Aiming at genetically modifying T cells with designed CAR, our scientists flexibly adopt versatile methodologies to transfect CAR construction into T cells, such as packing and transfection with retrovirus or lentivirus. Since the successful manufacture of CAR-T cells is largely dependent on T cell receptor (TCR) gene transfer, this genetic modification of T lymphocytes is one of the most critical steps to generate superior CAR-T cells. Although the T cells are difficult to modify using non-viral methods like lipid-based car t plasmid transfection due to the high toxicity and low efficiency, the electroporation and several virus-based transfection systems are much more robust to complete the T-cell transfection.
Figure 1 CAR-T cell transfection approaches (Creative Biolabs)
Electroporation is emerged as a powerful tool for the genetic modification of diverse cell types based on the transient disruption of cell membrane via exposure to an electric field, which allows charged molecules to enter the cell. For instance, the square-wave pulse-based new electroporation devices, such as Lonza Nucleofector II electroporation system, manifests a high efficiency in the genetic modification of T cells with proprietary electroporation buffers and electric parameters. In detail, up to 80 % of viability and 40-60 % of expression are achieved in human T cells. On the adverse side, the main potential disadvantage of electroporation is the excessive cell death and the low transfection efficiency, which is varied with cell type and electroporation conditions (voltage, wave profile, time of the pulse, and buffer composition). Meanwhile, the high cost of Lonza compatible electroporation kits and the dependence on manufacture reagents and devices also limit the application for large scale experiments.
Retroviral (simple retrovirus, γ-retrovirus, MLV) transfection is one of the mainstays of current gene therapy approaches, which contains a reverse transcriptase to enable the integration of artificial genes into the host genome in a stable status. Particularly, the retroviral vectors can either be replication-competent or replication-defective, and the latter is the most common choice for research since the viruses are competent for additional rounds of virion replication and packaging with other genes. With a typical maximum 8-10 kB length of an allowable DNA insertion in a replication-defective viral vector, the viruses are capable of infecting the target cells and delivering the specific viral payload. Meanwhile, the MLV fails to perform the typical lytic pathway and lead to cell lysis and death. Noticeably, MLV requires actively dividing cells for transduction.
Lentiviral (complex retrovirus) transfection is also one of the mainstays of current gene therapy approaches, which enable the integration of artificial genes into the host genome in a stable status for genetical modifications in actively dividing cells as a subclass of retroviruses. More powerfully, the lentivirus has the ability to integrate foreign genes into the genome of non-dividing cells. Generally comparing to the electroporation, the virus-based transfections are relatively time-consuming and expensive, which limit a broader application in the clinical usage.
At Creative Biolabs, our scientists are able to conduct versatile cell transfection in multiple cell lines, primary cells, stem cells, hematopoietic cells, and neuronal cells. Creative Biolabs offers the most professional CAR transfection in T cells and NK cells for the most cutting-edge genetically modified immunotherapies. The customized service plans are also welcome since we could offer every service and experiment kit for every step, including plasmid construction, virus packaging, cell transfection, etc.
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