Different Incubation Times of Cells After Gene Electrotransfer in Fetal Bovine Serum Affect Cell Viability, but Not Transfection Efficiency
- 308 Downloads
Electroporation as a delivery method is increasingly important in gene therapy, not only in vivo but also in in vitro experimental systems. Different applications of gene electrotransfer require high viability of cells and high transfection efficiency of gene electrotransfer. It was already demonstrated that the addition of fetal bovine serum (FBS) immediately after gene electrotransfer leads to improved cell survival and transfection efficiency. Therefore, the aim of the study was to determine whether prolonged incubation of cells in FBS, for more than standard 5 min, can lead to increased transfection efficiency and improved cell survival. Different murine melanoma and murine and human endothelial cell lines were transfected with plasmid encoding green fluorescent protein and then incubated for different periods of time in FBS (5–30 min). Transfection efficiency was determined by flow cytometry and fluorescence microscopy and cell survival by cell viability assay. Prolonged incubation of cells in FBS after gene electrotransfer had varying effect on cell survival, which was decreased in melanoma cell lines B16F1 and B16F10, minimally affected in SVEC4-10 and HUVEC cells and increased in 2H11 cell at 30 min of incubation time in FBS. On the other hand, transfection efficiency of gene electrotransfer was not affected by long incubation of cell in FBS, regardless of the cell line used. The results of our study emphasize the importance of optimization of gene electrotransfer protocol for particular cells and specific purposes of gene electrotransfer, taking into account the importance of transfection efficiency and cell survival.
KeywordsGene electrotransfer Fetal bovine serum Melanoma cells Endothelial cells Cell survival Transfection efficiency
The authors acknowledge the financial support from the state budget by the Slovenian Research Agency (program no. P3-0003, projects no. J3-4211, J3-4259). The research was conducted in the scope of LEA EBAM (French-Slovenian European Associated Laboratory: Pulsed Electric Fields Applications in Biology and Medicine) and is a result of networking efforts within COST TD1104 Action. We would like to thank Mira Lavric, Tanja Blagus, Tanja Dolinsek, Spela Kos and prof. Gregor Sersa (Institite of Oncology Ljubljana, Ljubljana, Slovenia) for all the valuable work they contributed to this research.
- Edhemovic I, Gadzijev EM, Brecelj E, Miklavcic D, Kos B, Zupanic A, Mali B, Jarm T, Pavliha D, Marcan M, Gasljevic G, Gorjup V, Music M, Vavpotic TP, Cemazar M, Snoj M, Sersa G (2011) Electrochemotherapy: a new technological approach in treatment of metastases in the liver. Technol Cancer Res Treat 10:475–485PubMedCentralPubMedGoogle Scholar
- Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, Billard V, Geertsen PF, Larkin JO, Miklavcic D, Pavlovic I, Paulin-Kosir SM, Cemazar M, Morsli N, Rudolf Z, Robert C, O’Sullivan GC, Mir LM (2006) Electrochemotherapy - An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. EJC Suppl 4:3–13CrossRefGoogle Scholar
- O′Hare MJ, Ormerod MG, Imrie PR, Peacock JH, Asche W (1989). Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells Electroporation and electrofusion in cell biology. E. S. A. E. J. C. A. Neumann. New York, Plenum PressGoogle Scholar
- Sedlar A, Dolinsek T, Markelc B, Prosen L, Kranjc S, Bosnjak M, Blagus T, Cemazar M, Sersa G (2012) Potentiation of electrochemotherapy by intramuscular IL-12 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity. Radiol Oncol 46:302–311PubMedCentralCrossRefPubMedGoogle Scholar
- Spanggaard I, Snoj M, Cavalcanti A, Bouquet C, Sersa G, Robert C, Cemazar M, Dam E, Vasseur B, Attali P, Mir LM, Gehl J (2013) Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: safety and efficacy results of a phase I first-in-man study. Hum Gene Ther Clin Dev 24:99–107CrossRefPubMedGoogle Scholar
- Testori A, Tosti G, Martinoli C, Spadola G, Cataldo F, Verrecchia F, Baldini F, Mosconi M, Soteldo J, Tedeschi I, Passoni C, Pari C, Di Pietro A, Ferrucci PF (2010) Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach. Dermatol Ther 23:651–661CrossRefPubMedGoogle Scholar
- Tozon N, Pavlin D, Sersa G, Dolinsek T, Cemazar M (2013). Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell carcinoma in cats, J Feline Med SurgGoogle Scholar