May Rahel D, Tekari Adel, Frauchiger Daniela A, Krismer Anna, Benneker Lorin M, Gantenbein Benjamin
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 .
Tissue Eng Part C Methods. 2017 Jan;23(1):30-37. doi: 10.1089/ten.TEC.2016.0355. Epub 2016 Dec 27.
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.
下腰痛（LBP）是一个日益严重的全球性健康问题，与椎间盘（IVD）创伤和退变相关。目前的治疗选择包括手术干预，但报告显示部分结果不尽人意，如未能缓解下腰痛、骨不连、神经损伤或相邻节段疾病。基于细胞的疗法以及补充了转染椎间盘细胞（这些细胞整合了增强基质合成的因子）的组织工程化IVD构建体，是一种有吸引力的椎间盘再生方法。通过电穿孔进行的瞬时非病毒基因治疗的基因递送方法具有很高的临床转化价值，因为导入的DNA在几代细胞后就会丢失，不会改变宿主基因组。与细胞系相比，从临床相关样本中分离出的人类原代细胞通常很难转染。在本研究中，我们展示了Neon转染系统的一系列参数（电压脉冲、数量和持续时间），用于高效转染人和牛的IVD细胞。为证明其效率，这些原代细胞用带有桡足类涡轮绿色荧光蛋白标签的市售质粒pCMV6-AC-GFP进行了示例性转染。随后应用流式细胞术对转染效率进行量化。我们的结果表明，对于人和牛的IVD细胞，1400 V的两个脉冲、持续20 ms可产生良好且可重复的结果，效率≥47%。所展示的参数可实现人和牛IVD细胞的成功转染，并为后续再生医学应用提供了机会。
