title: Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.
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 106to approximately 108cells 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.
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Research campus, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Kerstin F
authors.initials: KF
authors.lastname: Gerer
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Research campus, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Hoyer
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Research campus, Hartmannstraße 14, 91052, Erlangen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Dörrie
authors.affiliation: Department of Dermatology, Universitätsklinikum Erlangen, Research campus, Hartmannstraße 14, 91052, Erlangen, Germany. Niels.Schaft@uk-erlangen.de.
authors.forename: Niels
authors.initials: N
authors.lastname: Schaft
mesh_terms: D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology; D018274:Electroporation / Q000379:methods; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D012333:RNA, Messenger / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection / Q000379:methods
chemical_list: D000941:Antigens; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell
keywords: Antigen expression; B cell; CAR; GMP; Immunotherapy; Monocyte-derived DC (moDC); Protein expression; T cell; TCR; mRNA electroporation