Efficient non-viral transfection of primary human adult chondrocytes in a high-throughput format
Efficient non-viral transfection of primary human adult chondrocytes in a high-throughput format
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DOI：
10.1016/j.joca.2008.11.004
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发表时间：
2009-06-01
影响因子：
7
通讯作者：
Aigner, T.
中科院分区：
文献类型：
--
作者：
Haag, J.;Voigt, R.;Aigner, T.
目的：建立可靠的原代人关节软骨细胞高通量转染方案。方法：通过优化的酶消化方案从成人膝关节软骨中分离原代人软骨细胞，并在高密度单层培养物中培养3-5天。使用 amaxa 的 Nucleofector 96 孔 Shuttleg 系统，用表达绿色荧光蛋白 (GFP) 的报告构建体转染分离的软骨细胞。通过荧光激活细胞分选来测量转染效率，并通过 5'-三磷酸腺苷 (ATP) 测定来确定细胞活力。使用优化的核转染方案将 siRNA 寡核苷酸（针对 3-磷酸甘油醛脱氢酶 (GAPDH)）转染到细胞中，并通过分支 DNA 测定确定 mRNA 敲低值。 结果：使用本文中重新介绍的核转染方案，常规可实现超过 70% 的存活细胞的转染效率。转染后24小时细胞活力约为80%。每次转染使用的细胞数量减少至每个样品 2 x U。此外，该方案被证明非常适合将 siRNA 分子转移到原代人软骨细胞中，mRNA 水平的抑制率超过 95%（对于 GAPDH）。结论：我们使用微量板兼容格式成功地在原代人软骨细胞上使用核转染，首次允许同时高效转染多达 96 个样本。优化的核转染方案允许使用相同的参数集转移 DNA 和 siRNA 寡核苷酸，从而提供最大的底物灵活性。此外，转染程序所需的细胞数量比单比色皿方案少得多，因此非常适合需要多次实验重复的应用。 (C) 2008 年国际骨关节炎研究协会。由爱思唯尔有限公司出版。保留所有权利。
Objective: The development of a reliable high-throughput transfection protocol for primary human articular chondrocytes.Methods: Primary human chondrocytes were isolated from adult knee cartilage by an optimized enzymatic digestion protocol and cultivated in high-density monolayer culture for 3-5 days. Isolated chondrocytes were transfected with a green fluorescent protein (GFP)-expressing reporter construct using amaxa's Nucleofector 96-well Shuttleg System. Transfection efficiencies were measured by fluorescence activated cell sorting and cell viability was determined by an adenosine-5'-triphosphate (ATP) assay. siRNA oligonucleotides (against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) were transfected into the cells using the optimized nucleofection protocol and mRNA knockdown values were determined by a branched-DNA assay.Results: Transfection efficiencies of more than 70% of surviving cells were achieved routinely with the nucleofection protocol resented in this article. Cell viability 24 h post transfection was around 80%. The cell number used per transfection was reduced to 2 x U per sample. In addition, the protocol proved to be well suited for the transfer of siRNA molecules into primary human chondrocytes with suppression rates on the mRNA level of more than 95% (for GAPDH).Conclusions: We present the successful use of nucleofection on primary human chondrocytes using a microliter plate compatible format that for the first time allows the efficient transfection of up to 96 samples in parallel. The optimized nucleofection protocol is offering maximum substrate flexibility by allowing transfer of DNA and siRNA oligonucleotides with the same set of parameters. Moreover, the transfection procedure requires substantially lower cell numbers than single cuvette protocols and is therefore perfectly suited for applications requiring multiple experimental replicates. (C) 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.