Cryopreservation and post-thaw characterization of dissociated human islet cells.
Cryopreservation and post-thaw characterization of dissociated human islet cells.
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DOI：
10.1371/journal.pone.0263005
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Elliott JAW
中科院分区：
文献类型：
--
作者：
Marquez-Curtis LA;Dai XQ;Hang Y;Lam JY;Lyon J;Manning Fox JE;McGann LE;MacDonald PE;Kim SK;Elliott JAW
本研究的目的是优化分离的胰岛细胞的冷冻保存，并获得可用于糖尿病病理和治疗的单细胞转录组研究的功能细胞。采用迭代分级冷冻的方法，在10%二甲基亚砜和6%羟乙基淀粉中以1℃/min至-40℃的速度冷却，液氮中储存，快速解冻，并通过连续稀释去除冷冻保护剂后，获得了活细胞。上皮细胞黏附分子的表达在解冻后即刻下降，孵育过夜后恢复，而内分泌细胞标记物HPi2的表达在冷冻后仍保持较高水平。膜片钳电生理显示不同类型胰岛细胞的通道活动和胞吐作用不同；然而，低温保存后，胞吐反应和电压门控Na+和Ca~(2+)通道的生物物理特性保持不变。单细胞RNA测序表明，新鲜的和冷冻保存的分散的人胰岛细胞的总转录组和关键的胞吐功能基因是相似的。因此，我们报道了一种冷冻保存分散的胰岛细胞的优化程序，该程序保持了它们的膜完整性，以及它们的分子和功能表型。我们的发现不仅将为研究糖尿病的细胞机制提供现成的细胞来源，而且还将为模拟研究和潜在的移植应用提供生物工程假性胰岛和胰岛薄片。
The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to –40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+ and Ca2+ channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
6
作者：
Di Sanzo M;Quaresima B;Biamonte F;Palmieri C;Faniello MC
通讯作者：
Faniello MC
影响因子：
8.2
作者：
Fox, Jocelyn E. Manning;Lyon, James;MacDonald, Patrick E.
通讯作者：
MacDonald, Patrick E.
影响因子：
3.3
作者：
Kenmochi, Takashi;Asano, Takehide;Miyazaki, Mariko
通讯作者：
Miyazaki, Mariko
影响因子：
6.2
作者：
Lakey, JRT;Anderson, TJ;Rajotte, RV
通讯作者：
Rajotte, RV