Impact of cell density on transient transfection of HEK293 cells culture.

Recombinant proteins (r-proteins) are becoming increasingly popular both on the biotechnology market and in the pharmaceutical industry. An r-protein may be produced effectively through the transient transfection method, used also in this thesis. Cell culture 293HEK EBNA 1 was temporarily genetically modified (i.e. transiently transfected) by the gene of interest (AIM) using polyethyleneimine (PEI) as a transfection agent in two different transfection densities (i.e. “current” density and “test” density). The current transfection density was 20x106 cells/ml and the higher test density was 30x106 cells/ml. The aim of this Bachelor’s thesis was to determine whether the higher test transfection density of cells in transient transfection (as compared to the lower commonly used “current” transfection density) leads to higher yields of r-protein. A gene for the AIM protein (apoptotic inhibitor secreted by tissue macrophages) was temporarily inserted in cell culture 293 HEK EBNA. The successfully transfected cell cultures were purified on the Ni-NTA matrix and the amount of the r-protein obtained was quantified. It was ascertained that the r-AIM protein was always found in higher concentrations in the suspensions of the current transfection density of 20x106 cells/ml. Partial results showed that cultivation of cell culture 293HEK EBNA 1 in rectangular vessels leads to a higher cell density and viability in the course of the r-protein production as compared to the cultivation of the production culture in tubes