Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells
Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce...
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| Format: | Article |
| Language: | English |
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Razi Vaccine and Serum Research Institute
2021-11-01
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| Series: | Archives of Razi Institute |
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| Online Access: | https://archrazi.areeo.ac.ir/article_125475_4458223ab21d35a95faed7f94c727915.pdf |
| _version_ | 1818471124562870272 |
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| author | M Taghavi A Parham H Dehghani H Naderi-Meshkin |
| author_facet | M Taghavi A Parham H Dehghani H Naderi-Meshkin |
| author_sort | M Taghavi |
| collection | DOAJ |
| description | Conventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce antitumor agents. However, MSCs are difficult-to-transfect cells, and introducing the exogenous therapeutic gene into MSCs is challenging yet needs improvement. Transfection using chemical reagents, including Lipofectamine, is more convenient and less cytotoxic compared with different methods of introducing exogenous DNA into MSCs. Nonetheless, the major limitation of Lipofectamine is low transfection efficiency in MSCs. Therefore, the purpose of this study was to evaluate and suggest the optimum quantities of lipoplex components to enhance the transfection efficiency of human adipose tissue-derived MSCs (hASCs). Finding the best transgene expression time point and the optimum concentration of G-418 for antibiotic-based selection was another goal of this study. hASCs were transfected in a series of experiments with altering the quantities of Lipofectamine LTX® (Lip-LTX), the related “PLUS” reagent, and a plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (eGFP). After transfection, the percentage of eGFP-expressing cells was evaluated using fluorescence microscopy and ImageJ software in 12-hour intervals for 48 hours. Also, the viability of hASCs exposed to different concentrations of G-418 was measured using an MTT assay. The results demonstrated that a combination of 2 µL Lip-LTX, 0.75 µL of its “PLUS” reagent, and 0.75 g pDNA (6484 bp) improve the transfection efficiency of hASCs (23.75%), and the best period for evaluation of fluorescence for these cells is 12 to 24h post-transfection. Also, the optimum concentration of G-418 for antibiotic-based selection of hASCs was 0.25mg/mL. In conclusion, this study indicates that the setting up of optimized quantities of lipoplex components and the golden time of evaluation for transgene expression could increase the possibility of transgene expression in hASCs before beginning research and clinical application. Also, the definition of optimal dose of selection antibiotic for purification of transfected hASCs seems to be necessary for maximum transgene expression effects in the cell population. |
| first_indexed | 2024-04-13T21:47:30Z |
| format | Article |
| id | doaj.art-c5a5aac3538c460ab6b1a8ac67e0865a |
| institution | Directory Open Access Journal |
| issn | 0365-3439 2008-9872 |
| language | English |
| last_indexed | 2024-04-13T21:47:30Z |
| publishDate | 2021-11-01 |
| publisher | Razi Vaccine and Serum Research Institute |
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| series | Archives of Razi Institute |
| spelling | doaj.art-c5a5aac3538c460ab6b1a8ac67e0865a2022-12-22T02:28:32ZengRazi Vaccine and Serum Research InstituteArchives of Razi Institute0365-34392008-98722021-11-017651315132510.22092/ari.2021.354659.1645125475Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem CellsM Taghavi0A Parham1H Dehghani2H Naderi-Meshkin3Division of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, IranDivision of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, IranDivision of Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, IranStem Cells and Regenerative Medicine Research Group, Iranian Academic Center for Education, Culture, and Research (ACECR), Khorasan Razavi Branch, Mashhad, IranConventional cancer therapies, including surgery, radiotherapy, and chemotherapy, are not tumor site-specific and have cytotoxic and harmful side effects for normal cells. Mesenchymal stem cells (MSCs), due to their tumor-tropism migration property, are a promising alternative to deliver and produce antitumor agents. However, MSCs are difficult-to-transfect cells, and introducing the exogenous therapeutic gene into MSCs is challenging yet needs improvement. Transfection using chemical reagents, including Lipofectamine, is more convenient and less cytotoxic compared with different methods of introducing exogenous DNA into MSCs. Nonetheless, the major limitation of Lipofectamine is low transfection efficiency in MSCs. Therefore, the purpose of this study was to evaluate and suggest the optimum quantities of lipoplex components to enhance the transfection efficiency of human adipose tissue-derived MSCs (hASCs). Finding the best transgene expression time point and the optimum concentration of G-418 for antibiotic-based selection was another goal of this study. hASCs were transfected in a series of experiments with altering the quantities of Lipofectamine LTX® (Lip-LTX), the related “PLUS” reagent, and a plasmid DNA (pDNA) expressing the enhanced green fluorescent protein (eGFP). After transfection, the percentage of eGFP-expressing cells was evaluated using fluorescence microscopy and ImageJ software in 12-hour intervals for 48 hours. Also, the viability of hASCs exposed to different concentrations of G-418 was measured using an MTT assay. The results demonstrated that a combination of 2 µL Lip-LTX, 0.75 µL of its “PLUS” reagent, and 0.75 g pDNA (6484 bp) improve the transfection efficiency of hASCs (23.75%), and the best period for evaluation of fluorescence for these cells is 12 to 24h post-transfection. Also, the optimum concentration of G-418 for antibiotic-based selection of hASCs was 0.25mg/mL. In conclusion, this study indicates that the setting up of optimized quantities of lipoplex components and the golden time of evaluation for transgene expression could increase the possibility of transgene expression in hASCs before beginning research and clinical application. Also, the definition of optimal dose of selection antibiotic for purification of transfected hASCs seems to be necessary for maximum transgene expression effects in the cell population.https://archrazi.areeo.ac.ir/article_125475_4458223ab21d35a95faed7f94c727915.pdfgenetic engineeringmesenchymal stem cellstransfectionlipofectamine ltx |
| spellingShingle | M Taghavi A Parham H Dehghani H Naderi-Meshkin Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells Archives of Razi Institute genetic engineering mesenchymal stem cells transfection lipofectamine ltx |
| title | Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells |
| title_full | Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells |
| title_fullStr | Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells |
| title_full_unstemmed | Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells |
| title_short | Optimizing Lipofectamine LTX Complex and G-418 Concentration for Improvement of Transfection Efficiency in Human Mesenchymal Stem Cells |
| title_sort | optimizing lipofectamine ltx complex and g 418 concentration for improvement of transfection efficiency in human mesenchymal stem cells |
| topic | genetic engineering mesenchymal stem cells transfection lipofectamine ltx |
| url | https://archrazi.areeo.ac.ir/article_125475_4458223ab21d35a95faed7f94c727915.pdf |
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