A look at a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates.
Cell lines isolated from tumours are an important tool for studying cancer in vitro. They can be used for drug development as well as for understanding the basic mechanisms underlying cancer. Transfection of cancer cell lines with different molecules, such as plasmid DNA, siRNA or mRNA, is often an integral part of this kind of research.
4D-Nucleofector System is a modular system for the efficient transfection of primary cells and cell lines
Lonza's 4D-Nucleofector System (Fig. 1A) is a modular system for the efficient transfection of primary cells and cell lines with a variety of substrates including plasmid DNA and siRNA. The 4D-Nucleofector X Unit supports transfection in two different formats.
The aluminium-free 20µl Nucleocuvette Strip (Fig. 1B) allows the transfection of low cell numbers down to 2 x 104 cells per reaction. As 16 reactions can be performed in parallel, it is well suited for optimising Nucleofection Conditions for cells lacking a ready-to-use optimised protocol.
For higher cell numbers of up to 2 x 107 cells per reaction, the same Nucleofection Conditions can be applied in the 100µl single Nucleocuvette Vessel (Fig.1C). For higher throughput needs, the 96-well Shuttle Add-on can be connected to the 4D-Nucleofector System. With this add-on, six 20µl Nucleocuvette Strips can be processed in parallel, allowing for screening applications or accelerating the optimisation of transfection parameters for many cell types.
In this study, we used the 4D-Nucleofector System in combination with the 96-well Shuttle Add-on for optimising transfection conditions for more than 30 cancer cell lines. An exemplary optimisation process is depicted for the human prostate carcinoma cell line DU 145 and the human colorectal adenocarcinoma cell line COLO 205.
Materials and methods
During the initial optimisation of Nucleofection Conditions all cell lines were cultured according to standard protocols. Adherent cell lines were harvested using trypsin (Lonza, cat. no. 17-161E). Dependent on the cell type, 1-5 x 105 cells per sample were resuspended in 20µl of the respective Nucleofector Solution SE, SF or SG containing 0.4µg of pmaxGFP Vector. Samples were transferred into a 96-well Nucleocuvette Plate and processed in parallel with 31 different programs and a no program control in the 96-well Shuttle Add-on. After transfection, 80µl of equilibrated medium was added to each sample. Dependent on cell type, 2.5-10 x 104 cells were seeded into a standard 96-well cell culture plate.
Further fine-tuning of Nucleofection Conditions can be performed (optional). A) 10 minutes post-incubation: 2 x 105 cells per sample were resuspended in 20µl of the selected Nucleofector Solution containing 0.4µg pmaxGFP Vector and pulsed with the selected Nucleofector Program. Post transfection, samples were incubated for 10 minutes in Nucleofector Solution prior to adding 80µl of equilibrated cell culture medium. B) DNA Titration: 2 x 105 cells per sample were resuspended in 20µl of the selected Nucleofector Solution containing 0.4µg, 1µg or 5µg of pmaxGFP Vector. After transfection - if indicated by fine-tuning step A - samples were incubated for 10 minutes in Nucleofector Solution prior to adding 80µl of equilibrated cell culture medium.
Transfer to 100 µl Single Nucleocuvette Vessels. Based on the cell number used in the 20µl format, a five-fold increased cell number per sample was resuspended in 100µl of the respective Nucleofector Solution SE, SF or SG containing five times the amount of pmaxGFP Vector. Samples were transferred into the 100µl single Nucleocuvette Vessel and processed with the Nucleofector Program identified in the 20µl Nucleocuvette Strip. After transfection, 400µl of equilibrated medium was added to each sample. Dependent on cell type, 2.5-10 x 104 cells were seeded into a standard 96-well cell culture plate.
Analysis
Twenty-four hours after transfection, the percentage of maxGFP-positive, propidium-iodide negative cells was determined using flow cytometry (FACSCalibur, Becton Dickinson) according to standard procedures. Cell viability was determined using the ViaLight Plus BioAssay Kit (Lonza, cat. no. LT07-321), according to the protocol. Cell viability is expressed as percentage viability compared to the non-transfected no program controls.
Results
Using the 4D-Nucleofector System in combination with the 96-well Shuttle Add-on, we found that the human prostate carcinoma cell line DU 145 can be easily transfected. Best results were obtained with Nucleofector Solution SE and Program CA-137, resulting in a transfection efficiency of 78 per cent (Fig.2A) with a cell viability of 55 per cent (Fig.2B). These results show that efficient Nucleofection Conditions for cancer cell lines can often be easily determined in a single experiment.
For the human colorectal adenocarcinoma cell line COLO 205, the initial optimisation experiment achieved maximal transfection efficiencies of ~30 per cent (Fig.3A) with program-dependent cell viabilities (Fig.3B). Thus, we performed two further program fine-tuning rounds and identified Nucleofector Program DP-113, in combination with Nucleofector Solution SG and a cell seeding density of 1 x 105 cells in 96-well, as the best selections under the given experimental conditions (data not shown). However, transfection efficiency still remained below 30 per cent with a cell viability of 50 per cent.
In order to improve transfection efficiency and viability, further optimisation experiments were performed. Incubation of COLO 205 cells for 10 minutes in Nucleofector Solution post transfection increased transfection efficiency and cell viability.
An additional increase of transfection efficiency was observed when increasing the amount of plasmid DNA per reaction. Cell viability remained above 60 per cent compared to the no program control with DNA amounts up to 1µg per reaction. With a higher DNA concentration, a drop in cell viability was observed.
One important feature of the 4D-Nucleofector X Unit is the transferability of conditions from the 20µl Nucleocuvette Strips (for low cell numbers and higher throughput) to the 100µl single Nucleocuvette Vessels (for higher cell numbers).
For the latter, the same Nucleofection Conditions can be applied using a five times higher cell number and substrate amount.
Summary
Excellent transfection efficiencies of up to 99 per cent combined with high cell viability can be obtained with the 4D-Nucleofector System for different adherent and suspension cancer cell lines.
With the 96-well Shuttle Add-on, six 20µl Nucleocuvette Strips can be processed in parallel enabling screening applications as well as offering a convenient, effective and time-saving approach when optimising Nucleofection Conditions for multiple cells lacking a ready-to-use Optimised Protocol.
In many cases, the optimal Nucleofection Conditions can already be determined during the first optimisation round. The same protocol can be applied for 100µl and 20µl transfection volumes, allowing the transfection of variable cell numbers.
For more information at www.scientistlive.com/eurolab
The authors are Jenny Schroeder, Ludger Altrogge, Elke Lorbach, Sabine Schaepermeier, Meike Weigel, Gina Andretta-Beu, Stefanie Buesch, Tamara Grabeck, Alexandra Krumnow, Sonja Spicker and Andrea Toell who are with Lonza Cologne GmbH, Koeln, Germany. Srinivasan Kokatam, Sampada Kallol and Preeti Kapoor are with Lonza India PVT Ltd, Hyderabad, India
