The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells

Abstract

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.

Effective <i>in vitro</i> differentiation of adipose-derived stem cells into Schwann-like cells with folic acid supplementation

“…However, this study still has some shortcomings as theses SLCs (FA) generated by our modified protocol are still in conventional two-dimensional pattern. Many researchers including our team (8,9,11,43) have illustrated that the three-dimensional (3D) cell culture pattern could promote the differentiation efficiency of stem cells and improve the final therapeutic effect of cell transplantation and cell therapy, which is considered to be the trend of tissue engineering and regeneration medicine. Therefore, we decide and have already begun to apply this new protocol combined with 3D culture technology for the generation of more powerful SLCs and explore its potential on the nerve regeneration in vivo and in vitro.…”

“…Adipose-derived stem cells (ADSCs) have been focused on because of their multipotency and ease of harvest. We have investigated ADSC multipotency and reported scientific evidence of insulin producing cells (IPCs) for clinical applications (7)(8)(9)(10)(11). Thus, we hypothesized that ADSCs can also self-renew and differentiate into ectodermal lineage cells, including Schwann-like cells (SLCs).…”

The interaction between cancer associated fibroblasts and tumor associated macrophages via the osteopontin pathway in the tumor microenvironment of hepatocellular carcinoma

“…We used immunofluorescence procedures in our department that were previously reported [52]. Anti-OPN antibody (dilution 1:1000, ab8448; Abcam plc.…”

Syngeneically transplanted insulin producing cells differentiated from adipose derived stem cells undergo delayed damage by autoimmune responses in NOD mice

“…We have previously reported the generation of functional IPCs from ADSCs 5 with three-dimensional (3D) culture and a xenoantigen-free protocol 6 – 10 . The quality and functionality of IPCs generated in this way have been assessed using techniques such as the stimulation index (SI), which can be used to evaluate autonomous glucose response ability, and showed that the cells have satisfactory functionality for clinical use 5 – 10 .…”