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Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins

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RNA Vaccines

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1499))

Abstract

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 a decade 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, 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 for therapeutic vaccination 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 106 to approximately 108 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: Opti-MEM® 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 is altered. 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.

Kerstin F. Gerer, Stefanie Hoyer, Jan Dörrie, and Niels Schaft contributed equally to this manuscript.

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Acknowledgements

We thank Gerold Schuler and Beatrice Schuler-Thurner for their support during the establishment and improvement of mRNA electroporation. Furthermore, we thank the former and the current members of the RNA-group and our collaborators, who participated in the establishment of the mRNA electroporation protocol, or its adaption and improvement: Peter Thumann, Verena Wellner, Ina Müller, Stefanie Baumann, Tanja Moritz, Manuel Wiesinger, Michael Erdmann, Katrin Birkholz, Christian Hofmann, Thomas Harrer, Christian Wohn, Isabell Pfeiffer, Christian Krug, Sabrina Prommersberger, and Sandra Höfflin.

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    Authors and Affiliations

    1. Department of Dermatology, Universitätsklinikum Erlangen, Research campus, Hartmannstraße 14, 91052, Erlangen, Germany

      Kerstin F. Gerer, Stefanie Hoyer, Jan Dörrie & Niels Schaft

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    1. Kerstin F. Gerer
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    2. Stefanie Hoyer
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    3. Jan Dörrie
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    4. Niels Schaft
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    Correspondence to Niels Schaft .

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    Editors and Affiliations

    1. Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany

      Thomas Kramps

    2. Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany

      Knut Elbers

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    Gerer, K.F., Hoyer, S., Dörrie, J., Schaft, N. (2017). Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins. In: Kramps, T., Elbers, K. (eds) RNA Vaccines. Methods in Molecular Biology, vol 1499. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6481-9_10

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    • DOI: https://doi.org/10.1007/978-1-4939-6481-9_10

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