NDLI: Calcium phosphate transfection optimization for serum-free suspension culture

Content Provider	Springer Nature Link
Author	Girard, Philippe Porte, Luc Berta, Temugin Jordan, Martin Wurm, Florian M.
Copyright Year	2001
Abstract	Aim of this study was to identify optimal conditions for suspension transfection in the absence of serum. Transfection parameters for suspension culture can be very different to ones in adherent cells. Most transfection protocols have been developed and optimizedfor adherent culture. Using green fluorescent protein (GFP) as reporter, FCS was eliminated from the transfection process by altering critical parameters and by substituting serum with albumin. Using standard phosphate and calcium concentrations for transfection in the absence of serum resulted in titers of only 1% of those observed in the presence of serum. A reduction of the calcium concentration from 250 mM to 100 mM, yielded a 25-fold increase in the expression of the recombinant protein compared to the serum-free standard conditions. Altering the phosphate concentration, 1.4 mM in the transfection buffer, did not improve the protein expression. Interestingly, reduction of DNA quantity by half to a concentration of 0.5 μg per milliliter of culture volume resulted in a two-fold increase of protein production. Addition of albumin to serum-free medium protected the cells against the toxicity of the calcium phosphate transfection particles (CaPi) yielding higher protein expression. All the experiments were executed in a shaken multi-well system, allowing high multiplicity parameter screening to speed up optimizations. The culture system is inexpensive, simple and efficient, minimizing costs for labor and consumables.
Starting Page	175
Ending Page	180
Page Count	6
File Format	PDF
ISSN	09209069
Journal	Cytotechnology
Volume Number	35
Issue Number	3
e-ISSN	15730778
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	2001-01-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Biomedicine general Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Cell Biology Clinical Biochemistry Bioengineering Biomedical Engineering Biotechnology

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