Sauerer Tatjana, Albrecht Leoni, Sievers Nico M, Gerer Kerstin F, Hoyer Stefanie, Dörrie Jan, Schaft Niels
RNA-based Immunotherapy, Department of Dermatology, Universitätsklinikum Erlangen (UKER), Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Erlangen, Germany.
Comprehensive Cancer Center Erlangen European Metropolitan Area of Nuremberg (CCC ER-EMN), Erlangen, Germany.
Methods Mol Biol. 2024;2786:219-235. doi: 10.1007/978-1-0716-3770-8_10.
Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 10 to approximately 10 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique.
将mRNA电穿孔导入人细胞是一种广泛适用的方法，可在多种不同细胞类型中瞬时表达所需蛋白质。我们花了二十多年时间来优化和调整这种方法，最初用于树突状细胞（DC）的抗原加载，随后用于T细胞、B细胞、外周血单个核细胞（PBMC）以及多种细胞系。在这方面，抗原在MHC I类和II类的背景下被引入、加工和呈递。除此之外，粘附受体、T细胞受体（TCR）、嵌合抗原受体（CAR）、组成型活性信号转导子（即caIKK）等功能蛋白也成功表达。我们还在完全符合GMP的条件下建立了该方案，作为生产用于临床试验治疗应用的mRNA电穿孔DC和mRNA电穿孔T细胞的生产许可证的一部分。因此，我们在此希望分享我们通用的mRNA电穿孔方案以及我们用这种方法积累的经验。这里介绍的转染方法的优点包括：（1）易于适应不同细胞类型；（2）每次电穿孔可从10个细胞扩展到约10个细胞；（3）转染效率高（80 - 99%）；（4）蛋白质表达均匀；（5）如果在A级洁净室中进行电穿孔，则符合GMP要求；（6）无转基因整合到基因组中。所提供的方案包括：使用OptiMEM作为电穿孔培养基，500 V的方波脉冲，以及4 mm的比色皿。要使该方案适应不同大小的细胞，只需改变脉冲时间即可。因此，我们分享了已验证的电穿孔设置（包括复苏培养基）的概述，这些设置是我们为各种细胞类型建立的。除了基本方案外，我们还提供了一份详细的提示和技巧列表，我们认为这些对打算使用这种转染技术的每个人都非常有价值。
