Chubinskiy-Nadezhdin Vladislav I, Sudarikova Anastasia V, Shorokhova Mariia A, Vasileva Valeria Y, Khairullina Zuleikha M, Negulyaev Yuri A
Institute of Cytology RAS, 194064, Tikhoretsky Ave. 4, St. Petersburg, Russia.
Institute of Cytology RAS, 194064, Tikhoretsky Ave. 4, St. Petersburg, Russia.
Biochem Biophys Res Commun. 2022 Sep 3;619:22-26. doi: 10.1016/j.bbrc.2022.06.022. Epub 2022 Jun 15.
Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. Electrophysiological data on the features of ion channels functioning in 3D cell culture models are currently very limited in the literature. This gap of knowledge may be associated with technical difficulties that exist when researchers try to apply the standard patch clamp method for the registration of ion channels in cells aggregated in spheroids. In this regard, our study focuses on solving emerging technical difficulties and presents an example of their successful solution. Here, we developed a specific approach and have recorded the activity of mechanosensitive stretch-activated ion channels (SACs) in endometrial MSCs (eMSCs) assembled in spheroids. Moreover, we observed functional interplay of SACs with potassium channels of big conductance (BK) in the plasma membrane of eMSC spheroids consistently to revealed earlier in routine 2D cultured cells. Additionally, we observed a significant decrease in the frequency of SACs activation in spheroids that may indicate the differences in the level of functional expression of channels in 3D culture comparing to 2D culture of eMSCs.
三维（3D）细胞聚集体或球体被认为比传统的二维细胞培养更接近生理条件。聚集形成球体的间充质干细胞（MSCs）提高了移植细胞的存活率。干细胞在三维培养中的组织方式会影响细胞微环境及其力学性质。维持MSCs关键生理反应的生物学过程的调节与离子通道的功能密切相关。与二维培养相比，离子通道的表达模式、作用和调节机制在三维培养中可能有显著差异，因此需要在离子电流水平上进行适当分析。目前，关于三维细胞培养模型中离子通道功能特征的电生理数据在文献中非常有限。这种知识空白可能与研究人员试图应用标准膜片钳方法记录球体中聚集细胞的离子通道时存在的技术困难有关。在这方面，我们的研究重点是解决新出现的技术难题，并展示一个成功解决这些难题的例子。在这里，我们开发了一种特定的方法，并记录了聚集形成球体的子宫内膜间充质干细胞（eMSCs）中机械敏感拉伸激活离子通道（SACs）的活性。此外，我们观察到eMSC球体质膜中SACs与大电导钾通道（BK）之间的功能相互作用，这与在常规二维培养细胞中较早揭示的一致。此外，我们观察到球体中SACs激活频率显著降低，这可能表明与eMSCs的二维培养相比，三维培养中通道功能表达水平存在差异。
