Multipotent mesenchymal stromal cells: optimization and comparison of five cationic polymer-based gene delivery methods
Multipotent mesenchymal stromal cells: optimization and comparison of five cationic polymer-based gene delivery methods
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DOI：
10.1080/14653240802474307
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发表时间：
2008-01-01
期刊：
影响因子：
4.5
通讯作者：
Zeinali, S.
中科院分区：
文献类型：
--
作者：
Gheisari, Y.;Soleimani, M.;Zeinali, S.
背景多能间充质基质细胞（MSC）是再生医学领域有希望的候选者，并且在一些研究中已经进行了基因改造，以给这些细胞带来新的特性或增强现有的特性。此外，MSC已被用作基因递送载体。这些实验的成功取决于选择合适的基因传递至细胞的方法。方法MSC从大鼠骨髓中分离；通过分化实验以及细胞表面标记染色来检查它们的真实性。使用系统方法优化五种基于阳离子聚合物的基因递送方法（Lipofectamin2000、Effecten、Superfect、Polyfect 和 FuGENE HD）。 48小时后，使用9至12种不同比例和量的DNA/转染试剂，在3至6个单独的实验中测量每种方法的转染率和细胞活力。结果分离的MSC成功分化为成骨细胞、脂肪细胞和成软骨细胞。它们对大鼠 CD90 和 CD73 标记物呈阳性，对 CD31、CD45、CD11b 和 VEGFR2 标记物呈阴性。最佳条件下的平均转染率为 5.18 +/- 2.72 (FuGENE HD)、8.72 +/- 4.52 (Effecten)、9.59 +/- 3.12 (Superfect)、16.29 +/- 7.44 (Polyfect) 和 19.60 +/- 3.12 (Lipofectamine 2000)。所有试剂的毒性均低于 20%。讨论在优化条件下使用 Lipofectamine 2000 和 Polyfect 可以实现中等水平的转染和可接受的细胞活力。使用简单的 FSC-SSC 门控对活细胞进行门控和分选可以改善结果。
BackgroundMultipotent mesenchymal stromal cells (MSC) are promising candidates in the field of regenerative medicine and in several studies have been genetically modified to bring a new property to or enhance an existing one in these cells. Furthermore, MSC have been used as gene delivery vehicles. The success of these experiments depends on selecting an appropriate method for gene delivery to the cells.MethodsMSC were isolated from rat bone marrow; their authenticity was checked by differentiation experiments as well as staining for cell-surface markers. A systematic approach was used to optimize five cationic polymer-based gene delivery methods (Lipofectamin2000, Effecten, Superfect, Polyfect and FuGENE HD). The transfection yield and cell viability of each method was measured after 48 h in three to six separate experiments with nine to 12 different ratios and amounts of DNA/transfection reagent.ResultsThe isolated MSC were successfully differentiated to osteoblasts, adipocytes and chondroblasts. They were positive for rat CD90 and CD73 and negative for CD31, CD45, CD11b and VEGFR2 markers. The average transfection rates with optimum conditions were 5.18 +/- 2.72 (FuGENE HD), 8.72 +/- 4.52 (Effecten), 9.59 +/- 3.12 (Superfect), 16.29 +/- 7.44 (Polyfect) and 19.60 +/- 3.12 (Lipofectamine 2000). The toxicity was below 20% for all reagents.DiscussionModerate levels of transfection and acceptable cell viability could be achieved using Lipofectamine 2000 and Polyfect in optimized conditions. The results could be improved by gating and sorting live cells using a simple FSC-SSC gating.