Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency.
The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Helena
H
Sork
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Joel Z
JZ
Nordin
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Janne J
JJ
Turunen
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Oscar Pb
OP
Wiklander
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Burcu
B
Bestas
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.|Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, El-Khartoum square, Alexandria, Egypt.
Eman M
EM
Zaghloul
Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Helerin
H
Margus
Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Kärt
K
Padari
Department of Medicine, Center for Infectious Medicine, Novum, Karolinska Institutet, Huddinge, Sweden.|Cell Therapies Institute, Nova Southeastern University, Fort Lauderdale, Florida, USA.
Adil D
AD
Duru
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Giulia
G
Corso
Department of Biomedical Engineering, College of Engineering and Technology, Wentworth Institute of Technology, Boston, Massachusetts, USA.
Jeremy
J
Bost
Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
Pieter
P
Vader
Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Margus
M
Pooga
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.
Ci Edvard
CE
Smith
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
Matthew Ja
MJ
Wood
Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
Raymond M
RM
Schiffelers
Department of Neurochemistry, Stockholm University, Stockholm, Sweden.
Mattias
M
Hällbrink
Department of Laboratory Medicine, Clinical Research Center, Novum, Karolinska Institutet, Huddinge, Sweden.|Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
Samir El
SE
Andaloussi