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Sep 1, 2013 (Vol. 33, No. 15)

Transfection Methods Evolving

  • CHO Cell Bioproduction

    The majority of today’s biotherapeutic proteins are produced in CHO cell lines adapted to suspension growth in chemically defined media formulations. Transfecting cells that are cultured under these conditions introduces new hurdles.

    “While CHO cells have never been thought of as being a hard-to-transfect cell line, older techniques that worked well on adherent cell lines grown in serum have been found to be less efficient on suspension-adapted cells grown in serum-free, chemically defined medium,” noted Henry J. George, R&D manager at SAFC. “Electroporation routinely shows higher percentage transfection efficiencies for Sigma Aldrich CHOGS and CHO DHFR suspension-adapted cell lines.”

    While commercial production of biopharmaceuticals uses consistently productive cell lines that take many weeks to develop, the production of material from transient gene expression systems can be performed early within the drug development pipeline to generate milligram to gram amounts of representative therapeutic material for early preclinical and biochemical characterization studies. The challenge is optimizing reagents and processes for the 1–10 L (or larger) scale.

    In the past few years, there has been a significant increase in the number of commercially available reagents and protocols that can be used for larger-scale transient transfection of suspension cell lines cultured under chemically defined media conditions.

    Tempering the use of these reagents to date is the relative high cost at this scale and the need for reagent removal from the resulting biostream. Cell culture growth conditions and harvesting times for optimal production are not yet standardized protocols. Transfection efficiencies can vary significantly with the scale, cell line, expression vector or plasmid, purity of pDNA, and media.

  • Nonchemically Mediated Methods

    The age-old transfection pitfalls remain. Cells die after they are transfected, or cells remain healthy only because they are not transfected.

    “Increasing the transfection efficiency will simplify and clarify experimental answers. For example, if you have a population of cells that are only 50% affected, it can be difficult to determine the true response to treatment,” said Steve Kulisch, cell biology business unit manager at Bio-Rad.

    “Transfection efficiency rates with many immortalized cell lines are quite good. However, researchers continue to adopt additional, more challenging cell lines in their work, forcing the scientific-tools community to keep up.”

    The desire to get more efficient delivery of biomolecules forces innovation. With the vast number of cells researchers use today, the Holy Grail is to find some technology or technique that is efficacious for all cell lines with minimal amounts of optimization.

    Cellular variations include: varying lipid compositions in the plasma membranes and different cell sizes with ranging amounts of exposed surface areas. Adherent versus suspension cultures affect surface area as well. Getting the material into the cell is step one. Step two is getting that biomolecule imported to where it needs to be; some cells are more efficient at biomolecule transport than others.

    Bio-Rad pioneered electroporation as a transfection methodology and also offers biolistic devices. These devices coat small gold or tungsten microparticles with nucleic acids or proteins, then use a helium blast to accelerate the microparticles into the sample.

    Although the company’s technologies have not changed dramatically since their introduction, a recently introduced electroporation buffer assists with opening up the pores on the cells and also provides energy to help with recovery, improving electroporation transfection efficiency.

    Many basic research questions are still first addressed by transfecting easy-to-access and easy-to-culture, but also artificially immortalized or cancer-derived, cell lines. To confirm that insights gained from these “artificial” cell line systems reflect the in vivo situation, there is a need to switch to more biologically relevant models.

    For therapeutic approaches, the use of primary animal cells could reduce the number of animal tests, and use of primary human cells allows extrapolating findings from such animal models.

  • Click Image To Enlarge +
    Nucleofection™ of neurons in 24-well culture plates: Mouse cortical neurons were transfected after 6 DIV with pmaxGFP™ vector using the 4D-Nucleofector™ Y Unit. One day post Nucleofection, cells were stained by MAP2 antibody (red) and analyzed by fluorescence microscopy for maxGFP protein expression. [Lonza Pharma]

    “The challenge for transfection of biologically relevant cells, such as primary and stem cells, is to find an easy-to-use method that achieves reproducible high transfection efficiencies without major impacts on viability and functionality,” explained Andrea Toell, Ph.D., senior product manager at Lonza Pharma & Biotech-Bioscience Solutions.

    A technique that holds promise, viral transduction typically achieves high efficiencies in primary cells and allows for stable integration. But working with viruses is laborious. Some viruses may also have an influence on functionality due to their stable integration and induction of interferon responses.

    Lonza’s electroporation-based Nucleofector Technology is a nonviral technology. It achieves transfection efficiencies close to virus transduction for hard-to-transfect cells, according to a company official, who added that compared to classical electroporation, the technology achieves much higher viabilities for primary cells and higher efficiencies with smaller amounts of DNA.

    Transfection reagents require cell proliferation for transferring the DNA into the nucleus for expression, while the Lonza technology allows transfection of nondividing cells, such as unstimulated T cells or neurons, as the DNA is directly transferred to the nucleus.

  • Large-Scale Transfections

    Another key aspect of transient transfection innovation is the ability to simply and cost-effectively scale transfections without sacrificing performance or primary cell compatibility.

    “As more biologically relevant cell systems are developed, such as primary cells, researchers want to adopt these within biotherapeutic and drug development activities,” said Karen A. Donato, Ph.D., executive vp, global business development and marketing, at MaxCyte.

    MaxCyte flow electroporation was originally developed for ex vivo cell transfection, which requires reliable, high-efficiency transfection of primary cells (such as human embryonic or adult stem cells, human T cells, or dendritic cells) in large numbers with rigorous safety standards.

    Flow electroporation melds the benefits of electroporation with simple scalability, which is advantageous in the fields of small-molecule and therapeutic protein development, explained Dr. Donato.

    The MaxCyte electroporation system performs transfections of 5 × 105 cells in seconds to 2 × 1011 cells in less than 30 minutes, she continued, adding that the scalable approach results in high viability and high efficiency, which in turn leads to high expression of the protein of interest.

    “Speed, reliability, and reproducibility are all going to become increasingly important. Pharmaceutical companies are looking for technologies that can help them make better decisions up front in the development process with fewer resources, in less time. Transient transfection is becoming a significant part of the drug development toolkit,” concluded Dr. Donato.

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