Optimizing the immune system is nowadays a new tool to treat a broad range of diseases such as leukemia, solid-tumors or HIV. The establishment of recent techniques including the generation of Chimeric antigen receptor expressing cells and CRISPR/Cas9 based genome editing give the opportunity to improve or enhance the immune response fairly convenient, thus opening the door for novel immunotherapies.
Boosting the Immune System – Steps to Take for Successful Substrate Delivery
stable expression via insertion
use of optimized protocols
(Cytotoxicity)
use of optimized protocols
Table 1: Overview on advantages and disadvantages of different substrate delivery to primary immune cells
Step 1 – Choosing the transfection method
Figure 1: Cell functionality post Nucleofection. The bar graph displays the relative functionality of mouse dendritic cells, human macrophages and human T cells post Nucleofection (Sample). Functionality is given in percent related to non transfected control (Control). Functionality was analyzed by IL-6 specific ELISA for mouse dendritic cells, TNF-a specific ELISA for human macrophages, IFN-g specific ELISA for stimulated human T cells and by flow cytometry using a CD25 specific antibody for both human T cell states (resting and stimulated). Experiments were performed on a standard NucleofectorTM Device (mouse dendritic cells) or the 96-well ShuttleTM System (human macrophages and human T cells). [3]
But not only efficiency and viability are ensured. In view of CAR expressing T-cells [4] and NK-cells [5] as well as genome editing [6] also co-transfection of different substrates is possible – with a large flexibility in size and substrate type like DNA, RNA and proteins.
By using the 4D-NucleofectorTM Technology the transfection application is also closed and scalable. It can easily be transferred from small scale format of 20-100 ul for fundamental research or screening purposes to up to 20 ml for follow-up steps like ex-vivo modifications for cell therapy without any further optimization steps.
Step 2 – Get your immune cells to toe the line
Step 3 – Ready, Steady, Substrate Delivery

In regard to the recommended Nucleofection conditions please find to the right a graph on the development of cells of the immune system (Figure 2). Table 2 below summarizes the conditions for the majority of these cells with references for the 4D-NucleofectorTM System.

(Kit/pulse)
efficiency
CD34+ cell
P3 / EO-100 (op)
Genovese et al.
[8]
P3 / EO-117 (op)
Rappocciolo et al.
[9]
Natural Killer cell
P3 / FA-100 (ihd)
P3 / EK-100 (ihd)
32%
76%
Rady et al.
[10]
T-cell,
Unstimulated
P3 / FI-115 (op)
P3 / EO-115 (op)
68%
79%
Doherty et al.
[11]
T-cell,
Stimulated
P3 / EO-115 (op)
Park et al.
[6]
P3 / EA-100 (op)
Bharaj et al.
[12]
P3 / DP-148 (op)
Daj et al.
[13]
Dendritic cell,
Immature
P3 / CB-150 (ihd)
Gerdemann et al.
[14]
Table 2: Recommended Nucleofection conditions, results and references for primary human immune cells (op: optimized protocol available, ihd: based on in-house data)
Selected References
1 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
2 Lymphocyte apoptosis: induction by gene transfer techniques
3 Nucleofection – Combining High Transfection Performance with Superior Preservation of Functionality
5 Genetic manipulation of NK cells for cancer immunotherapy: Techniques and clinical implications
6 A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors
7 Human immune system variation
8 Targeted genome editing in human repopulating haematopoietic stem cells
9 Alterations in Cholesterol Metabolism Restrict HIV-1 Trans Infection in Nonprogressors
11 Hyperactive piggyBac Gene Transfer in Human Cells and In Vivo
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