Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy.
Utilization of commercial collagens for preparing well-differentiated human beta cells for confocal microscopy.
复制标题
DOI：
10.3389/fendo.2023.1187216
复制
发表时间：
2023
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
--
作者：
随着技术的进步，共聚焦和超分辨率显微镜已成为剖析细胞病理生理学的有力工具。细胞附着到与先进成像兼容的玻璃表面是关键的先决条件，但对于人类β细胞来说仍然是一个相当大的挑战。最近，Phelps等人报道，将人β细胞铺在IV型胶原（Col IV）上并在神经元培养基中培养，保留了β细胞的特征。我们研究了人胰岛细胞接种在两个商业来源的第四型胶原蛋白（C6745和C5533）和第五型胶原蛋白（Col V）的细胞形态的差异，通过共聚焦显微镜和分泌功能的葡萄糖刺激的胰岛素分泌（GSIS）。胶原蛋白通过质谱和荧光胶原蛋白结合粘附蛋白CNA 35进行鉴定。 所有三种制备物均允许具有NKX6.1的高核定位的β细胞附着，表明分化良好的状态。所有胶原蛋白制剂均支持稳健的GSIS。然而，3种制剂之间的胰岛细胞形态不同。C5533显示出作为成像平台的优选特征，具有最大的细胞扩散和有限的细胞堆积，其次是Col V和C6745。C6745附着行为的显著差异归因于该制剂的低胶原含量，表明涂层材料认证的重要性。接种在C5533上的人胰岛细胞显示出响应解偶联剂2-[2-[4-（三氟甲氧基）苯基]亚肼基]-丙二腈（FCCP）或高糖+油酸的线粒体和脂滴（LD）的动态变化。 Col IV的经认证的制剂提供了一个简单的平台，以应用先进的成像技术研究人类胰岛细胞的功能和形态。
With technical advances, confocal and super-resolution microscopy have become powerful tools to dissect cellular pathophysiology. Cell attachment to glass surfaces compatible with advanced imaging is critical prerequisite but remains a considerable challenge for human beta cells. Recently, Phelps et al. reported that human beta cells plated on type IV collagen (Col IV) and cultured in neuronal medium preserve beta cell characteristics. We examined human islet cells plated on two commercial sources of Col IV (C6745 and C5533) and type V collagen (Col V) for differences in cell morphology by confocal microscopy and secretory function by glucose-stimulated insulin secretion (GSIS). Collagens were authenticated by mass spectrometry and fluorescent collagen-binding adhesion protein CNA35. All three preparations allowed attachment of beta cells with high nuclear localization of NKX6.1, indicating a well-differentiated status. All collagen preparations supported robust GSIS. However, the morphology of islet cells differed between the 3 preparations. C5533 showed preferable features as an imaging platform with the greatest cell spread and limited stacking of cells followed by Col V and C6745. A significant difference in attachment behavior of C6745 was attributed to the low collagen contents of this preparation indicating importance of authentication of coating material. Human islet cells plated on C5533 showed dynamic changes in mitochondria and lipid droplets (LDs) in response to an uncoupling agent 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) or high glucose + oleic acid. An authenticated preparation of Col IV provides a simple platform to apply advanced imaging for studies of human islet cell function and morphology.
登录
查看更多内容
影响因子：
10.6
作者：
Kaufman, Brett A.;Li, Changhong;Soleimanpour, Scott A.
通讯作者：
Soleimanpour, Scott A.
影响因子：
8.2
作者：
Cottle L;Gan WJ;Gilroy I;Samra JS;Gill AJ;Loudovaris T;Thomas HE;Hawthorne WJ;Kebede MA;Thorn P
通讯作者：
Thorn P
影响因子：
--
作者：
Boudko SP;Danylevych N;Hudson BG;Pedchenko VK
通讯作者：
Pedchenko VK
DOI：
10.1038/nrm3902
发表时间：
2014-12
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Mouw JK;Ou G;Weaver VM
通讯作者：
Weaver VM
影响因子：
7.7
作者：
Friedlander, Mollie S. H.;Nguyen, Vy M.;Bevacqua, Romina J.
通讯作者：
Bevacqua, Romina J.