Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.
Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.
复制标题
用于电生理学研究和细胞疗法的生物合成兴奋组织的生成和定制。
DOI：
10.1038/nprot.2018.016
复制
发表时间：
2018
期刊：
影响因子：
14.8
通讯作者：
Bursac,Nenad
中科院分区：
文献类型：
--
作者：
Nguyen,HungX;Kirkton,RobertD;Bursac,Nenad
我们描述了一个两阶段协议，用于生成具有稳定和可定制的电生理表型的电兴奋和主动传导的细胞网络。使用这种方法，我们将单克隆衍生的可兴奋组织设计为一个强大且可重复的平台，以研究特定离子通道和突变如何影响动作电位（AP）形状和传导。在协议的第一阶段，我们结合计算建模、定点诱变和电生理技术来导出产生特定 AP 形状和传导特性的最佳哺乳动物和/或原核离子通道组。在该方案的第二阶段，通过病毒或非病毒传递的方式，选定的离子通道在不可兴奋的人类细胞中稳定表达，然后通过流式细胞术或抗生素选择来纯化所需的表型。该方案可与传统的异源表达系统或原代可兴奋细胞一起使用，并且将此方法应用于原代成纤维细胞可能会成为心脏细胞治疗的替代方法。与现有方法相比，该协议生成了明确的、相对均匀的可兴奋细胞电生理表型，有利于 AP 传导的实验和计算研究，并可以降低细胞移植时的心律失常风险。尽管基本的细胞培养和分子生物学技术足以使用所描述的协议产生可兴奋的组织，但需要膜片钳技术的经验来表征和优化衍生的细胞群。
We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.