title: Nonviral transfection strategies for keratinocytes, fibroblasts, and endothelial progenitor cells for ex vivo gene transfer to skin wounds.
abstract: In a search for the optimal nonviral gene transfer technique in epidermal and dermal supportive extracellular matrix studies, we investigated the efficiency of late generation liposomal transfection reagents and nucleofection of fibroblasts (FBs), endothelial progenitor cells (EPCs), and keratinocytes (KCs) as essential indicators of healing skin wounds. FBs, KCs, and EPCs were grown under serum-reduced conditions and manipulated according to optimized in vitro manufacturer protocols. Fugene HD, Effectene, PEI, and Lipofectin were compared to Amaxa Nucleofection. A green fluorescent protein (GFP)-encoded reporter gene plasmid was transfected, and transfection efficiencies were determined by green-fluorescence-activated cell sorting. Normal cell morphologies were observed after either transfection or nucleofection. For KC cell cultures, Fugene HD resulted in the highest transfection efficiency in human (41%) and porcine (42%) KCs. For EPCs, Effectene was optimal for human-derived cells (42%), whereas nucleofection was optimal (32%) for porcine cells. For FBs, however, nucleofection resulted in the highest transfection rates in human (46%) and porcine (60%) FBs. For specific epidermal cell studies, Fugene HD was the preferred gene transfer method, whereas Effectene appeared to be the optimal reagent for pro-angiogenic studies. Nucleofection in combination with FBs is the best combination to achieve the highest overall transfection rate and is thus the optimal combination for use in ex vivo gene transfer strategies of wound healing or skin tissue engineering.
authors.affiliation: Laboratory of Plastic Surgery and Tissue Engineering Research, Department of Plastic and Reconstructive Surgery-Biomedical Science Group, KUL Leuven University Hospitals, Leuven, Belgium.
authors.forename: Stijn
authors.initials: S
authors.lastname: Dickens
authors.forename: Stefaan
authors.initials: S
authors.lastname: Van den Berge
authors.forename: Benoit
authors.initials: B
authors.lastname: Hendrickx
authors.forename: Kristoff
authors.initials: K
authors.lastname: Verdonck
authors.forename: Aernout
authors.initials: A
authors.lastname: Luttun
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Vranckx
mesh_terms: D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D018014:Gene Transfer Techniques; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D008954:Models, Biological; D012867:Skin / Q000293:injuries* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D013552:Swine; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection / Q000379:methods*; D014945:Wound Healing / Q000235:genetics / Q000502:physiology; D014947:Wounds and Injuries / Q000628:therapy*