The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells.
The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells.
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DOI：
10.1177/0963689720954798
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发表时间：
2020-01
影响因子：
3.3
通讯作者：
Shimada M
中科院分区：
文献类型：
--
作者：
Tokuda K;Ikemoto T;Saito Y;Miyazaki K;Yamashita S;Yamada S;Imura S;Morine Y;Shimada M
本研究的目的是确定从脂肪组织来源的干细胞（ADSC）分化的胰岛素分泌细胞（IPC）是否可以冷冻保存。使用我们的两步方案将人ADSC分化为IPC，该方案包括三维培养和无异种抗原的方法。此后，使用三种不同的方法冷冻IPC。首先，IPC立即在-80 °C下冷冻（-80 °C组）。第二，IPC最初被置于Bicell冷冻容器中，然后在-80 °C下冷冻（BICELL组）。第三，使用卵母细胞和胚胎的玻璃化冷冻方法（CRYOTOP组）。CCK-8法检测细胞存活率显示，冻存后各组细胞存活率均下降（P < 0.01）。三磷酸腺苷含量在冻存后明显降低（P < 0.01）。免疫荧光染色显示，在所有冷冻保存组中，胰岛素阳性染色面积减少。此外，4′，6-diamidino-2-phenylindole和合并的免疫荧光图像显示，在−80°C组和CRYOTOP组中，冻存细胞似乎随机减少，而在BICELL组中，只有中心区域明显减少。使用免疫组织化学染色，冷冻保存后的IPC显示为阳性裂解的caspase-3抗体在所有组中。最后，冻存后各组IPC在葡萄糖刺激下的胰岛素分泌均显著减少（P < 0.01）。总之，IPC可能太脆弱，无法用成熟的方法进行冷冻保存，需要进一步研究合适的保存方法。
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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影响因子：
5.1
作者：
Guariguata, L.;Whiting, D. R.;Shaw, J. E.
通讯作者：
Shaw, J. E.
影响因子：
3.1
作者：
Nakamura, Kentaro;Iwazawa, Reiko;Yoshioka, Yasuhiro
通讯作者：
Yoshioka, Yasuhiro
影响因子：
6.2
作者：
Piemonti, L;Bertuzzi, F;Di Carlo, V
通讯作者：
Di Carlo, V
影响因子：
6.2
作者：
SCHARP, DW;LACY, PE;FLYE, MW
通讯作者：
FLYE, MW
影响因子：
2.7
作者：
TAYLOR, MJ;BANK, HL;BENTON, MJ
通讯作者：
BENTON, MJ