title: Transient gene expression in HEK293 and vero cells immobilised on microcarriers.
abstract: The upscale of transient gene expression (TGE) gained popularity over the last decade as it drastically shortens timelines for the production of recombinant proteins. Bottlenecks of the method turned out to be media composition and media exchange, which is usually required as conditioned medium drastically reduces the transfection efficiency. Media exchanges are typically done by centrifugation, which limits upscale, is prone to contamination or is a high cost factor when continuous centrifuges are used. In this work HEK/EBNA cells were grown and transfected on microcarriers. Cell immobilisation allows easy media exchange after sedimentation. The transfection method was optimised regarding polyethylenimine (PEI) concentration, optimal DNA:PEI ratio, type of PEI, incubation time and polyplex formation time. In addition to HEK, Vero cells were also transfected using the same protocol. The method was established in spinner flasks and scaled up to a 1.5 litre stirred tank reactor. Transfection efficiencies of up to 33% with pCEP4 and 98% with pMAX were reached. Additionally immobilisation on microcarriers was used to retain the cells during cultivation, thus allowing media replacement and prolonging cultivation time from one to two weeks with continuous expression of the recombinant protein.
authors.affiliation: Institute of Applied Microbiology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Fliedl
authors.forename: Christian
authors.initials: C
authors.lastname: Kaisermayer
mesh_terms: D000818:Animals; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D018914:Cells, Immobilized / Q000166:cytology / Q000378:metabolism; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008863:Microspheres; D011094:Polyethyleneimine / Q000494:pharmacology; D014162:Transfection; D014709:Vero Cells
chemical_list: D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine