title: Efficient Nonviral Transfection of Primary Intervertebral Disc Cells by Electroporation for Tissue Engineering Application.
abstract: Low back pain (LBP) is an increasing global health problem associated with intervertebral disc (IVD) trauma and degeneration. Current treatment options include surgical interventions with partial unsatisfactory outcomes reported such as failure to relieve LBP, nonunions, nerve injuries, or adjacent segment disease. Cell-based therapy and tissue engineered IVD constructs supplemented with transfected disc cells that incorporate factors enhancing matrix synthesis represent an appealing approach to regenerate the IVD. Gene delivery approaches using transient nonviral gene therapy by electroporation are of a high clinical translational value since the incorporated DNA is lost after few cell generations, leaving the host's genome unmodified. Human primary cells isolated from clinically relevant samples were generally found very hard to transfect compared to cell lines. In this study, we present a range of parameters (voltage pulse, number, and duration) from the Neon®Transfection System for efficient transfection of human and bovine IVD cells. To demonstrate efficiency, these primary cells were exemplarily transfected with the commercially available plasmid pCMV6-AC-GFP tagged with copepod turbo green fluorescent protein. Flow cytometry was subsequently applied to quantify transfection efficiency. Our results showed that two pulses of 1400 V for 20 ms revealed good and reproducible results for both human and bovine IVD cells with efficiencies ≥47%. The presented parameters allow for successful human and bovine IVD cell transfection and provide an opportunity for subsequent regenerative medicine application.
authors.affiliation: 1 Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern , Bern, Switzerland .
authors.forename: Rahel D
authors.initials: RD
authors.lastname: May
authors.affiliation: 1 Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern , Bern, Switzerland .
authors.forename: Adel
authors.initials: A
authors.lastname: Tekari
authors.affiliation: 1 Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern , Bern, Switzerland .
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Frauchiger
authors.affiliation: 1 Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern , Bern, Switzerland .|2 Department of Orthopedic Surgery and Traumatology, Inselspital, Bern University Hospital, University of Bern , Bern, Switzerland .
authors.forename: Anna
authors.initials: A
authors.lastname: Krismer
authors.affiliation: 2 Department of Orthopedic Surgery and Traumatology, Inselspital, Bern University Hospital, University of Bern , Bern, Switzerland .
authors.forename: Lorin M
authors.initials: LM
authors.lastname: Benneker
authors.affiliation: 1 Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern , Bern, Switzerland .
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein
mesh_terms: D000328:Adult; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D005260:Female; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology; D055959:Intervertebral Disc Degeneration / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D010957:Plasmids; D044968:Regenerative Medicine; D023822:Tissue Engineering / Q000379:methods*; D055815:Young Adult
keywords: DNA delivery; electroporation; intervertebral disc; nonviral gene therapy; tissue engineering applications