Heparin can improve the viability of transfected cystic fibrosis cell lines in vitro.
Cationic liposomes are widely used as gene transfer agents in in vitro and in vivo studies of cystic fibrosis. In this study we report comparative results of cationic mediated transfection in several cell lines. We have tested epithelial cell lines expressing the wild-type cystic fibrosis transmembrane protein CFTR (bronchial epithelium-16HBE14o-, submucosal gland-Calu3) and their cystic fibrosis counterparts (CFBE41o-, CFSMEo-), as well as baby hamster kidney fibroblast cell lines (BHK) heterologously expressing human CFTR. The cells were transfected with a green fluorescent protein plasmid complexed with commercial cationic liposome (Geneporter2, GP) and 25 kDa polyethylenimine (PEI). At the end of the incubation (2 hours), low molecular weight heparin was added in order to reduce the toxicity of the lipoplexes. Transfection efficiency and cell viability were measured by flow cytometry. Determination of fatty acid composition of cellular phospholipids was performed by capillary gas chromatography. The short incubation time was sufficient to obtain satisfactory transfection in all cell lines studied. Cells treated with PEI-complexes had lower transfection efficiency and viability compared to GP in all tested cell lines. DeltaF508 CFTR carrying airway epithelial cells were easier to transfect but had lower viability compared to their healthy counterparts. This was, however not the case for the BHK cells. The fatty acid analysis showed characteristic polyunsaturated fatty acid patterns, which correlated with the viability of the transfected cells. Low molecular mass heparin added at the end of the lipoplex incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency.
Dragomir
Anca
A
Department of Medical Cell Biology, University of Uppsala, Box 571, 751 23 Uppsala, Sweden. Anca.Dragomir@medcellbiol.uu.se
Hjelte
Lena
L
Hagenfeldt
Lars
L
Roomans
Godfried M
GM
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008854:Microscopy, Electron; D010743:Phospholipids / Q000378:metabolism; D011094:Polyethyleneimine / Q000494:pharmacology; D014162:Transfection
D000925:Anticoagulants; C505032:CFTR protein, human; D005227:Fatty Acids; D006495:Heparin, Low-Molecular-Weight; D008164:Luminescent Proteins; D010743:Phospholipids; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine