title: Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture.
abstract: Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10 ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2 x 10(8), 1.1 x 10(9) and 3.5 x 10(10) at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2mg at day 18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus a flexible and powerful tool for manufacturing recombinant proteins.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Pembroke Street, Cambridge CB2 3RA, UK.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Cheeks
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Edwards
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Arnot
authors.forename: Nigel K H
authors.initials: NK
authors.lastname: Slater
mesh_terms: D000180:Acrylic Resins / Q000494:pharmacology*; D000818:Animals; D019149:Bioreactors; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D020285:Cryoelectron Microscopy; D004247:DNA / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins / Q000096:biosynthesis; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D010477:Perfusion; D016062:Porosity / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D004247:DNA; D029721:Drosophila Proteins; D005947:Glucose; D049452:Green Fluorescent Proteins; D019344:Lactic Acid; D011994:Recombinant Proteins; D000099166:Polyacrylamides; C086179:spz protein, Drosophila