Peripheral blood derived mononuclear cells (PBMCs) are important mediators involved in immune surveillance and are actively explored as the foundation of a wide range of therapies . It is convenient to isolate the cells from patients and re-administer them after desired manipulations. There are various efforts to genetically modify the cells using vectors that permanently integrate their cargo into cellular genome. Transient expression of transgenes, on the other hand, is more desirable to exert some control over the expression kinetics . The messenger RNA (mRNA) has emerged as the reagent of choice for modifying PBMC since it can be processed in cytoplasm and yield therapeutic proteins in large quantities in a short period of time . Non-viral delivery of mRNA minimizes any effects on the host genome and leads to a safer intervention. Transfection reagents developed by RJH Biosciences are particularly suitable for this application since they are highly compatible with human cells and achieve elevated levels of delivery with mRNAs. The transfection reagents are effective in different tissue culture media as well as in high serum concentration (up to 50%), so that they can be effectively used with a range of immune cells. This application note summarizes experience with transfection of PBMC with plasmid DNA (pDNA) and mRNA and using the speciality reagents developed by RJH Biosciences.
To assess efficiency of mRNA-Fect in suspension-growing cells, lymphoblastic K562 cell line derived a female chronic myeloid leukemia patient and monocytic THP-1 cell line derived from a male acute myocytic leukemia patient were initially used. These are common cell lines that are easily propagated in culture for various experimental purposes. Using a GFP-expressing mRNA from a commercial vendor, cells were transfected with a commercial lipofection reagent and mRNA-Fect. GFP expression was investigated after 2 days. As shown in Figure 1, it was typical to obtain 40-60% transfection efficiency in the cell lines, which was higher for mRNA-Fect than a leading commercial lipofection reagent.
PBMCs from two sources (donors) were transfected without and with CD3/CD28 activation for 24 hours, after which the cells were transfected with pDNA or mRNA complexes formed with mRNA-Fect. Transfection without activation did not lead to significant level of transfection with either mRNA or pDNA complexes. With activation, PBMCs from both sources gave significant GFP expression, although some variation depending on the source was evident.
Figure 2. Microscopy images of PBMCs (from two separate donors) transfected with pGFP and mGFP complexes formed with mRNA-Fect. Top row shows the transfection results with CD3/CD28 activated cells, while the bottom row shows the results from inactivated cells. Pictures are overlays of DAPI-stained and GFP expressing cells.
In a separate experiment, PBMCs from another donor were activated with PMA/IO and CD3/CD28 and the extent of transfection was measured after 2 days of transfecting the cells with mGFP complexes of mRNA-Fect. The results are summarized in Figure 3. Similar to the results seen in the microscopy pictures in Figure 2, the inactivated cells did not give any transfection with the mGFP complexes. Both PMA/IO and CD3/CD28 activated cells gave significant transfection, with CD3/CD28 activated cells giving significantly higher levels of GFP expression in the modified cells.
Figure 3. Flow cytometry analysis of PBMCs transfected with mGFP complexes formed with mRNA-Fect. Left graph shows the percentage of GFP-positive cells while the right graph shows the extent of GFP fluorescence per cell. The cells were either inactivated, or activated with PMA/IO and CD3/CD28 combination. Note that PMA/IO activated cells gave some fluorescence even in the absence of transfection. The CD3/CD28 activated cells gave much higher GFP fluorescence per cell compared to PMA.IO activated cells.