Result 1: NK Expansion & Activation Experiment

In this particular experiment, three different conditions of NK cell activation were tested. Baseline had only the cytokine mix present. For the Cloudz, the Cloudz NK activation kit was used in addition to cytokine mix. For the Feeder-Cells condition, irradiated K562 cells were co-cultured with NK cells, also in addition to cytokine mix. Cells were stained with an antibody to CD56, a classical surface marker of NK cells and with an antibody to CD25 antigen, which is a marker of their activation. The result we got from this experiment is that NK cells activate best in the presence of feeder cells: Feeder-Cells>Baseline>Cloudz, so we decided to utilize K562 cells for our cell cultures.

Result 2: Transfection Experiment

Singlets (94%): This gate represents the percentage of cells that are considered single cells, meaning they are not clumped together or in aggregates. The fact that 94% of the cells fall into this category indicates that the majority of the cells analyzed are in a single-cell state, which is important for accurate flow cytometry measurements.

GFP Positive (18.1%): This gate represents the percentage of cells that express the green fluorescent protein (GFP), which is encoded by the transfected vector. These cells are considered GFP positive because they have successfully taken up the plasmid and expressed the GFP gene.

In summary, the flow cytometry analysis of HEK-293T cells transfected with the GFP vector indicates that the majority of the cells are in a single-cell state (94%), allowing for reliable individual cell analysis. Among these singlet cells, approximately 18.1% are GFP positive, demonstrating successful transfection and expression of the GFP gene. This information is essential for us because it proves that the plasmid we designed has some functionality and the IRES element does its work. It also proves that our transfection protocol is working although the transfection efficiency is not very big. The relatively low transfection efficiency may be due to the small concentration of plasmid used, as well as the transfection reagent. In future experiments we will modify the protocol using more plasmid and different transfection reagents to see which works best.

Result 3: Transduction Experiment

In a flow cytometry experiment aimed at assessing the transduction efficiency in iPSCs-NK, and NK-92 cells, it was found that the transduction was not successful. The flow cytometry results revealed the absence of GFP expression, indicating that the transduction process did not yield the desired outcome. This outcome suggests a need for further optimization of the transduction protocol or exploration of alternative methods to achieve the introduction and expression of the CAR-GFP synthetic gene in these cell types. A possible fact that led to this result is that the transfection efficiency was relatively low and thus not enough viral particles were produced. Furthermore, considering that the percentage of GFP-positive in transduced cells is almost zero, it is very possible that the virus could not be packaged well during the transfection of HEK-cells.