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.