When we initially began our project, we strived to prove our system works in difficult to transfect HPC-7 cells. These cells are notoriously difficult to work with and require a tremendous amount of care and diligence to culture. HPC-7 cells are not commonly used in research and there is very limited experimental data available for them. As a result, we had to use trial and error in order to determine even the most basic protocols.

We began by attempting to culture the cells, using slightly different conditions and mediums, until we found what worked best. The next challenge was transfecting the cells. For that purpose, we tried four different commercial chemical reagents: lipofectamine2000, LT1, polyJet, and TransIT2020. The transfection efficiency was very low so we decided to conduct an optimization experiment. This time we used only the two most promising regents: lipofectamine2000 and TransIT2020. We checked several parameters: Different DNA: reagent ratios, cell concentrations, exposure times to the reagents and different amounts of time before measurement, all based on a paper regarding similar cells ^[1] .

In parallel, we attempted different transfections methods as we wanted to be prepared incase chemical transfection proved to be impossible. Electroporation seemed to be a promising method to transfect this kind of cells based on many papers ^{[2] [3]} .

The first electroporator we used was the Eppendorf "Multiporator" since we had one in our lab. We did not pin our hopes on it since we knew exponential decay electroporators are less suitable for stem cells. The results were, unfortunately, in line with our expectations, and we were unsuccessful.

Undeterred, we contacted various companies in an attempt to acquire a more suitable electroporator. After a month, our attempts bore fruit and we got our hands on the NEPA21 electroporator, generously supplied by Almog Diagnostics, for three weeks. Unfortunately, the electroporation process was too aggressive for these sensitive cells. We managed to achieve modest transfection rates, but almost all of the cells died as a result of the electroporation process.

Accepting failure in the electroporation experiments was very difficult for us, especially after all the time and effort we had invested. Still determined, we pressed on, attempting once more to transfect these difficult cells with chemical reagents, but with the powerful EF-1a promoter, known for its high expression in stem cells. We conducted the experiment using lipofectamine2000 and TransIT2020 and transfected an Ef1a-GFP reporter plasmid to measure our efficiency.

You can see in figure 1 that the transfection was finally successful, and we got relatively high transfection efficiency.

Figure 1: Fold change of the transfection with lipofectamine 2000 and transit 2020.

Conclusion :

For the transfection of HPC-7 cell we recommend transfecting with chemical reagents, using lipofectamine 2000, and a DNA:Reagent ratio of 1:3.