Discussion
As we know, immune cells are among the key cell groups in biological studies and clinical applications but intracellular transfer of biomolecules such as DNA and siRNA to immune cells is difficult. In the case of T cells, it has been reported that RNA transfer to the cytoplasm is not difficult, but DNA transfer is difficult and challenging due to the need to cross the nucleus. In general, immune cells exhibit low tolerance and high resistance to plasmid entry regardless of gene delivery technique (Hendel et al., 2015). Electroporation is one of the techniques that works well in the transfer of SiRNA, mRNA to T lymphocytes but there are issues such as high killing, alteration of activation process, signaling pathways and expression profiles of essential genes that need to be addressed (Bhagat, Kuntaegowdanahalli, Kaval, Seliskar, & Papautsky, 2010).
numerous published papers about electroporation all agree that there is a linear relationship between efficiency and cell death as the main challenge in cell electroporation is long-term survival and post-pulse electrical activity. According to published reports, the lack of viability, growth and proliferation after electrical pulse induction in cells is one of the major disadvantages of this method for using as gene delivery approach, especially for primary and immune cells(Lenz, Bacot, Frazier-Jessen, & Feldman, 2003).
Concerns about efficiency and safety of various cell engineering approaches have limited cell-based therapies in the past. So far, the standards of manufacturing engineered cells for gene therapy have been with the aid of viral methods that are time consuming, laborious and expensive. In addition, cells produced by the viral method require very high safety equipment and conditions as well as follow-up and patient care for up to 15 years after treatment(Roodman, 1999).
Non-viral methods of gene transfer are a good alternative to produce engineered cells because they allow the transcellular delivery of various RNA, DNA, and macromolecules, such as proteins, temporarily or permanently. In addition, these methods have advantages over the current standard method, which can save time and cost as well as ease of scale. Physical transfection methods such as electroporation are easy, fast, efficient and 80% effective in gene transfer to T cells (Levine et al., 2017) But the low cell viability in this approach has limited the tendency for this method to be used in high-scale cell therapy that requires a large number of viable and active cells.
Using microfluidic knowledge is a lucrative solution to these problems that can save time and reduce concerns about virus safety and patient outcomes. This chipset designed by us is capable of producing large number of transfected cells at a designated speed without creating a clogging cell mass. In addition, according to published paper from Justin A. Jarrell, this technique had no negative effect on cell growth and viability compared to electroporation, which was less than 40% two to three days after transfection.(Bhagat et al., 2010)
Based on the percentage of cells transfected with serpentine devices, this study highlights the importance of microfluidic techniques for transferring genes to transfected- resistant blood cells.
As noted in the preceding paragraphs, the electroporation method or its improved form, nucleofection, is actively replacing method instead of viral systems for producing engineered cells. However, in addition to high levels of cell death, it results in increased cell activation, altered cellular gene expression profiles, and consequently altered cell function in the patient’s body, which resulted from severe pulse-induced stress. In an electroporation-based study, the PD-1 gene was suppressed in T lymphocytes by Crisper following transfection, which increased cytokine production and resulted in altered expression of genes associated with cell proliferation and survival. surprisingly, in addition to altering the expression of these genes, there was a disruption in the expression of genes responsible for DNA repair, growth factor production, cell proliferation, and decreased cellular ability to counteract the tumor (Tchou et al., 2017).However, in a study using microfluidics for T-cell gene transfer, unlike nucleofection No changes in expression of important surface markers in survival, activity and function of T lymphocytes were observed.
In general, studies and results indicate that the use of microfluidic technology, unlike nucleofection methods, will not negatively affect cell viability and expression of critical genes.
The microfluidic pattern employed in this study is less efficient than the compression and constriction pattern shown in several papers on different cells, possibly due to the greater impact of membrane disruption caused by the compression created with these chipsets (Jarrell et al., 2019). However, this decrease in the delivery efficiency in the serpentine device is compensated by the high cell viability, the ability to regulate high cell concentration at low volume and the plasmid without causing clogging cell (commonly seen in constriction-based chips) compared to constriction-based devices (Stewart, Langer, & Jensen, 2018). In addition, it takes less time for gene transfer by this pattern, resulting in less stress following shear stress and turbulency inside the device. While both nucleofection and cell squeezing rely on membrane disruption, our study indicates that mechanical membrane penetration coupled with diffusion-mediated delivery significantly detracts unintended negative consequences. In terms of genetic factor consumption (here the plasmid), two-dimensional systems such as nucleofection as well as virus production methods require a large amount of high OD plasmids.
For example, for each nucleofection reaction according to the Lunza instructions, 2 million myeloma cells need 5 μg gene, whereas in this chipset even low OD plasmids has no negative effect on outcome, because this technique unlike the routine methods used in two-dimensional systems such as chemical and nucleofection-based methods is independent of Ph.
Overall, our study provides a novel approach to the field of gene transfer to cells, especially transfection-resistant cells. here, we utilized a microfluidic system which can deliver plasmid DNA to myeloma cells with 55.7% efficiency. The model designed in this study, which was first applied to blood cells, consists of multiple U-shaped loops that cell is affected by the channel walls under the influence of forces such as drag and centrifugal force. The cell membrane become porous after consecutive hits, and the plasmid containing the gene enters the cell. This device has the potential to completely removes the two primary barriers to widespread gene-modified cell therapy: cost and scalability. The device may also reduce or even eliminate many of the challenges described in this paper.
In conclusion, Gene-modified cell therapies (GMCT) have brought hope for the treatment of an array of diseases. However, the development of GMCTs have been limited by issues surrounding the most critical step in their production, the introduction of the genetic material. In this paper, we compared nucleofection to a microfluidic transfection device and we understand that microfluidic technology had dramatically fewer side effects and higher efficiency. The significant differences in viability and rate of engineered cells underscores the importance of understanding the impact of intracellular delivery systems on cell function for research and clinical applications.