Genetic Engineering & Biotechnology News

JUL 2018

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10 | JULY 2018 | Genetic Engineering & Biotechnology News | GENengnews.com Single-Cell Cloning Remains a Challenge Patricia Fitzpatrick Dimond, Ph.D. Regulatory agencies say that recombinant proteins intended for therapeutic use must be produced from a cell line derived from a single progenitor cell. In addition, single-cell cloning has gained increasing importance as genome editing techniques have entered routine laboratory practice. Pure clonal isolation from a single progenitor cell is a criti- cal step in the genetic and functional characterization of mu- tations achieved by targeted genome editing methods such as those incorporating ZFNs, TALENs, and CRISPR-Cas9. These nuclease-driven genome editing tools can be used to introduce a change at a specific genomic location, either to cause a mutation or to insert an exogenous sequence. However, culturing edited cells results in a heterogeneous polyclonal cell population requiring generation of an edited cell line containing a homogeneous genetic background to retain the desired genotype and to facilitate downstream phe- notypic characterization. While traditionally the most laborious and time-consum- ing step in CRISPR-based genome engineering using cell models, the generation of clonal mutant cell lines, investiga- tors say, is absolutely required to draw any solid conclusions correlating a given mutation and cellular behavior. In the development of biopharmaceutical production cell lines, current workflows have drawbacks that include re- duced cell viability, inefficient single-cell isolation, and lim- ited evidence for clonality. Chinese hamster ovary (CHO) cells remain the major cells of choice for the manufacture of therapeutic recombinant proteins. However, one of the major challenges in CHO cell line generation remains efficient identification of high-pro- ducing clones among a large population of low-producing or non-productive clones. It is not unusual for several hundred individual clones to be screened for the identification of a commercial clonal cell line with acceptable productivity and a growth profile appropriate for large-scale production. Isolation Techniques Limiting dilution, the traditional method for isolating single cells, relies on statistical probabilities for monoclonal- ity that can vary significantly with slight changes to proto- cols. The technique, while highly inefficient at isolating single cells, preserves cell viability. Conversely, flow cytometry can provide strong evidence of monoclonality and high efficiency but negatively affects cell viability. Proponents of cloning in semisolid medium, in which cells are suspended in viscous medium, say it leads to the formation of easily isolated colonies and that it can be used to isolate monoclonal cell lines in less time and using fewer resources compared with limiting dilution selection and clon- ing. Solutions range from media tweeks to the use of systems that "print" single cells and produce sequential images to show that a cell colony did indeed arise from a single cell. For example, Molecular Devices' CloneSelect™ Single-Cell Printer™ (SCP) utilizes microfluidics technology and real-time image analysis to sort and deposit single cells into standard mi- croplates. The system can print a 96-well plate in 5 to 10 min- utes and works with 384-well plates. Five images capture single cells as they are dispensed, providing image-based evidence of monoclonality that can be used in regulatory filings. Cell imaging systems have been evolving to meet imaging demands, enabling better imaging that permits identification of single-cell clones, the ability to see a single cell on the day of seeding, and better visibility along well edges. "Single-cell cloning is, in part, a response to FDA guid- ance," says Tim Smith, technical product manager at Nex- celom. Nexcelom designs, manufactures, and markets its Cellometer and Celigo image-cytometry products for cell analysis in life sciences and biomedical research. Smith notes that guidance documents from the FDA and the International Conference on Harmonization of Techni- cal Requirements for Registration of Pharmaceuticals for Human Use state that the cloning procedure should be fully documented, with details of imaging techniques and/or ap- propriate statistics. For proteins derived from transfection with recombinant plasmid DNA technology, a single fully documented round of cloning is sufficient, provided that product homogeneity and consistent characteristics are "demonstrated throughout the production process and within a defined cell age beyond the production process," noted Genentech scientists (Shaw et al.) in Biotechnology Progress. Nexcelom's Celigo imaging system provides an image from day zero, showing only a single cell in the well and creating documented proof of single-cell clonality. "Single- cell cloning is becoming much more popular due to regula- tions and people have seen benefits," Smith asserts. "With the technology available now, it's a lot easier. You have an image in two minutes before you have to go through genetic characterization, allowing much higher throughput. "The unique thing about our instrument is the way the optics are set up. We can image an entire 96-well plate very fast without having to move the plate, capturing 16 images of a single well in milliseconds, and we can do 96 wells in two minutes." Iris Lindberg, Ph.D., professor of neurobiology and anatomy at the University of Maryland Medical School, tells GEN that her lab's method of picking desired clones arose from the need to speed up cloning of individual colonies de- rived from cell lines that are difficult to efficiently transfect, notably endocrine cells. Dr. Lindberg is particularly interested in neuropeptide and peptide hormone maturation in neural and endocrine cells; she first used the technique to isolate proenkephalin-overex- pressing AtT-20 cell clones (after antibiotic selection). Rather than use cloning rings—which Dr. Lindberg referred to as Drug Discovery Generation of clonal cell lines is an absolute requirement for investigators to draw solid conclusions when correlating induced mutations or cellular behavior. Kateryn Kon / Science Photo Library / Getty Images

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