Coming soon - transfection of cells in adherence using the Amaxa Nucleofector 96-well Shuttle system

Introduction

Primary cells or cell lines derived from solid tissue, eg endothelial, epithelial or neuronal tissue, typically grow in adherence when transferred into cell culture.

Transfection of such cells can be a challenge as commercially available transfection methods have their limitations. Lipid-mediated methods enable transfection of cells in adherence, but do not achieve high efficiencies for hard-to-transfect cell lines and primary cells. For the latter, viral transduction can be efficient, but also requires experience and effort in generating the viruses and furthermore, holds some safety concerns. The Amaxa Nucleofector Technology allows for very efficient transfection of a wide variety of hard-to-transfect cell lines and primary cells, such as stem cells and neurons, combined with high viability and functionality, but as an electroporation-based method, it has thus far required cells to be in suspension for transfection.

Now, following extensive R&D, the Nucleofector technology can also claim to be the first efficient non-viral method for transfection of cell lines and primary cells in adherence. Thus, cells can be kept in their physiological state for transfection.

Lonza’s new Amaxa 96-well Nucleofector AD Kits for use with the Nucleofector 96-well Shuttle System contain special 96-well Nucleocuvette AD Plates, a modified version of the currently available 96-well Nucleocuvette Plates. The modified plates allow for culturing of cells comparable to standard cell culture plates and transfection can be performed at any time point during this culturing period. Furthermore, cells can be analyzed by transmission light and fluorescence microscopy as well as by absorption, luminescence or fluorescence readers.

Cell culturing in 96-well Nucleocuvette Plates

Adherent cells are transferred into the 96-well Nucleocuvette AD Plate either from a normal culture plate after trypsinization or directly after cell isolation (e.g. neurons) and can be cultured up to 2 weeks in these plates. The modified geometry of the 96-well Nucleocuvette AD Plates would also enable culturing of neurons with glial co-culture using transwell inserts.

For culturing of primary neurons, 96-well Nucleocuvette AD Plates were coated with poly-D lysine overnight. Embryonic rat or mouse cortical neurons were isolated and seeded into a 96-well Nucleocuvette AD Plate at a density of 2 x 104 cells in 150 µl PNBMTM Primary Neuron Basal Medium containing PNGMTM Primary Neuron Growth Medium SingleQuots (L-glutamine, Penicillin/Streptomycin, and NSF-1, a supplement supporting neuronal growth and survival) to complement the basal medium. Half of the medium was replaced every 3-4 days. After approximately one week in culture, cells had developed a neuronal network and could be further cultured up to three weeks. Figure 1 shows cultured rat and mouse cortical neurons at 14 days stained against beta 3-tubulin and analysed by fluorescence microscopy.

Nucleofection of cells in adherence

Cells cultured in 96-well Nucleocuvette AD Plates can be transfected within the plate using the new 96-well Nucleofector AD Kits and corresponding Optimized Protocols.

As a cell line representative, HeLa cells (ATCC CCL-2TM) were seeded into a 96-well Nucleocuvette AD Plate at a density of 1 x 103 cells in 150 µl MEM Medium supplemented with 10% FCS, Penicillin and Streptomycin. Twenty-four hours after seeding, cells were transfected with pmaxGFP Vector in the 96-well Shuttle Device. Transfected cells were analyzed 24 hours post Nucleofection by fluorescence microscopy showing a typical HeLa morphology and high maxGFP Protein expression (Figure 2A).

For transfection of embryonic rat hippocampal neurons, 2 x 104 cells were cultured for 2 days after isolation in supplemented PNBMTM Medium. At 2 DIV the medium was replaced by Basic Neuron 96-well Nucleofector AD Solution and cells were transfected with 0.4 µg pmaxGFP Vector using the 96-well Shuttle Device. After Nucleofection, 160 µl medium was added to each well and replaced four hours later by fresh medium. Neurons were cultured for another 24 hours and then analysed for maxGFP Protein expression by fluorescence microscopy (Figure 2B). Transfection efficiency as determined by FACS analysis was approximately 50%.

Analysis of cells in 96-well Nucleocuvette Plates

The bottom of the Nucleocuvette AD Wells has been modified to allow analysis of cells by light and fluorescence microscopy or by plate readers for absorption, luminescence or fluorescence assays. Microscopic analysis allows for resolution of subcellular structures and assay qualities are comparable with specialized white-walled (for luminescence) or black-walled (for fluorescence) 96-well microtiter plates. No significant signal cross-talk between wells has been detected.

Figure 3 shows the performance of the 96-well Nucleocuvette AD Plate in a luminescence assay in comparison to a standard whitewalled 96-well microtiter plate. HeLa cells (ATCC CCL-2TM) were transfected with a firefly luciferase coding vector and lysed with Glo-Lysis-Buffer (Promega). A dilution series of cell lysates was applied into the wells of a 96-well Nucleocuvette AD Plate and the luciferase activity was determined using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer’s instructions.

Linearity, sensitivity and well-to-well variations were comparable to a standard white-walled 96-well microtiter plate.

Conclusion

The Nucleofector technology, known as an efficient transfection method for hard-to-transfect cells, now enters a new era allowing for direct Nucleofection of hard-to-transfect cell lines and primary cells, e.g. neurons, in adherence. Benefit from:

• Pre- and post Nucleofection culture in 96-well Nucleocuvette AD Plates which circumvents trypsinization and re-attachment of cells
• Nucleofection of neurons at a later developmental stage
• High efficiencies and viabilities
• 96-well Nucleocuvette AD Plates suited for cell analysis by microscopy or by absorption, luminescence or fluorescence assays

By Dietmar Lenz, Jenny Schroeder, and Andrea Toell; Lonza Cologne AG