Finding the Holy Grail of Cell Line Development: Viable, Monoclonal Cells

When it comes to manual laboratory procedures, single-cell selection is near the top of the list. In this blog post we talk about how an automated single-cell selection and retrieval platform - CellCelector - is simplifying workflows and freeing researchers’ time to tackle hard scientific questions and develop life-saving therapies.

^{This article is posted on our Science Snippets Blog}

Challenges in cell line development 

Cell line development is the foundation for 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 and cloning are limiting dilution, single-cell sorting by FACS, and single-cell printing. But these are labor-intensive and expensive.

• Limiting dilution: takes a lot of time processing many plates and often fails to produce viable, monoclonal colonies. 
• FACS and single-cell printing: compromise cell health and viability by stressing the cells, either with sheer force or isolation. 

A new method known as high-throughput nanowell-based image-verified cloning (HT-NIC) speeds up the cell line development process by producing colonies that are 100% verified monoclonal, with better outgrowth rates. 

Higher throughput speeds up cell-line development 

The key to the throughput advantage with HT-NIC is the nanowell plates. Nanowell plates are 6- or 24-well cell culture plates that have 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 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 more than two dozen 96-well plates. 

HT-NIC also increases throughput by simplifying the overall cell line development workflow. It not only automates and combines steps, like colony selection and transfer, but also uses automated imaging to verify monoclonality and viability. What you get are viable, monoclonal, productive colonies in less than a week. 

Depending on the plate type, there are between 4,300 to 22,000 nanowells per well, or between 100,000 to 130,000 nanowells per plate.

Verified monoclonality with automated imaging 

Monoclonality of the producer cell is extremely important when trying to manufacture a safe and reliable biologic. In traditional workflows, scientists check for monoclonal status either manually or retroactively after outgrowth. With HT-NIC, this is done automatically. 

After the initial seeding, each cell gets a unique ID and is tracked through growth, 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. Clones verified as monoclonal and healthy are automatically picked and transferred to multiwell plates for further expansion. Importantly, the process supports compliance with full documentation and images acquired both before and after colony selection. 

With the CellCelector HT-NIC approach, cells are separated within 200 µm sized nanowells and automatically identified by software.

Healthier cells with stronger outgrowth

Cultured cells are more likely to thrive together 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 crosstalk through shared medium inside the well. Using nanowell plates improves cell health and increases the likelihood of success with difficult-to-grow cell types, while maintaining the monoclonality of all single cells. 

In the CellCelector HT-NIC method, physically isolated single cells are grown to 20–75 cells per clone. Monoclonal, viable clones are automatically picked and transferred into 96- or 384-well plates for further analysis.

Conclusion

Cell line development is core to biomolecule development. However, it is 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 produces verified monoclonal colonies with better health and stronger outgrowth rates compared to traditional cell line development methods - all in less than one week.