Fabrication of functional rat pseudo‐islets after cryopreservation of pancreatic islets or dispersed islet cells
Fabrication of functional rat pseudo‐islets after cryopreservation of pancreatic islets or dispersed islet cells
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DOI：
10.1002/term.3219
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发表时间：
2021-05
影响因子：
3.3
通讯作者：
Kenyu Iwatsuki;M. Hirabayashi;S. Hochi
中科院分区：
文献类型：
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作者：
Kenyu Iwatsuki;M. Hirabayashi;S. Hochi
来自胰岛的分散的单细胞可以通过其聚集特性配置具有胰岛素分泌潜力和可控大小的三维胰岛样结构（假胰岛）。本研究旨在研究胰岛或胰岛细胞的冷冻保存是否有助于大鼠模型中有效的假胰岛制造。对照组（CT）取50-400 µm新鲜对照胰岛，经胰蛋白酶消化制备胰岛单细胞，然后在U形底微孔中培养3天，制备假胰岛。玻璃化-复温组（VW）取尼龙网玻璃化法冻存的复温后胰岛制备胰岛单细胞，培养3d。在冷冻组（FR）中，将来自新鲜胰岛的胰岛单细胞进行常规的Bicell®冷冻，并将解冻后的细胞培养3天。为了通过球体培养产生1个胰岛等效的假胰岛（150 μ π ι），将1250个CT细胞、1250个VW细胞和1500个FR细胞接种到每个微孔中。三组间假胰岛的活力相当（93.9%-96.9%）。此外，胰岛素分泌测定显示，这些假胰岛对高葡萄糖刺激有足够的反应。胰岛素和胰高血糖素的免疫染色显示，这些假胰岛的内分泌细胞排列与天然和分离的胰岛相似。这些胰岛/假胰岛具有核心中的β细胞和外套膜中的α细胞，这是啮齿动物胰岛的典型特征。然而，在FR假胰岛内观察到一些α细胞簇。有趣的是，VW假胰岛的α细胞显著少于CT或FR假胰岛。这些结果表明，即使在胰岛冷冻保存后，胰岛细胞的球形培养物也是产生具有定制尺寸和正常功能的假胰岛的有用工具。
Dispersed single cells from pancreatic islets can configure the three‐dimensional islet‐like architecture (pseudo‐islets) with insulin secretion potential and controllable size through their aggregation property. The present study was designed to investigate whether cryopreservation of islets or islet cells can contribute to the efficient pseudo‐islet fabrication in the rat model. In control group (CT), islet single cells were prepared by trypsin digestion of 50–400‐µm ø fresh control islets, and then cultured for 3 days in the U‐bottom microwell to fabricate pseudo‐islets. In vitrification‐warming group (VW), islet single cells were prepared from postwarm islets cryopreserved by vitrification on nylon mesh device, and then cultured for 3 days. In freezing group (FR), islet single cells originated from fresh islets were subjected to a conventional Bicell® freezing, and postthaw cells were cultured for 3 days. To generate 1 islet equivalent pseudo‐islets (150 µm ø) by the sphere culture, 1250 CT cells, 1250 VW cells, and 1500 FR cells were seeded to each microwell. The viability of the pseudo‐islets was comparable among the three groups (93.9%–96.9%). Furthermore, the insulin secretion assay showed that those pseudo‐islets responded sufficiently to the high glucose stimulation. Immunostaining for insulin and glucagon showed that the endocrine cell arrangement of those pseudo‐islets is similar to that of native and isolated islets. These islets/pseudo‐islets had the β‐cells in core and the α‐cells in mantle, which was typical characteristic of the rodent islets. However, some clusters of α‐cells were observed inside the FR pseudo‐islets. Interestingly, the VW pseudo‐islets had significantly fewer α‐cells than the CT or FR pseudo‐islets. These results suggest that the sphere culture of islet cells is useful tool to generate the pseudo‐islets with the customized size and normal functionality, even after islet cryopreservation.