基于多孔基底的电穿孔及跨上皮电阻监测。

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring.

机构信息

Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln; Department of Biomedical Engineering, Michigan State University.

Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln.

出版信息

J Vis Exp. 2024 Sep 27(211). doi: 10.3791/66971.

DOI:10.3791/66971

PMID:39400150

Abstract

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

摘要

多孔基底电穿孔（PSEP）是一种新兴的电穿孔方法，可提供高通量和一致的输送。与许多其他类型的细胞内输送一样，PSEP 严重依赖荧光标记物和荧光显微镜来确定输送是否成功。为了深入了解电穿孔过程的中间步骤，开发了一种带有集成跨上皮电阻（TEEI）监测的 PSEP 平台。细胞在具有多孔膜的市售插入物中培养。经过 12 小时的孵育期，以允许形成完全汇合的细胞单层后，将插入物放置在位于 PSEP 设备孔中的转染培养基中。然后，将细胞单层暴露于用户定义的波形下，并通过荧光显微镜确认输送效率。通过在脉冲和荧光显微镜之间进行 TEEI 测量，可以显著增强此工作流程，以收集有关 PSEP 过程的其他数据，并且此附加的 TEEI 数据与输送效率和活力等输送指标相关。本文描述了一种带有 TEEI 测量的 PSEP 协议。