2Department of Clinical Pathology, Faculty of Veterinary Medicine, Benha University, Qalioubeya, Egypt
We introduce a simple and cost-effective technique for the efficient transient transfection of suspension cells through the coating of cell culture plates with chicken egg white. The method is characterized by minimal toxicity and correct gene expression. Although established for the mast cell line C2 and the myeloid cell line HL-60 the technique is likely to be used for other suspension cell lines as well. Our improved protocol demonstrates that the overcoming of the non-adherent character of suspension cells substantially supports transfection efficiency, and that this can be achieved by a simple chicken egg white-based system.
Transfection of suspension cells has proven to be very difficult using conventional methods. Here, we present a simple and time-saving new transfection protocol wherein cell culture plates coated with chicken egg white are seeded with suspension cells prior to transfection. Our results demonstrate that coupling egg white coatings with commercially available transfection reagents leads to high transfection efficiency with suspension cell lines including canine mastocytoma C2 and the human myeloid cell line HL-60. This new approach, which should prove applicable to a wide range of cell lines, solves a crucial problem for researchers working with suspension cells.
Manipulation of cells via insertion of foreign DNA into them is of crucial importance for biomedical research and engineering. However, not all transfection methods can be applied to all types of cells, and there is a wide variation with respect to the achieved cell viability and the expression level of the gene of interest. The determination of the most appropriate transfection technique depends on several factors, such as the cell type or the adherence ability of the cells.
Suspension cell lines have traditionally proven to be very difficult to transfect. The common reason for this is a reduced attachment of the transfection complex, a mixture of the gene of interest with a mostly liposome-based transfection reagent, to the surface of the cells that results in a reduced uptake of the target DNA (1-3). Hence, there is a need for a simple and cost-effective technique to manipulate suspension cells. Here we present an efficient transfection method for suspension cell lines (please see our protocol for details).
The canine mast cell line C2 and the human myeloid cell line HL-60 were used. The mast cell line is derived from isolated canine mastocytoma tumor cells, which were passaged serially 30–50 times in athymic nude mice (4, 5). The characteristics of this cell line are comparable with other mast cell preparations like human lung mast cells, peritoneal- or bone marrow-derived mast cells of rats (4, 5). The human myeloid cell line HL-60, a predominantly neutrophilic promyelocyte precursor, was derived from a 36-year-old woman with acute promyelocytic leukemia (6). The gene of interest introduced was the enzyme phospholipase D (PLD) in its two isoforms PLD1 and PLD2. The C2 mast cells were transfected with the plasmids pEGFP-hPLD1 and pEGFP-mPLD2. The HL-60 cells were transfected with the plasmid pEGFP-hPLD1 and the empty plasmid pEGFP-C1. Transfection efficiency was monitored by confocal microscopy based on the enhanced green fluorescence protein (EGFP) tagging of the phospholipase (five independent counts per field for each well with 100 cells counted per field).
To overcome the nonadherent character of the cells, we coated the cell culture plates. Coating materials commonly used are collagen and poly-L-lysine (7). However, poly-L-lysine is reported to activate mast cells via a distinct Fc3RI-independent pathway (8) and is therefore not suitable for our purposes. Two different coating materials were tested: (i) chicken egg white (9) and (ii) collagen (collagen solution type 1 from calf skin; Sigma-Aldrich, Munich, Germany). To isolate the egg white, chicken eggs were disinfected superficially using 70% ethanol, then broken under a laminar flow hood and poured into a 50-mL Falcon tube. Cell culture plates (12-well; 22 mm2 surface area/well) were covered with egg white (0.75 mL/well) and carefully heated on a heat block at 58°C. After 60 min, the chicken egg white became semi-solid and adhered to the bottom of the wells. Excess egg white was washed out using PBS. For coating with collagen, the collagen was pipeted into the cell culture plates at a concentration of 6–10 µg/cm2 and incubated overnight at 37°C. Unbound collagen was removed, and wells were washed using PBS. The C2 cells were seeded at 5 × 105 cells/well, and the HL-60 cells were seeded at 4 × 105 cell/well regardless the coating material used.
Microscopic observations revealed that both chicken egg white and collagen promote cell attachment to the bottom of the cell culture plates. The cells attached to the surface, but did not invade the coating material. There was no effect of the coating on the morphological character of the cells regardless of the coating material used. To examine the impact of the coating on mast cell function, the ability of the C2 cells to degranulate following stimulation was investigated. Histamine release and the activity of the enzyme β-hexosaminidase were analyzed by means of high-performance liquid chromatography (HPLC) (10) and spectrophotometry (11), respectively. The wasp venom peptide toxin mastoparan was used for stimulation (10). There was no interfering effect of the coating on mast cell degranulation (Figure 1). Both spontaneous and mastoparan-stimulated histamine and β-hexosaminidase release were equal for C2 mast cells cultured on uncoated or coated cell culture plates regardless of the coating material used (Figure 1). In summary, coating of cell culture plates provides a simple and efficient technique to increase adherence of suspension cells. We found no qualitative differences between the two coating materials tested. However, we recommend chicken egg white for its cost-effectiveness.
In the next step, we evaluated the applicability of commercially available transfection reagents to efficiently incorporate a gene of interest into the C2 mast cells and the HL-60 cells. Five different transfection reagents were compared: Turbofect transfection reagent (Fermentas, Sankt Leon Rot, Germany), GenePORTER 3000 reagent (Genlantis, San Diego, CA, USA), GenePORTER Gold reagent (Genlantis, San Diego, CA, USA), X-tremeGENE DNA transfection reagent (Roche, Mannheim, Germany), and X-tremeGENE HP transfection reagent (Roche, Mannheim, Germany). All transfection reagents were used according to the manufacturer's instructions. Successfully transfected cells were identified by means of confocal microscopy based on their green fluorescence due to the incorporation of the GFP fusion plasmid. Effective transfection of the C2 mast cells with minimal cytotoxic effects was solely obtained with the use of Turbofect transfection reagent. For the HL-60 cells, the highest transfection efficiency combined with high cell viability was found for the X-tremeGENE HP transfection reagent. Nevertheless, for both cell lines tested, a high reagent to DNA ratio (3:1) was found to be considerably more effective than a low reagent to DNA ratio (2:1). Moreover, coating of cell culture plates significantly increased the number of successfully transfected cells for both cell lines (Figure 2). The positive effect of the coating was especially apparent for C2 cells and HL-60 cells, which had been cultured on chicken egg white–coated plates (Figure 2). For C2 cells, the evaluation of 20 independent transfection experiments revealed the percentage of successfully transfected cells to be 5%, 20%, and 50% in uncoated, collagen-coated, and egg white–coated cell culture plates, respectively (Figure 2). For HL-60 cells, the evaluation of 10 independent transfection experiments revealed the percentage of successfully transfected cell to be 2%, 35%, and 60% in uncoated, collagen-coated and egg white–coated cell culture, respectively (Figure 2). Transfection variability was low, regardless of the type of eggs used or the supplier. Of particular importance, the GFP fusion plasmids are expressed in a physiologically correct way. In unstimulated cells, PLD1 is known to be localized within intracellular vesicular structures, whereas PLD2 is known to be localized at the plasma membrane (12). Accordingly, transfection of the C2 cells and the HL-60 cells with the pEGFP-hPLD1 plasmid resulted in an intracellular distribution of the green fluorescence (Figure 3, A and C). Transfection of the mast cells with the pEGFP-mPLD2 plasmid, in contrast, led to a green fluorescence at the cell surface (Figure 3B). Moreover, we did not observe any differences in the responsiveness of the cells to stimulation. The stimulated histamine and β-hexosaminidase release of transfected C2 corresponded to the histamine and β-hexosaminidase release determined for nontransfected mast cells (data not shown).
Taken together, we introduce here a simple and cost-effective technique for the efficient transient transfection of the suspension mast cell lines C2 and HL-60. The method is characterized by minimal toxicity and correct gene expression. Although established for the cell lines C2 and HL-60, the technique is likely to be used for other suspension cell lines as well. Our improved protocol shows that overcoming the nonadherent character of suspension cells substantially supports transfection efficiency and that this can be achieved by a simple and cheap chicken egg white–based system. It can be speculated that the trapping of the cells at the bottom of the cell culture plate allows the transfection complex to accumulate on the cells, thus promoting DNA uptake and expression. So far, collagen has been the gold standard for cell culture plate coating (13). However, our data clearly show that coating of cell culture plates with chicken egg white yields better results combined with lower costs. Nevertheless, it is important to note that the cellular growth phase is of fundamental relevance for the success of the transfection experiment. A high transfection rate can only be achieved when the cells are in the logarithmic growth phase. Bearing in mind this aspect, our protocol might allow the directed manipulation of suspension cells in a reliable, simple, and cost-effective manner.
We would like to thank the DAAD (German Academic Exchange Service) for financial support of S.B., grant no. A/09/92498.
The authors declare no competing interests.
Address correspondence to Julia Schumann, Institute of Physiological Chemistry, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany. Email: [email protected]
1.) Montier, T., P. Delépine, K. Le Ny, Y. Fichou, M. Le Bris, E. Hardy, E. Picquet, J.C. Clément. 2004. KLN-5: a safe monocationic lipophosphoramide to transfect efficiently haematopoietic cell lines and human CD34 cells. Biochim. Biophys. 1665:118-133. 2.) Praveen, B.B., D.J. Stevenson, M. Antkowiak, K. Dholakia, and F.J. Gunn-Moore. 2011. Enhancement and optimization of plasmid expression in femtosecond optical transfection. J. Biophotonics 4:229-235. 3.) Landauer, K., M. Dürrschmid, H. Klug, S. Wiederkum, G. Blüml, and O. Doblhoff-Dier. 2002. Detachment factors for enhanced carrier to carrier transfer of CHO cell lines on macroporous microcarriers. Cytotechnology 39:37-45. 4.) DeVinney, R., and W.M. Gold. 1990. Establishment of 2 dog mastocytoma cell-lines in continuous culture. Am. J. Respir. Cell Mol. Biol. 3:413-420. 5.) Lazarus, S.C., L.J. Mccabe, J.A. Nadel, W.M. Gold, and G.D. Leikauf. 1986. Effects of mast cell derived mediators on epithelial cells in canine trachea. Am. J. Physiol. 251:387-394. 6.) Gallagher, R., S. Collins, J. Trujillo, K. McCredie, M. Ahearn, S. Tsai, R. Metzgar, G. Aulakh. 1979. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54:713-733. 7.) Zhang, Y., L. Wang, J. Zhu, Y. Hu, W. Xing, and J. Cheng. 2011. Real-time monitoring of extracellular matrix-mediated PC12 cell attachment and proliferation using an electronic biosensing device. Biotechnol. Lett. 34:397-404. 8.) Deng, Z., T. Zink, H.Y. Chen, D. Walters, F.T. Liu, and G.Y. Liu. 2009. Impact of actin rearrangement and degranulation on the membrane structure of primary mast cells: a combined atomic force and laser scanning confocal microscopy investigation. Biophys J. 96:1629-1639. 9.) Kaipparettu, B.A., I. Kuiatse, B. Tak-Yee Chan, M.B. Kaipparettu, A.V. Lee, and S. Oesterreich. 2008. Novel egg white-based 3-D cell culture system. BioTechniques 2:165-170. 10.) Gueck, T., J.R. Aschenbach, and H. Fuhrmann. 2002. Influence of vitamin E on mast cell mediator release. Vet. Dermatol. 13:301-305. 11.) Fowler, C.J., M. Sandberg, and G. Tiger. 2003. Effects of water-soluble cigarette smoke extracts upon the release of β-hexosaminidase from RBL-2H3 basophilic leukaemia cells in response to substance P, compound 48/80, concanavalin A and antigen stimulation. Inflamm. Res. 52:461-469. 12.) Sarri, E., R. Pardo, A. Fensome-Green, and S. Cockcroft. 2003. Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid. Biochem. J. 369:319-329. 13.) Haddow, D.B., S. MacNeil, and R.D. Short. 2006. A cell therapy for chronic wounds based upon a plasma polymer delivery surface. Plasma Process. Polym. 3:419-430.