Optimized DNA electroporation for primary human T cell engineering
Abstract Background Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared wi...
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| Format: | Article |
| Language: | English |
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BMC
2018-01-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://link.springer.com/article/10.1186/s12896-018-0419-0 |
| _version_ | 1818033806070775808 |
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| author | Zhang Zhang Shunfang Qiu Xiaopeng Zhang Wei Chen |
| author_facet | Zhang Zhang Shunfang Qiu Xiaopeng Zhang Wei Chen |
| author_sort | Zhang Zhang |
| collection | DOAJ |
| description | Abstract Background Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation. Here, we developed and optimized an electro-transfection method for the production of engineered chimeric antigen receptor (CAR)-T cells. Results Stimulation of T cells had the greatest effect on their transfection, with stimulation of cells for up to 3 days substantially improving transfection efficiency. Additionally, the strength of the external electric field, input cell number, and the initial amount of DNA significantly affected transfection performance. The voltage applied during electroporation affected plasmid permeation and was negatively correlated with the number of viable cells after electroporation. Moreover, higher plasmid concentration increased the proportion of positively transfected cells, but decreased cell viability, and for single-activated cells, higher cell density enhanced their viability. We evaluated the effects of two clinically relevant factors, serum supplementation in the culture medium and cryopreservation immediately after the isolation of peripheral blood lymphocytes. Our findings showed that our protocol performed well using xeno-free cultured, fresh T cells, with application resulting in a lower but acceptable transfection efficiency of cells cultured with fetal bovine serum or thawed cells. Furthermore, we described an optimized procedure to generate CAR-T cells within 6 days and that exhibited cytotoxicity toward targeted cells. Conclusions Our investigation of DNA electro-transfection for the use in human primary T cell engineering established and validated an optimized method for the construction of functional CAR-T cells. |
| first_indexed | 2024-12-10T06:29:07Z |
| format | Article |
| id | doaj.art-707ab13fcc434650b55e33a1ded7bfd7 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-10T06:29:07Z |
| publishDate | 2018-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-707ab13fcc434650b55e33a1ded7bfd72022-12-22T01:59:07ZengBMCBMC Biotechnology1472-67502018-01-011811910.1186/s12896-018-0419-0Optimized DNA electroporation for primary human T cell engineeringZhang Zhang0Shunfang Qiu1Xiaopeng Zhang2Wei Chen3Laboratory of Vaccine and Antibody Engineering, Beijing Institute of BiotechnologyLaboratory of Vaccine and Antibody Engineering, Beijing Institute of BiotechnologyLaboratory of Vaccine and Antibody Engineering, Beijing Institute of BiotechnologyLaboratory of Vaccine and Antibody Engineering, Beijing Institute of BiotechnologyAbstract Background Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation. Here, we developed and optimized an electro-transfection method for the production of engineered chimeric antigen receptor (CAR)-T cells. Results Stimulation of T cells had the greatest effect on their transfection, with stimulation of cells for up to 3 days substantially improving transfection efficiency. Additionally, the strength of the external electric field, input cell number, and the initial amount of DNA significantly affected transfection performance. The voltage applied during electroporation affected plasmid permeation and was negatively correlated with the number of viable cells after electroporation. Moreover, higher plasmid concentration increased the proportion of positively transfected cells, but decreased cell viability, and for single-activated cells, higher cell density enhanced their viability. We evaluated the effects of two clinically relevant factors, serum supplementation in the culture medium and cryopreservation immediately after the isolation of peripheral blood lymphocytes. Our findings showed that our protocol performed well using xeno-free cultured, fresh T cells, with application resulting in a lower but acceptable transfection efficiency of cells cultured with fetal bovine serum or thawed cells. Furthermore, we described an optimized procedure to generate CAR-T cells within 6 days and that exhibited cytotoxicity toward targeted cells. Conclusions Our investigation of DNA electro-transfection for the use in human primary T cell engineering established and validated an optimized method for the construction of functional CAR-T cells.http://link.springer.com/article/10.1186/s12896-018-0419-0ElectroporationT cell engineeringChimeric antigen receptor modified T cells |
| spellingShingle | Zhang Zhang Shunfang Qiu Xiaopeng Zhang Wei Chen Optimized DNA electroporation for primary human T cell engineering BMC Biotechnology Electroporation T cell engineering Chimeric antigen receptor modified T cells |
| title | Optimized DNA electroporation for primary human T cell engineering |
| title_full | Optimized DNA electroporation for primary human T cell engineering |
| title_fullStr | Optimized DNA electroporation for primary human T cell engineering |
| title_full_unstemmed | Optimized DNA electroporation for primary human T cell engineering |
| title_short | Optimized DNA electroporation for primary human T cell engineering |
| title_sort | optimized dna electroporation for primary human t cell engineering |
| topic | Electroporation T cell engineering Chimeric antigen receptor modified T cells |
| url | http://link.springer.com/article/10.1186/s12896-018-0419-0 |
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