Engineered cell lines have become the workhorse of many labs, including those seeking to better understand basic biology, model diseases, and develop therapeutics. “Cells allow researchers to more accurately reflect a disease state and then test drugs, stratify patients, or investigate mechanisms,” explains Jamie Freeman, product manager of bioproduction at Horizon Discovery.
But just because cell-based research is commonplace in life sciences studies doesn’t mean engineering cell lines is easy. Gail Seigel, research associate professor at the University at Buffalo, has been engineering cell lines since 1994, including an R28 retinal precursor cell line and a Mocha cochlear microglial cell line developed in 2017. She notes that cell-line generation can be difficult, costly, time-consuming, and not always successful. However, armed with a solid understanding of current cell biology and cell-characterization techniques along with the latest in genome-editing technologies, researchers can confidently create desired cell lines in less time and more accurately than ever before.
Image: Mocha cells are a rat cochlear microglial cell line. They were developed from an immunomagnetically enriched population of CD11b+ cells from P3 rat cochlea and immortalized with the 12S E1A gene of adenovirus in a replication-incompetent retroviral vector.
Understanding cell biology
Discussing the initial steps of cell-line engineering, Robert Newman, senior director of R&D for ATCC, emphasizes that “the molecular aspect of cell-line engineering is a significant challenge, but just as important is the cell biology aspect, which is affected by the starting cell lines, media, extracellular matrix components, and overall cell culture environment. This system should be optimized to increase the likelihood of creating a successful in vitro model.”
Before any cell line can be engineered effectively, it must be suitably characterized applying the fundamentals of cell biology. Cell properties, environment, and potential phenotypic alterations can influence the engineering process. Media conditions can affect cell growth rates and metabolism, which can be important in bioproduction to increase yield of biotherapeutic antibodies or growth factors. In disease modeling, cell culture environment is a consideration because cell lines used to model a physiological disease should mimic in vivo environments.
Still, one of the main challenges in cell-line engineering remains the transfectability or transductabilty of cells.
In addition to optimizing cell culture conditions to address cell growth and characterization, methods should be optimized to introduce genetic material into the cells. “Different cell types can be receptive to different types of transfection and transduction techniques,” explains Newman. By testing several methods early in the development process and optimizing conditions around the most effective method, researchers can increase the overall efficiency of their cell-engineering process. Still, one of the main challenges in cell-line engineering remains the transfectability or transductabilty of cells. Selecting the most compatible technique for a cell line, from lipid-based transfection and electroporation to viral delivery and naked genetic transfer, can mean the difference between progression and a stalled experiment.
Immortalizing cells enables further research on primary cell types, in particular those that usually have limited lifespans. Several techniques for immortalization, including hTERT and SV40, have been useful in developing cells that demonstrate comparable phenotypes with their primary cell counterpart. However, Siegel points out that “Some genes utilized to immortalize cell lines can cause malignant transformation (eg. SV40 T antigen), which makes them less optimal for in vivo studies, unless one is studying tumor biology. Also, the process of immortalization, regardless of method, may change the phenotype of the original primary cell. For that reason, one always needs to characterize a new cell line and compare with non-immortalized matching primary cells whenever possible.”
Genome editing takes the stage
Gene editing is a rapidly developing field, advancing techniques including random integration, gene amplification, homologous recombination, RNAi, zinc finger nucleases, TALENs, and, of great interest these days, CRISPR.
Successful creation of the initial double-strand break in chromosomes can deliver high efficiency and high specificity of editing, easing downstream processes. Thermo Fisher Scientific is working to improve both TALENS and CRISPR efficiency, developing TrueCut Cas9 V2 protein paired with TrueGuide synthetic guide RNA as editing tools and transfecting by electroporation using their Neon platform or a lipid-based approach like CRISPRMAX. Offering a complete toolbox to increase efficiency of double-strand breaks helps to adapt the process to numerous applications including array or knockout-based screening.
Seigel describes CRISPR genome-editing systems as “cost-effective, customizable, and reliable.” They can target multiple genes simultaneously, which allows for considerable flexibility. She adds, “CRISPR genome editing also allows for evaluation of non-coding genetic elements not possible by RNAi methods. These advantages allow investigators to develop cell and animal disease models relatively quickly to better understand gene function.”
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But can CRISPR do it all? It turns out CRISPR doesn’t work well for the entire genome. “For certain parts of the genome, TAL effectors can work better than CRISPR, particularly for knock-in editing,” says Jon Chesnut, senior director of synthetic biology R&D at Thermo Fisher Scientific. Relying on Protospacer adjacent motif (PAM) cleavage sites, CRISPR DNA insertion efficiency decreases the farther the edit is from a PAM site. Alternatively, TAL effectors can be designed to target any sequence within the genome, providing more surgical and precise cleavage. “We’ve found that between 20–40% of the genome does not have PAM sites close enough to the intended edit to make CRISPR efficient enough, so TALENs can be beneficial in those areas.” Chesnut adds that CRISPR is still optimal for larger inserts using standard length homology arms, where preciseness is not as critical.
Horizon Discovery provides custom-engineered isogenic cell lines that aim to more accurately reflect a disease state. Horizon’s original approach to cell-line engineering centered around rAAV, or recombinant adeno-associated virus, which uses homologous recombination-based mechanisms. While this technique is fairly low throughput, particularly compared to new techniques such as CRISPR, its precision offers base pair resolution engineering and, similar to TALENs, offers an alternative approach if CRISPR is not suitable.
Horizon is also adept at newer technologies such as CRISPR that achieve cell-line generation at a higher output. This has allowed the creation of thousands of edits across hundreds of cell lines, incorporating a large body of experience surrounding cell growth and transfection characteristics identified by Newman as critical to the success of an engineering project. “Combining engineering and screening knowledge has allowed the development of CRISPR-based screening, with CRISPRko, CRISPRi, and CRISPRa approaches allowing high-throughput objective interrogation of the genome,” explains Freeman.
As well as custom gene editing, Horizon also provides thousands of engineered lines available for immediate use. For example, they have developed a panel of cell lines ideal for modeling the effects of K-Ras mutations. K-Ras is a small GTP-binding protein and a common upstream activator of several signaling pathways, in which hyper-activation can result in key hallmarks of cancer such as growth factor independence and aberrant cell proliferation, survival, and motility. Ras mutations are very common in human cancers, so using these edited cell lines to understand the role of mutant K-Ras in modulating cellular processes can enable successful development of novel therapeutics.
Application explosion
Cell-line engineering can allow both the introduction of disease-causing mutations into wild-type parental backgrounds, as well as the reversion of putative disease-causing mutations to wild type, allowing very comprehensive studies to be done into the mechanisms of cell biology, disease progression, and therapeutic sensitivity.
In cell therapy applications, T-cell editing is growing immensely. Thermo Fisher has used its newest CRISPR RNP reagents to knock out a T-cell receptor in patient-derived cells with up to 95% efficiency in a single transfection. Seigel mentions that clinical trials are planned in the near future, in which CRISPR-edited human T cells will be used to augment cancer therapy, while CRISPR-mediated therapeutic approaches for sickle cell anemia and severe combined immune deficiency (SCID) are also in the pipeline. Primary cells that require the highest efficiency tools could also open a whole new world of engineering, even in patient-specific editing, suggests Chesnut.
Cell-line engineering has also done wonders for stem cell applications, particularly using iPSCs. These cells grow more slowly than other cell types, but technologies to engineer and analyze pluripotent stem cells provide the opportunity to edit at the stem cell stage and then differentiate edited cells into tissues for specific disease modeling. Chesnut postulates that these new editing technologies also might make possible personalized cell models, where one’s own iPSCs could be edited and differentiated to cell types such as neurons and cardiomyocytes.
We still have work to do
Cell-line engineering is a fascinating and exciting field, especially as these new technologies take hold. But of course, there is still work to be done. Streamlining the perpetually time-consuming process is underway, with innovations already being seen in pre-engineered cell lines and ready-to-use reagents Improvement remains sorely needed in post-editing clone isolation.
Dealing with off-target effects in gene editing, where additional non-specific double-strand breaks generate extraneous repair, can cause unrelated and potentially problematic issues such as deactivation of a tumor-suppressor gene or activation of a cancer-causing one. Researchers are now working to identify and map off-target effects by sequencing to alert users of potential issues. One such technology is Genome-wide Unbiased Identifications of DSBs Evaluated by Sequencing (
), a novel method developed to identify the off-target sites of CRISPR-Cas RNA-guided Nucleases by Keith Joung’s lab at Harvard Medical School.
Image: Micrograph of Neural Progenitor Cells (ATCC® ACS-5003™) differentiated into oligodendrocytes and stained with antibodies to the oligodendrocyte marker O4.