Cell line development (CLD) is an important means of producing proteins, biological therapeutics, and other biomolecules. A crucial step in the process is selecting robust, healthy colonies that originate from a single cell, and then verifying the monoclonal status.
Traditional methods of single-cell isolation, including the limiting dilution method, single-cell sorting by FACS, and single-cell printing, can be labor-intensive and expensive. The limiting dilution method requires significant time processing many plates and often fails to produce viable, monoclonal colonies. FACS for single-cell sorting and single-cell printing compromise cell health and viability by stressing the cells, namely with sheer force or isolation.
This article explains how a new CLD method known as high-throughput nanowell-based image-verified cloning (HT-NIC) addresses these limitations by speeding up the CLD process and producing colonies that are 100% verified monoclonal and have better outgrowth rates.
The HT-NIC method increases not just the speed, but also the overall throughput of CLD compared to traditional techniques. Both HT-NIC and the popular limiting dilution method begin by seeding multiwall culture plates with a single-cell suspension and allowing single cells to settle into the wells according to Poisson distribution. The key to the throughput advantage, however, lies in the plates.
HT-NIC uses nanowell plates, which are 6- or 24-well cell culture plates containing thousands of tiny (e.g., 200 µm) nanowells at the bottom of each well. Nanowells can effectively isolate single cells within a much smaller area. For example, a 24-well plate has about 4,000 nanowells per well. Therefore, depending on seeding conditions, one nanowell plate may contain up to 14,000 seeded single cells, with 400–600 single cells per well.
This means that a single well from a 24-well nanowell plate can yield up to 500 target clones. By comparison, getting this many clones using the limiting dilution method requires over two dozen 96-well plates.
The HT-NIC method also increases throughput by simplifying the overall CLD workflow. It automates and combines steps, such as colony selection and transfer, and integrates the verification of monoclonality and assessment of viability via automated imaging. This accelerates the process to give you viable, monoclonal, productive colonies in less than a week.
Monoclonality of the producer cell is essential to the efficacy and safety of a biologic. In traditional workflows, single cells often settle at the edge of a well. This precludes imaging, forcing researchers to check for monoclonal status either manually, or retroactively after outgrowth. The ability to verify monoclonality using automated imaging with HT-NIC spares researchers from wasting time and resources on polyclonal colonies.
Following the initial seeding, each cell receives a unique identifier and is tracked through growth, colony assessment, and colony picking. After a few days, nanowell plates are automatically scanned (e.g., for expression of fluorescent markers) and ranked for viability and monoclonal status. Importantly, the HT-NIC method captures all single cells, even those at the nanowell borders. Clones verified as monoclonal and healthy are automatically picked and transferred to multiwell plates for further expansion.
Quality control is a standard part of this process from start to finish. Images are acquired both before and after every colony selection event and complete workflow documentation is available for GLP and GMP compliance.
Cultured cells are more likely to thrive in each other’s company where they have access to natural growth factors shared through the medium. In contrast to traditional cell culture plates, the HT-NIC method supports cell health and outgrowth rates due to the unique architecture of its nanowell plates.
Although single cells are physically isolated within tiny nanowells, they can maintain chemical communication through the shared medium inside the well. Using nanowell plates enhances cell health and increases the likelihood of success with difficult-to-grow cell types, while maintaining the monoclonality of all single cells.
CLD is core to biomolecule development, but also a regular bottleneck due to the challenging nature of identifying viable, monoclonal cell lines. The HT-NIC method relieves three of the main pain points in this process: throughput, viability, and monoclonality. After one round of cloning, the HT-NIC method yields verified monoclonal colonies with better health and stronger outgrowth rates compared to results obtained with traditional CLD methods—and it achieves all this in less than one week.
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