Monoclonal Cell Line Development – What Method Should You Use?

Monoclonal cell lines are a powerful tool for biomedical research and drug development. The development and use of monoclonal cell lines have been accelerated due to the advent of breakthrough gene editing technologies such as CRISPR-Cas9.   Due to their ease of use and amenability to single cell cloning, there are several well-established and recognizable cell lines that are commonly used to create monoclonal colonies, including the CHO and HEK293 lines.  However, primary cells such as induced pluripotent stem cells (iPSCs), or immortalized cancer cell lines have proven more challenging. Historically, monoclonal cell lines have been developed using traditional methods, such as flow cytometry or manual limiting dilution.  Although widely utilized, these methods present both technical and biological challenges, and technologies such as the CellRaft Technology offer several profound advantages over traditional methods. In this blog post, we will provide an overview of available technologies for monoclonal cell line development and how researchers can overcome some of the limitations faced with these technologies.


Traditional Methods for Developing Monoclonal Cell Lines

Monoclonal cell lines can be generated with the incorporation of traditional methods such as limiting dilution, single cell dispensing, flow cytometry, cell sorting, and cell dispensers. A table comparing these methods can be found in the white paper titled Development of Monoclonal Cell Lines – Available Technologies and Overcoming Challenges. Unfortunately, each of these methods is not without limitations. While limiting dilution is seen as a lower-cost option, it is time-consuming, lacks proof of monoclonality, requires sample preparation, and drives a tremendous amount of nonbiodegradable recalcitrant plastic waste. Furthermore, two rounds are minimally recommended to be able to approach monoclonality and single cell confirmation. The incorporation of laboratory equipment over recent years has improved throughput and reduced the hands-on time that is required with limiting dilution, but many are expensive to incorporate, require extensive training, and can result in phenotypically perturbed cells that have poor viability and outgrowth.

Image 1 Monoclonal Cell Line Blog

Figure 1: Current methods for single cell cloning. The majority of technologies available on the market separate a heterogeneous population of cells into single cells within a microwell, either via manual pipetting or fluidics-based segregation.


Overcoming Limitations of Current Technologies

CellRaft Technology is an affordable and modern method for developing monoclonal cell lines that overcomes the limitations of traditional methods. It provides flask-like culture conditions at the resolution of a single cell, with gentle and automated isolation using image-based attributes for function, gene expression, and morphology. Within a single platform, researchers can grow, scan, analyze, and isolate single cell derived monoclonal colonies.

Image 2 Monoclonal Cell Line Blog

Figure 2: CellRaft Technology workflow.  The advantage of the CellRaft technology is that cells are seeded as a population while maintaining single-cell separation. Cells can grow in situ on individual CellRafts to form discrete colonies that can be identified using image-based software analysis tools.  After growth on the array, the monoclonal colony, rather than a single cell, is isolated intact and transferred to a downstream collection plate for continued growth, expansion, and downstream analysis.


Benefits of CellRaft Technology

The use of CellRaft Technology offers several advantages over traditional methods for developing monoclonal cell lines including:

  • proof of monoclonality
  • highly viable monoclonal colonies
  • cost savings
  • ease of use

Image 3 Monoclonal Cell Line Blog

Figure 3:  Track-and-trace colony growth from a single cell.  Three representative CellRafts containing single cells on day 1 and imaged on the CellRaft AIR System over a 4-day period until colony formation.  This serial imaging allows for precise monitoring of exponential growth and phenotypic characterization, as well as improved viability downstream.


One of the most critical components of cell line development is proof of monoclonality, and as the saying goes “a picture is worth a thousand words”. The CellRaft AIR can image an entire CellRaft array in as little as 6 minutes in brightfield, providing a saved image of every single CellRaft for impeccable record keeping and the ability to track and trace single cells from seeding to colony formation (Figure 3).  In addition, because the single cells are individually segregated within the array but share a contiguous media volume, viability and clonal growth are significantly improved compared to methods that put a single cell in a well. This key feature leads to hundreds to thousands of clonal colonies to choose from, ensuring that the researchers are able to get exactly the phenotypic characteristics they desire, rather than settling for whichever clones survive.  Lastly, the CellRaft technology is user-friendly, easy to learn, and requires a very low barrier to entry compared to instrumentation such as flow sorters.


Table 1:

Image 4 Monoclonal Cell Line Blog



In summary, monoclonal cell line development is an important step in biomedical research that has traditionally relied on time-consuming and labor-intensive techniques or high-cost alternatives that produce low-viability cell populations. However, CellRaft Technology offers significant advantages over traditional methods due to its affordability, speed, accuracy, and scalability capabilities, as well as its overall improvement of cell viability and clonal growth. For scientists struggling to find an efficient and successful method for growing and recovering high-quality monoclonal cell lines, CellRaft Technology is a novel solution that will improve overall research productivity and progress.

For a deeper look comparing technologies, download the white paper, Development of Monoclonal Cell Lines – Available Technologies and Overcoming Challenges.



Kap Kumar 2023
Kap Kumar, Ph.D., MBA
Vice President of Strategic Marketing |

Kap Kumar has over 25 years of experience in the life sciences tools and reagents industry. He started in R&D and product development, where he launched products for cell biology and imaging applications. For the last 15 years, Kap has led strategic marketing, market development, and product management for a variety of companies, including Thermo Fisher Scientific (Life Technologies), Danaher (Beckman Coulter Life Sciences), Cell Signaling Technologies, Nexcelom Biosciences, and Avantor-VWR. Kap has diverse experience managing complex portfolios, including instruments, consumables, and reagents, both in early-stage and mature companies. Kap has a Ph.D. in Cell and Molecular Biology from Kent State University, a post-doctorate from Harvard Medical School, and an MBA from Babson College.

Related Posts

Revolutionizing Electrophysiology: Embracing Automated Patch Clamp for Affordable High-Throughput Solutions
Revolutionizing Electrophysiology: Embracing Automated Patch Clamp for Affordable High-Throughput Solutions
Manual Patch Clamping: An Art Form In the 1990s, if you wanted to study the activity of ionotropic receptors across…
DIY breakdown: Advantages and Disadvantages of Do-it-yourself Flow Systems
DIY breakdown: Advantages and Disadvantages of Do-it-yourself Flow Systems
Mechanical forces have been shown to impact cell phenotype and function in vivo. The most prolific of these forces is…
Go with the Flow: Shear Stress Enhances Physiological Relevance
Go with the Flow: Shear Stress Enhances Physiological Relevance
In vivo, most cell types exist surrounded by moving fluid that influences their phenotype and function. The flow created from…