. Using immunohistochemical staining, IPCs after cryopreservation were shown to be positive for cleaved caspase-3 antibody in all groups. Finally, insulin secretion following glucose stimulation was significantly reduced in IPCs from all groups after cryopreservation (P < 0.01). In conclusion, IPCs may be too fragile for cryopreservation with accomplished methods and further investigations for a suitable preservation method are required.
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